Nucleic acid molecules, fusion proteins, and mRNA vaccines that enhance antigen presentation by mobilizing ligands

Abstract

The present invention belongs to the field of biomedicine and relates to a method for designing a vaccine with enhanced antigen presentation effect, which is mainly applied to the design and preparation of structural sequences of nucleic acid, protein, and polypeptide vaccines. In the present invention, a target antigen and a ligand, such as a polypeptide or protein domain having the function of binding or recruiting E3 ubiquitin ligase, are both encoded in the same nucleic acid sequence. After the nucleic acid molecule enters the cell, fusion expression of the antigen protein and the E3 ubiquitin ligase ligand is achieved, which promotes degradation of the antigen protein via the proteasome pathway, increases the number and abundance of antigen peptides containing the antigen epitope, forms more peptide-MHC (p-MHC) complexes, and is thus presented on the cell surface, enhancing the subsequent immune response and exerting efficient tumor immunotherapy. The nucleic acid, protein, and polypeptide vaccines provided by the present invention have efficient antigen presentation effect and strong immunogenicity, and have good prospects for clinical application.

Description

[Technical Field]

[0001] The present invention relates to the field of biomedicine, and specifically to nucleic acid molecules, fusion proteins, vaccines and uses thereof, particularly mRNA vaccines, that can enhance antigen presentation effects. [Background technology]

[0002] mRNA vaccines are a completely new class of vaccines, boasting advantages such as versatility, high efficacy, rapid construction, and ease of scalable production, demonstrating significant clinical value and future potential. The COVID-19 pandemic has significantly accelerated the development of mRNA vaccines, and new nucleic acid vaccines (mRNA or DNA) offer remarkable time-to-effect and advantages as emergency vaccines. Indeed, using mRNA vaccines to induce anti-tumor immunity in the body has shown great potential, with numerous research results currently entering clinical trials. mRNA tumor vaccines are a tumor treatment method that introduces tumor antigens into the patient's body in the form of mRNA, stimulating the patient's own T cells with the tumor antigens and activating the patient's own specific cellular immunity. While no mRNA tumor vaccines are currently commercially available, 19 clinical trials have been registered worldwide, with the fastest reaching phase II clinical trials. Nevertheless, conventional mRNA tumor vaccines still need to be optimized and improved in terms of enhancing antigen presentation efficiency and T cell immune responses, particularly in terms of mRNA sequence design and synthesis, translation efficiency and stability, antigen presentation efficiency, and T cell immune response strength, in order to further improve anti-tumor immunotherapeutic activity and accelerate clinical translation.

[0003] mRNA tumor vaccines primarily use the translational machinery of antigen-presenting cells (APCs) to translate mRNA into target antigen proteins, some of which are then ubiquitinated and degraded via the proteasome degradation pathway to generate antigen peptides containing numerous antigen epitopes. These antigen peptides are transported to the endoplasmic reticulum (ER) by the transporter associated with antigen processing (TAP), modified by N-terminal aminopeptidases ERAP1 and ERAP2, then loaded onto MHC class I proteins, transported to the cell surface, and recognized by antigen-specific CD8+ T cells, presenting the antigen epitope information to CD8+ T cells. Alternatively, secreted antigen proteins can be absorbed by professional antigen-presenting cells (APCs) located in tissues or lymph nodes, where they are degraded into large amounts of antigenic peptides via the lysosomal degradation pathway. These peptides are then substituted for CLIP (associated invariant peptide of MHC class II molecules) in the antigen-binding groove of MHC class II molecules, forming stable antigenic peptide-MHC class II complexes. These complexes are then transported to the cell membrane and used for recognition by CD4+ T cells, and can further present antigen epitope information to CD8+ T cells. APCs can also present internalized exogenous antigens via the MHC-I pathway on their cell surface, a process known as "cross-presentation." The cross-presentation process is crucial for the induction of cytotoxic T cell (CTL) and B cell responses by mRNA vaccines.

[0004] To effectively induce adaptive immune responses, antigens translated within antigen-presenting cells (especially DC cells) must be presented by MHC class I and MHC class II molecules. Therefore, the antigen presentation process is a critical factor affecting the immune efficacy of mRNA vaccines. All endogenous proteins are subject to MHC class I antigen presentation, while only a portion of intracellular antigens are presented by MHC class II molecules. However, the MHC class I antigen presentation process is usually inefficient, resulting in only one in ten thousand antigen peptides being presented even in response to high-affinity MHC class I ligands. Therefore, achieving efficient presentation of MHC class I and MHC class II antigens is of great significance for the development of mRNA vaccines. Currently, common strategies include co-delivering MHC molecules with antigens or targeting antigens to MHC molecules to enhance presentation efficiency. It has been reported that the combination of DNA molecules encoding HPV-16 E7 with DNA vaccines encoding heterologous MHC molecules can enhance the cross-presentation mechanism and significantly enhance E7-specific immune responses and antitumor effects.

[0005] The sequential steps of antigen processing and presentation affect the amount of epitope / MHC complexes presented on the cell surface. In addition to enhancing antigen presentation using MHC class I and MHC class II molecules, increasing the number of antigen peptide molecules containing degraded antigen epitopes is also an important and effective method for enhancing antigen presentation. Many MHC class I antigen peptides are typically produced in a proteasome-dependent manner. Strategies to increase antigen proteasome-dependent degradation have been shown to improve MHC class I presentation of antigens, thereby enhancing E7-specific CD8+ T cell immune responses and significantly improving vaccine efficacy. Therefore, targeting the proteasome to increase antigen proteasomal degradation may provide a new approach for nucleic acid drug development. Summary of the Invention

[0006] To solve the problems in the prior art, the present invention provides a vaccine design strategy for enhancing antigen presentation effect. This strategy is applicable to the structural sequence design and preparation of nucleic acid, protein, and polypeptide vaccines, providing a new approach to the immune effect of vaccines and new ideas for the development of new therapeutic methods.

[0007] The present invention is based on the following research conducted by the present inventors. Using OVA as the model antigen protein, we selected and coupled three peptide ligands for the E3 ligases MDM2, VHL, and Keap1, including PMI, VHL Ligand (hereinafter abbreviated as VHLL), and a Keap1 binding element (hereinafter abbreviated as Keap1 B). Based on the structural characteristics of the protein complex formed by MDM2 binding to the tumor suppressor protein P53, we selected the 17-26 helix residues of P53 as the E3 ligase ligand (hereinafter abbreviated as P53B). We further demonstrated that the E3 ubiquitin ligase binding or recruitment ligand enhances intracellular proteasome degradation of the OVA antigen protein, thereby improving the tumor suppressive and antitumor activities of mRNA tumor vaccines. The technical solution of the present invention can also be applied to the design of other mRNA tumor vaccines. Studies in various tumor models have demonstrated that it exerts antigen presentation-enhancing effects and activates specific T cells to kill tumor cells, resulting in effective tumor immunotherapy. The technical means of the present invention can also be applied to the design of prophylactic mRNA vaccines to obtain enhanced immunogenicity.

[0008] In one aspect, the invention provides a nucleic acid molecule.

[0009] The open reading frame of the nucleic acid molecule comprises at least one antigen element and at least one E3 ligand element, the E3 ligand element being a binding or recruiting ligand for an E3 ubiquitin ligase.

[0010] The E3 ubiquitin ligase in the nucleic acid molecule is one or more selected from Von Hippel-Lindau (VHL), MDM2, CRBN, IAPs, RNF, β-TrCP, DCAF, Keap1, or truncations or extensions thereof.

[0011] Furthermore, the E3 ubiquitin ligase is selected from the group consisting of RING-type, HECT-type, and RBR-type.

[0012] Furthermore, the E3 ubiquitin ligase in the nucleic acid molecule is preferably one or more selected from Von Hippel-Lindau (VHL), MDM2, β-TrCP, Keap1, or truncations or extensions thereof.

[0013] Furthermore, the E3 ubiquitin ligase binding or recruitment ligand binds to one or more E3 ubiquitin ligases.

[0014] Furthermore, the amino acid sequence corresponding to the binding or recruitment ligand of Keap1 is LDPETGEYL or a sequence having more than 60% identity thereto.

[0015] Furthermore, the amino acid sequence corresponding to the binding or recruiting ligand of said β-TrCP is DRHDSGLDSM or a sequence having greater than 60% identity thereto.

[0016] Furthermore, the amino acid sequence corresponding to the VHL binding or recruiting ligand is at least one of LAP(OH)YI and ALAPYIP or a sequence having greater than 60% identity thereto.

[0017] Furthermore, the amino acid sequence corresponding to the MDM2 binding or recruiting ligand is one or more of ETFSDLWKLL, TSFAEYWNLLSP, LTFEHYWAQLTS, TNWYANLEKLLR, TAWYANFEKLLR, DWWPLAFEALLR, CNCKAPETALCARRCQQH and CNCKAPETFLCYWRCLQH, or a sequence having greater than 60% identity thereto.

[0018] Furthermore, in the nucleic acid molecule, the antigen element and the E3 ligand element are linked in any one of the following forms: E3 ligand element-antigen element, antigen element-E3 ligand element, E3 ligand element-antigen element-E3 ligand element, or antigen element-E3 ligand element-antigen element.

[0019] Furthermore, the antigen element and the E3 ligand element are linked by a linker. Here, the amino acid sequence of the linker is GGGGS, (GGGGS)3, (GGGGS)6, (GGS) 10 and (GSG) 10 It is one or more of the following.

