Sequence construction and application of immunostimulatory sequence truncated poly A tail

Abstract

The present invention provides a nucleic acid molecule comprising: a first nucleic acid sequence that is transcribable or for introducing a transcribable nucleic acid sequence; the second nucleotide sequence comprises at least two A elements and at least one B element, each A element is defined as a nucleotide sequence consisting of 30 to 80 T nucleotides, and each B element is a sequence capable of activating PRRs after transcription; wherein the total number of A elements is one more than the total number of B elements, and wherein any two A elements are separated by one B element. The RNA generated by transcription of the nucleic acid molecule has higher immunogenicity and basically does not influence the expression of the protein.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular construction, and particularly relates to a nucleic acid molecule that can be transcribed into mRNA Background Art

[0002] mRNA technology is a promising tool, for example, for the synthesis of therapeutic proteins in cells. The advantage of using mRNA molecules is that only the mRNA needs to be introduced into the cytoplasm for protein translation. Compared with using the corresponding DNA sequence contained in an appropriate vector (such as a plasmid), the production process of mRNA molecules is standard, highly extensible, and avoids the risk of altering chromosomal DNA when the plasmid or part of the plasmid is incorporated into the genome. Initial challenges related to introducing mRNA molecules into cells, such as the instability of mRNA molecules and immune responses, have been successfully addressed using, for example, chemically modified nucleotides. So far, several determinants of mRNA efficiency have been identified and studied, including the efficiency of 5'-capping and the composition of the 5'-cap, the nature of the untranslated regions, codon optimization of the sequence encoding the protein, and 3'-polyA, etc.

[0003] To improve the efficiency of mRNA-based methods, it is crucial to optimize polyA. However, it is difficult in the prior art to directly generate mRNA molecules with a defined polyA length on a large scale. The mRNA molecules used for protein synthesis in vaccine / therapeutic applications are mainly generated by in vitro transcription. Such methods are based on DNA templates in which polyA has been encoded and amplified by cloning or polymerase chain reaction (PCR). Compared with adding polyA to the generated mRNA in an enzymatic catalytic manner, the DNA template-based method theoretically has the advantage of providing a defined and replicable polyA length, which can be used for the large-scale production of polyA mRNA molecules, and the cost of plasmid production is relatively low.

[0004] However, one of the challenges in bacterial amplification of the template DNA sequence using plasmids is that homopolymeric sequences (such as the sequence encoding polyA) can recombine during the bacterial amplification of plasmid DNA. Therefore, the sequence encoding polyA will shorten in an unpredictable manner over time. For example, compared with the sequence encoding polyA composed of at least 100 nucleotides, the sequence encoding 70-nucleotide-long polyA remains stable. Therefore, further optimization is required for the shortening of polyA caused by recombination. The 3'-polyA sequence of RNA plays an important role in nuclear export, RNA stability, and the translation efficiency of mRNA. The optimization of polyA should not affect the RNA stability and translation efficiency.

[0005] Immunity is divided into innate immunity and adaptive immunity, which work together to form an effective and continuous defense against pathogens. "Pattern recognition receptors" (PRRs) are a major component of innate immunity. PRRs are localized in multiple subcellular compartments and can detect specific pathogen-associated molecular patterns (PAMPs) that are unique to pathogens and not found in the host. Several PRRs, including toll-like receptors (TLRs), nod-like receptors (NLRs), and RIG-I-like receptors (RLRs), recognize different microbial components and directly activate immune cells. TLRs are transmembrane receptors, while NLRs and RLRs are intracellular molecules. Exposure of immune cells to the ligands of these receptors activates intracellular signaling cascades, rapidly inducing various overlapping and unique gene expressions involved in inflammation and immune responses.

