Novel mRNA of coronavirus S protein and application thereof

Abstract

The invention provides RNA (Ribonucleic Acid) for coding novel coronavirus S protein, a vaccine containing the RNA and application of the RNA. Also provided is a universal polynucleotide molecule comprising 5 '-UTR and / or 3'-UTR, and a nucleic acid sequence encoding a protein and / or polypeptide of interest, and optionally comprising polyA.

Description

Novel coronavirus S protein mRNA and its application Technical Field

[0001] The present invention belongs to the field of biopharmaceuticals. Specifically, the present invention relates to RNA encoding the novel coronavirus S protein, vaccines containing such RNA, and their uses. The present invention also relates to a universal polynucleotide molecule for preparing a novel mRNA vaccine, comprising a 5'-UTR and a nucleic acid sequence encoding a polypeptide of interest, and optionally comprising a 3'-UTR and / or polyA. Background Art

[0002] The novel coronavirus (SARS-CoV-2) can cause severe infectious disease and is highly contagious. The International Committee on Taxonomy of Viruses (ICTV) has named it severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Currently, some inactivated, live-attenuated, and recombinant protein subunit vaccines are available for this virus, with some success. However, SARS-CoV-2 continues to mutate and evolve, resulting in the emergence of many new strains with increased infectivity and pathogenicity. These new mutants evade the antibodies produced by existing vaccines. Therefore, developing an effective COVID-19 vaccine that can protect against these mutant strains remains the best and most pressing approach to controlling or even completely eradicating the epidemic.

[0003] SARS-CoV-2 belongs to the Coronaviridae family. Coronavirus particles are spherical or elliptical, with an envelope containing spikes. The genome within is single-stranded positive-sense RNA (ssRNA), resulting in the entire virus particle resembling a crown or sun corona under an electron microscope. The viral genome can be divided into two main categories based on their function: open reading frames (ORFs) ORF1a and ORF1b, which primarily encode non-structural proteins such as enzymes involved in viral replication and transcription; and gene sequences encoding structural proteins, such as the spike protein (S), envelope protein (E), membrane protein (M), and nucleocapsid protein (N). The S protein recognizes and binds to the host cell surface receptor angiotensin-converting enzyme 2 (ACE2), mediating fusion between the viral and host cell membranes. The S protein is also the primary antigen that induces both humoral and cellular immune responses, particularly the humoral immune response. Therefore, the S protein is suitable as a target for developing a vaccine against the new coronavirus.

[0004] Messenger RNA (mRNA) is a single-stranded RNA molecule. It is produced using genomic DNA sequences in the cell nucleus as a template and then exported to the cytoplasm, where it is translated by ribosomes to produce specific proteins, thereby exerting its biological effects. Mature mRNA primarily consists of several structures: a 5'-cap structure, a 5'-UTR, a coding region, a 3'-UTR, and a poly(A) tail. The 5'-cap structure is crucial for ribosome recognition and protection of the mRNA molecule from RNases. The untranslated regions, the 5'-UTR and 3'-UTR, control the stability and translation efficiency of the mRNA molecule, with the 3'-UTR even determining its cytoplasmic localization. The coding region, primarily composed of codons, is encoded by the ribosome and translated into protein. The start codon is typically AUG, and the stop codon in eukaryotic cells is typically UGA. The poly(A) tail, a long sequence of adenine nucleotides, facilitates nuclear export and translation and protects the mRNA from degradation.

[0005] Compared with recombinant protein subunit vaccines, inactivated vaccines, or DNA vaccines, mRNA vaccines have several distinct advantages. First, because mRNA does not infect the body or integrate into genomic DNA, its safety is greatly improved. Second, the use of modified bases can reduce the inherent immunogenicity of mRNA molecules and reduce their degradation by the body, further improving the safety of mRNA vaccines and the stability of mRNA. In addition, the yield of in vitro mRNA synthesis is very high, so mRNA vaccines have the potential to be efficient, rapidly developed, low-cost to manufacture, and easy to manage safely. For controlling new virus outbreaks, people can develop and mass-produce mRNA vaccines against new viruses more quickly, so compared with traditional vaccine products, mRNA vaccine research and development has great potential.

[0006] Since its emergence, SARS-COV-2 has mutated and evolved into many different variants, including Alpha (such as the B.1.1.7 variant and its descendant lineages), Beta (such as the B.1.351 variant and its descendant lineages), Gamma (such as the P.1 variant and its descendant lineages), Delta (such as the B.1.617.2 and AY lineages), and Omicron. With the widespread spread and prevalence of mutant lineages such as Delta and Omicron around the world, the efficiency of neutralizing antibodies in existing vaccines has been significantly reduced, resulting in a weakening of the protection induced by existing vaccines, increased transmissibility and disease severity, and has become a variant of concern (VOC) worldwide. Therefore, the rapid development of new mRNA vaccines against novel coronavirus variants is extremely challenging and of great significance.

[0007] The untranslated region (UTR) of mRNA controls the translation, degradation and localization of genes, including the stem-loop structure, upstream start codon, upstream open reading frame, internal ribosome entry site and various cis-acting elements that bind to RNA-binding proteins.

[0008] UTRs play a crucial role in post-transcriptional regulation of gene expression, including regulating mRNA nuclear export and translation efficiency, subcellular localization, and stability. UTRs may also play other roles, such as the specific incorporation of the modified amino acid selenocysteine ​​at the UGA codon in mRNAs encoding selenoproteins, a process mediated by a conserved stem-loop structure in the 3'-UTR.

[0009] Those skilled in the art know that the translation efficiency of mRNA molecules directly affects the dosage and dosing interval of mRNA drugs (especially mRNA vaccines), ultimately affecting the bioavailability of mRNA drugs and determining the clinical application value of mRNA drugs. Although there are technical solutions for increasing the translation efficiency and stability of mRNA molecules, such as by adding untranslated regions (UTRs) to improve the translation efficiency of mRNA molecules, there is still a need for further improvement in improving the translation efficiency of mRNA molecules.

[0010] Therefore, there is a need to identify or design UTRs that can achieve higher mRNA translation efficiency and / or stability.

[0011] Summary of the Invention

[0012] The inventors of this application unexpectedly discovered that by analyzing the mutation sites of the novel coronavirus, they designed a new RNA coding sequence for the novel coronavirus S protein and used it to prepare a novel mRNA vaccine. This mRNA vaccine has cross-protection against different strains, including the novel coronavirus wild-type SARS-CoV-2, as well as the Delta and Omicron variants, and has a strong immune effect against the Delta and Omicron variants, and can be developed and produced more quickly on a large scale.

[0013] The inventors of the present application also unexpectedly constructed a universal polynucleotide molecule for preparing a new mRNA vaccine, which comprises a 5'-UTR and a nucleic acid sequence encoding a protein and / or polypeptide of interest, and optionally comprises a 3'-UTR and / or polyA. These innovative designs improve the level of mRNA expression protein. The polynucleotide molecule can not only be used to prepare an mRNA vaccine with a good immune effect on the new coronavirus wild-type SARS-COV-2, Delta and Omicron variants, but can also be used to transfect cells or bacteria to produce corresponding antibodies or antigenic proteins, and is therefore a universal core component combination.

[0014] In addition, the inventors of the present application have identified a variety of 5'-UTRs and / or 3'-UTRs that can improve the translation efficiency and / or stability of mRNA molecules, as well as RNA (such as mRNA) containing the UTRs. The above-mentioned UTRs are universal core elements, which give RNA (such as mRNA) containing the UTRs enhanced translation efficiency, significantly improve the expression of target genes and / or mRNA stability, and have broad application value in the industrialization of RNA drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1A is a flow chart for the construction of plasmid H;

[0016] Figure 1B is a flowchart of engineering plasmid construction;

[0017] Figure 2 is a plasmid map of luciferase-pcDNA3;

[0018] FIG3 is a plasmid map of plasmid B of the present invention;

[0019] FIG4 is a plasmid map of plasmid C of the present invention;

[0020] FIG5 is a plasmid map of plasmid D of the present invention;

[0021] FIG6 is a plasmid spectrum of plasmid F in the present invention;

[0022] FIG7 is a plasmid spectrum of plasmid G in the present invention;

[0023] FIG8 is a plasmid spectrum of plasmid H in the present invention;

[0024] FIG9 is a spectrum of the engineered plasmid of the present invention;

[0025] Figure 10A is a schematic diagram of the construction and transformation of plasmid H;

[0026] Figure 10B is a schematic diagram of the engineering plasmid construction and transformation;

[0027] FIG11 shows the results of in vivo liver imaging in mice injected with different 5'-UTR mRNAs;

[0028] FIG12 is a summary of the average photon counts of in vivo liver imaging in mice injected with different 5'-UTR mRNAs;

[0029] FIG13 shows the results of in vivo imaging of mouse liver produced by fixed 5'-UTR and different 3'-UTR combinations;

[0030] FIG14 is a summary of the average photon counts of in vivo mouse liver imaging produced by fixed 5'-UTR and different 3'-UTR combinations;

[0031] FIG15 shows the results of in vivo liver imaging in mice screened for polyA;

[0032] FIG16 is a summary of the average photon counts of in vivo liver imaging in polyA-screened mice;

[0033] FIG17 is a western blot result of the antigen protein expressed in cells transfected with the engineered plasmid;

[0034] Figure 18 shows the FACS results of antigen protein expression in cells transfected with engineered plasmids and novel coronavirus mRNA;

[0035] Figure 19 shows the ELISA results of novel coronavirus mRNA expression levels in transfected cells;

[0036] Figure 20 shows the results of IgG antibody testing against the Delta variant S protein in mouse serum after immunization with the novel coronavirus mRNA vaccine preparation;

[0037] Figure 21 shows the results of an in vivo immunization experiment with the novel coronavirus mRNA and a neutralization test against a SARS-COV-2 pseudovirus;

[0038] Figure 22 shows the results of an in vivo immunization experiment with the novel coronavirus mRNA and a neutralization test against the Delta pseudovirus;

[0039] Figure 23 shows the results of IgG antibody testing against the S protein of the Omicron variant induced by the novel coronavirus mRNA vaccine in vivo in mice;

[0040] Figure 24 shows the results of neutralizing antibody testing against Omicron (BA.2) pseudovirus induced by the novel coronavirus mRNA vaccine in vivo in mice;

[0041] Figure 25 shows the results of Elispot (IFN-γ) testing of spleen lymphocytes in mice after in vivo immunization with the novel coronavirus mRNA vaccine;

[0042] FIG26 shows the results of in vivo liver imaging of mRNAs containing individual 3UTR-NO3, 3UTR-NO30, and 3UTR-NO50;

[0043] FIG27 contains the summary results of the average photon number of in vivo liver live imaging of individual 3UTR-NO3, 3UTR-NO30, and 3UTR-NO50 mRNAs. DETAILED DESCRIPTION

[0044] To make the objectives, technical solutions, and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other implementations obtained by those of ordinary skill in the art without inventive effort are within the scope of protection of the present invention.

[0045] definition

[0046] All patents, patent applications, scientific publications, manufacturer's instructions and guidelines, etc., cited herein, whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein should be construed as an admission that the present disclosure is not entitled to antedate such publication.

[0047] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, terms related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, and microbiology used herein are those widely used in the corresponding fields (see, for example, Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al., eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989). To better understand the present invention, definitions and explanations of relevant terms are provided below.

[0048] As used herein, the expressions "comprise," "include," "contain," and "have" are open ended and mean the inclusion of the listed elements, steps, or components but not the exclusion of other unlisted elements, steps, or components. The expression "consisting of excludes any element, step, or component not specified. The expression "consisting essentially of means that the scope is limited to the specified elements, steps, or components, plus optional elements, steps, or components that do not significantly affect the basic and novel properties of the claimed subject matter. It should be understood that the expressions "consisting essentially of" and "consisting of are encompassed within the meaning of the expression "comprising."

[0049] As used herein, unless the context indicates otherwise, the singular expressions "a" and "an / kind" and "the" and similar references used in the context of describing the present invention (particularly in the context of the claims) should be interpreted to cover both the singular and the plural. The terms "one or more" or "at least one" encompass 1, 2, 3, 4, 5, 6, 7, 8, 9 or more. The term "at least one" encompasses 1, 2, 3, 4, 5, 6, 7, 8, 9 or more. The term "at least two" encompasses 2, 3, 4, 5, 6, 7, 8, 9 or more. The term "at least two" encompasses 2, 3, 4, 5, 6, 7, 8, 9 or more.

[0050] The numerical ranges described herein should be understood to encompass any and all subranges contained therein. For example, the range "1 to 10" should be understood to include not only the explicitly stated values ​​of 1 and 10, but also any individual value (e.g., 2, 3, 4, 5, 6, 7, 8, and 9) and subranges (e.g., 1 to 2, 1.5 to 2.5, 1 to 3, 1.5 to 3.5, 2.5 to 4, 3 to 4.5, etc.) within the range of 1 to 10. This principle also applies to ranges that use only one value as a minimum or maximum value.

[0051] Unless otherwise stated, all methods described herein can be performed in any suitable order.

[0052] As used herein, the term "wild type" means that the sequence is naturally occurring and has not been artificially modified, including naturally occurring mutants. The term "fragment" or "fragment of a nucleic acid" refers to a portion of a nucleic acid. For example, a nucleic acid that is shortened at the 5' and / or 3' ends. A fragment of a nucleic acid comprises at least 50%, 60%, 70% or 80% of the nucleic acid. In some embodiments, a fragment of a nucleic acid comprises at least 70% or 80% of the nucleotide residues from the nucleic acid. Preferably, at least 90%, 95%, 96%, 97%, 98% or 99% of the nucleotide residues are present. Typically, this can be a shorter portion of the full length of the nucleic acid.

[0053] The term "variant" in reference to nucleic acids refers to a nucleic acid variant in which at least one nucleotide differs from a reference nucleic acid (or "parent"). Compared to a reference nucleic acid, a variant nucleic acid includes single or multiple nucleotide deletions, additions, mutations, and / or insertions, wherein: deletions include removal of one or more nucleotides from a reference nucleic acid; additions include fusion of one or more nucleotides (e.g., 1, 2, 3, 5, 10, 20, 30, 50, or more nucleotides) to the 5' and / or 3' ends of the reference nucleic acid; mutations may include, but are not limited to, substitutions (e.g., at least one nucleotide is removed and another nucleotide is inserted in its place (e.g., transversions and transitions)); and insertions include the addition of at least one nucleotide. In some embodiments, the nucleic acid variant is a 5'-UTR variant, a 3'-UTR variant, or a ployA variant. In some embodiments, the mutations introduced into the nucleic acid variant can prevent the nucleic acid variant from being recognized and cleaved by nucleases, from binding to microRNAs, and from forming complex secondary structures such as hairpin structures or G-quadruplexes. For example, the nucleic acid variant is derived from the 3'-UTR of the HDGFL1 gene (GenBank Accession No. NM_138574.4), in which two "C"s are mutated to two "A"s based on the 3'-UTR of the HDGFL1 gene to avoid recognition by nucleases and enzymatic cleavage.

[0054] As used herein, the term " nucleic acid variant " includes naturally occurring variants and engineered variants. Therefore, " nucleic acid variant " as defined herein can be derived from a reference nucleic acid, separated, relevant, based on or homologous to a reference nucleic acid sequence." nucleic acid variant " optionally has at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of a corresponding naturally occurring (wild type) nucleic acid or its homologue, fragment or derivative. In some embodiments, at least 70%, at least 80%, at least 85%, at least 90%, at least 95% or at least 97% sequence identity. It will be appreciated that for nucleic acid molecules, the term " variant " includes degenerate nucleic acid sequences, wherein the degenerate nucleic acid sequences according to the present invention are due to the degeneracy of genetic code and the nucleic acid different from the reference nucleic acid in codon sequence.

[0055] As used herein, the term "% identity" or "% identity" refers to the percentage of identical nucleotides or amino acids in an optimal alignment between the sequences to be compared. The differences between the two sequences can be distributed over local regions (segments) or over the entire length of the sequences to be compared. The identity between the two sequences is usually determined after optimal alignment of a segment or "comparison window." Optimal alignment can be performed manually or with the aid of algorithms known in the art. Algorithms known in the art include, but are not limited to, the local homology algorithm described by Smith and Waterman, 1981, Ads App. Math. 2, 482 and Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, the similarity search method described by Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 88, 2444, or the similarity search method described by computer programs such as GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis. For example, the percent identity of two sequences can be determined using the BLASTN or BLASTP algorithms publicly available on the National Center for Biotechnology Information (NCBI) website.

[0056] "% identity" or "% homology" can be obtained by determining the number of identical positions corresponding to the sequences to be compared, dividing this number by the number of positions compared (e.g., the number of positions in the reference sequence), and multiplying this result by 100 to obtain % identity. In some embodiments, the degree of identity is given for a region of at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%. In some embodiments, the degree of identity is given for the entire length of the reference sequence. Alignment to determine sequence identity can be performed using tools known in the art, preferably using optimal sequence alignment, for example, using Align, using standard settings, preferably EMBOSS::needle, Matrix::Blosum62, Gap Open 10.0, Gap Extend 0.5.

[0057] In some embodiments, a fragment or variant of a specific nucleic acid or a nucleic acid having a specific degree of identity with a specific nucleic acid has at least one functional property of the specific nucleic acid and is preferably functionally equivalent to the specific nucleic acid, e.g., a nucleic acid that exhibits properties that are the same or similar to those of the specific nucleic acid.

[0058] As used herein, "nucleotide" includes deoxyribonucleotides, ribonucleotides, deoxyribonucleotide derivatives, and ribonucleotide derivatives. As used herein, "ribonucleotide" is a constituent of ribonucleic acid (RNA), consisting of a base molecule, a pentose molecule, and a phosphate molecule. It refers to a nucleotide with a hydroxyl group at the 2' position of the β-D-ribofuranosyl group. "Deoxyribonucleotide" is a constituent of deoxyribonucleic acid (DNA), also consisting of a base molecule, a pentose molecule, and a phosphate molecule. It refers to a nucleotide in which the hydroxyl group at the 2' position of the β-D-ribofuranosyl group is replaced by hydrogen. It is the main chemical component of chromosomes.

[0059] "Nucleotides" are generally referred to by single letters representing the bases therein: "A" or "A nucleotide" refers to adenine deoxyribonucleotide or adenine ribonucleotide containing adenine, "C" or "C nucleotide" refers to cytosine deoxyribonucleotide or cytosine ribonucleotide containing cytosine, "G" or "G nucleotide" refers to guanine deoxyribonucleotide or guanine ribonucleotide containing guanine, "U" or "U nucleotide" refers to uracil ribonucleotide containing uracil, and "T" or "T nucleotide" refers to thymine deoxyribonucleotide containing thymine.

[0060] As used herein, the term "nucleic acid" generally refers to a polymer comprising deoxyribonucleotides (deoxyribonucleic acid, referred to as DNA) or a polymer comprising ribonucleotides (ribonucleic acid, referred to as RNA) or any compound of a combination thereof. In addition, nucleic acids herein also include derivatives of nucleic acids. The term "derivatives of nucleic acids" includes chemical derivatization of nucleic acids on the bases, sugars or phosphates of the nucleotides, as well as nucleic acids containing non-natural nucleotides and nucleotide analogs. In addition, herein, nucleic acids can be in the form of single-stranded or double-stranded linear or covalently closed circular molecules.

[0061] "Polynucleotide sequence," "nucleic acid sequence," and "nucleotide sequence" are used interchangeably to refer to the order of nucleotides in a polynucleotide. It will be understood by those skilled in the art that a DNA coding strand (sense strand) and the RNA it encodes can be considered to have the same nucleotide sequence, with deoxythymidylic acid in the DNA coding strand sequence corresponding to uridine nucleotides in the RNA sequence it encodes. An RNA encoded by a DNA refers to an RNA corresponding to the DNA, i.e., a polynucleotide in which all Ts in the DNA are replaced by Us. For example, an RNA encoded by a polynucleotide set forth in at least one of SEQ ID NOs: 47 to 67 refers to an RNA corresponding to the polynucleotide (DNA) set forth in at least one of SEQ ID NOs: 47 to 67, i.e., a polynucleotide in which all Ts in the polynucleotide set forth in at least one of SEQ ID NOs: 47 to 67 are replaced by Us. The RNA sequence corresponding to a DNA sequence refers to a nucleic acid sequence after all T in the DNA sequence is replaced by U. For example, the RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 9, 16, 17, 23, 24, 28, 29, 32, 37, 39 or 42 refers to a nucleic acid sequence after all T in the nucleic acid sequence shown in SEQ ID NO: 9, 16, 17, 23, 24, 28, 29, 32, 37, 39 or 42 is replaced by U.

[0062] The polynucleotide may comprise one or more segments (nucleic acid fragments) (e.g., 1, 2, 3, 4, 5, 6, 7, 8 segments). For example, the polynucleotide may comprise a segment encoding a polypeptide of interest (e.g., a polypeptide and polypeptide antigen described herein). In a specific embodiment, the polynucleotide may comprise a segment encoding a polypeptide of interest and a regulatory segment (including but not limited to a segment for transcriptional regulation and translational regulation). In one embodiment, the regulatory segment comprises a polynucleotide corresponding to one or more of the following regulatory elements: a promoter, a 5' untranslated region (5'-UTR), a 3' untranslated region (3'-UTR), and a poly(A) tail.

[0063] The term "promoter" refers to a polynucleotide located upstream of the 5' end of the coding region of a gene, which contains a conserved sequence required for RNA polymerase specific binding and transcription initiation, can activate RNA polymerase, enable RNA polymerase to accurately bind to template DNA and have the specificity of transcription initiation. Promoters can be derived from viruses, bacteria, fungi, plants, insects and animals. Representative examples of promoters include bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operator-promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, SV40 early promoter or SV 40 late promoter and CMV IE promoter.

[0064] As used herein, the term "5' untranslated region" or "5'-UTR" can be an RNA sequence in an mRNA that is upstream of the coding sequence and is not translated into protein. The 5'-UTR in a gene generally begins at the transcription start site and ends at the nucleotides upstream of the translation start codon of the coding sequence. The 5'-UTR can contain elements that control gene expression, such as a ribosome binding site, a 5'-terminal oligopyrimidine tract, and a translation initiation signal such as a Kozak sequence. mRNA can be post-transcriptionally modified by the addition of a 5' cap. Therefore, the 5'-UTR in a mature mRNA can also refer to the RNA sequence between the 5' cap and the start codon.

[0065] As used herein, the term "3' untranslated region" or "3'-UTR" can be an RNA sequence in an mRNA that is located downstream of a coding sequence and is not translated into protein. The 3'-UTR in an mRNA is located between the stop codon and the poly(A) sequence of the coding sequence, for example, starting from the nucleotides downstream of the stop codon and ending at the nucleotides upstream of the poly(A) sequence.

[0066] As used herein, "5' or 3'-UTR derived from gene A" refers to the 5' or 3'-UTR of the mRNA of gene A. The 5' or 3'-UTR derived from gene A may be the entire 5' or 3'-UTR of the mRNA of gene A, or may be a partial 5' or 3'-UTR of the mRNA of gene A. The partial 5' or 3'-UTR of the mRNA of gene A includes a partial 5'-UTR formed by splicing together multiple fragments of the 5'-UTR of the mRNA of gene A, or a partial 3'-UTR formed by splicing together multiple fragments of the 3'-UTR of the mRNA of gene A.

[0067] As used herein, the terms "poly(A)," "polyA," "poly(A) sequence," and "poly(A) tail" are used interchangeably. Naturally occurring poly(A) sequences are typically composed of adenine ribonucleotides. According to the present invention, the term "modified poly(A) sequence" refers to a poly(A) sequence that contains nucleotides or nucleotide segments other than adenine ribonucleotides. The poly(A) sequence is typically located at the 3' end of the mRNA, for example, at the 3' end (downstream) of the 3'-UTR.

[0068] As used herein, the term "5'-cap structure": The 5'-cap structure is generally located at the 5' end of the mature mRNA. In some embodiments, the 5'-cap structure is linked to the 5'-end of the mRNA via a 5'-5'-triphosphate bond. The 5'-cap structure is generally formed by modified (e.g., methylated) ribonucleotides (especially guanine nucleotide derivatives). For example, m7GpppN (cap 0 or "cap0" is a cap structure formed by the 5' phosphate group of the hnRNA reacting with the 5'-phosphate group of the m7GTP under the action of guanylyl transferase to form a 5', 5'-phosphodiester bond), wherein N is the terminal 5' nucleotide of the nucleic acid carrying the 5'-cap structure. In some embodiments, the 5'-cap structure includes but is not limited to cap 0, cap 1 (a cap structure formed by further methylating the 2'-OH of the first nucleotide sugar group of hnRNA on the basis of cap 0, or referred to as "cap 1"), cap 2 (a cap structure formed by further methylating the 2'-OH of the second nucleotide sugar group of hnRNA on the basis of cap 1, or referred to as "cap 2"), cap 4, cap 0 analogs, cap 1 analogs, cap 2 analogs or cap 4 analogs.

[0069] As used herein, the term "expression" includes transcription and / or translation of a nucleotide sequence. Thus, expression can involve the production of transcripts and / or polypeptides. The term "transcription" refers to the process of transcribing the genetic code in a DNA sequence into RNA (transcript). The term "in vitro transcription" refers to the in vitro synthesis of RNA, particularly mRNA, in a cell-free system (e.g., in an appropriate cell extract) (see, e.g., Pardi N., Muramatsu H., Weissman D., Karikó K. (2013). In: Rabinovich P. (eds) Synthetic Messenger RNA and Cell Metabolism Modulation. Methods in Molecular Biology (Methods and Protocols), vol 969. Humana Press, Totowa, NJ.). A vector that can be used to produce a transcript is also referred to as a "transcription vector," which contains regulatory sequences required for transcription. The term "transcription" encompasses "in vitro transcription."

[0070] As used herein, the term "polypeptide" refers to a polymer comprising two or more amino acids covalently linked by peptide bonds. A "protein" may comprise one or more polypeptides, wherein the polypeptides interact with each other by covalent or non-covalent means.

[0071] As used herein, the term "host cell" refers to a cell for receiving, maintaining, replicating, expressing a polynucleotide or a vector. The term "host cell" includes prokaryotic cells (e.g., Escherichia coli) or eukaryotic cells (e.g., yeast cells and insect cells). For example, cells from humans, mice, hamsters, pigs, goats, primates. The cell can be derived from a variety of tissue types and includes primary cells and cell lines. Some specific examples include keratinocytes, peripheral blood leukocytes, bone marrow stem cells, and embryonic stem cells. In other embodiments, the host cell is an antigen presenting cell, particularly a dendritic cell, a monocyte, or a macrophage. The nucleic acid can be present in a host cell in a single copy or in several copies. In some embodiments, the host cell can be a cell expressing a polypeptide of the present invention therein.

[0072] As used herein, the term "recombinant" or "recombinant" means "produced by genetic engineering." In some embodiments, in the context of the present invention, "recombinant material," such as a recombinant RNA molecule, is non-naturally occurring. As used herein, the term "naturally occurring" or "naturally occurring" refers to the fact that a material can be found in nature. For example, a peptide or nucleic acid that is present in an organism (including a virus) and can be isolated from a source in nature and has not been intentionally modified by man in an experiment is naturally occurring.

[0073] In the context of the present invention, the term "plasmid" generally refers to a circular DNA molecule, but the term can also encompass linearized DNA molecules. Specifically, the term "plasmid" also encompasses molecules obtained by, for example, digesting a circular plasmid with a restriction enzyme, thereby converting the circular plasmid molecule into a linear molecule and linearizing the circular plasmid. Plasmids can replicate, i.e., amplify the genetic information stored as chromosomal DNA in the cell, and can be used for cloning, i.e., for amplifying genetic information in bacterial cells. In some embodiments, the DNA plasmid is a medium copy or high copy plasmid. In some embodiments, the DNA plasmid is a high copy plasmid. Examples of such high copy plasmids include, for example, pUC and pTZ plasmids or any other plasmid (e.g., pMB1, pCoIE1) comprising a replication origin that supports high copies of the plasmid.

[0074] As used herein, the term "vaccine" is typically understood to mean a prophylactic or therapeutic material that provides at least one antigen or antigenic function that can stimulate the body's adaptive immune system to provide an adaptive immune response.

[0075] As used herein, the term "antigen" generally refers to a substance that can be recognized by the immune system, preferably recognized by the adaptive immune system, and can trigger an antigen-specific immune response (e.g., forming antibodies and / or antigen-specific T cells). Typically, an antigen can be or can comprise a peptide or protein that can be presented to a T cell by MHC. In the sense of the present disclosure, an antigen can be a translation product of a provided nucleic acid (e.g., RNA, RNA molecules, DNA herein). In addition, fragments, variants, and derivatives of peptides or proteins derived from peptides or proteins comprising at least one epitope or antigen (e.g., tumor antigens, viral antigens, bacterial antigens, protozoan antigens) can be understood as antigens.