[0020] Furthermore, the antigenic element is a naturally occurring antigenic protein, antigenic polypeptide or a truncation thereof.

[0021] Furthermore, the antigenic element comprises at least one antigenic epitope.

[0022] Further, the antigenic element comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 antigenic epitopes.

[0023] Furthermore, the antigenic epitope is a T cell antigenic determinant or a T cell antigen epitope.

[0024] The antigenic epitope is derived from an antigenic epitope peptide of a tumor, an antigenic epitope peptide of an autoimmune disease, or an antigenic epitope peptide of a pathogenic microorganism.

[0025] The tumor antigen or antigen epitope peptide is selected from antigens caused by genetic mutations, and / or tissue-specific differentiation antigens, and / or overexpressed antigens, and / or cancer-testis antigens, and / or universal antigens, and / or antigens derived from oncogenic viruses.

[0026] Furthermore, the antigen resulting from the gene mutation is one or more selected from p53, ras, β-catenin, CDK4, CDC27, and α-actinin-4.

[0027] Furthermore, the tissue-specific differentiation antigen is one or more selected from tyrosinase, TRP1 / gp75, TRP2, gp100, Melan-A / MART1, ganglioside, and PSMA.

[0028] Furthermore, the overexpressed antigen is one or more selected from HER2, WT1, EphA3, EGFR, and CD20.

[0029] Furthermore, the cancer / testis antigen is one or more antigens selected from MAGE, BAGE, GAGE, and NY-ESO-1.

[0030] Furthermore, the universal antigen is one or more selected from the group consisting of telomerase and survivin.

[0031] Furthermore, the oncogenic virus-derived antigen is one or more selected from EBV, HPV, HBV, human herpes virus, and Merkel cell polyomavirus.

[0032] In a specific embodiment, the nucleic acid molecule further comprises a signal peptide coding region.

[0033] The nucleic acid molecule further comprises a promoter, a 5'-end untranslated region, a 3'-end untranslated region, and polyA.

[0034] The promoter is a T7 or SP6 promoter.

[0035] Furthermore, in the nucleic acid molecule, the nucleic acid is at least one selected from DNA, ASO, siRNA, miRNA, mRNA, and aptamer.

[0036] Furthermore, in the nucleic acid molecule, the nucleic acid is mRNA.

[0037] In another aspect, the present invention provides a nucleic acid vaccine comprising the nucleic acid molecule described above and, optionally, a pharmaceutically acceptable adjuvant or component.

[0038] Furthermore, the nucleic acid vaccine is an mRNA vaccine, the auxiliary component is a nanocarrier that carries the mRNA, and the auxiliary agent is at least one selected from a buffer medium for injection, a lyoprotectant, and a cryoprotectant.

[0039] Furthermore, the nanocarrier is at least one selected from a liposome, a nanoparticle, a microsphere, and a lipid nanocarrier.

[0040] Furthermore, the nanocarrier The lipid composition is prepared using at least one lipid material selected from the group consisting of DOTAP, DOTMA, DOTIM, DDA, DC-Chol, CCS, diC14-amidine, DOTPA, DOSPA, DTAB, TTAB, CTAB, DORI, DORIE and its derivatives, DPRIE, DSRIE, DMRIE, DOGS, DOSC, LPLL, DODMA, DDAB, Dlin-MC3-DMA, CKK-E12, C12-200, DSPC, DMG-PEG, DOPE, phosphatidylethanolamine, phosphatidylcholine, and cholesterol.

[0041] Furthermore, in the mRNA vaccine, the mass ratio of the lipid material to the mRNA is (0.5-50):1, preferably (2-10):1.

[0042] Furthermore, the mRNA vaccine is formed by self-assembly of the mRNA and lipid materials using a microfluidic device; or The mRNA vaccine is formed by incubating the nanocarrier with mRNA.

[0043] In another aspect, the present invention provides a method for preparing the nucleic acid vaccine.

[0044] In another aspect, the invention provides a protein encoded by any one of the above described nucleic acid molecules.

[0045] In another aspect, the present invention provides a protein or polypeptide vaccine, wherein the protein is an antigenic component.

[0046] Furthermore, the protein or polypeptide vaccine may further comprise a pharmaceutically acceptable adjuvant or component, which is as defined above for any one of the nucleic acid molecules.

[0047] Additionally, the protein or polypeptide vaccine further comprises an immunoadjuvant.

[0048] Furthermore, the immunoadjuvant is one or more selected from Freund's incomplete adjuvant, Freund's complete adjuvant, aluminum hydroxide adjuvant, aluminum phosphate adjuvant, emulsion adjuvant, liposome adjuvant, and microbial adjuvant.

[0049] Furthermore, the present invention provides a vector carrying the above-mentioned nucleic acid molecule.

[0050] Furthermore, the vector may be a eukaryotic vector or a prokaryotic vector.

[0051] Furthermore, the vector is one or more selected from a plasmid vector, an adenovirus vector, a lentivirus vector, and an adeno-associated virus vector.

[0052] In another aspect, the present invention provides a vector vaccine comprising an active ingredient, said active ingredient being obtained by introducing a nucleic acid molecule as defined above into a vector as defined above.

[0053] In another aspect, the present invention provides a pharmaceutical composition comprising the nucleic acid molecule, the nucleic acid vaccine, the mRNA vaccine prepared by the method, the protein, the protein or polypeptide vaccine, or the vector vaccine, and a pharmaceutically acceptable adjuvant.

[0054] In another aspect, the present invention provides use of the above-mentioned nucleic acid molecule, the above-mentioned nucleic acid vaccine, the above-mentioned mRNA vaccine prepared by the above-mentioned method, the above-mentioned protein, the above-mentioned protein or polypeptide vaccine, or the above-mentioned vector vaccine, or the above-mentioned pharmaceutical composition in the preparation of a medicament for preventing or treating an associated disease.

[0055] Furthermore, the disease is a tumor.

[0056] The present invention has the following beneficial effects: The present invention provides a method for designing vaccines that enhance antigen presentation effect, which can be applied to the sequence design and preparation of nucleic acid, protein, and polypeptide vaccines.

[0057] In the present invention, by encoding both a target antigen and a ligand, such as a polypeptide or protein domain, capable of binding and recruiting E3 ubiquitin ligase in the same nucleic acid sequence, fusion expression of the antigen protein and the E3 ubiquitin ligase ligand is achieved after the nucleic acid molecule enters the cell, promoting the degradation of the antigen protein via the proteasome pathway and increasing the number and abundance of antigen peptides containing the antigen epitope, forming more peptide-MHC (p-MHC) complexes and presenting them on the cell surface, thereby enhancing the subsequent immune response and achieving efficient tumor immunotherapy.

[0058] The proteins or polypeptides encoded by the proteins, polypeptides, or nucleic acid molecules of the present invention depend on the ubiquitin-proteasome proteolytic pathway for antigen presentation in cells. By modifying and constructing them with different E3 ligase ligand sequences, screening for E3 ubiquitin ligase ligands, and efficiently recruiting E3 ligase, the proteins or polypeptides of the present invention can be ubiquitinated and proteolyzed, thereby improving the antigen presentation of vaccine immunogens. Therefore, the proteins, polypeptides, or nucleic acid vaccines provided by the present invention have efficient antigen presentation, high immunogenicity, and good prospects for clinical application. [Brief explanation of the drawings]

[0059] [Figure 1] FIG. 1 shows particle size and PDI results of mRNA-LNP in Example 3. [Figure 2] FIG. 10 shows the potential results of mRNA-LNP in Example 3. [Figure 3] 1 is a transmission electron microscope image of OVA-P53B mRNA lipid nanoparticles in Example 3. [Figure 4] 4A and 4B are diagrams showing the detection results of activation of lymph node T cells by mRNA-lipid nanoparticles in Example 4. Fig. 4A shows the detection data of early activated T cells, and Fig. 4B shows the detection data of late activated T cells. [Figure 5]5A and 5B are diagrams showing the detection results of mRNA-lipid nanoparticles on splenic T cell activation in Example 4. Fig. 5A shows the detection data for early activated T cells, and Fig. 5B shows the detection data for late activated T cells. [Figure 6] FIG. 1 is a schematic diagram of the immunization plan in Example 4. [Figure 7] E. Tumor growth curve of G7-OVA tumor-bearing mice. [Figure 8] Representative photographs of tumors from each treatment group are shown. [Figure 9] E. Weight-time curve of G7-OVA tumor-bearing mice. [Figure 10] FIG. 10 shows the results of detecting antigen presentation mediated in mice by each mRNA-lipid nanoparticle in Example 5. [Figure 11] These are diagrams showing the detection results of specific CTLs mediated in mice by each mRNA lipid nanoparticle in Example 5. Figure 11A shows the detection results of specific CTLs mediated in the lymph nodes of mice by mRNA lipid nanoparticles, Figure 11B shows the detection results of specific CTLs mediated in the spleen of mice by mRNA lipid nanoparticles, and Figure 11C shows the detection results of specific CTLs mediated in tumor tissue of mice by mRNA lipid nanoparticles. [Figure 12] 1 shows the detection results of the mRNA-lipid nanoparticles in Example 6 mediating the production of CTLs in mice. [Figure 13] FIG. 1 shows the detection data of ALT, AST, TP, CRE, LDH, and UREA in mouse serum in Example 7. DETAILED DESCRIPTION OF THE INVENTION

[0060] The following examples of the present invention are described in detail. The following examples are illustrative and are intended to illustrate the present invention only, but not to limit the present invention.