[0006] TLRs are type I transmembrane receptors that are evolutionarily conserved between insects and humans. Ten TLRs (TLRs1-10) have been discovered. TLRs have similar extracellular and intracellular domains. The extracellular domain has leucine-rich repeat sequences, and the intracellular domain is similar to the intracellular domain of interleukin-1 receptor (IL-1R). The intracellular domain of TLR can interact with the adaptor protein Myd88, leading to the activation of cytokines such as NF-κB. TLRs are mainly expressed on antigen-presenting cells (such as dendritic cells, macrophages, etc.). Activation of dendritic cells by stimulating TLRs causes the maturation of dendritic cells and the production of inflammatory cytokines such as IL-12. TLR agonists mainly come from bacteria and viruses. Each TLR subtype recognizes a different PAMP. For example, TLR2 recognizes peptidoglycan, TLR3 recognizes dsRNA, TLR4 recognizes lipopolysaccharide (LPS) of Gram-negative bacteria, TLR5 recognizes bacterial flagellin, TLR7 / 8 recognize imidazoquinoline and ssRNA, TLR9 recognizes CpG DNA of bacteria, viruses, and protozoa, as well as malarial pigment.

[0007] Retinoic acid-inducible gene I (RIG-I) receptor, melanoma differentiation-associated protein 5 (MDA5) receptor, and laboratory of genetics and physiology 2 (LGP2) receptor are three RLRs identified to date. All RLRs have a highly conserved DExD / H-box (Asp-Glu-Ala-Asp) helicase domain and a C-terminal domain (CTD). RIG-I and MDA5 also have caspase activation and recruitment domains (CARDs) that are crucial for signal transduction. RIG-I and MDA5 are intracellular viral sensors that recognize palindromic dsRNA with 5’ppp and long-chain dsRNA by their helicase domains, respectively, inducing ATP-dependent conformational changes leading to the formation of filamentous oligomers on the RNA. Their N-terminal CARDs form "lock-washer"-like tetramers with K63-linked ubiquitin chains and form signal-active oligomers with MAVS containing CARDs, ultimately activating several transcription factors, interferon regulatory factor 3 (IRF3), interferon regulatory factor 7 (IRF7), and nuclear factor kappa B (NF-κB), to induce type I interferons and pro-inflammatory cytokines. LGP2 does not have a CARD but only a DExD / H-box helicase domain and is reported to act as a negative regulator, especially in RIG-I- and MDA5-mediated pathways.

[0008] Targeting TLRs / RLRs is being investigated as a potential vaccine adjuvant. One of the advantages of using immunostimulatory RNAs as adjuvants compared to traditional adjuvants is the broad and well-defined immune responses elicited. Recent advances in nucleic acid technology have led to the emergence of in vitro transcribed (IVT) mRNA as an alternative platform for vaccine development. Compared to other vaccine strategies, mRNA vaccines are easy to manufacture and have good safety profiles. As effective vaccines, mRNA vaccines have two basic tasks: expressing antigens and eliciting strong immune responses against the expressed antigens. mRNA stability, protein expression, and balanced and effective immune responses remain challenges. Summary of the Invention

[0009] In view of this, the present invention provides a nucleic acid molecule that can encode an mRNA molecule and a modified polyA, wherein the nucleic acid molecule comprises: 1) a first nucleic acid sequence that can be transcribed or used to introduce a transcribable nucleic acid sequence; and 2) a second nucleic acid sequence, the second nucleotide sequence consisting of: a) at least two A elements, each A element being defined as a nucleotide sequence consisting of 40 to 65 T nucleotides, and b) at least one B element, each B element being a sequence that can activate PRRs post-transcriptionally or a combination thereof; wherein the total number of A elements is one more than the total number of B elements, and any two A elements are separated by one B element.

[0010] The nucleic acid molecule exhibits reduced recombination during amplification in a bacterial host cell, and the mRNA transcribed from the nucleic acid molecule has higher immunogenicity and basically does not affect the expression of its protein.

[0011] As used herein, the term "vaccine" refers to a composition suitable for administration to animals (including humans) that induces an immune response after administration, the intensity of which is sufficient to minimally assist in preventing, ameliorating, or curing a clinical disease caused by microbial infection.