[0076] The terms "treatment" and "treating" and the like are used herein to generally mean obtaining a desired pharmacological and / or physiological effect. Thus, the treatment of the present invention may relate to the treatment of a certain disease state, but may also relate to prophylactic treatment in terms of completely or partially preventing a disease or its symptoms. In some embodiments, the term "treatment" is understood to be therapeutic in terms of partially or completely curing a disease and / or attributing to the adverse effects and / or symptoms of the disease. Treatment may also be prophylactic or preventive treatment, i.e., measures taken to prevent a disease, for example, to prevent infection and / or the onset of a disease.

[0077] As used herein, the terms "subject" and "patient" can be used interchangeably. In certain embodiments, the subject is a mammal, such as a human, non-human primate (e.g., ape, chimpanzee, monkey, and orangutan), domesticated animal (including dog and cat and livestock (e.g., horse, cattle, pig, sheep, and goat)), or other mammal. Other mammals include, but are not limited to, mice, rats, guinea pigs, rabbits, hamsters, etc. In a specific embodiment, the subject is a human. In one embodiment, the subject is a mammal (e.g., a human) suffering from an infectious disease or a neoplastic disease. In another embodiment, the subject is a mammal (e.g., a human) at risk of developing an infectious disease or a neoplastic disease.

[0078] As used herein, the term "administer" refers to providing or administering a medicament to a subject by any effective route. Exemplary routes of administration include, but are not limited to, one or more of the following: injection (e.g., subcutaneous, intramuscular, intradermal, intraperitoneal, intrathecal, intracerebroventricular, or intravenous), oral, intracavitary, sublingual, rectal, transdermal, intranasal, vaginal, and inhalation. When used to treat a disease, disorder, condition, or symptom thereof, administration of the substance is typically performed after the onset of the disease, disorder, condition, or symptom. When used to prevent a disease, disorder, condition, or symptom, administration of the substance is typically performed before the onset of the disease, disorder, condition, or symptom.

[0079] Herein, some elements of the present invention will be described. These elements are listed together with specific embodiments, however, it should be understood that they can be combined in any manner and in any number to produce additional embodiments. The different described examples and preferred embodiments should not be interpreted as limiting the present invention to only the explicitly described embodiments. This specification should be understood to support and include embodiments that combine the explicitly described embodiments with any number of the disclosed and / or preferred elements. In addition, unless the context indicates otherwise, any permutation and combination of all described elements in the present invention should be deemed to be disclosed by the present specification. For example, in one embodiment, the 5'-UTR of the RNA molecule includes a polynucleotide with a sequence as shown in SEQ ID NO: 9, and in another embodiment, the 3'-UTR of the RNA molecule includes a polynucleotide with a sequence as shown in SEQ ID NO: 10. Then the following scheme is also an embodiment claimed in the present invention: the 5'-UTR of the RNA molecule includes a polynucleotide with a sequence as shown in SEQ ID NO: 9, and the 3'-UTR of the RNA molecule includes a polynucleotide with a sequence as shown in SEQ ID NO: 10.

[0080] In the first aspect, the present invention relates to an RNA encoding a novel coronavirus S protein, a vaccine comprising the RNA, and uses thereof.

[0081] In some embodiments, the present invention provides an RNA encoding a novel coronavirus S protein, wherein the S protein comprises the amino acid sequence shown in SEQ ID NO: 13. In some embodiments, the amino acid sequence of the S protein is shown in SEQ ID NO: 13; in some embodiments, the RNA is mRNA.

[0082] In some embodiments, the present invention provides an RNA encoding a novel coronavirus S protein, wherein the S protein comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identical to the amino acid sequence shown in SEQ ID NO: 13.

[0083] In some embodiments, the RNA comprises the nucleic acid sequence shown in SEQ ID NO: 14. In some embodiments, the nucleic acid sequence of the RNA is shown in SEQ ID NO: 14.

[0084] In some embodiments, some or all of the cytosine and / or uracil in the RNA are chemically modified to improve the stability of the RNA in vivo.

[0085] In some embodiments, the chemical modification comprises the following:

[0086] One or more uridine nucleosides in the RNA, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 uridine nucleosides or at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of the uridine nucleosides are replaced by at least one nucleoside selected from the group consisting of pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4′-thiouridine, 5-methylcytosine, 5-methyluridine, 2 -thio-1-methyl-1-deaza-pseudouridine, 2-thio-T-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine or 5-methoxyuridine and 2'-O-methyluridine. In an optional specific example, the nucleoside substitution is selected from the following: pseudouridine, N1-methylpseudouridine or N1-ethylpseudouridine; and / or

[0087] One or more cytosine nucleosides in the RNA, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 cytosine nucleosides or at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of the cytosine nucleosides are replaced by 5-methylcytosine nucleoside.

[0088] In some embodiments, all or part of the uridine nucleosides in the RNA are replaced by pseudouridine. In some embodiments, all or part of the uridine nucleosides in the RNA are replaced by N1-methylpseudouridine. In some embodiments, all or part of the uridine nucleosides in the nucleic acid sequence shown in SEQ ID NO: 14 are replaced by pseudouridine, preferably N1-methylpseudouridine.

[0089] In some embodiments, all uridine nucleosides in the nucleic acid sequence shown in SEQ ID NO: 14 are replaced by N1-methylpseudouridine.

[0090] In some embodiments, the RNA further comprises at least one of a 5'-cap structure, a 5'-UTR, a 3'-UTR, and polyA.

[0091] In some embodiments, the 5'-cap structure is selected from m 7 GpppG、m2 7，3′-O GpppG、m 7 Gppp(5′)N1 and m 7 Gppp(m 2′-O ) At least one of N1, preferably m 7 Gppp(5′)N1 or m 7 Gppp(m 2′-O )N1, where "m7G" represents 7-methylguanosine cap nucleoside, "ppp" represents the triphosphate bond between the 5' carbon of the cap nucleoside and the first nucleotide of the primary RNA transcript, N1 is the 5' most nucleotide, "G" represents guanosine nucleoside, "7" represents the methyl group at the 7-position of guanine, and "m 2′-O ” represents the methyl group at the 2′-O position of the nucleotide;

[0092] and / or the 5'-UTR is selected from the following (1)-(5):

[0093] (1) comprising a 5'-UTR of an RNA sequence corresponding to the nucleic acid sequence of SEQ ID NO: 9, 16, 17, 23, 24, 28, 29, 32, 37, 39 or 42, a homologue, fragment or variant thereof, wherein the homologue, fragment or variant has the same or better function of improving translation efficiency as the 5'-UTR of the RNA sequence corresponding to SEQ ID NO: 9, 16, 17, 23, 24, 28, 29, 32, 37, 39 or 42; in some embodiments, the nucleic acid sequence of the homologue has at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology to the RNA sequence corresponding to the nucleic acid sequence of SEQ ID NO: 9, 16, 17, 23, 24, 28, 29, 32, 37, 39 or 42;

[0094] (2) a 5′-UTR consisting of an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 9, 16, 17, 23, 24, 28, 29, 32, 37, 39 or 42;

[0095] (3) a 5'-UTR consisting of an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 9;

[0096] (4) A 5'-UTR obtained by concatenating two or more identical 5'-UTRs in (1) to (3) above; or

[0097] (5) 5'-UTR obtained by concatenating two or more different 5'-UTRs in (1)-(3) above;

[0098] And / or, the 3'-UTR is selected from the following (1)-(5):

[0099] (1) comprising a 3'-UTR of an RNA sequence corresponding to the nucleic acid sequence of SEQ ID NO: 47-67 or 10, a homologue, fragment or variant thereof; the homologue, fragment or variant having the same or better function of improving translation efficiency and / or stability as the 3'-UTR of the RNA sequence corresponding to SEQ ID NO: 47-67 or 10; in some embodiments, the nucleic acid sequence of the homologue has at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology to the RNA sequence corresponding to the nucleic acid sequence of SEQ ID NO: 47-67 or 10;

[0100] (2) a 3'-UTR consisting of an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 47-67 or 10;

[0101] (3) a 3'-UTR consisting of an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 10;

[0102] (4) A 3'-UTR obtained by concatenating two or more identical 3'-UTRs in (1) to (3) above; or

[0103] (5) 3'-UTR obtained by concatenating two or more different 3'-UTRs in (1)-(3) above;

[0104] And / or the polyA is a truncated polyA, i.e., a plurality of consecutive A nucleotides are followed by a 10 bp non-A linker sequence and then a plurality of consecutive A nucleotides; in some embodiments, the polyA is 30 consecutive A nucleotides followed by a 10 bp non-A linker sequence and then 70 consecutive A nucleotides;

[0105] In some embodiments, the polyA is selected from one of the following (1)-(3):

[0106] (1) A polyA comprising an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 68-70, 72, 75 or 11, a homologue, fragment or variant thereof, wherein the homologue, fragment or variant has the same or better function of improving translation efficiency and / or stability as the polyA shown in the RNA sequence corresponding to SEQ ID NO: 68-70, 72, 75 or 11; in some embodiments, the homologue comprises a nucleic acid sequence having at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology to the RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 68-70, 72, 75 or 11;

[0107] (2) a polyA consisting of an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 68-70, 72, 75 or 11;

[0108] (3) PolyA consisting of an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 11.

[0109] In some embodiments, the RNA comprises the nucleic acid sequence shown in SEQ ID NO: 15. In some embodiments, the nucleic acid sequence is shown in SEQ ID NO: 15. The sequence of SEQ ID NO: 15 itself already contains a cap structure: cap G 1 G 2 =m 7 G + -5′-ppp-5′-Gm 2′ -3′-p-[m 7 =7-CH3;m 2′ =2′-O-CH3; -ppp-=-PO2H-O-PO2H-O-PO2H)-; -p-=-PO2H-]. It should be noted that according to WIPO standard ST.26 for nucleotide or amino acid sequence listings, the t (thymine) in the RNA sequence (e.g., SEQ ID NO: 14, SEQ ID NO: 15) in the sequence listing is actually u (uracil).

[0110] In embodiments where the RNA comprises the nucleic acid sequence shown in SEQ ID NO: 15, all or part of the uridine nucleosides in the RNA are replaced by pseudouridine, preferably N1-methylpseudouridine; in some embodiments, all or part of the uridine nucleosides in the nucleic acid sequence shown in SEQ ID NO: 15 are replaced by pseudouridine, preferably N1-methylpseudouridine; in some embodiments, all uridine nucleosides in the nucleic acid sequence shown in SEQ ID NO: 15 are replaced by N1-methylpseudouridine.

[0111] In some embodiments, the present invention provides a protein expressed by any of the aforementioned RNAs; in some embodiments, the protein comprises the amino acid sequence shown in SEQ ID NO: 13, and in some embodiments, the amino acid sequence is shown in SEQ ID NO: 13.

[0112] In some embodiments, the present invention provides a DNA encoding the RNA of any of the above embodiments; in some embodiments, the DNA comprises the nucleic acid sequence shown in SEQ ID NO: 12; in some embodiments, the DNA comprises the nucleic acid sequence shown in SEQ ID NO: 8.

[0113] In some embodiments, the present invention provides a vector comprising the DNA of any of the above embodiments.

[0114] In some embodiments, the present invention provides a host cell comprising the vector of any of the above embodiments.

[0115] In some embodiments, the present invention provides a lipid nanoparticle comprising the RNA of any one of the above embodiments.

[0116] In some embodiments, the lipid nanoparticles further contain one or more of an ionizable cationic lipid, a helper lipid, a structural lipid, and a PEG-lipid.

[0117] In some embodiments, the ionizable cationic lipid is selected from one or more of Dlin-MC3-DMA, Dlin-KC2-DMA, DODMA, c12-200, or DlinDMA; and / or the auxiliary lipid is selected from one or more of DSPC, DOPE, DOPC, or DOPS; and / or the structural lipid is selected from at least one of cholesterol, cholesterol esters, steroid hormones, steroid vitamins, and bile acids; and / or the PEG-lipid is selected from PEG-DMG or PEG-DSPE, preferably PEG-DMG. PEG-DMG is a polyethylene glycol (PEG) derivative of 1,2-dimyristylglycerol. In some embodiments, the average molecular weight of PEG is about 2000 to 5000 Daltons. In an alternative specific example, the average molecular weight of PEG is about 2000 or 5000 Daltons.

[0118] In some embodiments, the present invention provides a pharmaceutical composition comprising the RNA, the protein, the DNA, the vector, the host cell or the lipid nanoparticle, and a pharmaceutically acceptable carrier and / or excipient.

[0119] In some embodiments, the present invention provides the use of the RNA, the protein, the DNA, the vector, the host cell or the lipid nanoparticle in the preparation of a vaccine for preventing novel coronavirus infection. In some embodiments, the novel coronavirus includes Alpha (such as B.1.1.7 variants and their progeny lineages), Beta (such as B.1.351 variants and their progeny lineages), Gamma (such as P.1 variants and their progeny lineages), Delta (such as B.1.617.2 and AY lineages) and Omicron, etc.

[0120] In some embodiments, the present invention provides a method for controlling, preventing or treating an infectious disease caused by the new coronavirus in a subject, comprising administering to the subject a therapeutically effective amount of the RNA, the protein, the DNA, the vector, the host cell, the lipid nanoparticle or the pharmaceutical composition.

[0121] In some embodiments, the subject is a human or non-human mammal. In some embodiments, the subject is an adult, an elderly person, a child, or a toddler. In some embodiments, the subject is at risk of or susceptible to infection with the novel coronavirus. In some embodiments, the subject has been diagnosed with a positive novel coronavirus infection or is asymptomatic.

[0122] In a second aspect, the present invention also relates to a universal polynucleotide molecule that can be used to prepare a novel RNA vaccine (eg, an mRNA vaccine), which comprises a 5'-UTR and / or a 3'-UTR.

[0123] In some embodiments, the present invention provides an artificial RNA molecule comprising a 5'-UTR and a nucleic acid sequence encoding a polypeptide and / or protein of interest, wherein the 5'-UTR is selected from the following (1)-(5):

[0124] (1) comprising a 5'-UTR of an RNA sequence corresponding to the nucleic acid sequence of SEQ ID NO: 9, 16, 17, 23, 24, 28, 29, 32, 37, 39 or 42, a homologue, fragment or variant thereof, wherein the homologue, fragment or variant has the same or better function of improving translation efficiency as the 5'-UTR of the RNA sequence corresponding to SEQ ID NO: 9, 16, 17, 23, 24, 28, 29, 32, 37, 39 or 42; in some embodiments, the nucleic acid sequence of the homologue has at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology to the RNA sequence corresponding to the nucleic acid sequence of SEQ ID NO: 9, 16, 17, 23, 24, 28, 29, 32, 37, 39 or 42;

[0125] (2) a 5′-UTR consisting of an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 9, 16, 17, 23, 24, 28, 29, 32, 37, 39 or 42;

[0126] (3) a 5'-UTR consisting of an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 9;

[0127] (4) A 5'-UTR obtained by concatenating two or more identical 5'-UTRs in (1) to (3) above; or

[0128] (5) A 5'-UTR obtained by concatenating two or more different 5'-UTRs in (1) to (3) above.

[0129] Herein, "A, a homologue, a fragment or a variant thereof" refers to A, a homologue of A, a fragment of A or a variant of A, for example, "a 5'-UTR, a homologue, a fragment or a variant thereof comprising an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 9, 16, 17, 23, 24, 28, 29, 32, 37, 39 or 42" refers to a 5'-UTR comprising an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 9, 16, 17, 23, 24, 28, 29, 32, 37, 39 or 42, a homologue of the 5'-UTR, a fragment of the 5'-UTR or a variant of the 5'-UTR.

[0130] In some embodiments, the RNA molecule further comprises a 3'-UTR and polyA.

[0131] In some embodiments, the 3'-UTR is selected from the following (1)-(5):

[0132] (1) comprising a 3'-UTR of an RNA sequence corresponding to the nucleic acid sequence of SEQ ID NO: 47-67 or 10, a homologue, fragment or variant thereof; the homologue, fragment or variant having the same or better function of improving translation efficiency and / or stability as the 3'-UTR of the RNA sequence corresponding to SEQ ID NO: 47-67 or 10; in some embodiments, the nucleic acid sequence of the homologue has at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology to the RNA sequence corresponding to the nucleic acid sequence of SEQ ID NO: 47-67 or 10;

[0133] (2) a 3'-UTR consisting of an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 47-67 or 10;

[0134] (3) a 3'-UTR consisting of an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 10;

[0135] (4) A 3'-UTR obtained by concatenating two or more identical 3'-UTRs in (1) to (3) above; or

[0136] (5) A 3'-UTR obtained by concatenating two or more different 3'-UTRs in (1) to (3) above.

[0137] In some embodiments, the polyA is a truncated polyA, that is, a 10 bp non-A linker sequence is added after a plurality of consecutive nucleotides A, and then a plurality of consecutive nucleotides A; in some embodiments, the polyA is 30 consecutive nucleotides A, followed by a 10 bp non-A linker sequence, and then 70 consecutive nucleotides A.

[0138] In some embodiments, the polyA is selected from the following (1)-(3):

[0139] (1) A polyA comprising an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 68-70, 72, 75 or 11, a homologue, fragment or variant thereof, wherein the homologue, fragment or variant has the same or better function of improving translation efficiency and / or stability as the polyA shown in the RNA sequence corresponding to SEQ ID NO: 68-70, 72, 75 or 11; in some embodiments, the homologue comprises a nucleic acid sequence having at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology to the RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 68-70, 72, 75 or 11;

[0140] (2) a polyA consisting of an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 68-70, 72, 75 or 11; or

[0141] (3) PolyA consisting of an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 11.

[0142] In some embodiments, the present invention also provides another RNA molecule comprising:

[0143] (1) a first nucleotide sequence encoding a polypeptide and / or protein of interest; and

[0144] (2) a second nucleotide sequence containing a 3'-untranslated region (3'-UTR);

[0145] The 3'-UTR comprises at least one of the following polynucleotides:

[0146] (a): a 3'-UTR derived from at least one of the genes APOA1, UL122, COP1, MYSM1, ASAH2B, NDUFAF2, APOD, HBB, TF, TMSB4X, CPAMD8, VIM, HDGFL1, TTR, SRM, HBA1, PRPF8, LMBRD1, IFNA1, CCDC146, and GHRL; in some embodiments, a 3'-UTR derived from at least one of the genes APOA1, IE1, COP1, MYSM1, ASAH2B, NDUFAF2, APOD, HBB, TF, TMSB4X, CPAMD8, VIM, HDGFL1, TTR, SRM, HBA1, PRPF8, LMBRD1, IFNA1, CCDC146, and GHRL;

[0147] (b): a fragment of the 3′-UTR described in (a);

[0148] (c): a variant of the 3'-UTR described in (a); and

[0149] (d): A variant of the fragment described in (b);

[0150] The first nucleotide sequence and the second nucleotide sequence do not naturally occur in the same RNA molecule.

[0151] In some embodiments, the gene is a eukaryotic gene. In some embodiments, the gene is an animal gene. In some embodiments, the gene is a chordate gene, such as a human or mouse gene.

[0152] In some embodiments, the gene is a human gene.

[0153] In some embodiments, the GenBank accession number of the APOA1 gene is NM_000039.3; the Gene ID of the UL122 gene is 3077563 (corresponding to 170689..174090 of the GenBank accession number NC_006273.2), the GenBank accession number of the COP1 gene is AY885669; the GenBank accession number of the MYSM1 gene is NM_001085487.3; the GenBank accession number of the ASAH2B gene is NM_001079516.4; the GenBank accession number of the NDUFAF2 gene is The accession number of the APOD gene is NG_008978.1; the accession number of the HBB gene is MK476504.1; the accession number of the TF gene is NM_001063.4; the accession number of the TMSB4X gene is BC001631.1; the accession number of the CPAMD8 gene is NM_015692.5; the accession number of the VIM gene is The GenBank accession number of the HDGFL1 gene is NM_138574.4; the GenBank accession number of the TTR gene is NM_000371; the GenBank accession number of the SRM gene is NM_003132.3; the GenBank accession number of the HBA1 gene is NM_000558.5; and the GenBank accession number of the PRPF8 gene is NM_006 445.4; the GenBank accession number of the LMBRD1 gene is NM_001367272.1; the GenBank accession number of the IFNA1 gene is NM_024013.3; the GenBank accession number of the CCDC146 gene is NM_020879.3; the GenBank accession number of the GHRL gene is NM_001134941.3; and the GenBank accession number of the GH1 gene is NM_000515.5.

[0154] In some embodiments, the second nucleotide sequence comprises at least one of the following polynucleotides:

[0155] (e): 3′-UTR derived from at least one of the genes COP1 and HDGFL1;

[0156] (f): a fragment of the 3′-UTR described in (e);

[0157] (g): A variant of the 3'-UTR described in (e); and

[0158] (h): A variant of the fragment described in (f).

[0159] In some embodiments, the second nucleotide sequence comprises at least one of the following polynucleotides: an RNA encoded by a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 47 to 67, a fragment of an RNA encoded by a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 47 to 67, a variant of an RNA encoded by a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 47 to 67, and a variant of a fragment of an RNA encoded by a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 47 to 67.

[0160] In some embodiments, variants of RNA encoded by polynucleotides having a sequence as set forth in at least one of SEQ ID NOs: 47-67, fragments of RNA encoded by polynucleotides having a sequence as set forth in at least one of SEQ ID NOs: 47-67, and variants of fragments of RNA encoded by polynucleotides having a sequence as set forth in at least one of SEQ ID NOs: 47-67 are at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the RNA encoded by polynucleotides having a sequence as set forth in at least one of SEQ ID NOs: 47-67.

[0161] In some embodiments, the fragment, variant, or variant of the RNA encoded by the polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 47 to 67 has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotide insertions, additions, deletions, or substitutions compared to the RNA encoded by the polynucleotide having a sequence as shown in one of SEQ ID NOs: 47 to 67.

[0162] It should be noted that, herein, "a fragment, variant, or variant of a fragment of A" is an abbreviation for a fragment of A, a variant of A, or a variant of a fragment of A. For example, "a fragment, variant, or variant of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 47 to 67" refers to a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 47 to 67, a variant of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 47 to 67, or a variant of a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 47 to 67. In some embodiments, a fragment of A, a variant of A, or a variant of a fragment of A has the same or better properties as A, such as improving the translation efficiency and / or stability of a nucleic acid (e.g., mRNA).

[0163] In some embodiments, the second nucleotide sequence comprises at least one of: 3'-UTRs of at least two of the genes described in (a), fragments of 3'-UTRs of at least two of the genes described in (a), variants of 3'-UTRs of at least two of the genes described in (a), and variants of fragments of 3'-UTRs of at least two of the genes described in (a). In some embodiments, the at least two genes are two genes, three genes, four genes, five genes, six genes, seven genes, eight genes, nine genes, or ten genes.

[0164] In some embodiments, the second nucleotide sequence comprises at least one of: at least two copies of the 3'-UTR in (a), at least two copies of a fragment of the 3'-UTR in (a), at least two copies of a variant of the 3'-UTR in (a), and at least two copies of a variant of a fragment of the 3'-UTR in (a). In some embodiments, the at least two copies are two copies, three copies, four copies, five copies, six copies, seven copies, eight copies, nine copies, or ten copies.

[0165] In some embodiments, the RNA molecule further comprises at least one of a promoter, a 5'-cap structure, a 5'-UTR, and polyA.

[0166] In some embodiments, the RNA molecule further comprises at least one of a 5'-cap structure, a 5'-UTR, and polyA.

[0167] In some embodiments, the 5'-cap structure comprises m 7 GpppG、m2 7，3′-O GpppG、m 7 Gppp(5′)N1 and m 7 Gppp(m 2′-O ) At least one of N1.

[0168] In some embodiments, the 5'-UTR comprises one of i) to iv):

[0169] i) at least one of a 5'-UTR, a fragment, a variant, or a variant of a fragment thereof, derived from at least one of the genes APOA1, CARD16, ALB, APOC1, EEF1A1, RBP4, GHRL, MPND, ASAH2B, FBX016, FBH1, SRM, NAAA, ACTB, MKNK2, ORM1, GADPH, NDUFAF2, FGFR2, MYCBPAP, CHMP2A, TSG101, PRPF8, NFKB2, NAE1, HDGFL1, GSDMD, FBXW12, FBXW10, FBXL8, and ATG4D;

[0170] ii) at least one of the following polynucleotides: an RNA encoded by the polynucleotide having a sequence as shown in SEQ ID NO: 9, a fragment of the RNA encoded by the polynucleotide having a sequence as shown in SEQ ID NO: 9, a variant of the RNA encoded by the polynucleotide having a sequence as shown in SEQ ID NO: 9, and a variant of a fragment of the RNA encoded by the polynucleotide having a sequence as shown in SEQ ID NO: 9.

[0171] iii) at least two copies of one of the polynucleotides of i) or ii).

[0172] iv) at least two polynucleotides according to i).

[0173] In some embodiments, the species or specific GenBank accession number of the source gene of the 5'-UTR can be referred to the relevant description of the third nucleotide sequence below, which will not be repeated here.

[0174] In some embodiments, the 5'-UTR in i) comprises or is one or more of the following: an RNA encoded by a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 16 to 46, a fragment of an RNA encoded by a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 16 to 46, a variant of an RNA encoded by a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 16 to 46, and a variant of a fragment of an RNA encoded by a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 16 to 46.

[0175] In some embodiments, variants of RNA encoded by polynucleotides having a sequence as set forth in at least one of SEQ ID NOs: 16-46, fragments of RNA encoded by polynucleotides having a sequence as set forth in at least one of SEQ ID NOs: 16-46, and variants of fragments of RNA encoded by polynucleotides having a sequence as set forth in at least one of SEQ ID NOs: 16-46, are at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the RNA encoded by polynucleotides having a sequence as set forth in at least one of SEQ ID NOs: 16-46.

[0176] In some embodiments, the variants of the RNA encoded by the polynucleotide with a sequence as shown in SEQ ID NO: 9, the fragments of the RNA encoded by the polynucleotide with a sequence as shown in SEQ ID NO: 9, and the variants of the fragments of the RNA encoded by the polynucleotide with a sequence as shown in SEQ ID NO: 9 in ii) have a sequence of at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the RNA encoded by the polynucleotide with a sequence as shown in SEQ ID NO: 9.

[0177] In some embodiments, the nucleotides constituting the polyA comprise at least 20, at least 40, at least 80, at least 100, or at least 120 A nucleotides; in some embodiments, the nucleotides constituting the polyA comprise at least 20, at least 40, at least 80, at least 100, or at least 120 A nucleotides consecutively.

[0178] In some embodiments, the nucleotides constituting the polyA include one or more nucleotides other than A nucleotides.

[0179] In some embodiments, the polyA is a truncated polyA; in some embodiments, after a plurality of consecutive A nucleotides, there is a 10 bp non-A linker sequence followed by a plurality of consecutive nucleotides A. In some embodiments, the polyA is 30 consecutive nucleotides A followed by a 10 bp non-A linker sequence followed by 70 consecutive nucleotides A.

[0180] In some embodiments, the polyA comprises at least one of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 68-76 and 11, a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 68-76 and 11, a variant of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 68-76 and 11, and a variant comprising a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 68-76 and 11. In some embodiments, the fragments, variants, and variants of the fragments of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 68-76 and 11 are at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an RNA encoded by a polynucleotide having a sequence as set forth in one of SEQ ID NOs: 68-76 and 11.

[0181] In some embodiments, the polyA is an RNA encoded by a polynucleotide having a sequence as shown in one of SEQ ID NOs: 68 to 70, 72, 75, and 11. In some embodiments, the polyA is an RNA encoded by a polynucleotide having a sequence as shown in SEQ ID NO: 11.

[0182] In some embodiments, the 3'-UTR is derived from one or more of the 3'-UTR of the COPl gene, fragments, variants, and variants of fragments thereof, and the 5'-UTR comprises one or more of the following polynucleotides: RNA encoded by the polynucleotide having a sequence as set forth in SEQ ID NO:9, fragments, variants, and variants of fragments thereof. In an alternative specific example, the 3'-UTR is one or more of RNA encoded by the polynucleotide having a sequence as set forth in SEQ ID NO:49, fragments, variants, and variants of fragments thereof. It should be noted that, herein, "A, fragments, variants, and variants of fragments" refers to A, fragments of A, variants of A, and variants of fragments of A. For example, "RNA encoded by the polynucleotide having a sequence as set forth in SEQ ID NO:9, fragments of the RNA encoded by the polynucleotide having a sequence as set forth in SEQ ID NO:9, variants of the RNA encoded by the polynucleotide having a sequence as set forth in SEQ ID NO:9, and variants of fragments of the RNA encoded by the polynucleotide having a sequence as set forth in SEQ ID NO:9."

[0183] In some embodiments, the 3'-UTR is derived from one or more of the 3'-UTR of the HDGFL1 gene, fragments, variants, and variants of fragments thereof, and the 5'-UTR comprises one or more of the following polynucleotides: RNA encoded by the polynucleotide having a sequence as shown in SEQ ID NO: 9, fragments, variants, and variants of fragments thereof. In an alternative specific example, the 3'-UTR is one or more of the RNA encoded by the polynucleotide having a sequence as shown in SEQ ID NO: 59, fragments, variants, and variants of fragments thereof.