[0061] In the specification, the terms "comprise" or "contain" are open-ended expressions that include the content specified in the present invention but do not exclude other content.

[0062] In the specification, the terms "optionally," "optional," or "optional" generally mean that the subsequently described event or circumstance may, but does not necessarily, occur, and that the description includes both cases where the event or circumstance occurs and cases where it does not occur.

[0063] In the specification, "(XXXX) n " means that n XXXX are bonded together, where X represents an amino acid. For example, "(GGGGS)3" represents GGGGSGGGSGGGGGS.

[0064] As used herein, the term "fragment" refers to a target protein or polypeptide, a target protein or polypeptide having an N-terminal (N-terminus) or C-terminal (C-terminus) truncation and / or an internal deletion.

[0065] As used herein, the terms "identity," "homology," or "similarity" are used to describe an amino acid sequence or a nucleic acid sequence compared to a reference sequence, and the percentage of identical amino acids or nucleotides between two amino acid or nucleic acid sequences is determined by conventional methods (e.g., Ausubel et al. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); the ALIGN program (Dayhoff (1978), Atlas of Protein Sequence and Structure 5:Suppl. 3 (National Biomedical Research Institute)) Foundation, Washington, DC). There are many algorithms for comparing sequences and determining sequence identity, including the compare identity algorithm of Needleman et al. (1970) J. Mol. Biol. 48:443; the local identity algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the similarity search algorithm of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85:2444; and the Smith-Waterman algorithm (Meth. Mol. Biol. 70:173-187 (1997)). and the BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J. Mol. Biol. 215:403-410). Computer programs using these algorithms are also available, including, but not limited to, ALIGN or Megalign (DNASTAR) software, WU-BLAST-2 (Altschul et al., Meth. Enzym., 266:460-480 (1996)); GAP, BESTFIT, and BLAST (Altschul et al., op. cit.); FASTA and TFASTA (Genetics Computing Group (GCG), version 8, Madison, Wisconsin, USA), and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0066] As used herein, the term "vector" generally refers to a vector into which a nucleic acid molecule that self-replicates in a suitable host can be inserted and which transfers the inserted nucleic acid molecule to a host cell and / or between host cells. The vector may include a vector primarily for inserting DNA or RNA into a cell, a vector primarily for replicating DNA or RNA, and a vector used for transcriptional and / or translational expression of DNA or RNA. The vector further includes various vectors having the above functions. The vector may be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Generally, the vector can produce a desired expression product by culturing a suitable host cell containing the vector.

[0067] In the specification, the term "pharmaceutical composition" generally refers to a unit dosage form and can be prepared by any of the methods well known in the pharmaceutical field. All methods include the step of combining an active ingredient with a carrier that constitutes one or more accessory ingredients. Typically, the composition is prepared by uniformly and intimately combining the active compound with a liquid carrier, a finely divided solid carrier, or both.

[0068] In the specification, the term "pharmaceutically acceptable excipient" can include any solvent, solid excipient, diluent, or other liquid excipient, etc., suitable for a specific target dosage form. The use of any conventional excipient is also contemplated by the present invention to the extent that it is incompatible with the compounds of the present invention, e.g., except for the occurrence of any adverse biological effects or interactions with any other components of the pharmaceutically acceptable composition in a deleterious manner.

[0069] As used herein, the term "administration" refers to the introduction of a predetermined amount of a substance into a patient by an appropriate means. The nucleic acid molecules, nucleic acid vaccines, vectors, vector vaccines, polypeptides, antibodies or antigen-binding fragments, recombinant proteins, multispecific antibodies, protein or polypeptide vaccines, conjugates, or pharmaceutical compositions of the present invention can be administered by any common route that delivers them to the desired tissue. Administration methods include intraperitoneal, intravenous, intramuscular, and subcutaneous injections, but the present invention is not limited to these exemplary administration methods. Preferably, the compositions of the present invention are administered by intravenous or subcutaneous injection.

[0070] As used herein, "E3 ubiquitin ligase" is involved in various physiological processes within cells by controlling the ubiquitination of regulatory proteins. All E3s have the ability to bind to target proteins and specific E2s. Known E3 ubiquitin ligases mainly include the HECT domain family, the RING domain family, and the U-box protein family. The HECT domain primarily exerts its action by forming a thioester bond with ubiquitin, which is essential for catalytic activity. The RING domain provides a docking site for both the E2 and the substrate, facilitating the transfer of ubiquitin from the E2 to the substrate.

[0071] As used herein, the term "E3 ubiquitin ligase binding or recruitment ligand" has the same meaning as "E3 ligand," "E3 ligand element," and "E3 ligase ligand," and refers to a naturally occurring or artificial polypeptide, protein, or truncation or extension thereof, or compound that is capable of binding to or recruiting an E3 ubiquitin ligase.

[0072] Because most peptides presented by MHC class I molecules are derived from the degradation of antigen proteins, strategies to increase antigen proteasomal degradation in vivo have been demonstrated to improve MHC class I presentation of antigens. In the present invention, for the first time, an antigen element is linked to a ligand for recruiting an E3 ubiquitin ligase to form a fusion protein. This fusion protein ubiquitinates itself via the ubiquitin-proteasome system (UPS) in vivo, causing its own ubiquitin degradation. This increases the amount of ubiquitination degradation of the antigen protein, improving the availability of effectively available antigens, enhancing the direct MHC-I presentation of target proteins or target antigenic determinants, and further enhancing the immune response induced by vaccines.

[0073] The sequence design of mRNA drugs is crucial for their ultimate therapeutic efficacy. Although the coding region of mRNA is not as flexible as the non-coding region, the excellent adaptability of mRNA molecules allows for minor functional modifications to the coding region during the design and synthesis process to enhance therapeutic efficacy. When mRNA vaccines function, they participate in a crucial step in the immune process: antigen presentation of degraded peptides. Most peptides presented by MHC class I molecules are derived from the degradation of antigen proteins, and the ubiquitin-proteasome degradation mechanism achieves targeted degradation of target proteins. Therefore, the present invention specifically increases the proteasome pathway degradation of target antigen proteins, thereby increasing the number of candidate peptides presented by MHC class I. Specifically, a polypeptide-based E3 ligase ligand is fused to an mRNA molecule, which recruits E3 ligase to induce rapid and efficient ubiquitination of the target antigen protein, and promotes the degradation of the target antigen protein via the proteasome pathway, thereby increasing the presence of pMHC complexes, thereby enhancing the antigen presentation effect of the mRNA drug, initiating an efficient immune response, and achieving effective tumor immunotherapy.

[0074] In the present invention, technical means that can be used to prepare a vaccine having the following components have been designed and verified. The open reading frame of the nucleic acid molecule comprises at least one antigen element and at least one E3 ligand element, wherein the E3 ligand element is a binding or recruitment ligand for an E3 ubiquitin ligase.

[0075] The open reading frame encodes the following structural elements: Antigen element-(linker)-ligand element recruited by E3 ubiquitin ligase

[0076] The recruitment ligand element of the E3 ubiquitin ligase of the present invention is not a ligand that binds to a target protein in cells directly or via a linker element, but rather an antigen element. Experiments have shown that the above structure of the present invention can effectively increase the degradation rate of antigen proteins, thereby improving the availability of effectively available antigens, enhancing direct presentation of target proteins by MHC class I, and further enhancing the immune response induced by vaccines.

[0077] The open reading frame of the nucleic acid molecule of the present invention comprises at least the following structure: E3 ligand-antigen element, antigen element-E3 ligand, E3 ligand-antigen element-E3 ligand or antigen element-E3 ligand-antigen element

[0078] When a linker element is present between each element, the structure is as follows: E3 ligand-linker-antigen element, antigen element-linker-antigen element-linker-antigen element-linker-E3 ligand, E3 ligand-linker-antigen element-linker-E3 ligand, or antigen element-linker-E3 ligand-linker-antigen element.

[0079] The linker element is GGGGS, (GGGGS)3, (GGGGS)6, (GGS) 10 and (GSG) 10Contains one or more of the sequences

[0080] In an embodiment of the present invention, the amino acid sequence of the linker is GGGGS, (GGGGS)3, (GGGGS)6, (GGS) 10 and (GSG) 10 At least one of the following is true.

[0081] Amino acid sequence of GGGGS: GGGGS (SEQ ID NO: 1) Amino acid sequence of (GGGGS)3: GGGGSGGGSGGGGS (SEQ ID NO: 2) Amino acid sequence of (GGGGS)6: GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 3) (GGS) 10 Amino acid sequence: GGSGGSGGSGGSGGSGGSGGSGGSGGSGGS (SEQ ID NO: 4) (GSG) 10 Amino acid sequence: GSGGSGGSGGSGGSGGSGGSGGSGGSGGSG (SEQ ID NO: 5)

[0082] The vaccine design strategy for enhancing antigen presentation efficiency according to the present invention can be applied to the design and preparation of structural sequences of nucleic acid, protein and polypeptide vaccines.

[0083] The recruitment ligand element of the E3 ubiquitin ligase refers to any polypeptide, protein, and its nucleic acid coding sequence that can bind to the E3 ubiquitin ligase.

[0084] The E3 ubiquitin ligase binding or recruitment ligand can bind to one or more E3 ubiquitin ligases. Common E3 ubiquitin ligase molecules include VHL, MDM2, CRBN, and IAPs. Common E3 ubiquitin ligase molecule ligands include VHL ligand (VHLL) (amino acid sequence: ALAPYIP) and Keap1 binding element (amino acid sequence: LDPETGEYI).