[0012] As used herein, the term "nucleotide" refers to an unmodified nucleotide or a modified nucleotide. Unmodified nucleotides are A, C, G, T, and U nucleotides. Modified nucleotides refer to any naturally occurring or chemically synthesized isomers of A, C, G, T, and U nucleotides, and refer to any naturally occurring or chemically synthesized analogs, alternatives, or modified nucleotides or isomers having, for example, chemically modified or substituted residues. Modified nucleotides may have base modifications and / or sugar modifications. Modified nucleotides may also have, for example, phosphate group modifications with respect to the five main caps of polynucleotides containing a sequence encoding a protein. Modified nucleotides also include nucleotides synthesized post-transcriptionally by covalent modification of nucleotides. In addition, any suitable mixture of unmodified and modified nucleotides is possible.

[0013] In a first aspect of the present invention, a nucleic acid molecule comprising a DNA sequence, the DNA sequence comprising: 1) a first nucleotide sequence encoding an mRNA molecule, and 2) a second nucleotide sequence;

[0014] Specifically, the second nucleotide sequence consists of:

[0015] a) at least two A elements, each A element being defined as a nucleotide sequence consisting of 40 to 65 T nucleotides, and

[0016] b) at least one B element, each B element being an immunostimulatory sequence capable of activating PRRs post-transcriptionally or a combination thereof;

[0017] wherein the total number of A elements is one more than the total number of B elements, and any two A elements are separated by one B element.

[0018] Specifically, the number of A elements is 2 - 6, and more preferably, the number of A elements is two, three, or four.

[0019] Specifically, the A element consists of 30 - 85 T nucleotides, the A element consists of 45 - 60 T nucleotides, more preferably, the A element consists of 50 - 60 T nucleotides, more preferably, the A element consists of 60 T nucleotides.

[0020] Specifically, the number of A elements is four. Preferably, the nucleotide sequences of the four A elements together have a total length of 240 nucleotides. More preferably, each A element has a length of 60 nucleotides.

[0021] Specifically, the number of A elements is three. Preferably, the nucleotide sequences of the three A elements together have a total length of 180 nucleotides. More preferably, each A element has a length of 60 nucleotides.

[0022] Specifically, the number of A elements is two. Preferably, the nucleotide sequences of the two A elements together have a total length of 120 nucleotides. More preferably, each A element has a length of 60 nucleotides.

[0023] Specifically, the B element can activate PRRs after transcription, preferably with a length of 5 - 250 bp; 5 - 200 bp; 5 - 150 bp; 5 - 100 bp; 5 - 80 bp, 5 - 60 bp, 5 - 50 bp, 5 - 40 bp, 5 - 30 bp, 5 - 20 bp, 5 - 20 bp, 10 - 80 bp, 10 - 60 bp, 10 - 50 bp, 10 - 40 bp, 10 - 30 bp, 10 - 20 bp, 12 - 80 bp, 12 - 60 bp, 12 - 50 bp, 12 - 40 bp, 12 - 30 bp, 12 - 25 bp, 12 - 22 bp.

[0024] Specifically, the B element is a TLRs or RLRs after transcription, more preferably TLR7 / 8 and / or RIG-I / MAD-5 or a combination thereof or a sequence with 95%, 90%, 85%, 80%, 75%, 70% similarity thereto.

[0025] Specifically, the sequence of the B element after transcription is a sequence that stimulates RIG-I / MAD-5 or a combination thereof or a sequence with 95%, 90%, 85%, 80%, 75%, 70% similarity thereto.

[0026] Specifically, the sequence of the B element after transcription is an immunostimulatory nucleotide sequence that stimulates TLR7 / 8, which contains a natural or modified base sequence, and the base sequence contains at least one natural or modified guanine and at least one natural or modified uracil, with the general formula NaGbNc. Preferably, G is a natural or modified guanine or uracil. More preferably, the sequence is any one of SEQ ID NO:1 to SEQ ID NO:20 or a combination thereof or a sequence with 95%, 90%, 85%, 80%, 75%, 70% similarity thereto.

[0027] Specifically, N is a nucleic acid sequence having a length of about 4 to 50 nucleic acids, preferably 4 to 30 nucleotides, more preferably 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 nucleotides, and each N is independently selected from natural or modified guanine, uracil, cytosine or adenine.