[0184] In some embodiments, fragments, variants, and variants of fragments of an RNA encoded by a polynucleotide having a sequence as set forth in SEQ ID NO: 9, 49, or 59 are at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an RNA encoded by a polynucleotide having a sequence as set forth in SEQ ID NO: 9, 49, or 59.

[0185] In some embodiments, the present invention also provides another RNA molecule comprising:

[0186] (1) a first nucleotide sequence encoding a polypeptide and / or protein of interest; and

[0187] (2) a third nucleotide sequence containing the 5'-untranslated region (5'-UTR);

[0188] The 5'-UTR comprises at least one of the following polynucleotides:

[0189] (a): 5'-UTR derived from at least one of the following genes: APOA1, CARD16, ALB, APOC1, EEF1A1, RBP4, GHRL, MPND, ASAH2B, FBX016, FBH1, SRM, NAAA, ACTB, MKNK2, ORM1, GADPH, NDUFAF2, FGFR2, MYCBPAP, CHMP2A, TSG101, PRPF8, NFKB2, NAE1, HDGFL1, GSDMD, FBXW12, FBXW10, FBXL8, and ATG4D;

[0190] (b): a fragment of the 5′-UTR described in (a);

[0191] (c): a variant of the 5'-UTR described in (a); and

[0192] (d): A variant of the fragment described in (b);

[0193] The first nucleotide sequence and the third nucleotide sequence do not naturally occur in the same RNA molecule.

[0194] In some embodiments, the gene is a eukaryotic gene. In some embodiments, the gene is an animal gene. In some embodiments, the gene is a chordate gene, such as a human or mouse gene. In some embodiments, the gene is a human gene.

[0195] In some embodiments, the GenBank accession number of the APOA1 gene is NM_000039.3; the GenBank accession number of the CARD16 gene is NM_052889.4; the GenBank accession number of the ALB gene is AH002596.2; the GenBank accession number of the APOC1 gene is NM_001645.5; the GenBank accession number of the EEF1A1 gene is NM_001402.6; the GenBank accession number of the RBP4 gene is BC020633.1; the GenBank accession number of the GHRL gene is NM_016362.5; the GenBank accession number of the MPND gene is N M_032868; the GenBank accession number of ASAH2B gene is NM_001079516.4; the GenBank accession number of FBX016 gene is NM_172366.4; the GenBank accession number of FBH1 gene is NG_047206.1; the GenBank accession number of SRM gene is M64231.1; the GenBank accession number of NAAA gene is NM_001363719.2; the GenBank accession number of ACTB gene is NM_001101.5; the GenBank accession number of MKNK2 gene is NM_017572.4; the GenBank accession number of ORM1 gene is The accession number of the GADPH gene is NG_007073; the accession number of the NDUFAF2 gene is NG_008978.1; the accession number of the FGFR2 gene is NM_000141.5; the accession number of the MYCBPAP gene is AK303041.1; the accession number of the CHMP2A gene is NM_198426.3; the accession number of the TSG101 gene is NM_006292.4; the accession number of the PRPF8 gene is NM_006445; the accession number of the NFKB2 gene is The GenBank accession number is NM_001322935; the GenBank accession number of the NAE1 gene is NM_001018159; the GenBank accession number of the HDGFL1 gene is HM005397; the GenBank accession number of the GSDMD gene is BC008904.2; the GenBank accession number of the FBXW12 gene is NM_207102.2; the GenBank accession number of the FBXW10 gene is XM_054314720.1; the GenBank accession number of the FBXL8 gene is NM_018378; and the GenBank accession number of the ATG4D gene is NM_032885.6.

[0196] In some embodiments, the third nucleotide sequence comprises at least one of the following polynucleotides: an RNA encoded by a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 16 to 46, a fragment of an RNA encoded by a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 16 to 46, a variant of an RNA encoded by a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 16 to 46, and a variant of a fragment of an RNA encoded by a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 16 to 46.

[0197] In some embodiments, variants of RNA encoded by polynucleotides having sequences as set forth in at least one of SEQ ID NOs: 16-46, fragments of RNA encoded by polynucleotides having sequences as set forth in at least one of SEQ ID NOs: 16-46, and variants of fragments of RNA encoded by polynucleotides having sequences as set forth in at least one of SEQ ID NOs: 16-46 are at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to RNA encoded by polynucleotides having sequences as set forth in at least one of SEQ ID NOs: 16-46.

[0198] In some embodiments, the fragment, variant, or variant of the RNA encoded by the polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 16 to 46 has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotide insertions, additions, deletions, or substitutions compared to the RNA encoded by the polynucleotide having a sequence as shown in one of SEQ ID NOs: 16 to 46.

[0199] In some embodiments, the third nucleotide sequence comprises at least one of: 5'-UTRs of at least two of the genes described in (a), fragments of 5'-UTRs of at least two of the genes described in (a), variants of 5'-UTRs of at least two of the genes described in (a), and variants of fragments of 5'-UTRs of at least two of the genes described in (a). In some embodiments, the at least two genes are two genes, three genes, four genes, five genes, six genes, seven genes, eight genes, nine genes, or ten genes.

[0200] In some embodiments, the third nucleotide sequence comprises at least one of: at least two copies of the 5'-UTR in (a), at least two copies of a fragment of the 5'-UTR in (a), at least two copies of a variant of the 5'-UTR in (a), and at least two copies of a variant of a fragment of the 5'-UTR in (a). In some embodiments, the at least two copies are two copies, three copies, four copies, five copies, six copies, seven copies, eight copies, nine copies, or ten copies.

[0201] In some embodiments, the RNA molecule further comprises at least one of a promoter, a 5'-cap structure, a 3'-UTR, and polyA.

[0202] In some embodiments, the RNA molecule further comprises at least one of a 5'-cap structure, a 3'-UTR, and polyA.

[0203] In some embodiments, the 5'-cap structure comprises m 7 GpppG、m2 7，3′-O GpppG、m 7 Gppp(5′)N1 and m 7 Gppp(m 2′-O ) At least one of N1;

[0204] In some embodiments, the 3'-UTR comprises one of i) to iii):

[0205] i) at least one of the 3'-UTR, fragments, variants and variants of fragments thereof, derived from at least one of the genes APOA1, UL122, COP1, MYSM1, ASAH2B, NDUFAF2, APOD, HBB, TF, TMSB4X, CPAMD8, VIM, HDGFL1, TTR, SRM, HBA1, PRPF8, LMBRD1, IFNA1, CCDC146, GHRL and GH1; or at least one of the 3'-UTR, fragments, variants and variants of fragments thereof, derived from at least one of the genes APOA1, IE1, COP1, MYSM1, ASAH2B, NDUFAF2, APOD, HBB, TF, TMSB4X, CPAMD8, VIM, HDGFL1, TTR, SRM, HBA1, PRPF8, LMBRD1, IFNA1, CCDC146, GHRL and GH1;

[0206] ii) at least two copies of one of the polynucleotides described in i).

[0207] iii) at least two polynucleotides according to i).

[0208] In some embodiments, the species or specific GenBank accession number of the source gene of the 3'-UTR can be referred to the relevant description of the second nucleotide sequence above, which will not be repeated here.

[0209] In some embodiments, the 3'-UTR in i) comprises or is one or more of: an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 47-67 and 10, a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 47-67 and 10, a variant of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 47-67 and 10, and a variant of a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 47-67 and 10;

[0210] In some embodiments, variants of RNA encoded by polynucleotides having sequences as set forth in at least one of SEQ ID NOs: 47-67 and 10, fragments of RNA encoded by polynucleotides having sequences as set forth in at least one of SEQ ID NOs: 47-67 and 10, and variants of fragments of RNA encoded by polynucleotides having sequences as set forth in at least one of SEQ ID NOs: 47-67 and 10 are at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to RNA encoded by polynucleotides having sequences as set forth in at least one of SEQ ID NOs: 47-67 and 10.

[0211] In some embodiments, the nucleotides constituting the polyA comprise at least 20, at least 40, at least 80, at least 100, or at least 120 A nucleotides; In some embodiments, the nucleotides constituting the polyA comprise at least 20, at least 40, at least 80, at least 100, or at least 120 A nucleotides consecutively;

[0212] In some embodiments, the nucleotides constituting the polyA include one or more nucleotides other than A nucleotides.

[0213] In some embodiments, the polyA is a truncated polyA; in some embodiments, after multiple consecutive A nucleotides, there is a 10 bp non-A linker sequence and then multiple consecutive nucleotides A; in some embodiments, the polyA is 30 consecutive nucleotides A, followed by a 10 bp non-A linker sequence and then 70 consecutive nucleotides A.

[0214] In some embodiments, the polyA comprises at least one of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 68-76 and 11, a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 68-76 and 11, a variant of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 68-76 and 11, and a variant comprising a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 68-76 and 11; in some embodiments, the fragments, variants, and variants of the fragments of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 68-76 and 11 are at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an RNA encoded by a polynucleotide having a sequence as set forth in one of SEQ ID NOs: 68-76 and 11.

[0215] In some embodiments, the polyA is an RNA encoded by a polynucleotide having a sequence as shown in one of SEQ ID NOs: 68 to 70, 72, 75, and 11; in some embodiments, the polyA is an RNA encoded by a polynucleotide having a sequence as shown in SEQ ID NO: 11.

[0216] In some embodiments, the present invention also provides another RNA molecule comprising:

[0217] (1) a first nucleotide sequence encoding a polypeptide and / or protein of interest; and

[0218] (2) a 5'-untranslated region (5'-UTR); wherein the 5'-UTR comprises at least one of the following polynucleotides: an RNA encoded by a polynucleotide having a sequence as shown in SEQ ID NO: 9, a fragment of an RNA encoded by a polynucleotide having a sequence as shown in SEQ ID NO: 9, a variant of an RNA encoded by a polynucleotide having a sequence as shown in SEQ ID NO: 9, and a variant of a fragment of an RNA encoded by a polynucleotide having a sequence as shown in SEQ ID NO: 9;

[0219] In some embodiments, variants of RNA encoded by the polynucleotide having a sequence as set forth in SEQ ID NO:9, fragments of RNA encoded by the polynucleotide having a sequence as set forth in SEQ ID NO:9, and variants of fragments of RNA encoded by the polynucleotide having a sequence as set forth in SEQ ID NO:9 have at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the RNA encoded by the polynucleotide having a sequence as set forth in SEQ ID NO:9.

[0220] In some embodiments, the fragment, variant, or variant of the RNA encoded by the polynucleotide having a sequence as shown in SEQ ID NO: 9 has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotide insertions, additions, deletions, or substitutions compared to the RNA encoded by the polynucleotide having a sequence as shown in SEQ ID NO: 9.

[0221] In some embodiments, the RNA molecule further comprises at least one of a promoter, a 5'-cap structure, a 3'-UTR, and polyA.

[0222] In some embodiments, the RNA molecule further comprises at least one of a 5'-cap structure, a 3'-UTR, and polyA.

[0223] In some embodiments, the 5'-cap structure comprises m 7 GpppG、m2 7，3′-O GpppG、m 7 Gppp(5′)N1 and m 7 Gppp(m 2′-O ) At least one of N1;

[0224] In some embodiments, the 3'-UTR comprises one of i) to iii):

[0225] i) at least one of the 3'-UTR, fragments, variants and variants of fragments thereof derived from at least one of the genes APOA1, UL122, COP1, MYSM1, ASAH2B, NDUFAF2, APOD, HBB, TF, TMSB4X, CPAMD8, VIM, HDGFL1, TTR, SRM, HBA1, PRPF8, LMBRD1, IFNA1, CCDC146, GHRL and GH1; or at least one of the 3'-UTR, fragments, variants and variants of fragments thereof derived from at least one of the genes APOA1, IE1, COP1, MYSM1, ASAH2B, NDUFAF2, APOD, HBB, TF, TMSB4X, CPAMD8, VIM, HDGFL1, TTR, SRM, HBA1, PRPF8, LMBRD1, IFNA1, CCDC146, GHRL and GH1.

[0226] ii) at least two copies of one of the polynucleotides described in i).

[0227] iii) at least two polynucleotides according to i).

[0228] In some embodiments, the species or specific GenBank accession number of the source gene of the 3'-UTR can be referred to the relevant description of the second nucleotide sequence above, which will not be repeated here.

[0229] In some embodiments, the 3'-UTR in i) comprises or is one or more of the following: an RNA encoded by a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 47 to 67 and 10, a fragment of an RNA encoded by a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 47 to 67 and 10, a variant of an RNA encoded by a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 47 to 67 and 10, and a variant of a fragment of an RNA encoded by a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 47 to 67 and 10.

[0230] In some embodiments, variants of RNA encoded by polynucleotides having sequences as set forth in at least one of SEQ ID NOs: 47-67 and 10, fragments of RNA encoded by polynucleotides having sequences as set forth in at least one of SEQ ID NOs: 47-67 and 10, and variants of fragments of RNA encoded by polynucleotides having sequences as set forth in at least one of SEQ ID NOs: 47-67 and 10 are at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to RNA encoded by polynucleotides having sequences as set forth in at least one of SEQ ID NOs: 47-67 and 10.

[0231] In some embodiments, the nucleotides constituting the polyA comprise at least 20, at least 40, at least 80, at least 100, or at least 120 A nucleotides; In some embodiments, the nucleotides constituting the polyA comprise at least 20, at least 40, at least 80, at least 100, or at least 120 A nucleotides consecutively;

[0232] In some embodiments, the nucleotides constituting the polyA include one or more nucleotides other than A nucleotides.

[0233] In some embodiments, the polyA is a truncated polyA; in some embodiments, after multiple consecutive A nucleotides, there is a 10 bp non-A linker sequence and then multiple consecutive nucleotides A; in some embodiments, the polyA is 30 consecutive nucleotides A, followed by a 10 bp non-A linker sequence and then 70 consecutive nucleotides A.

[0234] In some embodiments, the polyA comprises at least one of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 68-76 and 11, a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 68-76 and 11, a variant of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 68-76 and 11, and a variant comprising a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 68-76 and 11; in some embodiments, the fragments, variants, and variants of the fragments of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 68-76 and 11 are at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an RNA encoded by a polynucleotide having a sequence as set forth in one of SEQ ID NOs: 68-76 and 11.

[0235] In some embodiments, the polyA is an RNA encoded by a polynucleotide having a sequence as shown in one of SEQ ID NOs: 68 to 70, 72, 75, and 11; in some embodiments, the polyA is an RNA encoded by a polynucleotide having a sequence as shown in SEQ ID NO: 11.

[0236] In some embodiments, the RNA molecule of any of the above embodiments is a recombinant RNA molecule or an artificial RNA molecule.

[0237] In some embodiments, all or part of the uracils in the RNA molecule of any of the above embodiments are replaced by at least one nucleoside selected from the group consisting of pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4′-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-T-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio- dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine or 5-methoxyuridine and 2′-O-methyluridine; in some embodiments, all or part of the uridine nucleosides in the RNA molecule are replaced by pseudouridine, N1-methylpseudouridine or N1-ethylpseudouridine.

[0238] In some embodiments, one or more cytosine nucleosides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cytosine nucleosides), or at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% of the cytosine nucleosides in the RNA molecule are replaced with 5-methylcytosine.

[0239] In some embodiments, the RNA of any of the above embodiments, the RNA molecule of any of the above embodiments, or the artificial RNA molecule of any of the above embodiments comprises one or more modified nucleotides. In some embodiments, the modified nucleotides have base modifications and / or sugar modifications. For example, the modified nucleotides have base modifications.

[0240] In some embodiments, the RNA of any of the above embodiments, the RNA molecule of any of the above embodiments, or the artificial RNA molecule of any of the above embodiments comprises one or more modified nucleosides.

[0241] In some embodiments, the RNA of any of the above embodiments, the RNA molecule of any of the above embodiments, or the artificial RNA molecule of any of the above embodiments comprises at least one of a modified uridine, a modified cytidine, a modified adenosine, and a modified guanosine.

[0242] In some embodiments, the modified nucleoside is a modified uridine. In some embodiments, 0.1% to 100% of the uridines in the RNA of any of the above embodiments, the RNA molecule of any of the above embodiments, or the artificial RNA molecule of any of the above embodiments are modified. In some embodiments, 80% to 100% of the uridines are modified. In some embodiments, 100% of the uridines are modified. Exemplary modified uridines include, but are not limited to, one or more of the following: pseudouridine (ψ), N1-methyl pseudouridine, pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 4-thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho5U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), 3-methyl-uridine (m3U), 5-methoxy-uridine (mo5U), ), uridine-5-oxyacetic acid (cmo5U), uridine-5-oxyacetic acid methyl ester (memo5U), 5-carboxymethyl-uridine (cm5U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm5U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm5U), 5-methoxycarbonylmethyl-uridine (mcm5U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm5s2U), 5-aminomethyl-2-thio-uridine (nm5s2U), 5-methylaminomethyl-uridine (mnm5U), 5-methylamino Methyl-2-thio-uridine (mnm5s2U), 5-methylaminomethyl-2-seleno-uridine (mnm5se2U), 5-carbamoylmethyl-uridine (ncm5U), 5-carboxymethylaminomethyl-uridine (cmnm5U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm5s2U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (τm5U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio 1-methyl-pseudouridine (τm5s2U), 1-taurine methyl-4-thio-pseudouridine, 5-methyl-uridine (m5U, i.e., with the nucleobase deoxythymine), 1-methyl-pseudouridine (m1ψ), 5-methyl-2-thio-uridine (m5s2U), 1-methyl-4-thio-pseudouridine (m1s4ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m3ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m5D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp3U), 1-methyl-3-(3-amino-3 -carboxypropyl) pseudouridine (acp3ψ), 5-(isopentenylaminomethyl)uridine (inm5U), 5-(isopentenylaminomethyl)-2-thio-uridine (inm5s2U), α-thio-uridine, 2′-O-methyl-uridine (Um), 5,2′-O-dimethyl-uridine (m5Um), 2′-O-methyl-pseudouridine (ψm), 2-thio-2′-O-methyl- Uridine (s2Um), 5-methoxycarbonylmethyl-2′-O-methyl-uridine (mcm5Um), 5-carbamoylmethyl-2′-O-methyl-uridine (ncm5Um), 5-carboxymethylaminomethyl-2′-O-methyl-uridine (cmnm5Um), 3,2′-O-dimethyl-uridine (m3Um), and 5-(isopentenylaminomethyl)-2′-O-methyl-uridine (inm5Um), 1-thio-uridine, deoxythymidine, 2′-F-arabino-uridine (2′-F-ara-uridine), 2′-F-uridine, 2′-OH-ara-uridine, 5-(2-methoxyformylvinyl)uridine (5-(2-carbomethoxyvinyl)uridine) and 5-[3-(1-E-propenylamino)uridine. ,

[0243] In some embodiments, the modified nucleoside is a modified cytidine. In some embodiments, 0.1% to 100% of the cytidines in the RNA of any of the above embodiments, the RNA molecule of any of the above embodiments, or the artificial RNA molecule of any of the above embodiments are modified. In some embodiments, 80% to 100% of the cytidines are modified. In some embodiments, 100% of the cytidines are modified. Exemplary modified cytidines include, but are not limited to, one or more of the following: 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine (m3C), N4-acetyl-cytidine (ac4C), 5-formyl-cytidine (f5C), N4-methyl-cytidine (m4C), 5-methyl-cytidine (m5C), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm5C), 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine (s2C), 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularin. e), 5-aza-zebulin, 5-methyl-zebulin, 5-aza-2-thio-zebulin, 2-thio-zebulin, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, lysidine (k2C), α-thio-cytidine, 2′-O-methyl-cytidine (Cm), 5,2′-O -dimethyl-cytidine (m5Cm), N4-acetyl-2′-O-methyl-cytidine (ac4Cm), N4,2′-O-dimethyl-cytidine (m4Cm), 5-formyl-2′-O-methyl-cytidine (f5Cm), N4,N4,2′-O-trimethyl-cytidine (m42Cm), 1-thio-cytidine, 2′-F-ara-cytidine, 2′-F-cytidine and 2′-OH-ara-cytidine.

[0244] In some embodiments, the modified nucleoside is a modified adenosine. In some embodiments, 0.1% to 100% of the adenosines in the RNA of any of the above embodiments, the RNA molecule of any of the above embodiments, or the artificial RNA molecule of any of the above embodiments are modified. In some embodiments, 80% to 100% of the adenosines are modified. In some embodiments, 100% of the adenosines are modified. Exemplary modified adenosines include, but are not limited to, one or more of the following: 2-amino-purine, 2,6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenosine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyl-adenosine (m1A), 2-methyl- Adenine (m2A), N6-methyl-adenosine (m6A), 2-methylthio-N6-methyl-adenosine (ms2m6A), N6-isopentenyl-adenosine (i6A), 2-methylthio-N6-isopentenyl-adenosine (ms2i6A), N6-(cis-hydroxyisopentenyl)adenosine (io6A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine (ms2io6A), N6-glycylcarbamoyl-adenosine (g6A), N6-threonylcarbamoyl-adenosine (t6A), N6-methyl-N6-threonylcarbamoyl-adenosine (m6t6A), 2-methylthio- N6-threonylcarbamoyl-adenosine (ms2g6A), N6,N6-dimethyl-adenosine (m62A), N6-hydroxynorvalylcarbamoyl-adenosine (hn6A), 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenosine (ms2hn6A), N6-acetyl-adenosine (ac6A), 7-methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine, α-thio-adenosine, 2′-O-methyl-adenosine (Am), N 6,2′-O-dimethyl-adenosine (m6Am), N6,N6,2′-O-trimethyl-adenosine (m62Am), 1,2′-O-dimethyl-adenosine (m1Am), 2′-O-ribosyladenosine (phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1-thio-adenosine, 8-azido-adenosine, 2′-F-arabino-adenosine, 2′-F-adenosine, 2′-OH-arabino-adenosine and N6-(19-amino-pentaoxahoundadecanoyl)-adenosine.

[0245] In some embodiments, the modified nucleoside is a modified guanosine. In some embodiments, 0.1% to 100% of the guanosine in the RNA of any of the above embodiments, the RNA molecule of any of the above embodiments, or the artificial RNA molecule of any of the above embodiments is modified. In some embodiments, 80% to 100% of the guanosine is modified. In some embodiments, 100% of the guanosine is modified. Exemplary modified guanosines include, but are not limited to, one or more of the following: inosine (I), 1-methyl-inosine (m1I), wyosine (imG), methyl wyosine (mimG), 4-demethyl-wyosine (imG-14), iso-wyosine (imG2), yW, peroxyyW (o2yW), hydroxyyW (OHyW), undermodified hydroxyyW (OHyW*), 7-deaza-guanosine, queuosine ( Q), epoxy-braided glycoside (oQ), galactosyl-braided glycoside (galQ), mannosyl-braided glycoside (manQ), 7-cyano-7-deaza-guanosine (preQ0), 7-aminomethyl-7-deaza-guanosine (preQ1), archaeoside (G+), 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine (m7G), 6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine, 1 -methyl-guanosine (m1G), N2-methyl-guanosine (m2G), N2,N2-dimethyl-guanosine (m22G), N2,7-dimethyl-guanosine (m2,7G), N2,N2,7-dimethyl-guanosine (m2,2,7G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine, α-thio-guanosine, 2′-O-methyl-guanosine (Gm), N2-methyl-2′- O-methyl-guanosine (m2Gm), N2,N2-dimethyl-2′-O-methyl-guanosine (m22Gm), 1-methyl-2′-O-methyl-guanosine (m1Gm), N2,7-dimethyl-2′-O-methyl-guanosine (M2,7Gm), 2′-O-methyl-inosine (Im), 1,2′-O-dimethyl-inosine (m1Im), 2′-O-ribosylguanosine (phosphate) (Gr(p)), 1-thio-guanosine, O6-methyl-guanosine, 2′-F-arabino-guanosine and 2′-F-guanosine.

[0246] In some embodiments, modified nucleotides comprise isotope-containing nucleotides.

[0247] In some embodiments, the RNA, RNA molecule, or artificial RNA molecule of any of the above embodiments comprises nucleotides containing isotopes of hydrogen. Hydrogen isotopes are not limited to deuterium and tritium. In some embodiments, the RNA further comprises or contains nucleotides containing isotopes of elements other than hydrogen, including but not limited to carbon, oxygen, nitrogen, and phosphorus.

[0248] In some embodiments, the nucleotide sequence encoding a polypeptide of interest of any of the above embodiments or the first nucleotide sequence of any of the above embodiments encodes at least one polypeptide of interest. For example, one, two, three, four, five, six, seven, eight, nine or ten polypeptides of interest. In some embodiments, the nucleotide sequence encoding a polypeptide of interest of any of the above embodiments or the first nucleotide sequence of any of the above embodiments encodes at least one protein of interest. For example, one, two, three, four, five, six, seven, eight, nine or ten proteins of interest. In some embodiments, the nucleotide sequence encoding a polypeptide of interest and a protein of interest of any of the above embodiments or the first nucleotide sequence of any of the above embodiments encodes at least one polypeptide of interest and at least one protein of interest. For example, one, two, three, four, five, six, seven, eight, nine or ten polypeptides of interest and one, two, three, four, five, six, seven, eight, nine or ten proteins of interest.

[0249] In some embodiments, the protein of interest is the S protein of the novel coronavirus. In some embodiments, the amino acid sequence of the S protein of the novel coronavirus is as described above. In some embodiments, the sequence of the RNA encoding the S protein of the novel coronavirus is as described above. It is understood that the protein of interest is not limited to the S protein of the novel coronavirus, but may also include other proteins.

[0250] In some embodiments, the RNA of any of the above embodiments, the RNA molecule of any of the above embodiments, or the artificial RNA molecule of any of the above embodiments is no longer than 50,000 nt. In some embodiments, the RNA of any of the above embodiments, the RNA molecule of any of the above embodiments, or the artificial RNA molecule of any of the above embodiments is no longer than 40,000 nt, 30,000 nt, 20,000 nt, 10,000 nt, 9,000 nt, 8,000 nt, or 6,000 nt. In some embodiments, the RNA of any of the above embodiments, the RNA molecule of any of the above embodiments, or the artificial RNA molecule of any of the above embodiments is between 500 nt and 50,000 nt. In some embodiments, the RNA of any of the above embodiments, the RNA molecule of any of the above embodiments, or the artificial RNA molecule of any of the above embodiments is between 1,000 nt and 40,000 nt, between 1,000 nt and 30,000 nt, between 1,500 nt and 10,000 nt, or between 1,500 nt and 8,000 nt.

[0251] In some embodiments, the present invention further provides a DNA encoding the RNA of any of the above embodiments, a DNA encoding the artificial RNA molecule of any of the above embodiments, or a DNA encoding the RNA molecule of any of the above embodiments.

[0252] In some embodiments, the DNA of any of the above embodiments is no longer than 50,000 bp. In some embodiments, the DNA of any of the above embodiments is no longer than 40,000 bp, 30,000 bp, 20,000 bp, 10,000 bp, 9,000 bp, 8,000 bp, or 6,000 bp. In some embodiments, the DNA of any of the above embodiments is between 500 bp and 50,000 bp. In some embodiments, the DNA of any of the above embodiments is between 1,000 bp and 40,000 bp, between 1,000 bp and 30,000 bp, between 1,500 bp and 10,000 bp, or between 1,500 bp and 8,000 bp.

[0253] In some embodiments, the present invention also provides a vector comprising the DNA according to any one of the above embodiments.

[0254] In some embodiments, the present invention further provides another vector comprising a fourth nucleotide sequence encoding a 3'-UTR, wherein the fourth nucleotide sequence comprises at least one of the following polynucleotides:

[0255] (a): a polynucleotide encoding a 3'-UTR derived from at least one of the genes APOA1, UL122, COP1, MYSM1, ASAH2B, NDUFAF2, APOD, HBB, TF, TMSB4X, CPAMD8, VIM, HDGFL1, TTR, SRM, HBA1, PRPF8, LMBRD1, IFNA1, CCDC146, and GHRL; in some embodiments, a polynucleotide encoding a 3'-UTR derived from at least one of the genes APOA1, IE1, COP1, MYSM1, ASAH2B, NDUFAF2, APOD, HBB, TF, TMSB4X, CPAMD8, VIM, HDGFL1, TTR, SRM, HBA1, PRPF8, LMBRD1, IFNA1, CCDC146, and GHRL;

[0256] (b): a polynucleotide encoding a fragment of the 3'-UTR described in (a);

[0257] (c): a polynucleotide encoding a variant of the 3'-UTR described in (a); and

[0258] (d): A polynucleotide encoding a variant of the fragment described in (b). In some embodiments, the gene is a human gene.

[0259] In some embodiments, the vector does not comprise a polynucleotide encoding a protein and / or polypeptide of interest.

[0260] In some embodiments, the fourth nucleotide sequence comprises at least one of the following polynucleotides:

[0261] (e): a polynucleotide encoding a 3′-UTR derived from at least one of the genes COP1 and HDGFL1;

[0262] (f): a polynucleotide encoding the 3'-UTR fragment described in (e);

[0263] (g): a polynucleotide encoding a variant of the 3'-UTR described in (e); and

[0264] (h): A polynucleotide encoding a variant of the fragment described in (f).