[0085] Further classification and sequence information is provided in Table 1 below. [Table 1]

[0086] In a specific embodiment, the amino acid sequences of Keap1, β-TrCP, VHL, and MDM2 in Table 1 above may have an identity of more than 60%. In another specific embodiment, the amino acid sequences of Keap1, β-TrCP, VHL, and MDM2 in Table 1 above may have an identity of more than 70% thereto. In another specific embodiment, the amino acid sequences of Keap1, β-TrCP, VHL, and MDM2 in Table 1 above may have an identity of more than 80%. In another specific embodiment, the amino acid sequences of Keap1, β-TrCP, VHL, and MDM2 in Table 1 above may have more than 90% identity thereto. In another specific embodiment, the amino acid sequences of Keap1, β-TrCP, VHL, and MDM2 in Table 1 above may be sequences having more than 95% identity thereto.

[0087] In a specific embodiment, a signal peptide coding sequence is further attached to the N-terminus of the open reading frame. Generally, an MHC class I signaling signal sequence is used, but the signal peptide of the antigen-derived protein itself or other signal peptides commonly used in the art may also be used.

[0088] Here, the antigen element is at least one antigen epitope, known or unknown.

[0089] The antigenic epitope may be an antigenic epitope of any origin, for example, an antigenic epitope derived from a tumor, an antigenic epitope derived from an autoimmune disease, or an antigenic epitope derived from an infectious disease.

[0090] The antigenic element is an antigenic polypeptide or protein, known or unknown in the art, that contains at least one antigenic epitope.

[0091] In one embodiment of the present invention, an antigen protein is used as an antigen element as it is. The number of antigen proteins may be one.

[0092] In another embodiment, a polypeptide molecule obtained by de novo fusion with one or more antigenic epitope peptides is used as the antigenic element.

[0093] Here, the antigen epitope may be an antigen epitope of any origin, for example, an antigen epitope derived from a tumor, an antigen epitope derived from an autoimmune disease, or an antigen epitope derived from an infectious disease.

[0094] The antigen or antigenic epitope peptide is derived from a tumor antigen, a viral pathogen antigen, or any combination thereof.

[0095] The tumor antigen may be any tumor-specific antigen, tumor-associated antigen, or tumor neoantigen.

[0096] The tumors include breast cancer, ovarian cancer, breast cancer, testicular cancer, pancreatic cancer, liver cancer, colon cancer, colorectal cancer, thyroid cancer, lung cancer, prostate cancer, kidney cancer, melanoma, squamous cell carcinoma, gastrointestinal adenocarcinoma, chronic myeloid leukemia, acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, promyelocytic leukemia, multiple myeloma, B-cell lymphoma, bladder cancer, head and neck cancer, esophageal cancer, brain tumor, pharyngeal cancer, tongue cancer, synovial cell carcinoma, neuroblastoma, uterine cancer, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endothelial cell sarcoma, lymphoma, including, but not limited to, pancreatic cancer, synovial tumor, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, basal cell carcinoma, epidermoid carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, liver tumor, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, nephroblastoma, uterine cancer, cervical cancer, small cell lung cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, brain or intraspinal tumor, glioma, or retinoblastoma.

[0097] The one or more antigenic epitopes are one, two or more epitopes of one or more tumor-associated antigens. The tumor-associated antigens include kallikrein 4, papillomavirus binding factor (PBF), preferentially expressed melanoma antigen (PRAME), Wilms' tumor-1 (WT1), hydroxysteroid dehydrogenase-like 1 (HSDL1), mesothelin, cancer-testis antigen (NY-ESO-1), carcinoembryonic antigen (CEA), p53, human epidermal growth factor receptor 2 / neuroreceptor tyrosine kinase (Her2 / Neu), cancer-associated epithelial cell adhesion molecule (EpCAM), ovarian and uterine cancer antigen (CA125), folate receptor alpha, sperm protein 17, tumor-associated differentially expressed gene-12 (TADG-12), mucin-16 (MUC-16), L1 cell adhesion molecule (L1CAM), mannan-MUC-1, human endogenous retrovirus K (HERV-K-MEL), Kita-kyushu lung cancer antigen-1 (KK-LC-1), human cancer / Testis antigen (KM-HN-1), cancer testis antigen (LAGE-1), melanoma antigen-A1 (MAGE-A1), sperm surface zona pellucida-binding protein (Sp17), synovial sarcoma, X breakpoint 4 (SSX-4), transient axonal glycoprotein-1 (TAG-1), transient axonal glycoprotein-2 (TAG-2), activation homolog (ENAH), mammaglobin-A, breast cancer antigen (BAGE-1), NY-BR-1, B melanoma antigen, melanoma antigen-A1 (MAGE-A1), melanoma antigen-A2 (MAGE-A2), mucin k, synovial sarcoma, X breakpoint 2 (SSX-2), paclitaxel resistance-associated gene-3 (TRAG-3), avian cytomatosis viral oncogene (c-myc), cell cycle protein B1, mucin 1 (MUC1), p62, survivin, lymphocyte common antigen (CD45), DickkopfWNT signaling pathway inhibitor 1 (DKK1), telomerase, Kirsten rat sarcoma viral oncogene homolog (K-ras), G250, intestinal carboxylesterase, alpha-fetoprotein, macrophage colony-stimulating factor (M-CSF), prostate-specific membrane antigen (PSMA), caspase 5 (CASP-5), cytochrome c oxidase assembly factor 1 homolog (COA-1), O-linked β-N-acetylglucosamine transferase (OGT), osteosarcoma amplification 9, ER lectin (O S-9), transforming growth factor beta receptor 2 (TGF-βRII), murine leukemia glycoprotein 70 (gp70), calcitonin-related polypeptide alpha (CALCA), programmed cell death 1 ligand 1 (CD274), mouse double minute 2 homolog (mdm-2), alpha-actinin-4, elongation factor 2, malic enzyme 1 (ME1), nuclear transcription factor Y subunit C (NFYC), G antigen 1,3 (GAGE-1,3), melanoma antigen-A6 (MAGE-A6), cancer-testis antigen XAGE-1b, Six transmembrane epithelial antigens of the prostate 1 (STEAP1), PAP, prostate-specific antigen (PSA), fibroblast growth factor 5 (FGF5), heat shock protein hsp70-2, melanoma antigen-A9 (MAGE-A9), Arg-specific ADP-ribosyltransferase family C (ARTC1), B-Raf proto-oncogene (B-RAF), serine / threonine kinase, β-catenin, cell division cycle 27 homolog (Cdc27), cyclin-dependent kinase 4 (CDK4), and cyclin-dependent kinase CDK12, cyclin-dependent kinase inhibitor 2A (CDKN2A), casein kinase 1 alpha 1 (CSNK1A1), fibronectin 1 (FN1), specific growth inhibitory factor 7 (GAS7), glycoprotein non-metastatic melanoma protein B (GPNMB), HAUS-like augmin complex subunit 3 (HAUS3), LDLR-fucosylated melanoma antigen 2 (MART2), myostatin (MSTN), melanoma-associated antigen (mutated) 1 (MUM-1-2-3), Poly(A)Polymerase gamma (neo-PAP), myosin class I, protein phosphatase 1 regulatory subunit 3B (PPP1R3B), peroxidase-5 (PRDX5), receptor tyrosine protein phosphatase kappa (PTPRK), transforming protein N-Ras (N-ras), retinoblastoma-associated factor 600 (RBAF600), sirtuin-2 (SIRT2), SNRPD1, triosephosphate isomerase, oculocutaneous albinism type 1 protein (OA1), member of the RAS oncogene family (RAB38), tyrosinase-related protein 1-2 (TRP-1-2), melanoma antigen gp75 (gp75), tyrosinase, melanin A (M ART-1), glycoprotein 100 melanoma antigen (GP100), N-acetylglucosaminyltransferase V gene (GnTVf), lymphocyte antigen 6 complex locus K (LY6K), melanoma antigen-A10 (MAGE-A10), melanoma antigen-A12 (MAGE-A12), melanoma antigen-C2 (MAGE-C2), melanoma antigen NA88-A, paclitaxel resistance-associated protein 3 (TRAG-3), PDZ-binding kinase (pbk), caspase 8 (CASP-8), sarcoma antigen 1 (SAGE), breakpoint cluster region-Abelson oncogene (BCR-ABL), leukemia fusion protein, dek-can, elongation factor Tu The protein is selected from GTP-binding domain 2 (EFTUD2), ETS mutated gene 6 / acute myeloid leukemia fusion protein (ETV6-AML1), FMS-like tyrosine kinase-3 internal tandem repeat (FLT3-ITD), cyclin A1, fibronectin type III domain 3B (FDNC3B), promyelocytic leukemia / retinoic acid receptor alpha fusion protein (pml-RARα), melanoma antigen-C1 (MAGE-C1), alternative splicing isoform of membrane protein (D393-CD20), melanoma antigen-A4 (MAGE-A4), or melanoma antigen-A3 (MAGE-A3).

[0098] The infectious diseases include bacterial infections, viral infections, and diseases caused by infections with other pathogenic microorganisms such as rickettsia, chlamydia, and mycoplasma.