[0028] Specifically, a and c are integers from 1 to 40 independent of each other, preferably 1 to 30, more preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29. When a or c is 1, N is guanine or uracil or an analogue thereof. When it is greater than 1, at least 50% of these nucleotides are guanine or uracil or an analogue thereof; preferably 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%.

[0029] Specifically, b is an integer from 3 to 40, preferably 3 to 30, more preferably 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39.

[0030] Specifically, the ratio of guanine to uracil in the RNA sequence is between 1G:100U and 30G:1U. Preferably, it is between 1G:50U and 10G:1U. More preferably, it is between 1G:40U and 2G:1U, or between 1G:30U and 10G:1U, or between 1G:20U and 5G:1U, or between 1G:10U and 1G:1U, or between 1G:5U and 1G:1U.

[0031] In a second aspect of the present invention, a method for amplifying a nucleic acid molecule is provided, including: (1) providing the nucleic acid molecule as described above; (2) amplifying the bacterium including the nucleic acid molecule.

[0032] In a third aspect of the present invention, a method for obtaining RNA is provided, including amplifying the nucleic acid molecule according to the method as described above and in vitro transcribing the nucleic acid molecule into RNA using it as a template.

[0033] Specifically, the RNA includes linear and circular forms; preferably, the linear form is a conventional or self-replicating mRNA.

[0034] Specifically, the linear RNA further includes a 5' cap structure, a 5' non-coding region, and a 3' non-coding region.

[0035] In the fourth aspect of the present invention, it is provided that the RNA described above can be applied to the field of prophylactic vaccines, and the protein encoded by it can also have the potential to induce an immunogenic response, including but not limited to antigens in the following pathogens: antigens related to the novel coronavirus, antigens related to herpes simplex virus, antigens related to respiratory syncytial virus, antigens related to varicella-zoster virus, antigens related to influenza virus, antigens related to parainfluenza virus, antigens related to Epstein-Barr virus, antigens related to cytomegalovirus, antigens related to human metapneumovirus, antigens related to human papillomavirus, antigens related to rotavirus, antigens related to dengue virus, antigens related to malaria, antigens related to rabies virus or antigens related to monkeypox virus.

[0036] Specifically, it can be applied to the therapeutic field, including but not limited to the encoding of shared antigens such as KRAS, BRAF, EGFR and their mutants in the treatment of tumors such as lung cancer, pancreatic cancer or colon cancer, and personalized antigens screened, the encoding of tumor immunostimulants such as CD40L, CD70, TLR4, etc., and the systemic delivery of protein substitutes for rare single-gene diseases.

[0037] In the fifth aspect of the present invention, a method for obtaining a peptide or protein is provided, including obtaining RNA according to the method described above and translating the RNA into a peptide or protein sequence.

[0038] In the sixth aspect of the present invention, a system for generating eukaryotically translatable mRNA is provided, including 1) a vector or plasmid containing the nucleic acid molecule described above; 2) a host cell.

[0039] In the seventh aspect of the present invention, a composition is provided, comprising RNA, peptide or protein obtained by the method described above.

[0040] Specifically, the composition includes one or more of pharmaceutically acceptable excipients, carriers, buffers, protectants, stabilizers, surfactants, osmotic pressure regulators, adjuvants, preservatives, inactivators. The beneficial effects of the present invention compared with the prior art are as follows: the need for an in vitro transcription template is solved, and during the amplification in bacterial cells, the recombination of the sequence encoding the polyA tail is reduced, enabling the in vitro transcription template to encode an mRNA molecule with a defined polyA tail length; and at the same time, high immunogenicity is achieved with little impact on the expression of its protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 : Effects of the insertion of the second nucleotide sequence B element at different sites on protein expression.

[0042] Figure 2 : Effects of the combination of the second nucleotide sequence B element and different lengths of A element on polyA recombination.

[0043] Figure 3: Influence of B elements with different lengths on polyA recombination.