[0265] In some embodiments, the species or specific GenBank accession number of the source gene of the 3'-UTR can be referred to the relevant description of the second nucleotide sequence above, which will not be repeated here.

[0266] In some embodiments, the fourth nucleotide sequence comprises at least one of the following polynucleotides (1) to (3):

[0267] (1) At least one of a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 47 to 67, a fragment of a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 47 to 67, a variant of a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 47 to 67, and a variant of a fragment of a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 47 to 67.

[0268] (2) at least one of a polynucleotide encoding a 3'-UTR of at least two of the genes described in (a), a polynucleotide encoding a fragment of a 3'-UTR of at least two of the genes described in (a), a polynucleotide encoding a variant of a 3'-UTR of at least two of the genes described in (a), and a polynucleotide encoding a variant of a fragment of a 3'-UTR of at least two of the genes described in (a). In some embodiments, the at least two genes are two genes, three genes, four genes, five genes, six genes, seven genes, eight genes, nine genes, or ten genes.

[0269] (3) at least one of at least two copies of the 3'-UTR in (a), at least two copies of a fragment of the 3'-UTR in (a), at least two copies of a variant of the 3'-UTR in (a), and at least two copies of a variant of a fragment of the 3'-UTR in (a). In some embodiments, the at least two copies are two copies, three copies, four copies, five copies, six copies, seven copies, eight copies, nine copies, or ten copies.

[0270] In some embodiments, variants of the polynucleotides having a sequence as set forth in at least one of SEQ ID NOs: 47-67, fragments of the polynucleotides having a sequence as set forth in at least one of SEQ ID NOs: 47-67, and variants of fragments of the polynucleotides having a sequence as set forth in at least one of SEQ ID NOs: 47-67 are at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the polynucleotides having a sequence as set forth in at least one of SEQ ID NOs: 47-67.

[0271] In some embodiments, the fragment, variant, or variant of the fragment of a polynucleotide with a sequence as set forth in at least one of SEQ ID NOs: 47 to 67 has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotide insertions, additions, deletions, or substitutions compared to the polynucleotide with a sequence as set forth in one of SEQ ID NOs: 47 to 67.

[0272] In some embodiments, the vector further comprises at least one of a promoter, a polynucleotide encoding a 5'-UTR, and a polynucleotide encoding polyA.

[0273] In some embodiments, the 5'-UTR comprises one of i) to iv):

[0274] i) at least one of a 5'-UTR derived from at least one of the genes APOA1, CARD16, ALB, APOC1, EEF1A1, RBP4, GHRL, MPND, ASAH2B, FBX016, FBH1, SRM, NAAA, ACTB, MKNK2, ORM1, GADPH, NDUFAF2, FGFR2, MYCBPAP, CHMP2A, TSG101, PRPF8, NFKB2, NAE1, HDGFL1, GSDMD, FBXW12, FBXW10, FBXL8, and ATG4D, a fragment thereof, a variant thereof, and a variant of a fragment thereof.

[0275] ii) at least one of the following polynucleotides: an RNA encoded by the polynucleotide having a sequence as shown in SEQ ID NO: 9, a fragment of the RNA encoded by the polynucleotide having a sequence as shown in SEQ ID NO: 9, a variant of the RNA encoded by the polynucleotide having a sequence as shown in SEQ ID NO: 9, and a variant of a fragment of the RNA encoded by the polynucleotide having a sequence as shown in SEQ ID NO: 9.

[0276] iii) at least two copies of one of the polynucleotides of i) or ii).

[0277] iv) at least two polynucleotides according to i).

[0278] In some embodiments, the species or specific GenBank accession number of the source gene of the 5'-UTR can be referred to the relevant description of the third nucleotide sequence above, which will not be repeated here.

[0279] In some embodiments, the 5'-UTR in i) comprises or is one or more of the following: an RNA encoded by a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 16 to 46, a fragment of an RNA encoded by a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 16 to 46, a variant of an RNA encoded by a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 16 to 46, and a variant of a fragment of an RNA encoded by a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 16 to 46.

[0280] In some embodiments, variants of RNA encoded by polynucleotides having a sequence as set forth in at least one of SEQ ID NOs: 16-46, fragments of RNA encoded by polynucleotides having a sequence as set forth in at least one of SEQ ID NOs: 16-46, and variants of fragments of RNA encoded by polynucleotides having a sequence as set forth in at least one of SEQ ID NOs: 16-46 are at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the RNA encoded by polynucleotides having a sequence as set forth in at least one of SEQ ID NOs: 16-46.

[0281] In some embodiments, the variants of the RNA encoded by the polynucleotide with a sequence as shown in SEQ ID NO: 9, the fragments of the RNA encoded by the polynucleotide with a sequence as shown in SEQ ID NO: 9, and the variants of the fragments of the RNA encoded by the polynucleotide with a sequence as shown in SEQ ID NO: 9 in ii) have a sequence of at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the RNA encoded by the polynucleotide with a sequence as shown in SEQ ID NO: 9.

[0282] In some embodiments, the nucleotides constituting the polyA comprise at least 20, at least 40, at least 80, at least 100, or at least 120 A nucleotides; in some embodiments, the nucleotides constituting the polyA comprise at least 20, at least 40, at least 80, at least 100, or at least 120 A nucleotides consecutively.

[0283] In some embodiments, the nucleotides constituting the polyA include one or more nucleotides other than A nucleotides.

[0284] In some embodiments, the polyA is a truncated polyA; in some embodiments, after a plurality of consecutive A nucleotides, there is a 10 bp non-A linker sequence followed by a plurality of consecutive nucleotides A. In some embodiments, the polyA is 30 consecutive nucleotides A followed by a 10 bp non-A linker sequence followed by 70 consecutive nucleotides A.

[0285] In some embodiments, the polyA comprises at least one of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 68-76 and 11, a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 68-76 and 11, a variant of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 68-76 and 11, and a variant of an RNA encoded by a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 68-76 and 11; in some embodiments, the fragments, variants, and variants of the fragments of an RNA encoded by at least one of SEQ ID NOs: 68-76 and 11 are at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an RNA encoded by a polynucleotide having a sequence as set forth in one of SEQ ID NOs: 68-76 and 11.

[0286] In some embodiments, the polyAy is an RNA encoded by a polynucleotide having a sequence as shown in one of SEQ ID NOs: 68 to 70, 72, 75, and 11. In some embodiments, the polyA is an RNA encoded by a polynucleotide having a sequence as shown in SEQ ID NO: 11.

[0287] In some embodiments, the present invention further provides another vector comprising a fifth nucleotide sequence encoding a 5'-UTR, wherein the fifth nucleotide sequence comprises at least one of the following polynucleotides:

[0288] (a) a polynucleotide encoding the 5'-UTR of at least one of the following genes: APOA1, CARD16, ALB, APOC1, EEF1A1, RBP4, GHRL, MPND, ASAH2B, FBX016, FBH1, SRM, NAAA, ACTB, MKNK2, ORM1, GADPH, NDUFAF2, FGFR2, MYCBPAP, CHMP2A, TSG101, PRPF8, NFKB2, NAE1, HDGFL1, GSDMD, FBXW12, FBXW10, FBXL8, and ATG4D;

[0289] (b): a polynucleotide encoding a fragment of the 5'-UTR described in (a);

[0290] (c): a polynucleotide encoding a variant of the 5'-UTR described in (a); and

[0291] (d): A polynucleotide encoding a variant of the fragment described in (b).

[0292] In some embodiments, the species or specific GenBank accession number of the source gene of the 5'-UTR can be referred to the relevant description of the third nucleotide sequence above, which will not be repeated here.

[0293] In some embodiments, the vector does not comprise a polynucleotide encoding a protein and / or polypeptide of interest.

[0294] In some embodiments, the fifth nucleotide sequence comprises at least one of the following polynucleotides: a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 16 to 46, a fragment of a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 16 to 46, a variant of a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 16 to 46, and a variant of a fragment of a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 16 to 46.

[0295] In some embodiments, variants of a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 16-46, fragments of a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 16-46, and variants of a fragment of a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 16-46, are at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 16-46. In some embodiments, the fragment, variant, or variant of a fragment of a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 16-46 has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more insertions, additions, deletions, or substitutions of nucleotides compared to a polynucleotide having a sequence as set forth in one of SEQ ID NOs: 16-46.

[0296] In some embodiments, the fifth nucleotide sequence comprises at least one of: 5'-UTRs of at least two of the genes described in (a), fragments of 5'-UTRs of at least two of the genes described in (a), variants of 5'-UTRs of at least two of the genes described in (a), and variants of fragments of 5'-UTRs of at least two of the genes described in (a). In some embodiments, the at least two genes are two genes, three genes, four genes, five genes, six genes, seven genes, eight genes, nine genes, or ten genes.

[0297] In some embodiments, the fifth nucleotide sequence comprises at least one of: at least two copies of the 5'-UTR in (a), at least two copies of a fragment of the 5'-UTR in (a), at least two copies of a variant of the 5'-UTR in (a), and at least two copies of a variant of a fragment of the 5'-UTR in (a). In some embodiments, the at least two copies are two copies, three copies, four copies, five copies, six copies, seven copies, eight copies, nine copies, or ten copies.

[0298] In some embodiments, the vector further comprises at least one of a promoter, a polynucleotide encoding a 3'-UTR, and a polynucleotide encoding polyA.

[0299] In some embodiments, the 3'-UTR comprises one of i) to iii):

[0300] i) at least one of the 3'-UTR, fragments, variants and variants of fragments thereof derived from at least one of the genes APOA1, UL122, COP1, MYSM1, ASAH2B, NDUFAF2, APOD, HBB, TF, TMSB4X, CPAMD8, VIM, HDGFL1, TTR, SRM, HBA1, PRPF8, LMBRD1, IFNA1, CCDC146, GHRL and GH1; or at least one of the 3'-UTR, fragments, variants and variants of fragments thereof derived from at least one of the genes APOA1, IE1, COP1, MYSM1, ASAH2B, NDUFAF2, APOD, HBB, TF, TMSB4X, CPAMD8, VIM, HDGFL1, TTR, SRM, HBA1, PRPF8, LMBRD1, IFNA1, CCDC146, GHRL and GH1.

[0301] ii) at least two copies of one of the polynucleotides described in i).

[0302] iii) at least two polynucleotides according to i).

[0303] In some embodiments, the species or specific GenBank accession number of the source gene of the 3'-UTR can be referred to the relevant description of the second nucleotide sequence above, which will not be repeated here.

[0304] In some embodiments, the 3'-UTR in i) comprises or is one or more of the following: an RNA encoded by a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 47 to 67 and 10, a fragment of an RNA encoded by a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 47 to 67 and 10, a variant of an RNA encoded by a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 47 to 67 and 10, and a variant of a fragment of an RNA encoded by a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 47 to 67 and 10.

[0305] In some embodiments, variants of RNA encoded by polynucleotides having sequences as set forth in at least one of SEQ ID NOs: 47-67 and 10, fragments of RNA encoded by polynucleotides having sequences as set forth in at least one of SEQ ID NOs: 47-67 and 10, and variants of fragments of RNA encoded by polynucleotides having sequences as set forth in at least one of SEQ ID NOs: 47-67 and 10 are at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to RNA encoded by polynucleotides having sequences as set forth in at least one of SEQ ID NOs: 47-67 and 10.

[0306] In some embodiments, the nucleotides constituting the polyA comprise at least 20, at least 40, at least 80, at least 100, or at least 120 A nucleotides; in some embodiments, the nucleotides constituting the polyA comprise at least 20, at least 40, at least 80, at least 100, or at least 120 A nucleotides consecutively.

[0307] In some embodiments, the nucleotides constituting the polyA include one or more nucleotides other than A nucleotides.

[0308] In some embodiments, the polyA is a truncated polyA; in some embodiments, after multiple consecutive A nucleotides, there is a 10 bp non-A linker sequence and then multiple consecutive nucleotides A; in some embodiments, the polyA is 30 consecutive nucleotides A, followed by a 10 bp non-A linker sequence and then 70 consecutive nucleotides A.

[0309] In some embodiments, the polyA comprises at least one of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 68-76 and 11, a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 68-76 and 11, a variant of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 68-76 and 11, and a variant comprising a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 68-76 and 11; in some embodiments, the fragments, variants, and variants of the fragments of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 68-76 and 11 are at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an RNA encoded by a polynucleotide having a sequence as set forth in one of SEQ ID NOs: 68-76 and 11.

[0310] In some embodiments, the polyA is an RNA encoded by a polynucleotide having a sequence as shown in one of SEQ ID NOs: 68 to 70, 72, 75, and 11; in some embodiments, the polyA is an RNA encoded by a polynucleotide having a sequence as shown in SEQ ID NO: 11.

[0311] In some embodiments, the present invention further provides another vector comprising a polynucleotide sequence encoding a 5'-UTR; wherein the polynucleotide sequence encoding the 5'-UTR comprises at least one of the following polynucleotides: a polynucleotide having a sequence as shown in SEQ ID NO: 9, a fragment of the polynucleotide having a sequence as shown in SEQ ID NO: 9, a variant of the polynucleotide having a sequence as shown in SEQ ID NO: 9, and a variant of a fragment of the polynucleotide having a sequence as shown in SEQ ID NO: 9;

[0312] In some embodiments, variants of the polynucleotide with a sequence as set forth in SEQ ID NO: 9, fragments of the polynucleotide with a sequence as set forth in SEQ ID NO: 9, and variants of fragments of the polynucleotide with a sequence as set forth in SEQ ID NO: 9 have at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the polynucleotide with a sequence as set forth in SEQ ID NO: 9. In some embodiments, the fragments, variants, or variants of the fragments of the polynucleotide with a sequence as set forth in SEQ ID NO: 9 have at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more insertions, additions, deletions, or substitutions of nucleotides compared to the polynucleotide with a sequence as set forth in SEQ ID NO: 9.

[0313] In some embodiments, the vector further comprises at least one of a promoter, a polynucleotide encoding a 3'-UTR, and a polynucleotide encoding polyA.

[0314] In some embodiments, the 3'-UTR comprises one of i) to iii):

[0315] i) at least one of the 3'-UTR, fragments, variants and variants of fragments thereof derived from at least one of the genes APOA1, UL122, COP1, MYSM1, ASAH2B, NDUFAF2, APOD, HBB, TF, TMSB4X, CPAMD8, VIM, HDGFL1, TTR, SRM, HBA1, PRPF8, LMBRD1, IFNA1, CCDC146, GHRL and GH1; or at least one of the 3'-UTR, fragments, variants and variants of fragments thereof derived from at least one of the genes APOA1, IE1, COP1, MYSM1, ASAH2B, NDUFAF2, APOD, HBB, TF, TMSB4X, CPAMD8, VIM, HDGFL1, TTR, SRM, HBA1, PRPF8, LMBRD1, IFNA1, CCDC146, GHRL and GH1.

[0316] ii) at least two copies of one of the polynucleotides described in i).

[0317] iii) at least two polynucleotides according to i).

[0318] In some embodiments, the species or specific GenBank accession number of the source gene of the 3'-UTR can be referred to the relevant description of the second nucleotide sequence above, which will not be repeated here.

[0319] In some embodiments, the 3'-UTR in i) comprises or is one or more of the following: an RNA encoded by a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 47 to 67 and 10, a fragment of an RNA encoded by a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 47 to 67 and 10, a variant of an RNA encoded by a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 47 to 67 and 10, and a variant of a fragment of an RNA encoded by a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 47 to 67 and 10.

[0320] In some embodiments, variants of RNA encoded by polynucleotides having sequences as set forth in at least one of SEQ ID NOs: 47-67 and 10, fragments of RNA encoded by polynucleotides having sequences as set forth in at least one of SEQ ID NOs: 47-67 and 10, and variants of fragments of RNA encoded by polynucleotides having sequences as set forth in at least one of SEQ ID NOs: 47-67 and 10 are at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to RNA encoded by polynucleotides having sequences as set forth in at least one of SEQ ID NOs: 47-67 and 10.

[0321] In some embodiments, the nucleotides constituting the polyA comprise at least 20, at least 40, at least 80, at least 100, or at least 120 A nucleotides; in some embodiments, the nucleotides constituting the polyA comprise at least 20, at least 40, at least 80, at least 100, or at least 120 A nucleotides consecutively.

[0322] In some embodiments, the nucleotides constituting the polyA include one or more nucleotides other than A nucleotides.

[0323] In some embodiments, the polyA is a truncated polyA; in some embodiments, after multiple consecutive A nucleotides, there is a 10 bp non-A linker sequence and then multiple consecutive nucleotides A; in some embodiments, the polyA is 30 consecutive nucleotides A, followed by a 10 bp non-A linker sequence and then 70 consecutive nucleotides A.

[0324] In some embodiments, the polyA comprises at least one of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 68-76 and 11, a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 68-76 and 11, a variant of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 68-76 and 11, and a variant comprising a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 68-76 and 11; in some embodiments, the fragments, variants, and variants of the fragments of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 68-76 and 11 are at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an RNA encoded by a polynucleotide having a sequence as set forth in one of SEQ ID NOs: 68-76 and 11.

[0325] In some embodiments, the polyA is an RNA encoded by a polynucleotide having a sequence as shown in one of SEQ ID NOs: 68 to 70, 72, 75, and 11; in some embodiments, the polyA is an RNA encoded by a polynucleotide having a sequence as shown in SEQ ID NO: 11.

[0326] In any of the above embodiments, the polypeptide or protein of interest refers to a therapeutically or pharmaceutically active polypeptide or protein having a therapeutic or preventive effect, whose function in or near a cell is necessary or beneficial, for example, a protein whose deficiency or defective form leads to a disease, and whose provision can modulate or prevent the disease, or a protein whose presence in or near a cell is beneficial to the body. The polypeptide or protein may comprise the entire protein or a functional variant thereof.

[0327] In any of the above embodiments, the nucleotide sequence encoding the polypeptide and / or protein of interest or the peptide and / or protein expressed by the first nucleotide sequence comprises or is one or more of the following: (a) an antigen; (b) a therapeutic protein or polypeptide, a fragment, a fragment or a variant thereof; and (c) other polypeptides or proteins.

[0328] In some embodiments, the peptide and / or protein expressed by the nucleotide sequence encoding the polypeptide and / or protein of interest comprises or is an antigen.

[0329] In some embodiments, the antigen expressed by the nucleotide sequence encoding the polypeptide and / or protein of interest is derived from one or more of the following: (1) a pathogenic antigen, a fragment, a variant, or a variant of a fragment thereof, (2) a tumor antigen, a fragment, a variant, or a variant of a fragment thereof, (3) an allergic antigen, a fragment, a variant, or a variant of a fragment thereof, (4) an autoimmune self-antigen, a fragment, a variant, or a variant of a fragment thereof.

[0330] In some embodiments, pathogenic antigens are derived from pathogenic organisms that are capable of eliciting an immune response in a subject (e.g., a mammalian subject, further e.g., a human). In some embodiments, the pathogenic organisms include or are one or more of the following: bacteria, viruses, fungi, and protozoa (e.g., single-cell organisms, multicellular organisms).

[0331] In some embodiments, the pathogenic antigen comprises or is a surface antigen, fragment, variant, or variant of a fragment thereof, such as a protein, fragment (e.g., an external portion of a surface antigen), variant, or variant of a fragment thereof located on the surface of a virus, bacteria, or protozoa.

[0332] In some embodiments, the pathogenic antigen comprises or is derived from a polypeptide or protein of a pathogen associated with an infectious disease.

[0333] In some embodiments, the pathogenic antigen is selected from but not limited to the group consisting of antigens derived from pathogens described on pages 21 to 35 of WO2018 / 078053A1, antigens derived from pathogens described on page 57, paragraph 3 to page 63, paragraph 2 of WO2019 / 077001A1, antigens derived from pathogens described on page 32, line 26 to page 34, line 27 of WO2013 / 120628A1, and antigens described on page 34, line 29 to page 59, line 5 of WO2013 / 120628A1.

[0334] In some embodiments, the pathogen of the pathogenic antigen is selected from but not limited to one or more of the following: scabies, Babesia, Leishmania, Gnatostoma, Ancylostoma braziliensis, Ancylostoma duodenale, Strongyloides stercoralis, Trichuris trichuris, Toxocara canis, Toxocara felis, Toxoplasma gondii, Trypanosoma brucei, Trypanosoma cruzi, Brugia malayi, Onchocerca volvulus, Bancroftian filariasis, Tapeworm, Taenia solium, Echinococcus, Ascaris lumbricoides, Dikaryotic amoeba fragilis, Naegleria fowleri, fowleri), Necator americanus, Paragonimus (e.g., Paragonimus westermani), Clonorchis sinensis, Plasmodium (e.g., P. falciparum, P. vivax, P. malariae, or P. ovale), Pneumocystis jiroveci, Metagonid fluke, Trichinella spiralis, Trichomonas vaginalis, Giardia intestinalis, Schistosoma japonicum, Geotrichum candidum, human yeast, Bartonella henselae, Wernicke's black yeast (Hortaea Wernicke), Aspergillus, Malassezia, Vibrio cholerae, Acinetobacter baumannii, Paracoccidioides brasiliensis, Neisseria gonorrhoeae, Neisseria meningitidis, Pasteurella multocida, Nocardia asteroides, Nocardia schenckii, Sporothrix schenckii, Staphylococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Trichophyton, Yersinia enterocolitica, Yersinia pestis, Yersinia pseudotuberculosis, Ureaplasma urealyticum, Salmonella, Francisella tularensis, Fusobacterium, Mycobacterium leprae, Mycobacterium leprae disseminated, Mycobacterium tuberculosis, Mycobacterium ulcerans, Shigella, and Cryptococcus hemolyticus. Bacillus anthracis, Bacillus cereus, Histoplasma capsulatum, Blastomyces dermatitidis, Bordetella pertussis, Borrelia burgdorferi, Burkholderia, Brucella, Burkholderia (e.g., Burkholderia cepacia, Burkholderia mallei, Burkholderia pseudomallei), Campylobacter, Candida (e.g., Candida albicans), Chlamydomonas pneumoniae, Corynebacterium diphtheriae, Coxiella burnetii, Clostridium (e.g., Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium perfringens), Clostridium tetani, Coccidioides, Ehrlichia chaffeensis, Ehrlichia eiewingii), Ehrlichia, extraintestinal pathogenic Escherichia coli, Kingella, Klebsiella granulomatosa, Anaplasmas (e.g., Anaplasma phagocytophilum), Leptospira, Borrelia burgdorferi, Treponema pallidum, Rickettsiae (e.g., Rickettsia prowazekii, Rickettsia rickettsii, Rickettsia typhi), Chlamydia psittaci, Chlamydia trachomatis, kuru prion, Lassa virus (LASV), Legionella pneumophila, Listeria monocytogenes, Enterococci, dermatophytes, Escherichia coli O157:H7 and O104:H4, Fasciola hepatica and Fasciola gigantica, enteroviruses (e.g., Coxsackie virus Viruses include: A virus, enterovirus 71 (EV71), FFI prions, CJD prions, Epstein-Barr virus (EBV), feline immunodeficiency virus (FIV), flavivirus, GSS prions, Guanarito virus, Haemophilus ducreyi, Haemophilus influenzae, Helicobacter pylori, Bunyaviridae, Caliciviridae, Astroviridae, coronavirus, Congo hemorrhagic fever virus, Cryptococcus neoformans, Cryptosporidium spp., cytomegalovirus (CMV), BK virus, dengue virus, Ebola virus (EBOV), herpes simplex virus (HSV), human immunodeficiency virus (HIV), Human papillomavirus (HPV), influenza virus, rabies virus, norovirus, Nipah virus, Henlepa virus (Henkley virus-Nipah virus), hepatitis A virus, hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus, hepatitis E virus, human bocavirus (HBoV), human metapneumovirus (hMPV), human parainfluenza virus (HPIV), Japanese encephalitis virus, JC virus, Junin virus, yellow fever virus, MERS coronavirus, lymphocytic choriomeningitis virus (LCMV), Machupo virus, Marburg virus virus, measles virus, human molluscum contagiosum virus (MCV), mumps virus, parvovirus B19, Mycoplasma pneumoniae orthomyxovirus, poliovirus, rhinovirus, Rift Valley fever virus, rotavirus, rubella virus, Sabia virus, SARS coronavirus (e.g., SARS-CoV-2), nCoV-2019 coronavirus, Sin Nombre virus, hantavirus, vaccinia virus, respiratory syncytial virus (RSV), tick-borne encephalitis virus (TBEV), varicella-zoster virus (VZV), Venezuelan equine encephalitis virus, West Nile virus, western equine encephalitis virus, and Zika virus.

[0335] In some embodiments, the pathogenic antigen includes or is one or more of the following:

[0336] (1) one or more of the following proteins of SARS coronavirus 2 (SARS-CoV-2), nCoV-2019 coronavirus or SARS coronavirus (SARS-CoV): spike protein (S), envelope protein (E), membrane protein (M) or nucleocapsid protein (N); (2) one or more of the following proteins of MERS coronavirus: spike protein (S), spike S1 segment (S1), envelope protein (E), membrane protein (M) or nucleocapsid protein (N); (3) one or more of the following proteins of human papillomavirus (e.g. HPV16) One or more of the following proteins: replication protein E1, regulatory protein E2, protein E3, protein E4, protein E5, protein E6, protein E7, protein E8, major capsid protein L1 and minor capsid protein L2; (4) one or more of the following proteins of human parainfluenza virus (HPIV / PIV) (e.g., hPIV-1, hPIV-2, hPIV-3 or hPIV-4 serotype): fusion protein (F), hemagglutinin neuraminidase, glycoprotein (G), matrix protein (M), phosphoprotein (P), nucleocapsid protein, fusion glycoprotein F0, F1 or F2, recombinant PIV3 / PIV1 fusion glycoprotein, C protein, D protein, viral replicase (L) and nonstructural V protein; (5) one or more of the following proteins of human metapneumovirus (hMPV): fusion (F) glycoprotein, glycoprotein (G), phosphoprotein (P), and nucleocapsid protein; (6) one or more of the following proteins of influenza virus: hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), M1 protein, M2 protein, NS1 protein, NS2 protein (NEP protein: nuclear export protein), PA protein, PB1 protein (polymerase basic 1 protein), PB1-F2 protein and PB2 protein; (7) one or more of the following proteins of rabies virus: nucleoprotein (N), large structural protein (L), phosphoprotein (P), matrix protein (M) and glycoprotein (G); (8) one or more of the following proteins of human immunodeficiency virus: HIV p24 antigen, HIV envelope proteins (Gp120, Gp41, Gp160), polyprotein GAG, negative factor protein Nef, transcriptional transactivator Tat and Brec1; (9) one or more of the following proteins of Chlamydia trachomatis: major outer membrane protein MOMP, possible outer membrane protein PMPC, outer membrane complex protein B OmcB, heat shock protein Hsp60HSP10, protein IncA, type III secretion system protein, ribonucleotide reductase small chain protein NrdB, plasmid protein Pgp3, Chlamydia outer protein N CopN, antigen CT521, antigen CT425, antigen CT043, antigen TC0052, antigen TC0189, antigen TC0582, antigen TC0660, antigen TC0726, antigen TC0816, antigen TC0828;(10) One or more of the following proteins of cytomegalovirus (CMV / HCMV): pp65 antigen, membrane protein pp15, capsid proximal tegument protein pp150, protein M45, DNA polymerase UL54, helicase UL105, glycoprotein gM, glycoprotein gN, glycoprotein H, glycoprotein B gB, protein UL83, protein UL94, protein UL99, HCMV glycoprotein (selected from gH-gL, gB, gO, gN and gM), HCMV protein (selected from UL83, UL123, UL128, UL130 and UL131A), epidermal protein pp150 (pp150), tegument protein pp65 / submatrix phosphoprotein (pp65), envelope glycoprotein M (UL100), regulatory protein IE1 (UL123), envelope protein (UL128), envelope glycoprotein (130), envelope protein (UL131A), envelope glycoprotein B (UL55), structural glycoprotein N gpUL73 (UL73), structural glycoprotein O gpUL74 (UL74); (11) one or more of the following proteins of dengue virus: capsid protein C, pre-membrane protein prM, membrane protein M, envelope protein E (domain I, domain II, domain II), protein NS1, protein NS2A, protein NS2B, protein NS3, protein NS4A, protein 2K, protein NS4B, protein NS5; (12) one or more of the following proteins of EBOV virus: EBOV glycoprotein (GP), surface EBOV GP, wild-type EBOV pro GP, mature EBOV GP, secreted wild-type EBOV pro GP, secreted mature EBOV GP, EBOV nucleoprotein (NP), RNA polymerase L and EBOV matrix protein (selected from VP35, VP40, VP24 and VP30); (13) one or more of the following proteins of hepatitis B virus (HBV): hepatitis B surface antigen HBsAg, hepatitis B core antigen HbcAg, polymerase, protein Hbx, pre-S2 middle surface protein, surface protein L, large S protein, viral protein VP1, viral protein VP2, viral protein VP3, and viral protein VP4;(14) One or more of the following proteins of respiratory syncytial virus (RSV): fusion protein F, F protein, nucleoprotein N, matrix protein M, matrix protein M2-1, matrix protein M2-2, phosphoprotein P, small hydrophobic protein SH, major surface glycoprotein G, polymerase L, nonstructural protein 1 NS1, nonstructural protein 2 NS2, RSV attachment protein (G) (glycoprotein G), fusion (F) glycoprotein (glycoprotein F), nucleoprotein (N), phosphoprotein (P), large polymerase protein (L), matrix proteins (M, M2), small hydrophobic protein (SH), nonstructural protein 1 (NS1), nonstructural protein 2 (NS2), membrane-bound RSV F protein, membrane-bound DS Cavl (stable prefusion RSV F protein); (15) one or more of the following proteins of Mycobacterium tuberculosis: secretory antigen SssA (Staphylococcus spp., staphylococcal food poisoning); secretory antigen SssA (Staphylococcus spp., such as Staphylococcus aureus, staphylococcal infection); molecular chaperone DnaK, cell surface lipoprotein Mpt83, lipoprotein P23, phosphate transport system permeabilization protein pstA, 14 kDa antigen, fibronectin binding protein C FbpC1, alanine dehydrogenase TB43, glutamine synthetase 1, ESX-1 protein, protein CFP10, TB10.4 protein, protein MPT83, protein MTB12, protein MTB8, Rpf-like protein, protein MTB32, protein MTB39, crystal protein, heat shock protein HSP65, and protein PST-S; (16) one or more of the following proteins of yellow fever virus: genomic polyprotein, protein E, protein M, capsid protein C, protease NS3, protein NS1, protein NS2A, protein AS2B, protein NS4A, protein NS4B, protein NS5; (17) cyclosporin; and (18) one or more of the following proteins of Zika virus: Zika virus capsid protein (c), Zika virus premembrane protein (prM), Zika virus pr protein (pr), Zika virus membrane protein (M), Zika virus envelope protein (E), Zika virus nonstructural protein, Zika virus prME antigen, Zika virus capsid protein, premembrane / membrane protein, ZIKV envelope protein, ZIKV nonstructural protein 1, ZIKV nonstructural protein 2A, ZIKV nonstructural protein 2B, ZIKV nonstructural protein 3, ZIKV nonstructural protein 4A, ZIKV nonstructural protein 4B, ZIKV nonstructural protein 5, and Zika virus envelope protein (e). ;

[0337] In some embodiments, the tumor antigen is selected from but not limited to the group consisting of the tumor antigens described in WO2018 / 078053A1, pages 47-51.