[0099] The viral pathogen may be one or more selected from the group consisting of Dengue virus, Ebola virus, EBV, Hepitis A virus, Hepitis B virus, Hepitis C virus, Hepitis D virus, HIV, HSV1, HSV2, cytomegalovirus (CMV), Influenza A virus, Marburg virus, human respiratory syncytial virus (RSV), SARS coronavirus (SARS-CoV), West Nile virus, human papillomavirus (HPV), human rhinovirus (HRV), Epstein-Barr virus (EBV), human rabies virus (HRV), and Zika virus, but is not limited thereto.

[0100] The bacterial pathogens include Acinetobacter baumanii, Burkholderia cepacia, Bacterioides fragilis, Chlamydia trachomatis, Citrobacter freundii, Campylobacter jejuni, Escherichia coli, Enterobacter aerogenes, Enterobacter cloacae, Haemophilus influenzae type b, Helicobacter pylori, Klebsiella oxytoca, and the like. oxytoca, Klebsiella pneumoniae (MDR / CRE), Legionella pneumophila, Neisseria meningitides, Neisseria gonorrhoeae, Pseudomonas aeruginosa, Salmonella typhi, Salmonella paratyphi, Salmonella typhimurium, Serratia marcescens, Shigella flexneri, Stenotrophomonas maltophilia, Yersinia pseudotuberculosis, Bacillus subtilis, Clostridium neoformans neoformans), Clostridium difficile (C. difficile), Clostridium perfringens (C.perfringens, Corynebacterium spp., Enterococcus faecalis, Enterococcus faecium, Vancomycin-resistant Enterococcus (VRE), Listeria monocytogenes, Mycobacterium avium, M. tuberculosis, M. leprae, Nocardia farcinica, Propionibacterium acnes, Staphylococcus aureus aureus, methicillin-sensitive Staphylococcus aureus (MSSA), methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus epidermidis, Streptococcus pyogenes, Group A Streptococcus, Group B Streptococcus, and Group C Streptococcus, but are not limited to these.

[0101] The fungal pathogen may be one or more species selected from, but not limited to, Aspergillus spp., Blastomyces, Candida albicans, glabrata, guilliermondii, krusei, parapsilosis, tropicalis, Cryptococcus, Fusarium spp., Mucor spp., Saccharomyces, and Pneumocystis jirovecii (carinii).

[0102] In some embodiments, the tumor antigen or antigenic epitope peptide is selected from a genetically altered antigen, a tissue-specific differentiation antigen, an overexpressed antigen, a cancer-testis antigen, a universal antigen, and / or an antigen derived from an oncogenic virus.

[0103] In the tumor antigen, the antigen resulting from the gene mutation is one or more selected from p53, ras, β-catenin, CDK4, CDC27, and α-actinin-4. In tumor antigens, the tissue-specific differentiation antigen is one or more selected from tyrosinase, TRP1 / gp75, TRP2, gp100, Melan-A / MART1, ganglioside, and PSMA. In the tumor antigen, the overexpressed antigen is one or more selected from HER2, WT1, EphA3, EGFR, and CD20. In the tumor antigen, the cancer / testis antigen is one or more selected from MAGE, BAGE, GAGE, and NY-ESO-1. Among tumor antigens, the universal antigen is one or more selected from the group consisting of telomerase and survivin. In the tumor antigen, the oncogenic virus-derived antigen is one or more selected from EBV and HPV.

[0104] As will be understood by those skilled in the art, the nucleic acid molecules described herein include one or both of the complementary strands. For ease of explanation, the specification will generally show only one strand, but the complementary strand is also disclosed. Furthermore, the nucleic acid sequences of the present invention include DNA and RNA forms, and the disclosure of one of them also includes the other.

[0105] When the nucleic acid molecule is bound to a carrier, the nucleic acid molecule may be directly or indirectly bound to a control element on the carrier. These control elements may be capable of controlling the translation, expression, etc. of the nucleic acid molecule. These control elements may be derived directly from the carrier itself or may be exogenous (i.e., not derived from the carrier itself). The nucleic acid molecule may be operably linked to a control element. As used herein, "operably linked" means that by linking an exogenous gene to a vector, the control elements within the vector, such as transcriptional control sequences and translational control sequences, can perform the expected function of regulating the transcription and translation of the exogenous gene. Commonly used vectors may be, for example, plasmids, viral vectors, phages, etc. After being introduced into appropriate recipient cells, vectors according to some specific embodiments of the present invention can effectively express the protein, antibody, or complex under the control of a regulatory system, and can also enable in vitro mass production of the protein, antibody, or complex.

[0106] Furthermore, in the present invention, a series of nucleic acid molecules containing the above-mentioned open reading frames have been designed. These open reading frames may or may not contain a signal peptide sequence coding region in addition to the coding region encoding the antigen element-(linker)-E3 ubiquitin ligase recruitment ligand element structure.

[0107] The nucleic acid molecule may further comprise a 5'-untranslated region, an open reading frame, and a 3'-untranslated region, which are linked in this order, and may also comprise a promoter. mRNA can be obtained by transcription using a DNA template in which a promoter, a 5'-untranslated region, an open reading frame, and a 3'-untranslated region are linked in this order. The transcription process is carried out using conventional in vitro transcription methods and related kits. Furthermore, the nucleic acid molecule may further comprise a polyA fragment.

[0108] In one embodiment, the promoter is a T7 or SP6 promoter.

[0109] In one embodiment, the nucleotide sequence of the 5' untranslated region is set forth in SEQ ID NO:18 below. AGGCAAAAATCAAAATCAATCATCATCACAACATCAACAATCAATCATCAACACATCATCAAGACAGCCACC In one embodiment, the nucleotide sequence of the 3'-end untranslated region is set forth in SEQ ID NO:19 below. TGATGAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGCTG CGTCGAGAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAAGTCCAACTACTAAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAATAAAAAACATTTATTTTCATTGC In one embodiment, the nucleotide sequence of polyA is shown below in SEQ ID NO:20. AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGTCTTCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0110] The above nucleic acids, proteins, or polypeptides can be used as active ingredients in the preparation of drugs or vaccines for preventing and / or treating diseases. In general, those skilled in the art can use the above proteins as antigenic active ingredients in the preparation of protein or polypeptide vaccines, which contain the above proteins as antigenic components and pharmaceutically acceptable adjuvants or components.

[0111] When preparing a vaccine, an immunoadjuvant is added to enhance the immune response of the living body to the vaccine, such as Freund's incomplete adjuvant, Freund's complete adjuvant, aluminum hydroxide adjuvant, aluminum phosphate adjuvant, emulsion adjuvant, liposome adjuvant, microbial adjuvant, etc.

[0112] Those skilled in the art can obtain antibodies against the proteins described in the present invention based on the proteins. The antibodies may be polyclonal or monoclonal, preferably monoclonal. The antibodies are conjugated with a linking moiety. The linking moiety may be one or more selected from the group consisting of radionuclides, drugs, toxins, cytokines, enzymes, fluorescein, vector proteins, and biotin. The antibodies capable of specifically binding to the proteins can be used to prepare drugs and for related immunodetection of the proteins.

[0113] The present invention also includes genes encoding the above proteins. The genes encoding the above proteins can be used to express the proteins or antibodies and can be operatively incorporated into expression vectors to prepare vector vaccines or vector drugs. The expression vector can be selected from commonly used vectors such as plasmid vectors, adenovirus vectors, lentivirus vectors, or adeno-associated virus vectors. When an adenovirus vector is used, a replication-deficient adenovirus vector is generally used.

[0114] For example, if the nucleic acid molecule is DNA, the resulting mRNA can be transcribed with pharmaceutically acceptable adjuvants or components to prepare an mRNA vaccine. The adjuvant may be a nanocarrier that carries the mRNA. Lipid nanocarriers are commonly used as the nanocarrier. For example, lipid nanocarriers are prepared using at least one raw material, such as DOTAP, DOTMA, DOTIM, DDA, DC-Chol, CCS, diC14-amidine, DOTPA, DOSPA, DTAB, TTAB, CTAB, DORI, DORIE and its derivatives, DPRIE, DSRIE, DMRIE, DOGS, DOSC, LPLL, DODMA, DDAB, Dlin-MC3-DMA, CKK-E12, C12-200, DSPC, DMG-PEG, DOPE, phosphatidylethanolamine (PE), phosphatidylcholine (PC), and cholesterol (Chol).

[0115] Preferably, the lipid material used to prepare the nanocarrier is an amphiphilic lipid material or a cationic lipid material. Such lipid materials have a positively charged surface under acidic conditions and can encapsulate mRNA molecules by electrostatic adsorption with the phosphate groups of nucleic acids to form mRNA-lipid complexes. The mRNA-lipid complexes are adsorbed by the negatively charged cell membrane, and the mRNA is delivered to cells via membrane fusion or cellular endocytosis for expression, thereby exerting the immune effect of the vaccine.

[0116] Generally, in preparing the above-mentioned mRNA vaccine, mRNA and lipid materials can be self-assembled using a conventional microfluidic device to form an mRNA-lipid complex, or nanocarriers can be prepared and then incubated with mRNA to form an mRNA-lipid complex.

[0117] The present invention will be described in detail below with reference to examples.

[0118] Example 1: Construction of mRNA transcription vector template for E3 ubiquitin ligase ligand antigen element In this example, the mRNA transcription template DNA is obtained by sequentially linking a promoter, a 5'-terminal untranslated region, a signal peptide sequence, an antigen coding region sequence, a linker sequence, an E3 ubiquitin ligase recruitment ligand sequence, a 3'-terminal untranslated region, and Poly(A), introducing the resulting sequence into the corresponding position of a plasmid, and verifying that the sequence is correct by sequencing. Here, the signal peptide sequence, the antigen coding region sequence, the linker sequence, and the E3 ubiquitin ligase recruitment ligand sequence may each be provided as two sequences, or may be provided as being located in the same sequence.