[0044] Figure 4 : Detection of HAI antibody titer.

[0045] Figure 5 : Detection of the frequencies of CD4+ T cells specifically secreting TNFα, IFNγ and IL-2 by ICS method.

[0046] Figure 6 : Detection of the frequencies of CD8+ T cells specifically secreting TNFα, IFNγ and IL-2 by ICS method. Specific implementation mode

[0047] To better understand the present invention, specific embodiments will be given to further illustrate the present invention. However, it should be understood that the described embodiments are exemplary embodiments, and the present invention can be implemented in various forms and should not be limited by the embodiments described herein. On the contrary, these embodiments are provided to be able to more thoroughly understand the present invention and to be able to fully convey the scope of the present invention to those skilled in the art.

[0048] Example 1 Influence of the insertion of the B element of the second nucleotide sequence at different sites on protein expression

[0049] To confirm the immune response effect of the insertion of the B element in the second nucleotide sequence at different sites, the following experiment was designed. Synthesize a DNA vector. The vector contains a T7 promoter, 5'UTR, a coding sequence for EGFP protein, 3'UTR and a poly-A tail (a 100bp T nucleotide sequence). The B element of the second nucleotide sequence (SEQ ID NO:8) was inserted respectively before the 5'UTR, after the 5'UTR, before the 3'UTR, after the 3'UTR and at 40bp of the poly-A tail.

[0050] Transcribe with a T7 in vitro transcription kit to obtain uncapped mRNA. Digest the transcription template with DNaseI respectively, and purify the mRNA by precipitation method. Cap the mRNA with a Cap1 capping kit, and purify the capped mRNA with an mRNA purification kit respectively.

[0051] Transfect the mRNA stock solution into HEK293 cells with Lip2000 transfection reagent, and observe the expression of EGFP protein under a fluorescence microscope 24h later (as Figure 1As shown in the figure. The results showed that the fluorescence intensity decreased significantly when the immune-stimulating sequence was inserted before the 5'UTR or after the 3'UTR, while there was no significant difference in fluorescence intensity compared with the control group when it was inserted after the 5'UTR, before the 3'UTR, and at the 40bp of the poly-A tail, indicating that the positions after the 5'UTR, before the 3'UTR, and at the 40bp of the poly-A tail are suitable for the insertion of the immune-stimulating sequence, especially before the 3'UTR and at the 40bp of the poly-A tail.

[0052] Example 2 Effect of the combination of the second nucleotide sequence B element and different lengths of A element on polyA recombination

[0053] To test the effect of different lengths of A element on polyA recombination after transformation into Escherichia coli, we designed to construct the open reading frame sequences of the GFP gene with four different lengths of polyA into the pUC57-kanamycin vector respectively (the A elements were a 120A fragment, two 60A fragments, two 40A fragments, and three 40A fragments, and the B element was the stimulating sequence SEQ ID NO:8 or the control sequence SEQ ID NO:21: GAUAUC). After transformation into Escherichia coli, positive clones containing the target gene and polyA were selected. For plasmids with different poly-A tails, 50 clones were selected for each, amplified and cultured, and plasmids were extracted for sequencing.

[0054] The recombination ratio refers to the proportion of the number of clones containing an unexpected poly-A tail.

[0055]

[0056]

[0057] The results showed that compared with the absence of the B element, the insertion of the B element and six random nucleotide sequences (SEQ ID NO:21) could significantly reduce the recombination of different A elements in Escherichia coli; and the efficiency of reducing recombination by inserting the B element was significantly higher than that of inserting six random nucleotide sequences.

[0058] Example 3 Effect of different lengths of B element on polyA recombination

[0059] To test the effect of plasmids with different B elements on polyA recombination after transformation into Escherichia coli, we designed to construct the open reading frame sequences of the GFP gene with polyA of different lengths of B elements into the pUC57-kanamycin vector respectively, and the A element was three 40A fragments. The B elements were set as follows in the table.