[0338] In some embodiments, the antigens expressed by the nucleotide sequence encoding the polypeptide and / or protein of interest include or are allergic antigens and autoimmune self-antigens. In some embodiments, the allergic antigens and autoimmune self-antigens are derived from or selected from the group of antigens described on pages 59 to 73 of WO2018 / 078053A1, but are not limited thereto.

[0339] In some embodiments, the antigens expressed by the nucleotide sequences encoding the polypeptides and / or proteins of interest are listed on pages 48 to 51 of WO 2018 / 078053A1.

[0340] In some embodiments, the polypeptide and / or protein expressed by the nucleotide sequence encoding the polypeptide and / or protein of interest comprises or is a therapeutic protein or polypeptide.

[0341] In some embodiments, the therapeutic protein or polypeptide includes or is one or more of the following:

[0342] (1) Enzyme replacement therapy for the treatment of metabolic, endocrine or amino acid disorders or therapeutic proteins or polypeptides for replacing missing, defective or mutated proteins; (2) Therapeutic proteins or polypeptides for the treatment of blood diseases, circulatory system diseases, respiratory system diseases, infectious diseases or immune deficiencies; (3) Therapeutic proteins or polypeptides for the treatment of cancer or tumor diseases; (4) Therapeutic proteins or polypeptides for hormone replacement therapy; (5) Therapeutic proteins or polypeptides for reprogramming somatic cells into pluripotent stem cells or totipotent stem cells; (6) Therapeutic proteins or polypeptides used as adjuvants or immunostimulants; (7) Therapeutic proteins or polypeptides as therapeutic antibodies; (8) Therapeutic proteins or polypeptides as gene editing agents; (9) Therapeutic proteins or polypeptides for the treatment or prevention of liver diseases selected from the group consisting of liver fibrosis, cirrhosis and liver cancer; and (10) Therapeutic proteins or polypeptides for the treatment or prevention of rare diseases.

[0343] In some embodiments, the enzyme replacement therapy for treating metabolic, endocrine or amino acid disorders or the therapeutic protein or polypeptide for replacing a missing, deleted or mutant protein comprises or is one or more of the following: acid sphingomyelinase, fatty acid, aglycosidase beta, leucosidase, α-galactosidase A, α-glucosidase, α-L-iduronidase, α-N-acetylglucosaminidase, amphiregulin, angiopoietin (Ang1, Ang2, Ang3, Ang4, ANGPTL2, ANG PTL3, ANGPTL4, ANGPTL5, ANGPTL6, ANGPTL7), ATPase, Cu(2+)-transporting β polypeptide (ATP7B), argininosuccinate synthetase (ASS1), beta-cells, β-glucuronidase, bone morphogenetic proteins (BMPs) (BMP1, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP10, BMP15), CLN6 protein, epidermal growth factor (EG F), epigenetic proteins, epigenetic regulators, fibroblast growth factors (FGF, FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, FGF-16, FGF-17, FGF-17, FGF-18, FGF-19, FGF-20, FGF-21, FGF-22, FGF-2 3), fumarylacetoacetate hydrolase (FAH), sulfatase, growth hormone-releasing peptide, glucocerebrosidase, GM-CSF, heparin-binding EGF-like growth factor (HB-EGF), hepatocyte growth factor HGF, hepatopoietin, human albumin, increased albumin loss, iduronate sulfatase (iduronate-2-sulfatase), integrins αVβ3, αVβ5, and α5β1, uduronate sulfatase, laronidase, N-acetylgalactosamine-4-sulfatase (rhASB;Sulfatase, arylsulfatase A (ARSA), arylsulfatase B (ARSB)), N-acetylglucosamine-6-sulfatase, nerve growth factor (NGF, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin 4 / 5 (NT-4 / 5), neuregulins (NRG1, NRG2, NRG3, NRG4), neuropilin (NRP-1, NRP-2), obestatin, phenylalanine hydroxylase (PAH), phenylalanine aminohydrolase (PAL), platelet-derived growth factor (PDGF (PDFF-A, PDGF-B, PDGF-C, PDGF-D)), TGFβ receptors (endothelin, TGFβ1 receptor, TGFβ2 receptor, TGFβ3 receptor), thrombopoietin (THPO) (megakaryocyte growth and development factor (MGDF)), transforming growth factors (TGF (TGF-a, TGF-β (TGFβ1, TGFβ2 and TGFβ3))), VEGF (VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F and PIGF) , nesiritide, trypsin, adrenocorticotropic hormone (ACTH), atrial natriuretic peptide (ANP), cholecystokinin, gastrin, leptin, oxytocin, somatostatin, vasopressin (antidiuretic hormone), calcitonin, exenatide, growth hormone (GH), somatropin, insulin, insulin-like growth factor IIGF-1, mecarsiphene axetil, IGF-1 analog, pegvisomant, pramlintide, teriparatide (human parathyroid hormone residues 1-34), becaplermin, dibotermin-α (bone morphogenetic protein 2), histrelin acetate (gonadotropin-releasing hormone GnRH), octreotide, hepatocyte nuclear factor 4 alpha (HNF4A), CCAAT / enhancer binding protein alpha (CEBPA), fibroblast growth factor 21 (FGF21), extracellular matrix protease or human collagenase MMP1, hepatocyte growth factor (HGF), TNF-related apoptosis-inducing ligand (TRAIL), opioid growth factor receptor-like 1 (OGFRL1), clostridial type II collagenase, relaxin 1 (RLN1), relaxin 2 (RLN2), relaxin 3 (RLN3), and palifermin (keratinocyte growth factor; KGF).

[0344] In some embodiments, the therapeutic protein or polypeptide for treating metabolic or endocrine diseases is selected from the proteins or polypeptides described in Table A (in combination with Table C) of WO2017 / 191274.

[0345] In some embodiments, the therapeutic protein or polypeptide for treating a blood disorder, a circulatory system disorder, a respiratory system disorder, a cancer or tumor disorder, an infectious disease, or an immunodeficiency comprises or is one or more of the following: alteplase (tissue plasminogen activator; tPA), anistreplase, antithrombin III (AT-III), bivalirudin, darbepoetin-α, drotrecogin-α (activated protein C), erythropoietin, epoetin alfa, erythropoietin, erthropoyetin, factor IX, factor VIIa, factor VIII, recombinant hirudin, protein C concentrate, reteplase (a deletion mutant of tPA), streptokinase, tenecteplase, urokinase, angiostatin, anti-CD22 immunotoxin, denileukin, immunocyanine, MPS (zinc finger protein), aflibercept, endostatin, collagenase, human deoxyribonuclease I, deoxyribonuclease, hyaluronidase, papain, L-aspartate Aminase, PEG-asparaginase, rasburicase, human chorionic gonadotropin (hCG), human follicle-stimulating hormone (FSH), luteinizing hormone-alpha, prolactin, alpha-1-proteinase inhibitor, lactase, pancreatic enzymes (lipase, amylase, protease), adenosine deaminase (bovine peg deaminase, PEG-ADA), abatacept, alefacept, anakinra, etanercept, interleukin-1 (IL-1 receptor antagonist), anakinra, thymosin nonapeptide, TNF-alpha antagonist, enfuvirtide, and thymosin alpha 1.

[0346] In some embodiments, the therapeutic protein or polypeptide for treating cancer or tumor disease includes or is one or more of the following: cytokines, chemokines, suicide gene products, immunogenic proteins or peptides, apoptosis inducers, angiogenesis inhibitors, heat shock proteins, tumor antigens, β-catenin inhibitors, STING pathway activators, checkpoint regulators, innate immune activators, antibodies, dominant negative receptors and decoy receptors, myeloid-derived suppressor cell (MDSCs) inhibitors, IDO pathway inhibitors, and proteins or peptides that bind to apoptosis inhibitors;

[0347] In some embodiments, the hormones in the therapeutic protein or polypeptide for hormone replacement therapy include one or more of the following: estrogen, progesterone, progesterone, and testosterone.

[0348] In some embodiments, therapeutic proteins for reprogramming somatic cells into pluripotent or totipotent stem cells include one or more of the following: Oct-3 / 4, Sox gene family (e.g., Sox1, Sox2, Sox3, and Sox15), Klf family (e.g., Klf1, Klf2, Klf4, and Klf5), Myc family (e.g., c-Myc, L-Myc, and N-Myc), Nanog, and LIN28.

[0349] In some embodiments, the therapeutic protein or polypeptide used as an adjuvant or immunostimulatory protein includes or is one or more of the following: human adjuvant proteins, in particular pattern recognition receptors TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11; NOD1, NOD2, NOD3, NOD4, NOD5, NALP1, NALP2, NALP3, NALP4, NALP5, NALP6, NALP6, NALP7, NALP7, NALP8, NALP9, NALP10, NALP11, NALP12, NALP13, NALP14; IPAF, NAIP, CIITA, RIG-I, MDA5 and LGP2, signal transducers of TLR signals (including adaptor proteins (such as Trif and Cardif), components of small GTPases signals (such as RhoA, Ras, Rac1, Cdc42, Rab, etc.), components of PIP signals (such as PI3K, Src kinase, etc.), components of MyD88-dependent signals (such as MyD88, IRAK1, IRAK2, IRAK4, TIRAP, TRAF6, etc.), components of MyD88-independent signals (such as TICAM1, TICAM2, TRAF6, TBK1, IRF3, TAK1, IRAK1, etc.); activated kinases (such as Akt, ME KK1, MKK1, MKK3, MKK4, MKK6, MKK7, ERK1, ERK2, GSK3, PKC kinase, PKD kinase, GSK3 kinase, JNK, p38MAPK, TAK1, IKK, TAK1, etc.); activated transcription factors (e.g., NF-kB, c-Fos, c-Jun, c-Myc, CREB, AP-1, Elk-1, ATF2, IRF-3, IRF-7, heat shock proteins (e.g., HSP10, HSP60, HSP65, HSP70, HSP75, and HSP90), gp96, fibrinogen, type III repeat extra domain of fibronectin, etc.); components of the complement system (e.g., C1q, MBL, C1r, C1 s, C2b, Bb, D, MASP-1, MASP-2, C4b, C3b, C5a, C3a, C4a, C5b, C6, C7, C8, C9, CR1, CR2, CR3, CR4, C1qR, C1INH, C4bp, MCP, DAF, H, I, P, CD59, etc.); cell surface proteins that induce target genes (e.g., β-defensins). In some embodiments, the human auxiliary proteins include one or more of the following: trif, flt-3 ligand, Gp96 or fibronectin, cytokines that induce or enhance innate immune responses (e.g., IL-1α, IL-1R1, IL1β, IL-2, IL-6, IL-7, IL-8, IL-9, IL-12, IL-13, IL-15, IL-16, IL-17, IL-18, IL-21, IL-23, TNFα, IFNα, IFNβ, IFNγ, GM-CSF, G-CSF, M-CSF), chemokines (e.g., IL-8, IP-10, MCP-1, MIP-1α, RANTES, Eotaxin, CCL21), cytokines released by macrophages (e.g., IL-1, IL-6, IL-8, IL-12, TNF-α, etc.).

[0350] In some embodiments, therapeutic proteins or polypeptides used as adjuvants or immunostimulators include one or more of the following: bacterial (adjuvant) proteins, protozoan (adjuvant) proteins, viral (adjuvant) proteins, fungal (adjuvant) proteins, and animal-derived proteins.

[0351] In some embodiments, the bacterial (adjuvant) protein includes one or more of the following: bacterial heat shock proteins or chaperones (including Hsp60, Hsp70, Hsp90, Hsp100); Gram-negative bacterial OmpA (outer membrane protein); OspA; bacterial porins (e.g., OmpF); bacterial toxins (e.g., pertussis toxin (PT) of Bordetella pertussis, pertussis adenylate cyclase toxins CyaA and CyaC of Bordetella pertussis, pertussis toxin PT-9K / 129G mutant, pertussis adenylate cyclase toxins CyaA and CyaC of Bordetella pertussis, tetanus toxin, cholera toxin (CT), cholera toxin The B subunit of toxin, cholera toxin CTK63 mutant, CTE112K mutant of CT, Escherichia coli heat-labile enterotoxin (LT), heat-labile enterotoxin with reduced toxicity (LTB), B subunit of Escherichia coli heat-labile enterotoxin mutant (e.g., LTK63, LTR72); phenol-soluble regulatory protein; Helicobacter pylori neutrophil-activating protein (HP-NAP); surfactant protein D; Borrelia burgdorferi outer surface protein A lipoprotein, Ag38 (38 kDa antigen) of Mycobacterium tuberculosis; bacterial fimbriae proteins (e.g., fimbriae proteins of Gram-negative bacteria) and surfactant protein A, and bacterial flagellin.

[0352] In some embodiments, the protozoan (adjuvant) proteins include one or more of the following: Tc52 from Trypanosoma cruzi, PFTG from Trypanosoma gondii, protozoan heat shock proteins, LeIF from Leishmania, and spectra-like proteins from Toxoplasma gondii.

[0353] In some embodiments, the viral (adjuvant) proteins include one or more of the following: respiratory syncytial virus fusion glycoprotein (F protein), MMT virus envelope protein, mouse leukemia virus protein, and wild-type measles virus hemagglutinin protein.

[0354] In some embodiments, the fungal (adjuvant) protein comprises a fungal immunomodulatory protein (FIP, eg, LZ-8).

[0355] In some embodiments, the animal-derived protein comprises keyhole limpet hemocyanin (KLH).

[0356] In some embodiments, the polypeptide and / or protein expressed by the nucleotide sequence encoding the polypeptide and / or protein of interest comprises or is a therapeutic protein or polypeptide that is a therapeutic antibody, for example, one or more of the cytokines, chemokines, suicide enzymes and gene products, apoptosis inducers, endogenous angiogenesis inhibitors, heat shock proteins, tumor antigens, innate immune activators, and antibodies against proteins associated with tumor or cancer development listed in Table 1, Table 2, Table 3, Table 4, Table 5, Table 6, Table 7, Table 8, Table 9, Table 10, Table 11, and Table 12 of WO2016 / 170176A1.

[0357] In some embodiments, the present invention provides a host cell comprising the vector of any of the above embodiments, the RNA of any of the above embodiments, the artificial RNA molecule of any of the above embodiments, or the RNA molecule of any of the above embodiments.

[0358] In some embodiments, the present invention also provides a lipid nanoparticle comprising the RNA of any of the above embodiments, the artificial RNA molecule of any of the above embodiments, the RNA molecule of any of the above embodiments, the protein of any of the above embodiments, the DNA of any of the above embodiments, or the vector of any of the above embodiments.

[0359] In some embodiments, the lipid nanoparticles further contain one or more of an ionizable cationic lipid, a helper lipid, a structural lipid, and a PEG-lipid.

[0360] In some embodiments, the lipid nanoparticles contain ionizable cationic lipids.

[0361] In some embodiments, the lipid nanoparticles further contain one or more of a helper lipid, a structural lipid, and a PEG-lipid (polyethylene glycol-lipid). In an alternative specific example, the lipid nanoparticles contain an ionizable cationic lipid, a helper lipid, a structural lipid, and a PEG-lipid.

[0362] In some embodiments, the ionizable cationic lipid is selected from one or more of Dlin-MC3-DMA, Dlin-KC2-DMA, DODMA, c12-200, and DlinDMA.

[0363] In some embodiments, the helper lipid is a phospholipid. The phospholipid is typically semi-synthetic, but may also be of natural origin or chemically modified. The phospholipid includes, but is not limited to, DSPC (distearoylphosphatidylcholine), DOPE (dioleoylphosphatidylethanolamine), DOPC (dioleoylphosphatidylcholine), DOPS (dioleoylphosphatidylserine), DSPG (1,2-dioctadecanoyl-sn-glycero-3-phospho-(1'-rac-glycerol)), DPPG (dipalmitoylphosphatidylglycerol), DPPC (dipalmitoylphosphatidylcholine), DGTS (1,2-dipalmitoyl-sn-glycero-3-O-4'-(N,N,N-trimethyl)homoserine), lysophospholipids, and the like. In some embodiments, the helper lipid is one or more selected from DSPC, DOPE, DOPC, and DOPS. In some embodiments, the helper lipid is DSPC and / or DOPE.

[0364] In some embodiments, the structured lipid is a sterol substance, including but not limited to cholesterol, cholesterol ester, sterol hormones, sterol vitamins, bile acid, cholesterol, ergosterol, β-sitosterol and oxidized cholesterol derivatives. In some embodiments, the structured lipid is at least one selected from cholesterol, cholesterol ester, sterol hormones, sterol vitamins and bile acids. In some embodiments, the structured lipid is cholesterol, preferably high-purity cholesterol, especially injection-grade high-purity cholesterol. For example, CHO-HP (produced by AVT).

[0365] As used herein, the term PEG-lipid (polyethylene glycol-lipid) is a conjugate of polyethylene glycol and a lipid structure. In some embodiments, the PEG-lipid is selected from PEG-DMG and PEG-distearoylphosphatidylethanolamine (PEG-DSPE). The PEG-DMG is a polyethylene glycol (PEG) derivative of 1,2-dimyristyl glycerol.

[0366] In some embodiments, the PEG-lipid is selected from PEG-DMG or PEG-DSPE.

[0367] In some embodiments, the average molecular weight of PEG is about 2000 Daltons to 5000 Daltons. More preferably, the average molecular weight of PEG is about 2000 Daltons or 5000 Daltons.

[0368] In some embodiments, the PEG-lipid is PEG2000-DMG.

[0369] In some embodiments, the present invention also provides a pharmaceutical composition comprising the artificial RNA molecule of any of the above embodiments, the RNA molecule of any of the above embodiments, the DNA of any of the above embodiments, the vector of any of the above embodiments, the host cell of any of the above embodiments, or the lipid nanoparticle of any of the above embodiments, and a pharmaceutically acceptable carrier.

[0370] In some embodiments, the present invention also provides a use of an artificial RNA molecule according to any of the above embodiments, an RNA molecule according to any of the above embodiments, a DNA according to any of the above embodiments, a vector according to any of the above embodiments, a host cell according to any of the above embodiments, or a lipid nanoparticle according to any of the above embodiments, or a pharmaceutical composition according to any of the above embodiments in the preparation of a drug.

[0371] In some embodiments, the medicament is for gene therapy, genetic vaccination, protein replacement therapy, antisense therapy, or treatment by interfering RNA.

[0372] Gene vaccines are vaccines used for genetic vaccination and are generally considered "third-generation" vaccines. They are typically composed of genetically engineered nucleic acid molecules that allow for the in vivo expression of peptide or protein (antigen) fragments specific to pathogens or tumor antigens. Gene vaccines are administered to patients and taken up by target cells. Expression of the administered nucleic acid results in the production of the encoded protein. When the patient's immune system recognizes these proteins as foreign, an immune response is triggered.

[0373] In some embodiments, the present invention also provides a use of an artificial RNA molecule according to any of the above embodiments, an RNA molecule according to any of the above embodiments, a DNA according to any of the above embodiments, a vector according to any of the above embodiments, a host cell according to any of the above embodiments, or a lipid nanoparticle according to any of the above embodiments, or a pharmaceutical composition according to any of the above embodiments in the preparation of a drug for nucleic acid transfer.

[0374] In some embodiments, the nucleic acid is RNA, messenger RNA (mRNA), antisense oligonucleotide, DNA, plasmid, ribosomal RNA (rRNA), microRNA (miRNA), transfer RNA (tRNA), small inhibitory RNA (siRNA), and small nuclear RNA (snRNA).

[0375] In some embodiments, the medicament is for gene therapy, genetic vaccination, or protein replacement therapy.

[0376] In some embodiments, the drug is used to treat and / or prevent a disease. In some embodiments, the drug is a vaccine; in some embodiments, the drug is a vaccine for preventing infection with the novel coronavirus.

[0377] In some embodiments, the disease is selected from the group consisting of a rare disease, an infectious disease, cancer, a genetic disease, an autoimmune disease, diabetes, a neurodegenerative disease, a cardiovascular disease, a renal vascular disease, and a metabolic disease.

[0378] In some embodiments, the cancer comprises one or more of lung cancer, stomach cancer, liver cancer, esophageal cancer, colon cancer, pancreatic cancer, brain cancer, lymphoma, blood cancer, or prostate cancer. The genetic disease comprises one or more of hemophilia, thalassemia, and Gaucher's disease. The rare disease comprises one or more of the following: osteogenesis imperfecta, Wilson's disease, spinal muscular atrophy, Huntington's disease, Rett syndrome, amyotrophic lateral sclerosis, Duchenne muscular dystrophy, Friedreich's ataxia, methylmalonic acidemia, cystic fibrosis, glycogen storage disease 1a, glycogen storage disease III, Crigler-Najjar syndrome, ornithine transcarbamylase deficiency, propionic acidemia, phenylketonuria, hemophilia A, hemophilia B, β-thalassemia, Lafora disease, Dravet syndrome, Alexander disease, Leber congenital amaurosis, myelodysplastic syndrome, and CBS-deficiency homocystinuria.

[0379] In some embodiments, the drug is a nucleic acid drug, wherein the nucleic acid comprises at least one of RNA, messenger RNA (mRNA), DNA, plasmid, ribosomal RNA (rRNA), single-stranded guide RNA (sgRNA), and cas9 mRNA.

[0380] As used herein, the term "transfer" refers to the delivery of a nucleic acid, such as a therapeutic and / or prophylactic agent, to a target. For example, delivering a therapeutic and / or prophylactic agent to a subject may involve administering a lipid nanoparticle comprising the therapeutic and / or prophylactic agent to the subject (e.g., via intravenous, intramuscular, intradermal, or subcutaneous routes, etc.).

[0381] In one embodiment, the polypeptide of interest refers to a therapeutically or pharmaceutically active polypeptide or protein having a therapeutic or preventive effect, whose function in or near a cell is necessary or beneficial, for example, a protein whose deficiency or defective form causes a disease, and providing such a protein can regulate or prevent the disease, or a protein whose presence in or near a cell is beneficial to the body. The polypeptide or protein may comprise a complete protein or a functional variant thereof. In some embodiments, the polypeptide or protein of interest is described above.

[0382] In some embodiments, the present invention also provides a method for treating and / or preventing a disease, which comprises administering to a subject in need thereof an artificial RNA molecule according to any of the above embodiments, an RNA molecule according to any of the above embodiments, a DNA according to any of the above embodiments, a vector according to any of the above embodiments, a host cell according to any of the above embodiments, or a lipid nanoparticle according to any of the above embodiments, or a pharmaceutical composition according to any of the above embodiments.

[0383] In some embodiments, the disease or disorder is described above in the context of the use of an artificial RNA molecule, RNA molecule, DNA, vector, host cell or lipid nanoparticle or pharmaceutical composition in the preparation of a medicament for nucleic acid transfer.

[0384] In some embodiments, the artificial RNA molecule, RNA molecule, DNA, vector, host cell or lipid nanoparticle or pharmaceutical composition is used as a vaccine to prevent a disease. In some embodiments, the artificial RNA molecule, RNA molecule, DNA, vector, host cell or lipid nanoparticle or pharmaceutical composition is used to produce an antigen or portion thereof of a pathogen.

[0385] In some embodiments, an artificial RNA molecule, RNA molecule, DNA, vector, host cell or lipid nanoparticle or pharmaceutical composition is used to produce the protein associated with the genetic disease.

[0386] In some embodiments, an artificial RNA molecule, RNA molecule, DNA, vector, host cell, or lipid nanoparticle or pharmaceutical composition is used to produce an antibody, such as a scFV or nanobody.

[0387] In a third aspect, the present invention provides a method for preparing the above-mentioned RNA, the method comprising the following steps:

[0388] (1) Extract the engineered plasmid and ensure that the supercoil rate of the plasmid is above 90%;

[0389] (2) Plasmid linearization and purification;

[0390] (3) in vitro transcription and purification; and

[0391] (4) Capping and purification.

[0392] In a fourth aspect, the present invention provides a method for preparing plasmid H, comprising:

[0393] (1) Construction of plasmid D: Using the luciferase-pcDNA3 plasmid as the backbone, new restriction sites HindIII and BamHI were added before the Kozack sequence, and new restriction sites KpnI and ApaI were added after the stop codon of luciferase to obtain plasmid B; 5'-UTR was inserted between the HindIII and BamHI restriction sites of plasmid B to obtain plasmid C; 3'-UTR was inserted between the KpnI and ApaI restriction sites of plasmid C to obtain plasmid D;

[0394] (2) Construction of plasmid G: Using plasmid B as the backbone, the ampicillin resistance gene sequence was replaced with the kanamycin sulfate resistance gene sequence, and the neo / KanR sequence from nucleotide position 3746 to 4540 (corresponding to the position numbering of plasmid B) was removed to obtain plasmid F; the polyA sequence was inserted into plasmid F to obtain plasmid G;

[0395] (3) Construction of plasmid vector H: Plasmid G and plasmid D were double-digested with HindIII and ApaI, respectively. The product of double-digestion of plasmid G was a fragment with a large molecular weight, and the product of double-digestion of plasmid D was a fragment with a small molecular weight. The two fragments were ligated with T4 enzyme to obtain plasmid H. The plasmids used in the preparation of plasmid H, as shown in the nucleotide sequences of SEQ ID NO: 2 (plasmid B), SEQ ID NO: 3 (plasmid C), SEQ ID NO: 4 (plasmid D), SEQ ID NO: 5 (plasmid F), and SEQ ID NO: 6 (plasmid G), are also part of the present invention.

[0396] Plasmid H is double-digested with BamHI and KpnI to extract a large molecular weight fragment, which is homologously recombined with the S protein coding sequence of the new coronavirus of the present invention to obtain an mRNA vaccine engineering plasmid. Therefore, the present invention also provides an engineered plasmid for preparing an mRNA vaccine, which comprises a nucleotide sequence that can transcribe mRNA, specifically comprising a template nucleotide sequence for the mRNA vaccine of the present invention. The template nucleotide sequence refers to a DNA sequence in the engineered plasmid, which serves as a template for in vitro synthesis of mRNA to guide the synthesis of mRNA molecules; more specifically, the template nucleotide sequence of the mRNA vaccine of the present invention is the S protein coding sequence of the new coronavirus of the present invention. In one embodiment, the engineered plasmid is shown in SEQ ID NO: 8.