[0119] The promoter is a T7 or SP6 promoter.

[0120] Chicken ovalbumin (OVA) was used as a model antigen. Three E3 ubiquitin ligases, PMI, VHL, and Keap1, recruiting ligands P53B, PMI, VHLL, and Keap1B, were used to construct and produce mRNAs encoding the full-length OVA protein (OVA-mRNA (control), OVA-P53B mRNA, OVA-PMI mRNA, OVA-VHLL mRNA, and OVA-keap1B mRNA, respectively). A positive control, OVA-H2Db mRNA, was also constructed. The antigen-encoding region was ligated into the corresponding position in a plasmid vector and verified by sequencing. The results are shown in Table 2.

[0121] Table 2: Information on mRNA sequences containing E3 ubiquitin ligase ligand antigen elements [Table 2]

[0122] Example 2: In vitro transcription of mRNA containing E3 ubiquitin ligase ligand antigen elements The mRNA transcription template DNA from Example 1 was inserted into a pUC57 vector to prepare plasmid DNA. The plasmid DNA was then enzymatically digested and processed according to the "Molecular Cloning Laboratory Guide (4th Edition)" and the instructions for use of commercially available restriction enzymes and DNA purification kits to obtain linearized plasmid DNA templates. The linearized plasmid DNA templates were then purified. The concentrations and purity of the plasmid DNA and linearized DNA templates were determined by spectrophotometry and gel electrophoresis, and complete linearization was confirmed by electrophoresis.

[0123] mRNA was synthesized using RNA polymerase, NTPs, and cap analogs. In vitro transcription of precursor mRNA was performed according to the instruction manual of the kit (Novesan TR101-02). Specifically, the relevant reactants were placed in 1.5 mL centrifuge tubes in the amounts and order shown in Table 3. Mix thoroughly by pipetting three times or gently tapping the bottom of the tube with a finger. Then, briefly centrifuged to concentrate the reaction mixture at the bottom. After 4 h of incubation in a 37°C water bath, 1 μL of DNase was added, followed by 15 min of incubation at 37°C. 179 μL of nuclease-free water was added, and then 200 μL of phenol / chloroform / isoamyl alcohol (25:24:1) solution was added to extract mRNA. After mixing thoroughly, the mixture was centrifuged at 15,000 rpm for 10 min, and the aqueous phase was removed. Next, 200 μL of 5 M ammonium acetate solution was added, and the mixture was left overnight at 4°C to precipitate the mRNA. The mixture was then removed and centrifuged at 15,000 rpm for 10 min at 4°C, and the supernatant was carefully removed. The precipitate was washed with 1 mL of 70% ethanol, centrifuged at 15,000 rpm at 4°C for 10 minutes, the supernatant was carefully removed, and the precipitate was air-dried. Then, 20 μL of nuclease-free water was added to redissolve the precipitate, yielding an mRNA solution.

[0124] The purity of mRNA was detected by agarose gel electrophoresis or a nucleic acid fragment analyzer.

[0125] Table 3: mRNA transcription reaction mixture [Table 3]

[0126] Example 3: Preparation of mRNA-lipid nanoparticles (mRNA-LNPs) As shown in Table 4, mRNA lipid nanoparticles can be prepared by selecting an appropriate formulation depending on the mRNA, target organ, etc. Specifically, the lipid materials in the formulation were dissolved in an ethanol solution, then mixed with the corresponding mRNA solution, which self-assembled to form mRNA-lipid nanoparticles, after which the ethanol was removed using a tangential flow system and the mRNA-lipid nanoparticles were obtained by sterilization filtration. Specifically, the process included the following steps:

[0127] (1) Solution preparation: Cationic lipids (MC3, DTAB, DC-Chol, CTAB, DOTMA, DDA, DOTAP), co-lipids (DSPC or DOPE), cholesterol (Chol), and DMG-PEG2000 were dissolved in absolute ethanol to obtain a lipid solution with an ionizable lipid concentration of 10 mg / mL. The molar ratio of cationic lipid, co-lipid, cholesterol (Chol), and DMG-PEG2000 was 50:10:38.5:1.5. The mRNA was diluted to an appropriate concentration with PBS buffer (prepared with RNase-free water).

[0128] (2) Preparation of LNP: The lipid solution obtained in step (1) was mixed with mRNA solutions of different concentrations to obtain samples with different mass ratios of cationic lipid to mRNA as shown in Table 4. Mixing was performed in a microfluidic control device (Shanghai Minnan Instrument Technology Co., Ltd.), and the mixing process parameters were a volume ratio of lipid solution to mRNA solution of 1:3 and a total flow rate of 9 mL / min.

[0129] (3) Ultrafiltration: The LNP crude product prepared using the microfluidic control device was diluted 25-fold with PBS buffer and ultrafiltered to the initial volume in an ultrafiltration cup to obtain the final LNP formulation. During the ultrafiltration process, ethanol was removed to obtain mRNA-lipid nanoparticles. The process parameters for ultrafiltration were a 100 kDa membrane, a 0.2 MPa pressure, and a 100-200 rpm rotation speed.

[0130] Using the above method, lipid nanoparticles containing six types of mRNA (OVA-mRNA, OVA-H2Db mRNA, OVA-P53B mRNA, OVA-PMI mRNA, OVA-VHLL mRNA, and OVA-keap1B mRNA) obtained in Example 1 were prepared according to the lipid formulation and mass ratio of Formulation 1 shown in Table 4, as well as blank lipid nanoparticles containing no mRNA. The particle size, electrical potential, and mRNA encapsulation rate of the seven types of mRNA lipid nanoparticles prepared were measured. The specific detection method is described below.

[0131] A predetermined volume of the mRNA-lipid nanoparticle colloidal solution was taken and diluted with purified water to an mRNA concentration of 0.01 mg / ml, and the particle size and potential of the mRNA-lipid nanoparticles were measured in a laser particle size analyzer (n=3, i.e., three measurements were performed for each formulation). As shown in the detection results, the particle size of the seven types of mRNA-lipid nanoparticles prepared was approximately 100 nm (Figure 1 - mRNA-LNP particle size and PDI result diagram), the PDI was approximately 0.2, and the potential was approximately 25 mV (Figure 2 - mRNA-LNP potential result diagram), demonstrating good uniformity.

[0132] Encapsulation rate detection: Quant-iT TM RiboGreen TM The encapsulation rate was measured using a kit. The detection results are shown in Table 5. The prepared mRNA-LNPs had good nanoformulation properties, with encapsulation rates all exceeding 95%, indicating good protection for mRNA.

[0133] The morphology of the resulting OVA-P53B mRNA lipid nanoparticles was observed under a transmission electron microscope (TEM) and photographed. As shown in Figure 3, the TEM results for the OVA-P53B mRNA lipid nanoparticles were spherical, with a round appearance, and the particle size was essentially consistent with the particle size measurement results.

[0134] Observation of the morphology of LNPs using a transmission electron microscope (TEM) for other OVA-PMI mRNA, OVA-VHLL mRNA, and OVA-keap1B mRNA lipid nanoparticles showed similar effects to those described above.

[0135] Table 4: Formulation of mRNA-lipid nanoparticles [Table 4]

[0136] Table 5: Detection results of mRNA-LNP encapsulation rate [Table 5]

[0137] Example 4: Activation of mRNA-lipid nanoparticles containing E3 ubiquitin ligase ligand antigen elements on T cells in vivo Lipid nanoparticles encapsulating OVA-mRNA and OVA-P53B mRNA prepared in Example 3 were administered to mice. The mRNA content in the lipid nanoparticle sample was 0.1 mg / mL. Specifically, C57BL / 6 mice were conditioned for one week and then divided into control, OVA, and OVA-P53 groups (four mice per group) and immunized intravenously. The control group received 100 μL saline, while the OVA and OVA-P53 groups received 100 μL of the corresponding mRNA-lipid nanoparticle samples. 36 h after administration, all mice were euthanized and dissected. Spleen and lymph node tissues were collected, processed, and filtered to obtain single-cell suspensions. After centrifugation at 400 g, red blood cell lysis solution was added, lysed at room temperature, and centrifuged again. The supernatant was removed. T cell protein antibodies (including anti-CD45, anti-CD3ε, anti-CD4, anti-CD8, anti-CD25, and anti-CD69) were added, stained at room temperature, centrifuged, and the supernatant was discarded. The cells were resuspended and detected using a flow cytometer.

[0138] The results are shown in Figures 4 and 5.

[0139] Figure 4 shows the results of immune cell detection in lymph nodes. Figure 4A shows early activated CD8 + and CD4 + Figure 4B shows the T cell detection data. + and CD4 + 4 shows the detection data of T cells. As can be seen from FIG. 4, in the lymph node region, the lipid nanoparticles of OVA-P53B mRNA to which the E3 ubiquitin ligase recruitment ligand P53B according to this example is bound showed a higher CD8 T cell count than the lipid nanoparticles of OVA-mRNA to which the E3 ubiquitin ligase recruitment ligand P53B is not bound. + T cells and CD4 + It can activate T cells better.