[0060] The results showed that B elements with different lengths could effectively reduce the recombination ratio of A elements (taking A element sequence 40A*3 as an example). When the length of the B element was 12 - 22 bp, the recombination ratio of the A element was slightly higher. When the length of the B element was 37 bp or more, the recombination ratio of the A element was slightly lower.

[0061]

[0062] Example 4 Effect of transfection of polyA mRNA containing different B element sequences on protein expression

[0063] To test whether polyA post-transcriptional mRNA containing different B element sequences affects protein expression and mRNA stability, we designed a DNA vector for in vitro transcription of linear RNA. This vector sequentially included a T7 promoter, 5' UTR, a protein sequence encoding d2EGFP, 3' UTR, and polyA. Specifically, it was the 11 types mentioned in Example 3, and a total of 11 DNA transcription templates were constructed.

[0064] Transcription was carried out using a T7 in vitro transcription kit. By selecting to use natural uracil or 1-methylpseudouracil when preparing the transcription reaction solution, uncapped natural nucleoside mRNA and modified nucleoside mRNA were obtained respectively. The transcription template was digested with DNaseI, and mRNA was purified by the precipitation method. The mRNA was capped with a Cap1 capping kit, and the capped mRNA was purified with an mRNA purification kit respectively. The purified mRNA was dissolved in acidic sodium citrate buffer for later use.

[0065] HEK293 cells were inoculated into a 96-well plate at a density of 4 * 10 4 cells / well, and after culturing for 24 h, they were transfected with LIP2000 (250 ng / well mRNA), and FACS measurement was performed. The cells were washed with PBS, digested with trypsin, and resuspended in FACS buffer. Propidium iodide staining was added during detection to distinguish live cells and dead cells. The fluorescence intensity of d2EGFP was detected 4 h, 24 h, 48 h, and 72 h after transfection.

[0066] The results showed that compared with the polyA tail mRNA sequence without the addition of the B element, when the length of the inserted B element was 12 - 22 bp, there was no obvious difference in fluorescence intensity. However, when the length of the B element was 37 bp or more, the fluorescence intensity decreased significantly after insertion, indicating that when the length of the inserted B element was 12 - 22 bp, it would not affect protein expression, but when it was greater than 37 bp, it would reduce the protein expression level.

[0067]

[0068] Application of polyA mRNA Containing Different B Element Sequences in Influenza Vaccines

[0069] To test whether polyA post-transcriptional mRNA containing different B element sequences can enhance the immune response, we designed a DNA vector for in vitro transcription of linear RNA. This vector sequentially contains a T7 promoter, 5'UTR, a sequence encoding an influenza virus antigen protein, 3'UTR, and polyA. Specifically, it is groups 1-9 mentioned in Example 3, and a total of 9 DNA transcription templates were constructed.

[0070] Transcription was carried out using a T7 in vitro transcription kit. By choosing to use natural uracil or 1-methylpseudouracil when preparing the transcription reaction solution, uncapped natural nucleoside mRNA and modified nucleoside mRNA were obtained respectively. The transcription templates were digested with DNaseI, and the mRNA was purified by precipitation. The mRNA was capped with a Cap1 capping kit, and the capped mRNA was purified with an mRNA purification kit respectively. The purified mRNA was dissolved in acidic sodium citrate buffer for later use. Cationic lipid: neutral phospholipid: sterol lipid: polyethylene glycol (PEG)-lipid were dissolved and mixed in ethanol at a molar ratio of 45:10:43:2. The total flow rate of the nano-drug manufacturing equipment was set at 12 ml / min. The mRNA solution was encapsulated with the lipid mixed solution at a flow rate ratio of 3:1. After encapsulation, the sample was collected by ultrafiltration and buffer exchange using a tangential flow filtration system, and a sucrose solution was added to obtain mRNA-LNP. BALB / c mice were randomly divided into groups of 8 and immunized with mRNA-LNP at 5 μg / mouse on days 0 and 14. Blood was collected on day 14 (before the second immunization) and day 28 to detect the antibody titer ( Figure 4 ). The mice were sacrificed on day 28, and spleen cells were harvested. Then, they were stimulated with a full-length influenza virus HA overlapping peptide library, and the frequencies of antigen-specific CD4+ and CD8+ cytokine-secreting T cells were evaluated by the ICS method ( Figure 5 and Figure 6 ).