[0397] Based on the above, the present invention has designed a novel coronavirus S protein, encoding DNA, and corresponding mRNA. Sera obtained from mice immunized with this mRNA vaccine can simultaneously neutralize SARS-CoV-2 pseudoviruses, Delta pseudoviruses, and Omicron pseudoviruses, demonstrating that this mRNA vaccine can achieve cross-protection against different strains. The sequence was expressed and applied to prepare a novel mRNA vaccine targeting the Delta and Omicron variants of the novel coronavirus, demonstrating robust immune responses against these strains and enabling faster development and large-scale production.

[0398] The present invention also constructs a polynucleotide molecule for preparing a novel mRNA vaccine. Through the scientific design of the 5'-UTR, 3'-UTR, and polyA regions, it increases the level of mRNA protein expression. This polynucleotide molecule can not only be used to prepare mRNA vaccines that protect against wild-type SARS-CoV-2, Delta, and Omicron variants, but can also be used to transfect cells or bacteria to produce corresponding antibodies or antigenic proteins. It is a versatile core component combination with great significance for advancing the development of the biopharmaceutical industry.

[0399] Example

[0400] Example 1 Construction and preparation of the novel coronavirus S protein coding sequence

[0401] We analyzed the mutation sites of the novel coronavirus and designed a nucleic acid sequence encoding the novel coronavirus S protein, as shown in SEQ ID NO: 12. The encoded amino acid sequence is shown in SEQ ID NO: 13, and the mRNA sequence is shown in SEQ ID NO: 14, in which all uridine nucleosides are replaced with N1-methylpseudouridine. This nucleic acid sequence (SEQ ID NO: 12) was commissioned for synthesis by Nanjing GenScript Biotechnology Co., Ltd. and was released by GenScript QC.

[0402] The characterization data is: GenScript QC release results show that the sequence is correct.

[0403] Example 2 Construction of Plasmid H

[0404] The construction flowchart of plasmid H is shown in Figure 1A , and the construction and transformation schematic is shown in Figure 10A .

[0405] The specific construction methods include:

[0406] Construction of plasmid B:

[0407] Using the luciferase-pcDNA3 plasmid (purchased from Addgene, plasmid catalog #18964) as the backbone, new restriction sites HindIII and BamHI were added before the Kozack sequence, and new restriction sites KpnI and ApaI were added after the luciferase stop codon to generate plasmid B. The nucleotide sequence of the luciferase-pcDNA3 plasmid is shown in SEQ ID NO: 1. The plasmid map of luciferase-pcDNA3 is shown in Figure 2. The plasmid map of plasmid B is shown in Figure 3. The nucleotide sequence of plasmid B is shown in SEQ ID NO: 2.

[0408] Construction of plasmid C:

[0409] The 5'-UTR sequence shown in SEQ ID NO: 9 was inserted between the HindIII and BamHI restriction sites of plasmid B to obtain plasmid C.

[0410] Specifically, plasmid B was double-digested with HindIII and BamHI, and a fragment with a molecular weight of approximately 7 kb was recovered and purified from the gel to obtain the vector fragment. The 5'-UTR sequence shown in SEQ ID NO: 9 was introduced into the homology arm sequence by PCR to form the insert fragment. The vector fragment and insert fragment were homologously recombined to generate plasmid C. The plasmid map of plasmid C is shown in Figure 4. The nucleotide sequence of plasmid C is shown in SEQ ID NO: 3.

[0411] Construction of plasmid D:

[0412] The 3'-UTR sequence shown in SEQ ID NO: 10 was inserted between the KpnI and ApaI restriction sites of plasmid C to obtain plasmid D. The plasmid map of plasmid D is shown in Figure 5. The nucleotide sequence of plasmid D is shown in SEQ ID NO: 4.

[0413] Construction of plasmid F:

[0414] The Amp (ampicillin) resistance gene in plasmid B was replaced with the kana (kanamycin) resistance gene, and the neo / KanR sequence from 3746 to 4540 was deleted to obtain plasmid F. The plasmid map of plasmid F is shown in Figure 6. The nucleotide sequence of plasmid F is shown in SEQ ID NO: 5.

[0415] Construction of plasmid G:

[0416] Plasmid F was inserted with the polyA sequence shown in SEQ ID NO: 11. Plasmid F was digested with ApaI, and the product was purified. The purified product and the polyA sequence shown in SEQ ID NO: 11 were then reconstructed by homologous recombination. The product was transformed into DH5α, and clones were screened and sequenced to identify plasmids that had been correctly sequenced. A map of plasmid G is shown in Figure 7. The nucleotide sequence of plasmid G is shown in SEQ ID NO: 6.

[0417] Construction of plasmid H:

[0418] Plasmid D was double-digested with HindIII and ApaI, and a fragment with a molecular weight of approximately 1.6 kb was recovered from a gel (axygen). Plasmid G was double-digested with HindIII and ApaI, and a fragment with a molecular weight of approximately 4.5 kb was recovered from a gel (axygen). Fragments A and B were ligated using quick ligase T4 (NEB) at 25°C for 5 minutes. DH5α (takara) competent cells were transformed with the above reaction system, plated (with 50 μg / mL kanamycin), and cultured for 16 hours. Three to four single colonies were picked and cultured in medium containing 50 μg / mL kanamycin for 8 hours. The plasmid was then sent to Shanghai Biotechnology for sequencing. The plasmid map of plasmid H is shown in Figure 8. The nucleotide sequence of plasmid H is shown in SEQ ID NO: 7.

[0419] Example 3 Synthesis of 5'-UTR

[0420] Design the following 5'-UTR sequence.

[0421] Table 1: 5'-UTR sequence list

[0422] The above 5'-UTR sequence was synthesized by Sangon Biotech (Shanghai) Co., Ltd. According to the company's quality analysis report, the 5'-UTR sequence is consistent with the theoretical design sequence.

[0423] Example 4 Synthesis of 3'-UTR

[0424] Design the following 3'-UTR sequence.

[0425] Table 2: 3'-UTR sequence list

[0426] The above 3'-UTR sequence was synthesized by Shanghai Sangon Biotechnology Co., Ltd. According to the company's quality analysis report, the 3'-UTR sequence is consistent with the theoretical design sequence.

[0427] Example 5 Synthesis of polyA

[0428] Design the following polyA sequence.

[0429] Table 3: PolyA sequence list

[0430] The above polyA sequence was synthesized by IDT (intergrated DNA technologies) Co., Ltd. According to the company's quality analysis report, the sequence was consistent with the theoretical design sequence.

[0431] Example 6 Construction of Engineering Plasmid

[0432] By PCR, homology arm sequences were introduced at both ends of the novel coronavirus S protein coding sequence as shown in SEQ ID NO: 12. This PCR product was then digested with BamHI and KpnI by plasmid H, and the macromolecular fragments recovered by gel run were homologously recombined. The competent cells DH5α were transformed, single clones were selected, and sequencing was performed to finally obtain the correct engineering plasmid. The 5'-UTR used in this engineering plasmid is 5UTR-NO54 as shown in SEQ ID NO: 9, the 3'-UTR is 3UTR-NO50 as shown in SEQ ID NO: 10, and the polyA sequence used is shown in SEQ ID NO: 11.

[0433] Data Characterization: Sequencing verification was performed starting with plasmid H. The results showed that the nucleotide sequence was completely inserted into the correct position and was completely consistent with the theoretical sequence. The plasmid map of the engineered plasmid is shown in Figure 9, the construction flow chart is shown in Figure 1B, and the construction and transformation schematic is shown in Figure 10B.

[0434] Example 7 Preparation of Novel Coronavirus Vaccine mRNA

[0435] 1. Extract the engineered plasmid template prepared in Example 6 and ensure that the supercoil rate of the plasmid is above 90%.

[0436] 2. Plasmid Linearization

[0437] (1) Enzyme linearization: Take 20 μg of the engineered plasmid and use the corresponding enzyme to linearize the plasmid.

[0438] Note: Adjust the reaction system according to actual conditions. Due to the influence of the purification kit column, the plasmid in each reaction system should not exceed 20μg.

[0439] (2) Prepare the system in a 0.2 mL tube, mix well, and place in a 37°C incubator for overnight or 2 hours for enzyme digestion reaction.

[0440] (3) Take 1 μL of the digested product and run electrophoresis together with the original plasmid to check whether the digestion is complete.

[0441] 3. Linearized plasmid purification: Use Takara recovery kit to recover

[0442] (1) Add 3 times the amount of Buffer DC to the PCR reaction solution (or other enzymatic reaction solution) (if the amount of Buffer DC to be added is less than 100 μL, add 100 μL) and mix evenly.

[0443] (2) Place the Spin Column in the kit on the Collection Tube.

[0444] (3) Transfer the solution from step (1) above to a spin column, centrifuge at 12,000 rpm for 1 minute at room temperature, and discard the filtrate.

[0445] (4) Add 700 μL of Buffer WB to the spin column, centrifuge at 12000 rpm for 30 seconds at room temperature, and discard the filtrate.

[0446] 4. Phenol-chloroform extraction (to remove RNAse, proteins, etc.)

[0447] (1) Dilute the plasmid to be extracted to 300 μL or 500 μL, add an equal volume of phenol chloroform (be careful to separate the layers and do not absorb the upper layer), and centrifuge at 12,000 rpm for 10 min.

[0448] (2) After centrifugation, aspirate the upper liquid as much as possible, add an equal volume of phenol chloroform, and centrifuge at 12000 rpm for 10 min.

[0449] (3) After centrifugation, aspirate the upper layer of liquid as much as possible (be careful not to aspirate the lower layer of liquid this time), add 0.1×5M NaCl and 0.7×isopropanol, and then ice-bathe for 15 minutes. Centrifuge at 12000 rpm for 10 minutes and discard the supernatant.

[0450] (4) Wash once with 200 μL of 70% ethanol (pre-cooled), centrifuge at 12,000 rpm for 1 min, discard the supernatant, centrifuge again at 12,000 rpm for 1 min, and thoroughly aspirate with a small pipette tip.

[0451] (5) After drying at room temperature, add an appropriate amount of UItraPure DNase / RNase-Free-Distilled Water and mix well.

[0452] (6) Use OneDrop to determine the concentration of the purified sample.

[0453] Identification: Take 100 ng of the purified plasmid and perform electrophoresis together with the original plasmid. After confirming that the plasmid is completely linearized and has no impurity bands, it can be used for mRNA synthesis.

[0454] 5. In vitro transcription of mRNA (refer to Novozymes IVT reaction kit)

[0455] (1) Cleaning the experimental area: First, sterilize the biosafety cabinet with UV light for 0.5 h. Then, wipe the cabinet clean with an alcohol swab and spray it with RNase inhibitor. After 5 min, wipe the inhibitor clean with an alcohol swab and begin the RNA experiment.

[0456] (2) System configuration: Before preparing the system, take out each component reagent, vortex mix on an oscillator, centrifuge, and place in an ice box for later use.

[0457] Note: During the preparation of the system, the entire process was performed on ice, and the plasmid template and IVT system were prepared separately.

[0458] (3) Preparation of IVT system: All components except the plasmid template were added below the liquid level of the system, and finally T7 RNA polymerase was added and vortexed to mix. Uracil (U) was replaced with N1-methyl pseudouridine.

[0459] (4) Take out the required amount of plasmid into a new PCR tube and incubate it with the IVT system for 5 minutes. Finally, mix the two systems together, vortex to mix, centrifuge and incubate at 37°C for 2 hours.

[0460] (5) Prepare the system in a 0.2 mL flat-top thin-walled tube, mix thoroughly, and place in a PCR instrument for reaction at 37°C for 2 h. Note: The reaction system can also be scaled up for simultaneous production based on the desired production volume.

[0461] (6) Use DNase to remove the linearized plasmid template. Add 1 μL (20 μL system) or 5 μL (100 μL system) DNase to the reaction system and incubate at 37°C for 15 min.

[0462] 6. Purification method: (refer to Thermo MEGAclear Kit)

[0463] Column purification

[0464] (1) Transfer each reaction sample to a 1.5 mL EP tube (if the volume is less than 100 μL, add Elution Solution to make it up to 100 μL) and mix gently.

[0465] (2) Add 350 μL of Binding Solution Concentrate and mix gently with a pipette;

[0466] (3) Add 250 μL of anhydrous ethanol and mix gently with a pipette;

[0467] (4) Insert the filter cartridge provided with the kit into the collection tube, add 700 μL of the mixture to the filter cartridge, combine at room temperature for 10 min, centrifuge at 12,000 g for 1 min, discard the filtrate, reuse the collection tube, and then wash the RNA;

[0468] (5) Add 500 μL of Wash Solution and filter through a filter (centrifuge at 12000 g for 1 min);

[0469] (6) Repeat step 5;

[0470] (7) Remove the wash solution and continue centrifugation at the highest speed for 1 min to remove the residual wash solution;

[0471] (8) Add an appropriate amount (80-100 μL) of preheated RNase-free distilled water to the filter cartridge, cover the filter cartridge, incubate at 70°C for 10 min, and centrifuge at 12,000 g for 1 min. Elution times can be increased as needed.

[0472] (9) Concentration determination: use onedrop or Qubit to determine the concentration (determine after 10-fold dilution).

[0473] 7. Identification: Mix 5 μL of the diluted purified sample with NorthernMax Formaldehyde Load Dye and incubate at 75°C for 10 minutes, then store on ice. Perform gel electrophoresis on a 1% agarose gel to confirm that the mRNA is of the correct size and that the bands are not smeared. Proceed to the capping reaction.

[0474] 8. Capping reaction

[0475] mRNACap1 capping reaction:

[0476] The Cap1 type cap structure and reaction principle are as follows:

[0477] pppN1(p)Nx-OH(3′)→ppN1(pN)x-OH(3′)+Pi

[0478] ppN1(pN)x-OH(3′)+GTP→G(5′)ppp(5′)N1(pN)x-OH(3′)+PPi

[0479] G(5′)ppp(5′)N1(pN)x-OH(3′)+AdoMet→m7G(5′)ppp(5′)N1(pN)x-OH(3′)+AdoHyc

[0480] m7GpppN1(pN)x-OH(3′)+AdoMet→m7Gppp[m2'-O]N1(pN)x-OH(3′)+AdoHyc

[0481] 5'-Cap1 type cap structure:

[0482] cap G 1 G 2 =m 7 G + -5′-ppp-5′-Gm 2′ -3′-p-[m 7 =7-CH3;m 2′ =2′-O-CH3; -ppp-=-PO2H-O-PO2H-O-PO2H)-; -p-=-PO2H-], 37°C for 5 min or 65°C for 5 min (CELL SCRIPT, the reaction system is shown in Table 4 below).

[0483] Table 4: Cap1 type capping reaction system

[0484] Note: Due to the influence of the system, the amount of capped mRNA per time (100μL system) should not exceed 60μg.

[0485] Capping (100 μL system) (refer to CELL SCRIPT Capping Reaction Kit) is shown in Table 5 below.

[0486] Table 5: 100 μL capping system

[0487] The pre-heated mRNA was mixed with the above system and incubated at 37°C for 1 h.

[0488] 9. Purification method: the same as the purification method of in vitro transcription products

[0489] Characterization data: Determine concentration using onedrop or Qubit.

[0490] Experimental results: The sequence of the final mRNA product encoding the novel coronavirus S protein is the sequence obtained by replacing all uridine (U) nucleosides in SEQ ID NO: 15 with N1-methylpseudouridine. The experiment confirmed the acquisition of this final mRNA product.

[0491] Example 8 5'-UTR screening test

[0492] The effects of 5'-UTR on luciferase expression were compared using in vivo imaging (IVIS) data.

[0493] The mRNA synthesis process is as follows:

[0494] The different 5'-UTRs in Example 3 were inserted between the HindIII and BamHI regions of plasmid G to obtain a series of plasmids with identical sequences in other parts but different 5'-UTR sequences. These plasmids replaced the engineered plasmids, and a series of mRNAs with identical sequences in other parts but different 5'-UTR sequences were prepared according to the method described in Example 7.

[0495] The details of the mouse in vivo imaging experiment (IVIS) are as follows:

[0496] The specific process for encapsulating mRNA products containing different 5'-UTRs is as follows:

[0497] The batch size was 1.5 mL, the mRNA:lipid mass ratio was 1:10, and the final aqueous phase concentration of HAc-NaOAc buffer (0.2 M, pH 5.0) was 0.025 M. Samples for each formulation were prepared using a microfluidic device (MPE-L2) and chip (SN.000035) at a flow rate of 9 mL / min for aqueous phase and 3 mL / min for alcohol phase. The two phases were then injected into the microfluidic chip for mixing according to the interface requirements. The experimental design details are shown in Table 6 below.

[0498] Table 6: Experimental design

[0499] Table 7: Water-phase alcohol mixture formula

[0500] The chemical structure of lipid SM-102 is shown below:

[0501] SM-102

[0502] The encapsulated preparation samples were dialyzed and exchanged for liquid as follows:

[0503] Dialysis fluid preparation:

[0504] 1×PBS + 8% sucrose solution: Take 2 packets of 1×PBS powder injection and place them in a beaker. Dissolve and mix thoroughly with 2L of DEPC water. Add 160g of sucrose and mix thoroughly to obtain 1×PBS + 8% sucrose solution.

[0505] Each group of drug solutions was placed in a 100 kD dialysis bag, immersed in a beaker containing 1 L of dialysis solution, wrapped in aluminum foil and dialyzed at 100 rpm and room temperature for 1 hour, then the dialysate was replaced and dialysis continued for 1 hour.

[0506] The obtained mRNA preparations containing different 5'-UTRs were commissioned to Nanjing Yunqiao Pure Biotechnology Co., Ltd. for animal imaging experiments. The specific process is as follows:

[0507] Male BALB / C mice, 5 to 7 weeks old, were housed and tested in a SPF laboratory environment. The encapsulated mRNA formulation was injected via the tail vein. Three mice were used for each 5'-UTR assay, with each mouse receiving 20 μg of the mRNA formulation. After 16-18 hours, the mice were anesthetized using isoflurane inhalation anesthesia and injected with 150 mg / kg of D-Luciferin luciferase substrate. The animals were placed in a supine position, and the signal distribution and expression intensity of the luciferin in the living animals were observed using an IVIS in vivo imaging system.

[0508] Observation of relevant indicators:

[0509] (1) Images observed by IVIS, including the original images and the comparison images after unifying the photon number bar values;

[0510] (2) Fluorescent signal distribution and expression intensity analysis data.

[0511] The experimental results are shown in Figures 11 and 12: the 5'-UTRs represented by 5UTR-NO1, 5UTR-NO3, 5UTR-NO21, 5UTR-NO24, 5UTR-NO31, 5UTR-NO32, 5UTR-NO36, 5UTR-NO43, 5UTR-NO45, and 5UTR-NO48 have better effects, and the 5'-UTR represented by 5UTR-NO54 has the best effect.

[0512] Example 9 3'-UTR screening test

[0513] The effects of 3'-UTR on luciferase expression levels were screened and compared using in vivo imaging (IVIS) data.

[0514] 1. Does not contain 5'-UTR

[0515] Referring to Example 7, mRNAs containing no 5'-UTR and containing 3'-UTRs represented by 3UTR-NO3, 3UTR-NO30, or 3UTR-NO50 were prepared. Then, these mRNAs were used to prepare finished mRNA preparations according to Example 8. Animal imaging experiments were conducted by Nanjing Yunqiao Pure Biotechnology Co., Ltd. The specific process was as follows:

[0516] Male BALB / C mice, 5-7 weeks old, were raised and tested in an SPF laboratory environment. The encapsulated mRNA preparation product was injected into the tail vein. Three mice were used to test each mRNA preparation product, and 20 μg of the mRNA preparation product was injected into the tail vein of each mouse. After 16-18 hours, the mice were anesthetized by isoflurane inhalation anesthesia and injected with D-Luciferin luciferase development substrate (150 mg / kg). The animals were placed in a supine position, and the signal distribution and expression intensity of the luciferin in the living animals were observed under the IVIS in vivo imaging system.

[0517] Observation of relevant indicators:

[0518] (1) Images observed by IVIS, including the original images and the comparison images after unifying the photon number bar values;

[0519] (2) Fluorescent signal distribution and expression intensity analysis data.

[0520] The experimental results are shown in Figures 26 and 27. mRNA that does not contain a 5'-UTR but contains a single 3'-UTR represented by 3UTR-NO3, 3UTR-NO30, or 3UTR-NO50 can increase the expression level of the protein. Among them, the mRNA containing a single 3'-UTR represented by 3UTR-NO3 or 3UTR-NO30 has a better effect than the mRNA containing a single 3'-UTR represented by 3UTR-NO50.

[0521] 2. Contains 5'-UTR

[0522] The mRNA synthesis process is as follows:

[0523] 5'-UTR-NO54 was inserted between HindIII and BamHI of blank plasmid G. After the 5'-UTR sequence was fixed, the different 3'-UTRs described in Example 4 were then inserted between ApaI and KpnI of this plasmid. This yielded a series of plasmids containing the 5'-UTR-NO54 sequence, with the remaining sequences identical but with different 3'-UTRs. These plasmids were substituted for the engineered plasmids, and a series of mRNAs containing the 5'-UTR-NO54 sequence, with the remaining sequences identical but with different 3'-UTRs, were prepared according to the method described in Example 7.

[0524] The details of the mouse in vivo imaging experiment (IVIS) are as follows:

[0525] The specific process for encapsulating mRNA products containing different 3'-UTRs is similar to that for encapsulating mRNA products containing different 5'-UTRs in Example 8, except that the mRNA products containing different 3'-UTRs are used instead of the mRNA products containing different 5'-UTRs.

[0526] The obtained mRNA preparations containing different 3'-UTRs were commissioned to Nanjing Yunqiao Pure Biotechnology Co., Ltd. for animal imaging experiments. The specific process is as follows:

[0527] Male BALB / C mice, 5-7 weeks old, were raised and tested in an SPF laboratory environment. The encapsulated mRNA preparation product was injected into the tail vein. Three mice were used for each 3'-UTR test, and 20μg of the mRNA preparation product was injected into the tail vein of each mouse. After 16-18h, the mice were anesthetized by isoflurane inhalation anesthesia and injected with D-Luciferin luciferase development substrate (150mg / kg). The animals were placed in the supine position, and the signal distribution and expression intensity of the luciferin in the living animals were observed under the IVIS in vivo imaging system.

[0528] Observation of relevant indicators:

[0529] (1) Images observed by IVIS, including the original images and the comparison images after unifying the photon number bar values;

[0530] (2) Fluorescent signal distribution and expression intensity analysis data.

[0531] The experimental results are shown in Figures 13 and 14. After fixing the 5'-UTR to 5UTR-NO54, different 3'-UTRs were replaced and it was found that 3UTR-NO2, 3UTR-NO3, 3UTR-NO9, 3UTR-NO21, 3UTR-NO27, 3UTR-NO28, 3UTR-NO30, 3UTR-NO35, and 3UTR-NO50 had better effects.

[0532] Example 10 Screening test of polyA

[0533] Preparation of mRNA: After inserting the different polyA sequences in Example 5 into the ApaI site of plasmid F, a series of plasmids with the same other sequences but different polyA sequences were prepared. These plasmids were then used to replace the engineered plasmid, and a series of mRNAs with the same other sequences but different polyA sequences were prepared according to the method described in Example 7.

[0534] The effects of PolyA on mRNA were compared using in vivo IVIS imaging data.

[0535] The details of the mouse in vivo imaging experiment (IVIS) are as follows:

[0536] The specific process of encapsulating mRNA products containing different Poly A is similar to the specific process of encapsulating mRNA products containing different 5'-UTRs in Example 8, except that the mRNA products containing different Poly A are used to replace the mRNA products containing different 5'-UTRs.

[0537] The final different Poly A mRNA preparations obtained were commissioned to Nanjing Yunqiao Pure Biotechnology Co., Ltd. for animal imaging experiments. The specific process is as follows:

[0538] Male BALB / C mice, 5-7 weeks old, were raised and tested in an SPF laboratory environment. The encapsulated mRNA preparation product was injected into the tail vein. Three mice were used for each 5UTR test, and each mouse was injected with 12μg of the mRNA preparation product into the tail vein. After 16-18h, the mice were anesthetized by isoflurane inhalation anesthesia and injected with D-Luciferin luciferase development substrate (150mg / kg). The animals were placed in the supine position, and the signal distribution and expression intensity of the luciferin in the living animals were observed under the IVIS in vivo imaging system.

[0539] Observation of relevant indicators:

[0540] (1) Images observed by IVIS, including the original images and the comparison images after unifying the photon number bar values;

[0541] (2) Fluorescent signal distribution and expression intensity analysis data.

[0542] The results showed that, as shown in Figures 15 and 16, polyAV1, polyAV2, polyAV3, polyAV4, polyAV6, and polyAV9 could increase the expression of protein levels in vivo compared to other polyA sequences, among which polyAV1 had the highest protein expression level in vivo.

[0543] Example 11 Preparation of Novel Coronavirus Vaccine mRNA Vaccine and Immune Serum in Mice

[0544] The mRNA used in this example was prepared according to the method described in Example 7, and its sequence was obtained by replacing all uridine (U) nucleosides in SEQ ID NO: 15 with N1-methylpseudouridine.

[0545] The formulation systems for encapsulating the novel coronavirus vaccine mRNA in the in vivo immunization experiment are shown in Tables 8 and 9 below.

[0546] Table 8: Encapsulation formulation system

[0547] Table 9: Water-phase alcohol mixture formula

[0548] M-DMG2000 is PEG2000-DMG

[0549] Dialysis fluid preparation:

[0550] 1×PBS + 8% sucrose solution: Take 2 packets of 1×PBS powder injection and place them in a beaker. Dissolve and mix thoroughly with 2L of DEPC water. Add 160g of sucrose and mix thoroughly to obtain 1×PBS + 8% sucrose solution.

[0551] Each group of drug solution was placed in a 100KD dialysis bag, immersed in a beaker containing 1L of dialysis solution, wrapped in aluminum foil and dialyzed at 100 rpm and room temperature for 1 hour, then the dialysis solution was replaced and dialysis was continued for 1 hour.

[0552] The dialyzed preparation sample was sterilized and filtered as follows: sterilized and filtered through a 0.22 μm disposable filter membrane; the encapsulation efficiency of the final preparation was tested.

[0553] In vivo immunization experiment grouping of the new coronavirus vaccine mRNA vaccine, female mice, 16g~18g, SPF environment adaptability breeding. Divided into three groups of experiments, namely: normal saline group (blank control, 6 mice), mRNA vaccine-5μg test group (6 mice), mRNA vaccine-10μg test group (6 mice). The test samples were injected intramuscularly, and the time of the first injection of the vaccine was recorded as day 1; on the 26th day, the second immunization was performed (the same as the first immunization dosage method). On the 7th, 14th, 33rd and 44th days after vaccine immunization, orbital blood was collected to test for delta-specific IgG antibodies (for testing in Example 12). On the 14th, 37th and 44th days, orbital blood was collected from mice for neutralization experiments of wild-type SARS-COV-2 and mutant delta pseudovirus (for testing in Example 13).

[0554] Serum collection steps: Collect the blood in a non-anticoagulant tube, place on ice for 30 minutes, and centrifuge at 4°C, 3500 rpm for 10 minutes. After stratification, carefully aspirate the upper light yellow liquid with a pipette.

[0555] Example 12 IgG antibody titration experiment against S protein in mouse immune serum of novel coronavirus mRNA vaccine

[0556] The immune serum required for the specific S protein IgG antibody titration test is provided by the experiment in Example 11.

[0557] Consumables and reagents: SARS-CoV-2 Spike Trimer (T19R, G142D, EF156-157del, R158G, L452R, T478K, D614G, P681R, D950N, purchased from acrobiosystems, catalog number SPN-C52He), Goat anti-Mouse IgG (H+L) HRP Conjugated, Ms mAb to SARS spike glycoprotein [IA9], 96-well ELISA plate, PBS, bovine serum albumin, one-component TMB colorimetric solution, and stop solution.

[0558] The first step is antigen coating: dilute the antigen SARS-CoV-2 Spike Trimer to 1μg / mL with 1×PBS, add 100μL of antigen to each well, seal the plate with sealing film, incubate at 37℃ for 4h, or 4℃ for 16h, discard the liquid in the well, and wash three times with washing solution.

[0559] Second blocking: add 230 μL / well of blocking solution (3% BSA, PBST) to the ELISA plate, seal the plate with a sealing film, incubate at 37°C for 40-60 minutes, and wash three times after the incubation.

[0560] Sample Preparation: Dilute the mouse immune serum sample to be tested (provided in Example 11) to an appropriate concentration gradient (1:10 to 1:31250, starting at 1:10, and 5-fold serial dilutions). Use a large dilution volume, generally ensuring that the sample volume is >20 μL. For the positive control, the Ms mAb to SARS spike glycoprotein [IA9], dilute 1:50,000. Incubate at 37°C for 60 min. Wash three times after completion of the incubation.

[0561] Add enzyme-labeled secondary antibody (Goat anti-Mouse IgG (H+L) HRP Conjugated) at a dilution of 1:10,000. Incubate at 37°C for 40 min. Wash three times after incubation.

[0562] Substrate color development (TMB color development substrate): 100 μL per well, incubate at room temperature in the dark for 5 minutes.