[0140] Figure 5 shows the results of detecting immune cells in the spleen. Figure 5A shows early activated CD8 + and CD4 + Figure 4B shows the T cell detection data. + and CD4 +5 is a diagram showing the detection data of T cells. As can be seen from Fig. 5, in the spleen region, the lipid nanoparticles of OVA-P53B mRNA to which the E3 ubiquitin ligase recruitment ligand P53B according to this example is bound showed a higher CD8 T cell count than the lipid nanoparticles of OVA-mRNA to which the E3 ubiquitin ligase recruitment ligand P53B is not bound. + T cells and CD4 + It can activate T cells better.

[0141] The mRNA lipid nanoparticles containing the E3 ubiquitin ligase mobilizing ligand P53B according to this example were able to mediate T cell activation in mice, and the effect was much greater than that of mRNA lipid nanoparticles without the E3 ubiquitin ligase ligand antigen element.

[0142] Example 5: In vivo anti-E.G7-OVA tumor efficacy of mRNA lipid nanoparticles containing E3 ubiquitin ligase ligand antigen elements Lipid nanoparticles containing OVA mRNA, OVA-H2Db mRNA, OVA-P53B mRNA, OVA-PMI mRNA, OVA-VHLL mRNA, and OVA-keap1B mRNA, prepared in Example 3, were administered to mice. The mRNA content in the lipid nanoparticle sample was 0.1 mg / mL.

[0143] 1. Investigation of the effect of the mRNA-lipid nanoparticles of the present invention on tumor volume Male C57BL / 6 mice aged 5–6 weeks were randomly assigned to groups of six. The antitumor effects of immunizing mice bearing E.G7-OVA tumors with OVA mRNA lipid nanoparticles containing the E3 ubiquitin ligase ligand antigen element were investigated.

[0144] The specific method is as follows. After adaptive feeding of C57BL / 6 mice for 1 week, the density of the EG7-OVA tumor cell suspension was increased to 7 × 10 6The concentration of mRNA-LNP was adjusted to 100 cells / mL, and 100 μL of the solution was subcutaneously injected into the upper right axilla of C57BL / 6 mice. Seven days after inoculation, the mice were immunized intravenously according to the experimental protocol (Figure 6: Schematic diagram of the immunization protocol). The mRNA-LNP dose was 10 μg mRNA / mouse (the control group received the same volume of saline).

[0145] On the day of the first administration, tumor diameters were measured using electronic calipers, and then recorded every other day. Tumor volumes were calculated according to the experimental design, and tumor growth volume-time curves for the tumor-bearing mice were plotted. The resulting curves are shown in Figure 7 (Tumor growth curves for G7-OVA tumor-bearing mice).

[0146] As can be seen from the above experimental results, compared with the control group and the positive control OVA-H2Db group, the OVA mRNA vaccine containing the E3 ubiquitin ligase ligand antigen element according to the present invention had a significantly higher inhibitory effect on tumor growth, with OVA-P53B showing the highest tumor inhibitory effect.

[0147] 2. Investigation of the inhibition rate of the mRNA lipid nanoparticles of the present invention against mouse tumors After the immunization experiment, all mice were euthanized and dissected. Tumor tissues were collected, photographed, weighed, and the tumor inhibition rate was calculated. The results are shown in Figure 8 (representative photographs of tumors from each treatment group).

[0148] As can be seen from the above experimental results, the tumor inhibition rate of OVA-H2Db was 38.8%, demonstrating a certain degree of tumor inhibition effect. The tumor inhibition rates of OVA-PMI, OVA-Keap1B, and OVA-VHLL were 51.1%, 66.8%, and 70.5%, respectively, demonstrating good tumor inhibition effects. The tumor inhibition rate of OVA-P53B reached 84.8%, demonstrating excellent antitumor activity.

[0149] 3. The safety of administration of the mRNA-lipid nanoparticles of the present invention was evaluated by investigating the effect on mouse body weight. On the day of the first administration, the weight of the tumor-bearing mice was measured using an electronic balance, and then recorded every other day. The weight-time curve of the tumor-bearing mice was created to evaluate toxicity. The weight-time curve of the tumor-bearing mice obtained is shown in Figure 9 (weight-time curve of E.G7-OVA tumor-bearing mice).

[0150] As can be seen from the experimental results, during the treatment process, each group tended to gain weight, but there was no significant weight loss, indicating that each preparation had no significant toxic side effects and was safe for internal administration.

[0151] 4. The effects of the mRNA lipid nanoparticles of the present invention on antigen presentation and the production of specific CTL in mice were investigated. The specific procedures are as follows: After the above immunization experiments were completed, all mice were euthanized and dissected. Lymph node, spleen, and tumor tissues were collected, processed, and filtered to obtain single-cell suspensions. After centrifugation, an appropriate amount of red blood cell lysis solution was added and lysed at room temperature. The cells were then centrifuged again and the supernatant was discarded. Antibodies against various immune cell types, such as DCs, macrophages, and effector T cells, were added and stained at room temperature. These antibodies included anti-CD45, anti-CD11c, anti-CD3ε, anti-CD4, anti-CD8, anti-H-2Kb bound to SIINFEKL, and H2Kb OVA tetramer. The cells were then resuspended and analyzed using a flow cytometer.

[0152] The results are shown in Figures 10 and 11. Figure 10 is a graph showing the results of detecting antigen presentation mediated by mRNA lipid nanoparticles in mouse bodies. Figure 11 is a graph showing the results of detecting specific CTLs mediated by mRNA lipid nanoparticles in mouse bodies. Figure 11A is a graph showing the results of detecting specific CTLs mediated by mRNA lipid nanoparticles in mouse lymph nodes. Figure 11B is a graph showing the results of detecting specific CTLs mediated by mRNA lipid nanoparticles in mouse spleens. Figure 11C is a graph showing the results of detecting specific CTLs mediated by mRNA lipid nanoparticles in mouse tumor tissues.

[0153] 10 and 11, compared with OVA-mRNA without the E3 ubiquitin ligase ligand antigen element, the mRNA lipid nanoparticles containing the E3 ubiquitin ligase ligand antigen elements OVA-P53B mRNA, OVA-PMI mRNA, OVA-VHLL mRNA, and OVA-keap1B mRNA according to this example all mediated stronger antigen presentation in mice and produced more specific CTLs, with OVA-P53B showing the greatest effect.

[0154] Example 6: Effect of P53 on the in vivo anti-EBV tumor efficacy of EBV LMP2A@mRNA lipid nanoparticles The amino acid sequence of the EBV virus LMP2A antigen is shown in SEQ ID NO: 21 below. MGSLEMVPMGAGPPSPGGDPDGYDGGNNSQYPSASGSSGNTPTPPNDEERESNEEPPPPYEDPYWGNGDRHSDYQPLGTQDQSLYLGLQHDGNDGLPPPPYSPRDDSSQHIYEEAGRGSMNPVC LPVIVAPYLFWLAAIAASCFTASVSTVVTATGLALSLLLLAAVASSYAAAQRKLLTPVTVLTAVVTFFAICLTWRIEDPPFNSLLFALLAAAGGLQGIYVLVMLVLLILAYRRRWRRLTVCGGI MFLACVLVLIVDAVLQLSPLLGAVTVVSMTLLLLAFVLWLSSPGGLGTLGAALLTLAAALALLASLILGTLNLTTMFLLMLLWTLVVLLICSSCSSCPLSKILLARLFLYALALLLLASALIAG GSILQTNFKSLSSTEFIPNLFCMLLLIVAGILFILAILTEWGSGNRTYGPVFMCLGGLLTMVAGAVWLTVMSNTLLSAWILTAGFLIFLIGFALFGVIRCCRYCCYYCLTLESEERPPTPYRNTV

[0155] mRNA containing the above-mentioned LMPA2 antigen sequence was prepared according to the methods of Examples 1 and 2, and lipid nanoparticles encapsulating LMP2A-mRNA, LMP2A-P53B mRNA, and LUC mRNA were prepared according to the method of Example 3 at a cationic lipid to mRNA mass ratio of 15:1. The mRNA content was 0.1 mg / mL. The in vivo antitumor effect of LMP2A-P53B mRNA lipid nanoparticles containing the E3 ubiquitin ligase ligand antigen element P53B was investigated according to the method of Example 5.

[0156] The antitumor effect of LMP2A-P53B mRNA lipid nanoparticles containing E3 ubiquitin ligase ligand antigen elements on the generation of specific T cells in vivo was investigated using the ELISpot method.

[0157] The specific method is as follows. After the immunization experiment, all mice were euthanized and dissected. The spleens were collected, processed, and filtered to obtain single-cell suspensions. After centrifugation, red blood cell lysing was added at room temperature, and the supernatant was discarded. After re-centrifugation, the splenic lymphocyte suspension was obtained and analyzed.

[0158] The results are shown in Figure 12. mRNA lipid nanoparticles containing the LMP2A antigen can mediate the production of specific CTLs in mice. LMP2A-P53B-mRNA lipid nanoparticles containing the E3 ubiquitin ligase ligand antigen element sequence have a more pronounced effect.

[0159] As can be seen from the above results, compared with the control group, the mode mRNA group (LUC mRNA), and the group containing only the LMP2A antigen (LMP2A mRNA), the LMP2A-P53B mRNA lipid nanoparticles containing the E3 ubiquitin ligase ligand antigen element P53B according to this embodiment can induce the production of more CTLs, have a stronger inhibitory effect on tumor growth, and have a higher tumor inhibition rate.