[0071] The results showed that compared with the absence of the B element, the antibody titer increased at different levels after the insertion of the B element, and it induced a Th1-biased response and could also significantly activate the CD8+ T cell response. When the length of the B element was 20 bp, it could better exert the immune-stimulating effect.

[0072] In summary, the nucleic acid molecule provided by the present invention can not only greatly reduce the proportion of polyA recombination, but also enhance the humoral and cellular immune responses.

[0073] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0074] In addition, it should be noted that, in the various specific technical features described in the above specific embodiments, they can be combined in any appropriate manner without conflict. To avoid unnecessary repetition, the present invention will not separately describe various possible combination manners.

Claims

1. A nucleic acid molecule, characterized in that, The nucleic acid molecule comprises: 1) a first nucleic acid sequence that can be transcribed or used to introduce a transcribable nucleic acid sequence; and 2) a second nucleic acid sequence, the second nucleotide sequence comprising: a) at least two A elements, each A element being defined as a nucleotide sequence consisting of 30 to 80 T nucleotides, and b) at least one B element, each B element being a sequence that can activate PRRs post-transcriptionally; wherein the total number of A elements is one more than the total number of B elements, and wherein any two A elements are separated by one B element.

2. The nucleic acid molecule according to claim 1, wherein the number of A elements is 2 - 6, and more preferably, the number of A elements is two, three, or four.

3. The nucleic acid molecule according to claim 1, wherein the A element consists of 45 - 60 T nucleotides, more preferably, the A element consists of 50 - 60 T nucleotides, more preferably, the A element consists of 60 T nucleotides.

4. The nucleic acid molecule according to any one of claims 1 - 2, wherein the number of A elements is four, preferably, the nucleotide sequences of the four A elements together have a total length of 240 nucleotides, and more preferably, each A element has a length of 60 nucleotides.

5. The nucleic acid molecule according to any one of claims 1 - 2, wherein the number of A elements is three, preferably, the nucleotide sequences of the three A elements together have a total length of 180 nucleotides, and more preferably, each A element has a length of 60 nucleotides.

6. The nucleic acid molecule according to any one of claims 1 - 2, wherein the number of A elements is two, preferably, the nucleotide sequences of the two A elements together have a total length of 120 nucleotides, and more preferably, each A element has a length of 60 nucleotides.

7. The nucleic acid molecule according to any one of claims 1 - 6, wherein the B element can activate PRRs post-transcriptionally, preferably with a length of 5 - 250 bp; 5 - 200 bp; 5 - 150 bp; 5 - 100 bp; 5 - 80 bp, 5 - 60 bp, 5 - 50 bp, 5 - 40 bp, 5 - 30 bp, 5 - 20 bp, 5 - 20 bp, 10 - 80 bp, 10 - 60 bp, 10 - 50 bp, 10 - 40 bp, 10 - 30 bp, 10 - 20 bp, 12 - 80 bp, 12 - 60 bp, 12 - 50 bp, 12 - 40 bp, 12 - 30 bp, 12 - 25 bp, 12 - 22 bp.

8. The nucleic acid molecule according to any one of claims 1 - 7, wherein the B element is a TLRs or RLRs post-transcriptionally, more preferably TLR7 / 8 and / or RIG-I / MAD-5 or a combination thereof or a sequence having 95%, 90%, 85%, 80%, 75%, 70% similarity thereto.

9. The nucleic acid molecule according to any one of claims 1 - 8, wherein the post-transcriptional sequence of the B element is a sequence that stimulates RIG-I / MAD-5 or a combination thereof or a sequence having 95%, 90%, 85%, 80%, 75%, 70% similarity thereto.