[0563] Termination reaction: Add 50 μL of stop solution to each well to terminate the reaction and measure the experimental results within 20 minutes.

[0564] Detection index: ELISA results are expressed in OD at wavelength nm (450nm-630nm)

[0565] The test results are shown in Figure 20: The new coronavirus mRNA vaccine produces higher IgG antibodies against the Delta S protein (10μg mRNA vaccine produces an average IgG antibody titer of 1.3+e6 on day 44).

[0566] Example 13: Pseudovirus Neutralization Experiment of Mouse Immune Serum with Novel Coronavirus mRNA Vaccine

[0567] The immune serum required for the pseudovirus neutralization test was provided by the experiment in Example 11.

[0568] Reagents and consumables: Pseudovirus SARS-CoV-2Delta strain-Fluc: 1~2×10 4 TCID50 / mL

[0569] Inactivated mouse serum: 56°C, 30 min, Vero cells, DMEM, FBS, luciferase reporter gene assay reagent.

[0570] The initial dilution of serum samples (diluted with serum-free DMEM): 1:20 (serum samples need to be inactivated in advance at 56°C for 30 min).

[0571] 100 μL of DMEM medium containing 10% FBS was added to B2 to G2 of a 96-well white plate as a cell control (CC), and 100 μL of DMEM medium containing 10% FBS was added to B3 to G3 as a virus control (VC).

[0572] 150 μL of diluted serum sample (provided in Example 11) was added to wells B4 to B12 of a 96-well white plate as the initial diluent of the sample, and 100 μL of DMEM serum-free medium was added to wells C4 to G12.

[0573] Take 50 μL of liquid from B4 to B12 and transfer it to the next row, mix well, and perform a 3-fold gradient dilution for a total of 7 gradients. Discard the excess 50 μL of liquid in the last row.

[0574] Except for B2 to G2, 50 μL of diluted virus was added to each well, and the virus titer was diluted to 13000 TCID50 / mL and incubated at 37°C for 1 hour; 100 μL of 0.5×10 6 Vero cells per well; after culturing for 24 hours, the 96-well white plate was removed and equilibrated to room temperature. 100 μL of culture medium was aspirated from the plate, and 100 μL of Bio-Lite reporter gene detection reagent equilibrated at room temperature was added. The plate was shaken for 2 minutes, and the chemiluminescence value (RLU) was detected using a microplate reader after standing at room temperature for 5 minutes.

[0575] The pseudovirus neutralization test experiment was commissioned to Nanjing Novozymes Biotechnology Co., Ltd., and the specific experimental steps were carried out as described above.

[0576] The results are shown in Figures 21 and 22: The new coronavirus mRNA vaccine can produce a good immune protection effect against wild-type SARS-COV-2 (neutralizing antibody IC50 of 10μg mRNA vaccine on day 44 is 7952) and Delta (neutralizing antibody IC50 of 10μg mRNA vaccine on day 44 is 3966) pseudoviruses.

[0577] Example 14 Western blot activity experiment of engineered plasmids

[0578] Reagents and consumables: HEK293T, DMEM basic (1×), Opti-MEM TM I reduced serum medium, 2000 Reagent, 6-well plate, sample buffer & leafli 2× concentrate, 8% SDS-PAGE Color Preparation Kit, Ms mAb to SARS spike glycoprotein [IA9], Goat anti-Mouse IgG (H+L) HRP Conjugated, PageRuler prestained Protein Ladder, bovine serum albumin, ultrasensitive ECL luminescence reagent, methanol.

[0579] One day in advance, follow the 2×10 6 293T cells were plated in 60 mm culture dishes and transfected when the cell confluence reached 70% to 90% on the next day.

[0580] Prepare the transfection reagent according to the transfection ratio required by the experiment, transfect 5 μg of the engineered plasmid into each well of cells, incubate in a 37°C CO2 incubator for 24 h, and then perform western blot experiments.

[0581] Cells were lysed and fully denatured by heating at 95-100°C for 10 minutes. After returning to room temperature, the samples were loaded onto the gel and run. After transfer, the membrane was transferred for half an hour and blocked with 5% skim milk powder for 1 hour at room temperature. The primary antibody (Ms mAb to SARS spike glycoprotein [IA9], 1:2000) was diluted in 1% BSA and incubated at room temperature for 1 hour, followed by three washes with TBST. The secondary antibody (Goat anti-Mouse IgG (H+L) HRP conjugated, 1:5000) was diluted in 1% BSA and incubated at room temperature for 1 hour on a shaker, followed by three washes with TBST. Chemiluminescence was detected using an ECL colorimeter.

[0582] The results are shown in FIG17 : The engineered plasmid vector can successfully express the corresponding antigen protein at a high level in cells.

[0583] Example 15 Cell FACS activity experiment of engineered plasmids and corresponding mRNA

[0584] mRNA preparation: The novel coronavirus mRNA preparation process is detailed in Example 7.

[0585] Reagents and consumables: HEK293T, 2000 Reagent, DMEM basic (1×), 24-well plates, TrypLE Express, flow cytometry tubes (BD), primary flow cytometry antibody ACE2-Fc (acrobiosystems), secondary flow cytometry antibody PE anti-human IgG Fc Recombinant Antibody (Biolegend).

[0586] Transfect HEK293T with engineering plasmid or corresponding mRNA one day in advance at 1×10 5 HEK293T cells were plated in 24-well plates and transfected when the cell confluence reached 70% to 90% on the next day.

[0587] Prepare the transfection reagent according to the transfection ratio required by the experiment, add 100 μL of transfection complex per well, transfect 3 μg DNA or 3 μg mRNA per well, incubate in a 37°C CO2 incubator for 24 hours, and then perform FACS.

[0588] FACS analysis: Remove the cell supernatant and add 100 μL / well cell digestion buffer to each well. Digest at room temperature for 1 min. Resuspend in 1 mL of 1× PBS and centrifuge at 2000 rpm for 2 min. Remove the supernatant. A blank control group is a non-transfected group (an isotype control group is also included in the presence of an isotype control antibody).

[0589] Primary antibody incubation: dilute the primary antibody ACE2-Fc (acrobiosystems) to 1 μg / mL in 1× PBS, add 100 μL to each sample, incubate at 4°C for 30 min, centrifuge at 2000 rpm for 2 min, remove the supernatant, and add 1 mL of 1× PBS to wash three times.

[0590] Secondary antibody incubation: dilute the secondary antibody PE anti-human IgG Fc Recombinant Antibody (Biolegend) in 1× PBS.

[0591] 1 μg / mL, add 100 μL to each sample, incubate at 4°C for 30 min, centrifuge at 2000 rpm for 2 min, remove the supernatant, add 1 mL 1× PBS to wash 3 times, then add 100 μL 1× PBS to resuspend and detect.

[0592] The experimental results are shown in Figure 18: both the engineered plasmid and the mRNA product can highly express the correct S protein in the transfected cells.

[0593] Example 16 ELISA activity test of novel coronavirus mRNA

[0594] mRNA preparation: The mRNA preparation process is detailed in Example 7.

[0595] Reagents and consumables: DMEM basic (1×), fetal bovine serum FBS, Lipofectamine TM MessengerMAX TM Opti-MEM TM I reduced serum medium, 0.25% Trypsin-EDTA (1×), lysis buffer, cocktail, Anti-nCoV-RBD Neutralizing Antibody, HRP conjugated Anti-nCoV-RBD-1 Neutralizing antibody, PBS buffer, ELISA developer, stop solution, 96-well ELISA plate.

[0596] Press 6×10 5 The cells were plated in 96-well plates at a density of 6 × 10 cells / mL. 4Three replicate wells were set up for each sample. 24 hours after plating, mRNA was transfected, and the transfection complex was prepared. Transfection was performed according to the mRNA transfection gradient (μg / well). Three replicate wells were set up, and the transfection gradient was 0.05μg, 0.015μg, 0.03μg, 0.06μg, 0.18μg, 0.54μg, 1.62μg, and 4.86μg. 24 hours after transfection, the culture medium was discarded, the cells were washed once with PBS, and 120μL NP40 lysis buffer (with 100× cocktail) was used for lysis on ice for 10 minutes, centrifuged at 4000rpm for 10 minutes, and then 100μL was aspirated for subsequent ELISA experiments. Anti-nCoV-RBD Neutralizing Antibody was diluted to 1μg / mL with coating solution (PBS buffer), 100μL / well, and allowed to stand overnight at 4 degrees. After washing, add 240 μL / well of blocking buffer (3% w / v BSA / PBS buffer) to the ELISA plate, seal the plate with sealing film, and incubate at 37°C for 1 hour. After washing, pipette 100 μL of the harvested cell supernatant into a 96-well plate and incubate at room temperature for 2.5 hours. After washing, dilute the HRP-conjugated anti-nCoV-RBD-1 neutralizing antibody 8000-fold in sample diluent (0.5% w / v BSA / wash buffer (wash buffer is PBS buffer with 0.05% Tween 20)) and incubate at room temperature for 1 hour. After washing, add 100 μL / well of ELISA colorimetric solution for color development and incubate at room temperature for 10 minutes. Terminate the reaction with stop solution and read on a microplate reader.

[0597] The results are shown in Figure 19: The novel coronavirus mRNA product can express S protein in cells in a dose-dependent manner.

[0598] Example 17 Synthesis of Compound (I)

[0599] Step 1): Synthesis of 8-((3-hydroxycyclohexyl)amino)nonyl octanoate

[0600] In a 100 mL reaction bottle, 8-bromooctanoic acid 1-octylnonyl ester (3.50 g, 10 mmol), 3-aminocyclohexanol (11.5 g, 100 mmol), and 30 mL of ethanol were added in sequence. After stirring and dissolving, N, N-diisopropylethylamine (2.58 g, 20 mmol) was added and reacted at room temperature for 24 h. 100 mL of dichloromethane was added, and the mixture was washed with water 3 times, dried over anhydrous sodium sulfate, concentrated, and purified using a flash column chromatography system (dichloromethane). ∶ Methanol = 20:1 to 5:1) to give 8-((3-hydroxycyclohexyl)amino)octanoic acid nonyl ester (2.34 g, 61%).

[0601] 1 H NMR (600MHz, CDCl3) δ4.20-4.13 (m, 0.5H), 4.05 (t, 2H), 3.83 (m, 0.5H), 3.09 (m, 0.5H), 2.87 (m, 0.5H), 2.76-2.59 (m, 2H), 2.29(t, 2H), 2.00(m, 0.5H), 1.93-1.78(m, 1.5H), 1.78-1.65(m, 2H), 1.65-1.46(m, 8H), 1.40-1.18(m, 20H), 0.88(t, 3H).

[0602] LCMS: 384.3 [M+H] + .

[0603] Step 2): Synthesis of compound (I)

[0604] In a 100 mL reaction flask, 8-((3-hydroxycyclohexyl)amino)octanoic acid nonyl ester (383 mg, 1 mmol), 8-bromooctanoic acid 1-octyl nonyl ester (554 mg, 1.2 mmol), and 20 mL of acetonitrile were added in sequence. After stirring and dissolving, potassium carbonate (276 mg, 2 mmol) and potassium iodide (166 mg, 1 mmol) were added. The mixture was reacted at room temperature for 24 h. 100 mL of dichloromethane was added, and the mixture was washed with water three times, dried over anhydrous sodium sulfate, concentrated, and purified using a flash column chromatography system (dichloromethane: methanol = 20: 1 to 5: 1) to obtain compound (I) (596 mg, 78%).

[0605] 1 H NMR (600MHz, CDCl3) δ4.95-4.81(m, 1H), 4.30-4.12(m, 0.5H), 4.07(t, 2H), 3.72-3.61(m, 0.5H), 2.97(m, 0.5H), 2.64-2.52(m , 0.5H), 2.48-2.37(m, 4H), 2.30(q, 4H), 1.93-1.81(m, 2H), 1.72-1.57(m, 8H), 1.51(t, 4H), 1.43-1.18(m, 56H), 0.89(m, 9H).

[0606] LCMS: 765.3 [M+H] + .

[0607] Example 18: Experimental study on the protective activity of the novel coronavirus mRNA vaccine against the Omicron variant

[0608] The in vivo immunization experiment group of the new coronavirus mRNA vaccine was divided into three groups: female mice, 16g to 18g, and raised in an SPF environment. The experiments were divided into three groups: normal saline group (blank control, 5 mice), mRNA vaccine-5μg test group (5 mice), and mRNA vaccine-10μg test group (5 mice). The test samples were injected intramuscularly, and the time of the first injection of the vaccine was recorded as day 1; on the 21st day, the second immunization was performed (the dosage method was consistent with the first immunization). On the 7th, 14th, 28th, and 35th days after vaccination, orbital blood was collected to test IgG antibodies specific to Omicron. On the 35th day, blood was collected from mice for the Omicron (BA.2) pseudovirus neutralization experiment, and spleen lymphocytes were taken for IFNγ-Elispot assay to test the cellular immune response of the vaccine.

[0609] Serum collection steps: Collect the blood in a non-anticoagulant tube, place on ice for 30 minutes, and centrifuge at 4°C, 3500 rpm for 10 minutes. After stratification, carefully aspirate the upper light yellow liquid with a pipette.

[0610] 1. Detection of IgG antibody titers specifically binding to Omicron S protein after immunization of mice with the novel coronavirus mRNA vaccine

[0611] Consumables and reagents: Omicron Spike Trimer (purchased from acrobiosystems, catalog number SPN-C52Hz), Goat anti-Mouse IgG (H+L) HRP Conjugated, Ms mAb to SARS spike glycoprotein [IA9], 96-well ELISA plate, PBS, bovine serum albumin, one-component TMB colorimetric solution, and stop solution.

[0612] The experimental procedures refer to Example 12.

[0613] The results are shown in Figure 23: The new coronavirus mRNA vaccine produces higher IgG antibodies against the Omicron S protein (5μg mRNA vaccine produces an average IgG antibody titer of 2.67+e5 on day 35).

[0614] 2. Neutralizing antibody titer detection against Omicron pseudovirus after immunization of mice with novel coronavirus mRNA vaccine

[0615] Reagents and consumables: pseudovirus SARS-CoV-2 Omicron (BA.2) strain-Fluc: 1-2×10 4 TCID50 / mL

[0616] Inactivated mouse serum: 56°C, 30 min, Vero cells, DMEM, FBS, luciferase reporter gene assay reagent.

[0617] The experimental procedures refer to Example 13.

[0618] The results are shown in Figure 24: The new coronavirus mRNA vaccine can produce a good immune protection effect against the Omicron pseudovirus (the neutralizing antibody IC50 of 10μg mRNA vaccine on the 35th day is 7952).

[0619] 3. IFN-γ Elispot detection of spleen lymphocytes

[0620] Reagents and consumables: SARS-CoV-2 Spike S1 Peptide Pool (PP003-A), SARS-CoV-2 Spike S2 ​​Peptide Pool (PP003-B), Mouse Lymphocyte Separation Medium (Dakoway, 7211011), Mouse IFN-γ ELISPOT Kit (Dakoway, CT317-PR2)

[0621] (1) Lymphocyte separation: Place 5 mL of mouse lymphocyte separation medium that has returned to room temperature in a 60 mm culture dish and grind the spleen. Immediately transfer the separation medium containing the spleen cells to a 15 mL centrifuge tube and cover with 1 mL of RPMI 1640 culture medium (keep the liquid surface boundary clear). Centrifuge at room temperature at 800 g for 30 minutes in a horizontal rotor. Pay attention to setting a slower acceleration and deceleration. After the centrifugation, separate the cells, aspirate the lymphocyte layer, add 10 mL of RPMI 1640 culture medium, and invert to wash. Centrifuge at room temperature at 250 g for 10 minutes to collect the cells. Pour off the supernatant, resuspend the cells in culture medium, and count.

[0622] (2) Peptide library stimulation Elispot experiment:

[0623] Positive control wells: Add positive stimulator working solution (dilute the PMA stock solution 10-fold with serum-free medium or RPMI 1640 to a concentration of 10 μg / mL, and then add 10 μL / well to each well).

[0624] Negative control wells: add the culture medium used to resuspend cells;

[0625] Experimental wells: Add the experimenter's own stimuli (dilute the S1+S2 peptide library with serum-free culture medium or RPMI 1640. The final concentration of the working solution is 1 μg / mL).

[0626] The specific process was carried out according to the operating instructions of the kit.

[0627] The results are shown in Figure 25: After mice are immunized with the new coronavirus mRNA vaccine, lymphocytes can be activated to produce a strong cellular immune response.

[0628] The above examples demonstrate that the inventors have successfully designed a novel RNA coding sequence for the novel coronavirus S protein and a novel mRNA vaccine derived therefrom. The mRNA vaccine exhibits cross-protection against different strains, including the wild-type SARS-CoV-2, as well as the Delta and Omicron variants, and exhibits strong immune responses against the Delta and Omicron variants, and can be developed and produced more rapidly on a large scale.

[0629] The above examples also show that the inventors of the present application have successfully constructed a universal polynucleotide molecule (e.g., 3'-UTR, 5'-UTR, vector, etc.) for preparing a new RNA vaccine (e.g., mRNA vaccine), which increases the level of mRNA expression protein. The polynucleotide molecule can not only be used to prepare mRNA vaccines with good immune effects on the new coronavirus wild-type SARS-COV-2, Delta and Omicron variants, but can also be used to transfect cells or bacteria to produce corresponding antibodies or antigenic proteins, thus being a universal core component combination.

[0630] The above examples also demonstrate that the inventors have successfully constructed a universal polynucleotide molecule (e.g., 3'-UTR, 5'-UTR, vector, etc.) that increases the level of protein expression from mRNA. This polynucleotide molecule can be used not only for in vitro production of proteins and / or polypeptides but also for in vivo production of corresponding proteins and / or polypeptides, representing a universal core component.

Claims

1. An RNA encoding a novel coronavirus S protein, wherein the S protein comprises the amino acid sequence shown in SEQ ID NO: 13; preferably, the RNA comprises the nucleic acid sequence shown in SEQ ID NO: 14, and more preferably, the nucleic acid sequence of the RNA is as shown in SEQ ID NO:

14.

2. The RNA of claim 1 , wherein one or more uridine nucleosides, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 uridine nucleosides or at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of the uridine nucleosides in the RNA are replaced by at least one nucleoside selected from the group consisting of pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4′-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1- deaza-pseudouridine, 2-thio-T-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine or 5-methoxyuridine and 2′-O-methyluridine, preferably pseudouridine or N1-methylpseudouridine or N1-ethylpseudouridine, more preferably N1-methylpseudouridine; and / or One or more cytosine nucleosides in the RNA, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 cytosine nucleosides or at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of the cytosine nucleosides are replaced by 5-methylcytosine nucleoside.

3. The RNA according to claim 1 or 2, wherein all or part of the uridine nucleosides in the RNA are replaced by pseudouridine, preferably N1-methylpseudouridine; preferably, all or part of the uridine nucleosides in the nucleic acid sequence shown in SEQ ID NO: 14 are replaced by pseudouridine, preferably N1-methylpseudouridine; more preferably, all uridine nucleosides in the nucleic acid sequence shown in SEQ ID NO: 14 are replaced by N1-methylpseudouridine. 4 . The RNA according to claim 1 , further comprising at least one of a 5′-cap structure, a 5′-UTR, a 3′-UTR, and polyA.

5. The RNA according to claim 4, wherein the 5'-cap structure is selected from m 7 GpppG、m2 7，3′-O GpppG、m 7 Gppp(5′)N1 or m 7 Gppp(m 2′-O ) At least one of N1, preferably m 7 Gppp(5′)N1 or m 7 Gppp(m 2′-O )N1, where "m7G" represents 7-methylguanosine cap nucleoside, "ppp" represents the triphosphate bond between the 5' carbon of the cap nucleoside and the first nucleotide of the primary RNA transcript, N1 is the 5'most nucleotide, "G" represents guanosine nucleoside, "7" represents the methyl group at the 7-position of guanine, and "m 2′-O ” represents the methyl group at the 2′-O position of the nucleotide; and / or the 5'-UTR is selected from the following (1)-(5): (1) comprising a 5'-UTR of an RNA sequence corresponding to the nucleic acid sequence of SEQ ID NO: 9, 16, 17, 23, 24, 28, 29, 32, 37, 39 or 42, a homologue, fragment or variant thereof, wherein the homologue, fragment or variant has the same or better function of improving translation efficiency as the 5'-UTR of the RNA sequence corresponding to SEQ ID NO: 9, 16, 17, 23, 24, 28, 29, 32, 37, 39 or 42; preferably, the nucleic acid sequence of the homologue has at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology to the RNA sequence corresponding to the nucleic acid sequence of SEQ ID NO: 9, 16, 17, 23, 24, 28, 29, 32, 37, 39 or 42; (2) a 5′-UTR consisting of an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 9, 16, 17, 23, 24, 28, 29, 32, 37, 39 or 42; (3) a 5'-UTR consisting of an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 9; (4) A 5'-UTR obtained by concatenating two or more identical 5'-UTRs in (1) to (3) above; or (5) 5'-UTR obtained by concatenating two or more different 5'-UTRs in (1)-(3) above; and / or the 3'-UTR is selected from the following (1)-(5): (1) comprising a 3'UTR of an RNA sequence corresponding to the nucleic acid sequence of SEQ ID NO: 47-67 or 10, a homologue, fragment or variant thereof; said homologue, fragment or variant having the same or better function of improving translation efficiency and / or stability as the 3'UTR of the RNA sequence corresponding to SEQ ID NO: 47-67 or 10; preferably, said nucleic acid sequence of said homologue has at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology with the RNA sequence corresponding to the nucleic acid sequence of SEQ ID NO: 47-67 or 10; (2) a 3'-UTR consisting of an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 47-67 or 10; (3) a 3'-UTR consisting of an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 10; (4) A 3'-UTR obtained by concatenating two or more identical 3'-UTRs in (1) to (3) above; or (5) 3'-UTR obtained by concatenating two or more different 3'-UTRs in (1)-(3) above; And / or the polyA is a truncated polyA, i.e., a plurality of consecutive A nucleotides are followed by a 10 bp non-A linker sequence and then a plurality of consecutive A nucleotides; preferably, the polyA is 30 consecutive A nucleotides followed by a 10 bp non-A linker sequence and then 70 consecutive A nucleotides; Preferably, the polyA is selected from the following (1)-(3): (1) A polyA comprising an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 68-70, 72, 75 or 11, a homologue, fragment or variant thereof, wherein the homologue, fragment or variant has the same or better function of improving translation efficiency and / or stability as the polyA shown in the RNA sequence corresponding to SEQ ID NO: 68-70, 72, 75 or 11; preferably, the homologue comprises a nucleic acid sequence having at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology to the RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 68-70, 72, 75 or 11; (2) a polyA consisting of an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 68-70, 72, 75 or 11; or (3) PolyA consisting of an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:

11.

6. The RNA according to any one of claims 1 to 5, comprising the nucleic acid sequence shown in SEQ ID NO: 15, more preferably, the nucleic acid sequence thereof is shown in SEQ ID NO:

15.

7. The RNA according to claim 6, wherein all or part of the uridine nucleosides in the RNA are replaced by pseudouridine, preferably N1-methylpseudouridine; preferably, all or part of the uridine nucleosides in the nucleic acid sequence shown in SEQ ID NO: 15 are replaced by pseudouridine, preferably N1-methylpseudouridine; more preferably, all uridine nucleosides in the nucleic acid sequence shown in SEQ ID NO: 15 are replaced by N1-methylpseudouridine.

8. A protein expressed by the RNA according to any one of claims 1 to 7, preferably, the protein comprises the amino acid sequence shown in SEQ ID NO: 13, more preferably, the amino acid sequence shown in SEQ ID NO:

13.

9. A DNA encoding the RNA according to any one of claims 1 to 7; preferably, the DNA comprises the nucleic acid sequence shown in SEQ ID NO: 12; preferably, the DNA comprises the nucleic acid sequence shown in SEQ ID NO:

8.

10. A vector comprising the DNA according to claim 9. A host cell comprising the vector according to claim 10 .

12. A lipid nanoparticle comprising the RNA of any one of claims 1-7.

13. The lipid nanoparticle of claim 12, further comprising one or more of an ionizable cationic lipid, a helper lipid, a structural lipid, and a PEG-lipid.

14. The lipid nanoparticle of claim 13, wherein the ionizable cationic lipid is selected from one or more of Dlin-MC3-DMA, Dlin-KC2-DMA, DODMA, c12-200, or DlinDMA; and / or the auxiliary lipid is selected from one or more of DSPC, DOPE, DOPC, or DOPS; and / or the structural lipid is selected from at least one of cholesterol, cholesterol esters, steroid hormones, steroid vitamins, and bile acids; and / or the PEG-lipid is selected from PEG-DMG or PEG-DSPE, preferably PEG-DMG, more preferably, PEG-DMG is a polyethylene glycol (PEG) derivative of 1,2-dimyristyl glyceride, and further preferably, the average molecular weight of PEG is about 2000 or 5000 Daltons, preferably 2000 Daltons.

15. A pharmaceutical composition comprising the RNA of any one of claims 1-7, the protein of claim 8, the DNA of claim 9, the vector of claim 10, the host cell of claim 11 or the lipid nanoparticle of any one of claims 12-14, and a pharmaceutically acceptable carrier and / or excipient.

16. Use of the RNA of any one of claims 1 to 7, the protein of claim 8, the DNA of claim 9, the vector of claim 10, the host cell of claim 11, or the lipid nanoparticles of any one of claims 12 to 14, or the pharmaceutical composition of claim 15 in the preparation of a vaccine for preventing novel coronavirus infection.

17. An artificial RNA molecule comprising a 5'-UTR and a nucleic acid sequence encoding a protein and / or polypeptide of interest, wherein the 5'-UTR is selected from the following (1)-(5): (1) A 5'-UTR comprising an RNA sequence corresponding to the nucleic acid sequence of SEQ ID NO: 9, 16, 17, 23, 24, 28, 29, 32, 37, 39 or 42, a homologue, fragment or variant thereof, wherein the homologue, fragment or variant has the same or better function of improving translation efficiency as the 5'-UTR of the RNA sequence corresponding to SEQ ID NO: 9, 16, 17, 23, 24, 28, 29, 32, 37, 39 or 42; preferably, the nucleic acid sequence of the homologue has at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 15 94%, 95%, 96%, 97%, 98% or 99% homology; (2) a 5′-UTR consisting of an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 9, 16, 17, 23, 24, 28, 29, 32, 37, 39 or 42; (3) a 5'-UTR consisting of an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 9; (4) A 5'-UTR obtained by concatenating two or more identical 5'-UTRs in (1) to (3) above; or (5) A 5'-UTR obtained by concatenating two or more different 5'-UTRs in (1) to (3) above.

18. The artificial RNA molecule according to claim 17, further comprising a 3'-UTR and polyA; Preferably, the 3'-UTR is selected from the following (1)-(5): (1) comprising a 3'UTR of an RNA sequence corresponding to the nucleic acid sequence of SEQ ID NO: 47-67 or 10, a homologue, fragment or variant thereof; said homologue, fragment or variant having the same or better function of improving translation efficiency and / or stability as the 3'UTR of the RNA sequence corresponding to SEQ ID NO: 47-67 or 10; preferably, said nucleic acid sequence of said homologue has at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology with the RNA sequence corresponding to the nucleic acid sequence of SEQ ID NO: 47-67 or 10; (2) a 3'-UTR consisting of an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 47-67 or 10; (3) a 3'-UTR consisting of an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 10; (4) A 3'-UTR obtained by concatenating two or more identical 3'-UTRs in (1) to (3) above; or (5) 3'-UTR obtained by concatenating two or more different 3'-UTRs in (1)-(3) above; And / or the polyA is a truncated polyA, i.e., a plurality of consecutive A nucleotides are followed by a 10 bp non-A linker sequence and then a plurality of consecutive A nucleotides; preferably, the polyA is 30 consecutive A nucleotides followed by a 10 bp non-A linker sequence and then 70 consecutive A nucleotides; Preferably, the polyA is selected from the following (1)-(3): (1) A polyA comprising an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 68-70, 72, 75 or 11, a homologue, fragment or variant thereof, wherein the homologue, fragment or variant has the same or better function of improving translation efficiency and / or stability as the polyA shown in the RNA sequence corresponding to SEQ ID NO: 68-70, 72, 75 or 11; preferably, the homologue comprises a nucleic acid sequence having at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology to the RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 68-70, 72, 75 or 11; (2) a polyA consisting of an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 68-70, 72, 75 or 11; or (3) PolyA consisting of an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO:

11.

19. An RNA molecule comprising: (1) a first nucleotide sequence encoding a polypeptide and / or protein of interest; and (2) a second nucleotide sequence containing a 3'-untranslated region (3'-UTR); The 3'-UTR comprises at least one of the following polynucleotides: (a): 3'-UTR derived from at least one of the genes APOA1, IE1, COP1, MYSM1, ASAH2B, NDUFAF2, APOD, HBB, TF, TMSB4X, CPAMD8, VIM, HDGFL1, TTR, SRM, HBA1, PRPF8, LMBRD1, IFNA1, CCDC146, and GHRL; (b): a fragment of the 3′-UTR described in (a); (c): a variant of the 3'-UTR described in (a); and (d): A variant of the fragment described in (b); The first nucleotide sequence and the second nucleotide sequence do not naturally occur in the same RNA molecule.