[0160] Example 7: Safety evaluation of mRNA lipid nanoparticles containing E3 ubiquitin ligase ligand antigen elements In this example, the safety of the mRNA lipid nanoparticles prepared in Example 3 in mice was investigated.

[0161] The specific operations are as follows: After one week of adaptive breeding, C57BL / 6 mice were intravenously immunized according to the experimental design (Figure 6 - Immunization design diagram). The dose was 10 μg mRNA per mouse. Seven days after the end of administration, blood samples were collected from the mice to prepare mouse plasma, and ALT, AST, TP, CRE, LDH, and UREA were detected in the mouse serum using a blood biochemistry analyzer.

[0162] The results are shown in Figure 13. Compared to the control group, after immunization with each mRNA lipid nanoparticle, the data for ALT, AST, TP, CRE, LDH, and UREA in mouse serum did not change significantly. As can be seen from these results, after administration of each mRNA lipid nanoparticle, there was no significant change in the indicators related to the liver function and kidney function of the mice, and each mRNA lipid nanoparticle containing the E3 ubiquitin ligase ligand antigen element of this example has good safety.

[0163] The particular features, structures, materials, or characteristics described herein may be combined in any suitable manner in one or more embodiments, and those skilled in the art may combine different embodiments and features of different embodiments described herein, provided that they are not mutually inconsistent.

Claims

1. A nucleic acid molecule comprising at least one open reading frame, the open reading frame comprises at least one antigen element and at least one E3 ligand element; The nucleic acid molecule, wherein the E3 ligand element is an E3 ubiquitin ligase binding or recruitment ligand, and the E3 ubiquitin ligase binding or recruitment ligand binds to one or more E3 ubiquitin ligases.

2. The nucleic acid molecule according to claim 1, wherein the E3 ubiquitin ligase is selected from the group consisting of RING type, HECT type, and RBR type.

3. the E3 ubiquitin ligase is one or more selected from Von Hippel-Lindau (VHL), MDM2, CRBN, IAPs, RNF, β-TrCP, DCAF, Keap1, or truncations or elongations thereof; The nucleic acid molecule of claim 1, wherein the E3 ligand binds to one or more E3 ubiquitin ligases.

4. The nucleic acid molecule according to claim 3, wherein the amino acid sequence corresponding to the binding or recruitment ligand of Keap1 is LDPETGEYL or a sequence having more than 60% identity thereto.

5. The nucleic acid molecule according to claim 3, wherein the amino acid sequence corresponding to the binding or recruitment ligand of β-TrCP is DRHDSGLDSM or a sequence having more than 60% identity thereto.

6. The nucleic acid molecule of claim 3, wherein the amino acid sequence corresponding to the VHL binding or recruitment ligand is at least one of LAP(OH)YI and ALAPYIP or a sequence having greater than 60% identity thereto.

7. The nucleic acid molecule of claim 3, wherein the amino acid sequence corresponding to the MDM2 binding or recruitment ligand is at least one of ETFSDLWKLL, TSFAEYWNLLSP, LTFEHYWAQLTS, TNWYANLEKLLR, TAWYANFEKLLR, DWWPLAFEALLR, CNCKAPETALCARRCQQH, and CNCKAPETFLCYWRCLQH, or a sequence having greater than 60% identity thereto.

8. The nucleic acid molecule according to any one of claims 1 to 7, wherein the antigen element and the E3 ligand element are linked in any one of the following forms: E3 ligand element-antigen element, antigen element-E3 ligand element, E3 ligand element-antigen element-E3 ligand element, and antigen element-E3 ligand element-antigen element, and the antigen element and the E3 ligand element are linked by a linker.

9. The amino acid sequence of the linker is GGGGS, (GGGGS) ３ , (GGGGS) ６ , (GGS) １０ and (GSG) １０ The nucleic acid molecule according to claim 8, characterized in that it is at least one of the following:

10. The nucleic acid molecule according to any one of claims 1 to 9, characterized in that the antigenic element comprises at least one antigenic epitope.

11. The nucleic acid molecule according to claim 10, wherein the antigen element is a naturally occurring antigenic protein, antigenic polypeptide or a truncated form thereof.

12. The nucleic acid molecule of claim 10, wherein the antigen element comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 antigenic epitopes.

13. The nucleic acid molecule according to claim 10, wherein the antigenic epitope is a T cell antigenic determinant or a T cell antigen epitope.

14. The nucleic acid molecule according to claim 10, wherein the antigenic epitope is derived from an antigenic epitope peptide of a tumor, an antigenic epitope peptide of an autoimmune disease, or an antigenic epitope peptide of a pathogenic microorganism.

15. The tumor antigen or antigen epitope peptide is selected from an antigen caused by genetic mutation, and / or a tissue-specific differentiation antigen, and / or an overexpressed antigen, and / or a cancer / testis antigen, and / or a universal antigen, and / or an antigen derived from an oncogenic virus; the antigen resulting from a gene mutation is one or more selected from p53, ras, β-catenin, CDK4, CDC27, and α-actinin-4; the tissue-specific differentiation antigen is one or more selected from tyrosinase, TRP1 / gp75, TRP2, gp100, Melan-A / MART1, ganglioside, and PSMA; the overexpressed antigen is one or more selected from HER2, WT1, EphA3, EGFR, and CD20; the cancer / testis antigen is one or more selected from MAGE, BAGE, GAGE, and NY-ESO-1; the universal antigen is one or more selected from the group consisting of telomerase and survivin; The nucleic acid molecule according to claim 14, wherein the oncogenic virus-derived antigen is one or more selected from the group consisting of EBV, HPV, HBV, HCV, human herpes virus, and Merkel cell polyomavirus.

16. 16. The nucleic acid molecule according to any one of claims 1 to 15, characterized in that the nucleic acid molecule further comprises a signal peptide coding region.

17. the nucleic acid molecule further comprises a promoter, a 5'-end untranslated region, a 3'-end untranslated region, and polyA; 17. The nucleic acid molecule according to any one of claims 1 to 16, characterized in that the promoter is a T7 or SP6 promoter.

18. The nucleic acid molecule according to any one of claims 1 to 17, characterized in that the nucleic acid is at least one selected from DNA, ASO, siRNA, miRNA, mRNA, and an aptamer.

19. 19. A nucleic acid vaccine comprising the nucleic acid molecule of any one of claims 1 to 18 and a pharmaceutically acceptable adjuvant or component, wherein the nucleic acid vaccine is an mRNA vaccine and the component is a nanocarrier that delivers the mRNA; and / or The nucleic acid vaccine, wherein the adjuvant comprises at least one selected from a buffer medium for injection, a lyoprotectant, and a cryoprotectant.

20. The nucleic acid vaccine of claim 19, wherein the nanocarrier comprises at least one selected from liposomes, nanoparticles, microspheres, and lipid nanocarriers.

21. The nucleic acid vaccine of claim 19, wherein the nanocarrier is made of at least one lipid material selected from the group consisting of DOTAP, DOTMA, DOTIM, DDA, DC-Chol, CCS, diC14-amidine, DOTPA, DOSPA, DTAB, TTAB, CTAB, DORI, DORIE and derivatives thereof, DPRIE, DSRIE, DMRIE, DOGS, DOSC, LPLL, DODMA, DDAB, Dlin-MC3-DMA, CKK-E12, C12-200, DSPC, DMG-PEG, DOPE, phosphatidylethanolamine, phosphatidylcholine, and cholesterol.

22. the mRNA vaccine is formed by self-assembly of the mRNA and lipid material using a microfluidic device; or The nucleic acid vaccine of claim 19, wherein the mRNA vaccine is formed by incubating the nanocarrier and mRNA.

23. A protein, characterized in that it is encoded by a nucleic acid molecule according to any one of claims 1 to 18.

24. A protein or polypeptide vaccine, comprising the protein of claim 23 as an antigen component.

25. 25. A protein or polypeptide vaccine according to claim 24, characterized in that it comprises a pharmaceutically acceptable auxiliary agent or auxiliary component and an immunoadjuvant.

26. 26. The protein or polypeptide vaccine of claim 25, wherein the immunoadjuvant comprises one or more selected from Freund's incomplete adjuvant, Freund's complete adjuvant, aluminum hydroxide adjuvant, aluminum phosphate adjuvant, emulsion adjuvant, liposome adjuvant, and microbial adjuvant.

27. A vector carrying a nucleic acid molecule according to any one of claims 1 to 18, A vector characterized in that it carries a eukaryotic or prokaryotic vector.

28. The vector according to claim 27, wherein the vector comprises one or more selected from the group consisting of a plasmid vector, an adenovirus vector, a lentivirus vector, and an adeno-associated virus vector.

29. A vector vaccine, comprising an active ingredient obtained by introducing a nucleic acid molecule according to any one of claims 1 to 18 into a vector according to claim 27 or 28.

30. A nucleic acid molecule according to any one of claims 1 to 18, a nucleic acid vaccine according to any one of claims 19 to 22, a protein according to claim 23, a protein or polypeptide vaccine according to any one of claims 24 to 26, or a vector vaccine according to claim 29; and pharmaceutically acceptable adjuvants. A pharmaceutical composition comprising:

31. Use of a nucleic acid molecule described in any one of claims 1 to 18, a nucleic acid vaccine described in any one of claims 19 to 22, a protein described in claim 23, a protein or polypeptide vaccine described in any one of claims 24 to 26, or a vector vaccine described in claim 29, or a pharmaceutical composition described in claim 30 in the preparation of a medicament for preventing or treating an associated disease.

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