20. The RNA molecule of claim 19, wherein the gene is a human gene.

21. The RNA molecule according to claim 19 or 20, wherein the second nucleotide sequence comprises at least one of the following polynucleotides: (e): 3′-UTR derived from at least one of the genes COP1 and HDGFL1; (f): a fragment of the 3′-UTR described in (e); (g): A variant of the 3'-UTR described in (e); and (h): A variant of the fragment described in (f).

22. The RNA molecule according to any one of claims 19 to 21, wherein the second nucleotide sequence comprises at least one of the following polynucleotides: RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 47-67, fragments of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 47-67, variants of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 47-67, and variants of a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 47-67; Preferably, variants of RNA encoded by polynucleotides having a sequence as set forth in at least one of SEQ ID NOs: 47 to 67, fragments of RNA encoded by polynucleotides having a sequence as set forth in at least one of SEQ ID NOs: 47 to 67, and variants of fragments of RNA encoded by polynucleotides having a sequence as set forth in at least one of SEQ ID NOs: 47 to 67 have at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the RNA encoded by polynucleotides having a sequence as set forth in at least one of SEQ ID NOs: 47 to 67.

23. The RNA molecule according to any one of claims 19 to 22, wherein the second nucleotide sequence comprises at least one of: 3'-UTRs of at least two of the genes described in (a), fragments of 3'-UTRs of at least two of the genes described in (a), variants of 3'-UTRs of at least two of the genes described in (a), and variants of fragments of 3'-UTRs of at least two of the genes described in (a).

24. The RNA molecule according to any one of claims 19 to 23, wherein the second nucleotide sequence comprises: at least two copies of at least one of the 3'-UTR in (a), at least two copies of a fragment of the 3'-UTR in (a), at least two copies of a variant of the 3'-UTR in (a), and at least two copies of a variant of the fragment of the 3'-UTR in (a). 25 . The RNA molecule according to claim 19 , further comprising at least one of a promoter, a 5′-cap structure, a 5′-UTR, and polyA.

26. The RNA molecule according to claim 25, (1) The 5'-cap structure includes m 7 GpppG、m2 7，3′-O GpppG、m 7 Gppp(5′)N1 and m 7 Gppp(m 2′-O ) At least one of N1; (2) The 5'-UTR comprises: i) at least one of the 5'-UTR of at least one of the genes APOA1, CARD16, ALB, APOC1, EEF1A1, RBP4, GHRL, MPND, ASAH2B, FBX016, FBH1, SRM, NAAA, ACTB, MKNK2, ORM1, GADPH, NDUFAF2, FGFR2, MYCBPAP, CHMP2A, TSG101, PRPF8, NFKB2, NAE1, HDGFL1, GSDMD, FBXW12, FBXW10, FBXL8, and ATG4D, fragments, variants, and variants of fragments thereof; preferably, an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 16 to 46, a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 16 to 46, a variant of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 16 to 46, and a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 16 to 46. A variant of the RNA fragment encoded by at least one of the polynucleotides shown in NO: 16 to 46; Preferably, said variants of RNA encoded by the polynucleotides having a sequence as set forth in at least one of SEQ ID NOs: 16-46, fragments of RNA encoded by the polynucleotides having a sequence as set forth in at least one of SEQ ID NOs: 16-46, and variants of fragments of RNA encoded by the polynucleotides having a sequence as set forth in at least one of SEQ ID NOs: 16-46 are at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the RNA encoded by the polynucleotides having a sequence as set forth in at least one of SEQ ID NOs: 16-46; ii) at least one of the following polynucleotides: an RNA encoded by the polynucleotide having a sequence as set forth in SEQ ID NO: 9, a fragment of the RNA encoded by the polynucleotide having a sequence as set forth in SEQ ID NO: 9, a variant of the RNA encoded by the polynucleotide having a sequence as set forth in SEQ ID NO: 9, and a variant of a fragment of the RNA encoded by the polynucleotide having a sequence as set forth in SEQ ID NO: 9; Preferably, variants of RNA encoded by the polynucleotide having a sequence as set forth in SEQ ID NO:9, fragments of RNA encoded by the polynucleotide having a sequence as set forth in SEQ ID NO:9, and variants of fragments of RNA encoded by the polynucleotide having a sequence as set forth in SEQ ID NO:9 are at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the RNA encoded by the polynucleotide having a sequence as set forth in SEQ ID NO:9; iii) at least two copies of one of the polynucleotides in i) or ii); or iv) at least two polynucleotides according to i); (3) the nucleotides constituting the polyA contain at least 20, at least 40, at least 80, at least 100 or at least 120 A nucleotides; preferably, the nucleotides constituting the polyA contain at least 20, at least 40, at least 80, at least 100 or at least 120 A nucleotides continuously; Preferably, the nucleotides constituting the polyA include one or more nucleotides other than A nucleotides; preferably, the polyA is a truncated polyA; preferably, after a plurality of consecutive A nucleotides, there is a 10 bp non-A linker sequence followed by a plurality of consecutive A nucleotides; preferably, the polyA is 30 consecutive A nucleotides followed by a 10 bp non-A linker sequence and then 70 consecutive A nucleotides; Preferably, the polyA comprises at least one of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 68-76 and 11, a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 68-76 and 11, a variant of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 68-76 and 11, and a variant comprising a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 68-76 and 11; preferably, the fragments, variants, and variants of the fragments of the RNA encoded by at least one of SEQ ID NOs: 68-76 and 11 are at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the RNA encoded by a polynucleotide having a sequence as set forth in one of SEQ ID NOs: 68-76 and 11; More preferably, the polyA is an RNA encoded by a polynucleotide having a sequence as one of SEQ ID NOs: 68 to 70, 72, 75 and 11; preferably, the polyA is an RNA encoded by a polynucleotide having a sequence as SEQ ID NO:

11.

27. An RNA molecule comprising: (1) a first nucleotide sequence encoding a polypeptide and / or protein of interest; and (2) a third nucleotide sequence containing the 5'-untranslated region (5'-UTR); The 5'-UTR comprises at least one of the following polynucleotides: (a): 5'-UTR derived from at least one of the following genes: APOA1, CARD16, ALB, APOC1, EEFIA1, RBP4, GHRL, MPND, ASAH2B, FBX016, FBH1, SRM, NAAA, ACTB, MKNK2, ORM1, GADPH, NDUFAF2, FGFR2, MYCBPAP, CHMP2A, TSG101, PRPF8, NFKB2, NAE1, HDGFL1, GSDMD, FBXW12, FBXW10, FBXL8, and ATG4D; (b): a fragment of the 5′-UTR described in (a); (c): a variant of the 5'-UTR described in (a); and (d): A variant of the fragment described in (b); The first nucleotide sequence and the third nucleotide sequence do not naturally occur in the same RNA molecule.

28. The RNA molecule of claim 27, wherein the gene is a human gene.

29. The RNA molecule according to claim 27 or 28, wherein the third nucleotide sequence comprises at least one of the following polynucleotides: an RNA encoded by a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 16 to 46, a fragment of an RNA encoded by a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 16 to 46, a variant of an RNA encoded by a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 16 to 46, and a variant of a fragment of an RNA encoded by a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 16 to 46; Preferably, variants of RNA encoded by polynucleotides having sequences as set forth in at least one of SEQ ID NOs: 16 to 46, fragments of RNA encoded by polynucleotides having sequences as set forth in at least one of SEQ ID NOs: 16 to 46, and variants of fragments of RNA encoded by polynucleotides having sequences as set forth in at least one of SEQ ID NOs: 16 to 46, have at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the RNA encoded by polynucleotides having sequences as set forth in at least one of SEQ ID NOs: 16 to 46.

30. The RNA molecule according to any one of claims 27 to 29, wherein the third nucleotide sequence comprises at least one of: 5'-UTRs of at least two of the genes described in (a), fragments of 5'-UTRs of at least two of the genes described in (a), variants of 5'-UTRs of at least two of the genes described in (a), and variants of fragments of 5'-UTRs of at least two of the genes described in (a); And / or, the third nucleotide sequence comprises: at least two copies of the 5'-UTR in (a), at least two copies of a fragment of the 5'-UTR in (a), at least two copies of a variant of the 5'-UTR in (a), and at least two copies of a variant of the fragment of the 5'-UTR in (a).

31. An RNA molecule comprising: (1) a first nucleotide sequence encoding a polypeptide and / or protein of interest; and (2) a 5'-untranslated region (5'-UTR); wherein the 5'-UTR comprises at least one of the following polynucleotides: an RNA encoded by a polynucleotide having a sequence as shown in SEQ ID NO: 9, a fragment of an RNA encoded by a polynucleotide having a sequence as shown in SEQ ID NO: 9, a variant of an RNA encoded by a polynucleotide having a sequence as shown in SEQ ID NO: 9, and a variant of a fragment of an RNA encoded by a polynucleotide having a sequence as shown in SEQ ID NO: 9; Preferably, variants of RNA encoded by the polynucleotide having a sequence as shown in SEQ ID NO: 9, fragments of RNA encoded by the polynucleotide having a sequence as shown in SEQ ID NO: 9, and variants of fragments of RNA encoded by the polynucleotide having a sequence as shown in SEQ ID NO: 9 have at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the RNA encoded by the polynucleotide having a sequence as shown in SEQ ID NO:

9.

32. The RNA molecule according to any one of claims 27 to 31, further comprising at least one of a promoter, a 5'-cap structure, a 3'-UTR, and polyA; Preferably, (1) the 5'-cap structure comprises m 7 GpppG、m2 7，3′-O GpppG、m 7 Gppp(5′)N1 and m 7 Gppp(m 2′-O ) At least one of N1; (2) The 3'-UTR comprises: i) at least one of a 3'-UTR derived from at least one of the genes APOA1, IE1, COP1, MYSM1, ASAH2B, NDUFAF2, APOD, HBB, TF, TMSB4X, CPAMD8, VIM, HDGFL1, TTR, SRM, HBA1, PRPF8, LMBRD1, IFNA1, CCDC146, GHRL, and GH1, a fragment thereof, a variant thereof, and a variant of a fragment thereof; Preferably, an RNA encoded by a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 47 to 67 and 10, a fragment of an RNA encoded by a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 47 to 67 and 10, a variant of an RNA encoded by a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 47 to 67 and 10, and a variant of a fragment of an RNA encoded by a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 47 to 67 and 10; Preferably, variants of RNA encoded by polynucleotides having sequences as set forth in at least one of SEQ ID NOs: 47-67 and 10, fragments of RNA encoded by polynucleotides having sequences as set forth in at least one of SEQ ID NOs: 47-67 and 10, and variants of fragments of RNA encoded by polynucleotides having sequences as set forth in at least one of SEQ ID NOs: 47-67 and 10 are at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the RNA encoded by polynucleotides having sequences as set forth in at least one of SEQ ID NOs: 47-67 and 10; ii) at least two copies of one of the polynucleotides described in i); or iii) at least two polynucleotides according to i); (3) the nucleotides constituting the polyA contain at least 20, at least 40, at least 80, at least 100 or at least 120 A nucleotides; preferably, the nucleotides constituting the polyA contain at least 20, at least 40, at least 80, at least 100 or at least 120 A nucleotides continuously; Preferably, the nucleotides constituting the polyA include one or more nucleotides other than A nucleotides; preferably, the polyA is a truncated polyA; preferably, after a plurality of consecutive A nucleotides, there is a 10 bp non-A linker sequence followed by a plurality of consecutive A nucleotides; preferably, the polyA is 30 consecutive A nucleotides followed by a 10 bp non-A linker sequence and then 70 consecutive A nucleotides; Preferably, the polyA comprises at least one of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 68-76 and 11, a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 68-76 and 11, a variant of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 68-76 and 11, and a variant comprising a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 68-76 and 11; preferably, the fragments, variants, and variants of the fragments of the RNA encoded by at least one of SEQ ID NOs: 68-76 and 11 are at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the RNA encoded by a polynucleotide having a sequence as set forth in one of SEQ ID NOs: 68-76 and 11; More preferably, the polyA is an RNA encoded by a polynucleotide having a sequence as one of SEQ ID NOs: 68 to 70, 72, 75 and 11; preferably, the polyA is an RNA encoded by a polynucleotide having a sequence as SEQ ID NO:

11.

33. The RNA molecule according to any one of claims 19 to 32, wherein all or part of the uracil in the RNA molecule is replaced by at least one nucleoside selected from the group consisting of pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4′-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-T-methyl-pseudouridine, 2-thio-5-aza-uridine uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine or 5-methoxyuridine and 2′-0-methyluridine; preferably, all or part of the uridine nucleosides in the RNA molecule are replaced by pseudouridine, N1-methylpseudouridine or N1-ethylpseudouridine; Preferably, one or more cytosine nucleosides or at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of the cytosine nucleosides in the RNA molecule are replaced by 5-methylcytosine.

34. A DNA encoding the artificial RNA molecule according to any one of claims 17 to 18 or a DNA encoding the RNA molecule according to any one of claims 19 to 33. A vector comprising the DNA according to claim 34 .

36. A vector comprising a fourth nucleotide sequence encoding a 3'-UTR, wherein the fourth nucleotide sequence comprises at least one of the following polynucleotides: (a): a polynucleotide encoding the 3'-UTR of at least one of the genes APOA1, IE1, COP1, MYSM1, ASAH2B, NDUFAF2, APOD, HBB, TF, TMSB4X, CPAMD8, VIM, HDGFL1, TTR, SRM, HBA1, PRPF8, LMBRD1, IFNA1, CCDC146, and GHRL; (b): a polynucleotide encoding a fragment of the 3'-UTR described in (a); (c): a polynucleotide encoding a variant of the 3'-UTR described in (a); and (d): A polynucleotide encoding a variant of the fragment described in (b).

37. The vector according to claim 36, wherein the gene is a human gene; and / or the vector does not comprise a polynucleotide encoding a protein and / or polypeptide of interest.

38. The vector according to claim 36 or 37, wherein the fourth nucleotide sequence comprises at least one of the following polynucleotides: (e): a polynucleotide encoding a 3′-UTR derived from at least one of the genes COP1 and HDGFL1; (f): a polynucleotide encoding the 3'-UTR fragment described in (e); (g): a polynucleotide encoding a variant of the 3'-UTR described in (e); and (h): A polynucleotide encoding a variant of the fragment described in (f).

39. The vector according to any one of claims 33 to 38, wherein the fourth nucleotide sequence comprises at least one of the following polynucleotides: (1) at least one of a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 47 to 67, a fragment of a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 47 to 67, a variant of a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 47 to 67, and a variant of a fragment of a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 47 to 67; Preferably, variants of the polynucleotides having a sequence as set forth in at least one of SEQ ID NOs: 47-67, fragments of the polynucleotides having a sequence as set forth in at least one of SEQ ID NOs: 47-67, and variants of fragments of the polynucleotides having a sequence as set forth in at least one of SEQ ID NOs: 47-67 are at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an RNA encoded by the polynucleotides having a sequence as set forth in at least one of SEQ ID NOs: 47-67; (2) at least one of a polynucleotide encoding the 3'-UTR of at least two genes among the genes described in (a), a polynucleotide encoding a fragment of the 3'-UTR of at least two genes among the genes described in (a), a polynucleotide encoding a variant of the 3'-UTR of at least two genes among the genes described in (a), and a polynucleotide encoding a variant of the fragment of the 3'-UTR of at least two genes among the genes described in (a); and (3) at least one of at least two copies of the 3'UTR in (a), at least two copies of a fragment of the 3'UTR in (a), at least two copies of a variant of the 3'UTR in (a), and at least two copies of a variant of the fragment of the 3'UTR in (a). 40 . The vector according to claim 36 , further comprising at least one of a promoter, a polynucleotide encoding a 5′-UTR, and a polynucleotide encoding polyA.

41. The carrier according to claim 40, which satisfies at least one of the following (1) to (2): (1) The 5'-UTR comprises: i) at least one of the 5'-UTR of at least one of the genes APOA1, CARD16, ALB, APOC1, EEF1A1, RBP4, GHRL, MPND, ASAH2B, FBX016, FBH1, SRM, NAAA, ACTB, MKNK2, ORM1, GADPH, NDUFAF2, FGFR2, MYCBPAP, CHMP2A, TSG101, PRPF8, NFKB2, NAE1, HDGFL1, GSDMD, FBXW12, FBXW10, FBXL8, and ATG4D, fragments, variants, and variants of fragments thereof; preferably, an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 16 to 46, a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 16 to 46, a variant of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 16 to 46, and a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 16 to 46. A variant of the RNA fragment encoded by at least one of the polynucleotides shown in NO: 16 to 46; Preferably, said variants of RNA encoded by the polynucleotides having a sequence as set forth in at least one of SEQ ID NOs: 16-46, fragments of RNA encoded by the polynucleotides having a sequence as set forth in at least one of SEQ ID NOs: 16-46, and variants of fragments of RNA encoded by the polynucleotides having a sequence as set forth in at least one of SEQ ID NOs: 16-46 are at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the RNA encoded by the polynucleotides having a sequence as set forth in at least one of SEQ ID NOs: 16-46; ii) at least one of the following polynucleotides: an RNA encoded by the polynucleotide having a sequence as set forth in SEQ ID NO: 9, a fragment of the RNA encoded by the polynucleotide having a sequence as set forth in SEQ ID NO: 9, a variant of the RNA encoded by the polynucleotide having a sequence as set forth in SEQ ID NO: 9, and a variant of a fragment of the RNA encoded by the polynucleotide having a sequence as set forth in SEQ ID NO: 9; Preferably, variants of RNA encoded by the polynucleotide having a sequence as set forth in SEQ ID NO:9, fragments of RNA encoded by the polynucleotide having a sequence as set forth in SEQ ID NO:9, and variants of fragments of RNA encoded by the polynucleotide having a sequence as set forth in SEQ ID NO:9 are at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the RNA encoded by the polynucleotide having a sequence as set forth in SEQ ID NO:9; iii) at least two copies of one of the polynucleotides in i) or ii); or iv) at least two polynucleotides according to i); (2) the nucleotides constituting the polyA contain at least 20, at least 40, at least 80, at least 100 or at least 120 A nucleotides; preferably, the nucleotides constituting the polyA contain at least 20, at least 40, at least 80, at least 100 or at least 120 A nucleotides continuously; Preferably, the nucleotides constituting the polyA include one or more nucleotides other than A nucleotides; preferably, the polyA is a truncated polyA; preferably, after a plurality of consecutive A nucleotides, there is a 10 bp non-A linker sequence followed by a plurality of consecutive A nucleotides; preferably, the polyA is 30 consecutive A nucleotides followed by a 10 bp non-A linker sequence and then 70 consecutive A nucleotides; Preferably, the polyA comprises at least one of an RNA encoded by a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 68 to 76 and 11, a fragment of an RNA encoded by a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 68 to 76 and 11, a variant of an RNA encoded by a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 68 to 76 and 11, and a variant of a fragment of an RNA encoded by a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 68 to 76 and 11; preferably, the fragment, variant and variant of the fragment of the RNA encoded by at least one of SEQ ID NOs: 68 to 76 and 11 are the same as the fragment of SEQ ID NOs: 68 to 76 and 11. The RNA encoded by the polynucleotide shown in one of NOs: 68 to 76 and 11 has at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity; more preferably, the polyAy is an RNA encoded by a polynucleotide shown in one of SEQ ID NOs: 68 to 70, 72, 75 and 11; preferably, the polyA is an RNA encoded by a polynucleotide shown in SEQ ID NO:

11.

42. A vector comprising a fifth nucleotide sequence encoding a 5'-UTR, wherein the fifth nucleotide sequence comprises at least one of the following polynucleotides: (a) a polynucleotide encoding the 5'-UTR of at least one of the following genes: APOA1, CARD16, ALB, APOC1, EEF1A1, RBP4, GHRL, MPND, ASAH2B, FBX016, FBH1, SRM, NAAA, ACTB, MKNK2, ORM1, GADPH, NDUFAF2, FGFR2, MYCBPAP, CHMP2A, TSG101, PRPF8, NFKB2, NAE1, HDGFL1, GSDMD, FBXW12, FBXW10, FBXL8, and ATG4D; (b): a polynucleotide encoding a fragment of the 5'-UTR described in (a); (c): a polynucleotide encoding a variant of the 5'-UTR described in (a); and (d): A polynucleotide encoding a variant of the fragment described in (b).

43. The vector according to claim 42, wherein the gene is a human gene; and / or the vector does not comprise a polynucleotide encoding a protein or polypeptide of interest.

44. The vector according to claim 42 or 43, wherein the fifth nucleotide sequence comprises at least one of the following polynucleotides: a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 16 to 46, a fragment of a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 16 to 46, a variant of a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 16 to 46, and a variant of a fragment of a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 16 to 46; Preferably, the fifth nucleotide sequence comprises at least one of the following polynucleotides: variants of the polynucleotides as shown in at least one of SEQ ID NOs: 16 to 46, fragments of the polynucleotides as shown in at least one of SEQ ID NOs: 16 to 46, and variants of the fragments of the polynucleotides as shown in at least one of SEQ ID NOs: 16 to 46, which have a sequence identity of at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% to the polynucleotides as shown in at least one of SEQ ID NOs: 16 to 46.

45. The vector according to any one of claims 42 to 44, wherein the fifth nucleotide sequence comprises at least one of: 5'-UTRs of at least two of the genes described in (a), fragments of 5'-UTRs of at least two of the genes described in (a), variants of 5'-UTRs of at least two of the genes described in (a), and variants of fragments of 5'-UTRs of at least two of the genes described in (a); And / or, the fifth nucleotide sequence comprises: at least two copies of the 5'-UTR in (a), at least two copies of a fragment of the 5'-UTR in (a), at least two copies of a variant of the 5'-UTR in (a), and at least two copies of a variant of the fragment of the 5'-UTR in (a).

46. ​​A vector comprising a polynucleotide sequence encoding a 5'-UTR; wherein the polynucleotide sequence encoding the 5'-UTR comprises at least one of the following polynucleotides: a polynucleotide having a sequence as shown in SEQ ID NO: 9, a fragment of the polynucleotide having a sequence as shown in SEQ ID NO: 9, a variant of the polynucleotide having a sequence as shown in SEQ ID NO: 9, and a variant of a fragment of the polynucleotide having a sequence as shown in SEQ ID NO: 9; Preferably, variants of the polynucleotide with a sequence as set forth in SEQ ID NO: 9, fragments of the polynucleotide with a sequence as set forth in SEQ ID NO: 9, and variants of fragments of the polynucleotide with a sequence as set forth in SEQ ID NO: 9 have at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the polynucleotide with a sequence as set forth in SEQ ID NO:

9.

47. The vector according to any one of claims 42 to 46, further comprising at least one of a promoter, a polynucleotide encoding a 3'-UTR, and a polynucleotide encoding polyA; Preferably, (1) the 3'-UTR comprises: i) at least one of a 3'-UTR derived from at least one of the genes APOA1, IE1, COP1, MYSM1, ASAH2B, NDUFAF2, APOD, HBB, TF, TMSB4X, CPAMD8, VIM, HDGFL1, TTR, SRM, HBA1, PRPF8, LMBRD1, IFNA1, CCDC146, GHRL, and GH1, a fragment thereof, a variant thereof, and a variant of a fragment thereof; Preferably, an RNA encoded by a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 47 to 67 and 10, a fragment of an RNA encoded by a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 47 to 67 and 10, a variant of an RNA encoded by a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 47 to 67 and 10, and a variant of a fragment of an RNA encoded by a polynucleotide having a sequence as shown in at least one of SEQ ID NOs: 47 to 67 and 10; Preferably, variants of RNA encoded by polynucleotides having sequences as set forth in at least one of SEQ ID NOs: 47-67 and 10, fragments of RNA encoded by polynucleotides having sequences as set forth in at least one of SEQ ID NOs: 47-67 and 10, and variants of fragments of RNA encoded by polynucleotides having sequences as set forth in at least one of SEQ ID NOs: 47-67 and 10 are at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the RNA encoded by polynucleotides having sequences as set forth in at least one of SEQ ID NOs: 47-67 and 10; ii) at least two copies of one of the polynucleotides described in i); or iii) at least two polynucleotides according to i); (2) The nucleotides constituting the polyA include at least 20, at least 40, at least 80, at least 100 or at least 120 A nucleotides; preferably, the nucleotides constituting the polyA include at least 20, at least 40, at least 80, at least 100 or at least 120 A nucleotides consecutively. or at least 120 A nucleotides; Preferably, the nucleotides constituting the polyA include one or more nucleotides other than A nucleotides; preferably, the polyA is a truncated polyA; preferably, after a plurality of consecutive A nucleotides, there is a 10 bp non-A linker sequence followed by a plurality of consecutive A nucleotides; preferably, the polyA is 30 consecutive A nucleotides followed by a 10 bp non-A linker sequence and then 70 consecutive A nucleotides; Preferably, the polyA comprises at least one of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 68-76 and 11, a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 68-76 and 11, a variant of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 68-76 and 11, and a variant comprising a fragment of an RNA encoded by a polynucleotide having a sequence as set forth in at least one of SEQ ID NOs: 68-76 and 11; preferably, the fragments, variants, and variants of the fragments of the RNA encoded by at least one of SEQ ID NOs: 68-76 and 11 are at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the RNA encoded by a polynucleotide having a sequence as set forth in one of SEQ ID NOs: 68-76 and 11; More preferably, the polyA is an RNA encoded by a polynucleotide having a sequence as one of SEQ ID NOs: 68 to 70, 72, 75 and 11; preferably, the polyA is an RNA encoded by a polynucleotide having a sequence as SEQ ID NO:

11. A host cell comprising the vector according to any one of claims 35 to 47.

49. A lipid nanoparticle comprising the artificial RNA molecule of claim 17 or 18, or the RNA molecule of any one of claims 19 to 33; Preferably, the lipid nanoparticles further contain one or more of ionizable cationic lipids, helper lipids, structural lipids and PEG-lipids; The ionizable cationic lipid is selected from one or more of Dlin-MC3-DMA, Dlin-KC2-DMA, DODMA, c12-200 and DlinDMA; and / or the auxiliary lipid is selected from one or more of DSPC, DOPE, DOPC and DOPS; and / or the structural lipid is selected from at least one of cholesterol, cholesterol ester, steroid hormones, steroid vitamins and bile acids; and / or the PEG-lipid is selected from PEG-DMG or PEG-DSPE; preferably, the average molecular weight of PEG is about 2000 Daltons to 5000 Daltons; further preferably, the average molecular weight of PEG is about 2000 Daltons or 5000 Daltons.

50. A pharmaceutical composition comprising the artificial RNA molecule of claim 17 or 18, the RNA molecule of any one of claims 19 to 33, the DNA of claim 34, the vector of claims 35 to 47, the host cell of claim 48 or the lipid nanoparticle of claim 49, and a pharmaceutically acceptable carrier.

51. Use of the artificial RNA molecule according to claim 17 or 18, the RNA molecule according to any one of claims 19 to 33, the DNA according to claim 34, the vector according to claims 35 to 47, the host cell according to claim 48, the lipid nanoparticle according to claim 49, or the pharmaceutical composition according to claim 50 in the preparation of a medicament; Preferably, the medicament is used for gene therapy, gene vaccination, protein replacement therapy, antisense therapy or treatment by interfering RNA.

52. Use of the artificial RNA molecule of claim 17 or 18, the RNA molecule of any one of claims 19 to 33, the DNA of claim 34, the vector of claims 35 to 47, the host cell of claim 48, the lipid nanoparticle of claim 49, or the pharmaceutical composition of claim 50 in the preparation of a medicament for nucleic acid transfer; Preferably, the nucleic acid is RNA, messenger RNA (mRNA), antisense oligonucleotide, DNA, plasmid, ribosomal RNA (rRNA), microRNA (miRNA), transfer RNA (tRNA), small inhibitory RNA (siRNA) and small nuclear RNA (snRNA). Preferably, the drug is used for gene therapy, gene vaccination or protein replacement therapy; Preferably, the drug is used for the treatment and / or prevention of a disease; preferably, the drug is a vaccine; more preferably, the drug is a vaccine for preventing novel coronavirus infection; Preferably, the disease is selected from the group consisting of rare diseases, infectious diseases, cancer, genetic diseases, autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular diseases, renal vascular diseases, and metabolic diseases; Preferably, the cancer includes one or more of lung cancer, stomach cancer, liver cancer, esophageal cancer, colon cancer, pancreatic cancer, brain cancer, lymphoma, blood cancer or prostate cancer; the genetic disease includes one or more of hemophilia, thalassemia and Gaucher's disease; The drug is a nucleic acid drug, wherein the nucleic acid comprises at least one of the following: RNA, messenger RNA (mRNA), DNA, mitochondria, ribosomal RNA (rRNA), single-stranded guide RNA (sgRNA) and cas9 mRNA.

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