10. The nucleic acid molecule according to any one of claims 1-9, wherein the post-transcriptional sequence of the B element is an immunostimulatory nucleotide sequence that stimulates TLR7 / 8, characterized in that, It contains a natural or modified base sequence, and the base sequence contains at least one natural or modified guanine and at least one natural or modified uracil, and its general formula is N a G b N c , preferably, wherein G is a natural or modified guanine or uracil, more preferably, the sequence is any one of SEQ ID NO: 1 to SEQ ID NO: 20 or a combination thereof or a sequence having 95%, 90%, 85%, 80%, 75%, 70% similarity thereto.

11. The nucleic acid molecule according to claim 10, wherein N is a nucleic acid sequence having a length of about 4 to 50 nucleic acids, preferably 4 to 30 nucleotides, more preferably 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 nucleotides, and each N is independently selected from natural or modified guanine, uracil, cytosine or adenine.

12. The nucleic acid molecule according to any one of claims 10 - 11, characterized in that, a and c are integers from 1 to 40 independent of each other, preferably 1 to 30, more preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29. When a or c is 1, N is guanine or uracil or its analog. When it is greater than 1, at least 50% of these nucleotides are guanine or uracil or its analog; preferably 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%.

13. The nucleic acid molecule according to any one of claims 10-12, characterized in that, b is an integer from 3 to 40, preferably 3 to 30, more preferably 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39.

14. The nucleic acid molecule according to any one of claims 10-13, characterized in that, The ratio of guanine to uracil in the RNA sequence is between 1G:100U and 30G:1U, preferably between 1G:50U and 10G:1U, more preferably between 1G:40U and 2G:1U, or between 1G:30U and 10G:1U or between 1G:20U and 5G:1U or between 1G:10U and 1G:1U or between 1G:5U and 1G:1U.

15. A method for amplifying a nucleic acid molecule, comprising: (1) providing a nucleic acid molecule as described in any one of claims 1-14 (2) amplifying a bacterium comprising the nucleic acid molecule.

16. A method for obtaining RNA, comprising amplifying the nucleic acid molecule according to the method described in claim 15 and in vitro transcribing the nucleic acid molecule into RNA using it as a template.

17. The method for obtaining RNA according to claim 16, wherein, The RNA includes linear and circular forms; preferably, the linear form is a conventional or self-replicating mRNA.

18. The method for obtaining RNA according to claim 17, wherein, The linear RNA also includes a 5' cap structure, a 5' untranslated region, and a 3' untranslated region.

19. Use of the RNA obtained according to the method described in claims 16-18 in the preparation of a prophylactic vaccine, wherein the protein encoded by the RNA has the potential to induce an immunogenic response. Preferably, the antigen is selected from: a new coronavirus-related antigen, a herpes simplex virus-related antigen, a respiratory syncytial virus-related antigen, a varicella-zoster virus-related antigen, an influenza virus-related antigen, a parainfluenza virus-related antigen, an Epstein-Barr virus-related antigen, a cytomegalovirus-related antigen, a human metapneumovirus-related antigen, a human papillomavirus-related antigen, a rotavirus-related antigen, a dengue virus-related antigen, a malaria-related antigen, a rabies virus-related antigen or a monkeypox virus-related antigen. Use of RNA obtained by the method according to claims 16-18 in the preparation of a therapeutic drug, preferably, the drug is a tumor therapeutic drug, more preferably, the tumor is lung cancer, pancreatic cancer or colon cancer. A method for obtaining a peptide or protein, comprising obtaining RNA by the method according to any one of claims 16-18, and translating the RNA into a peptide or protein sequence.

22. A system for generating eukaryotic translatable mRNA, characterized in that, Comprising 1) a vector or plasmid containing the nucleic acid molecule according to claims 1-14; 2) a host cell.

23. A composition, characterized in that, Comprising RNA obtained by the method according to any one of claims 16-18, or a peptide or protein obtained by the method according to claim 21.

24. The composition according to claim 23, wherein The composition comprises a pharmaceutically acceptable excipient, preferably, the excipient is one or more of a carrier, a buffer, a protecting agent, a stabilizing agent, a surfactant, an osmotic pressure regulator, an adjuvant, a preservative or an inactivating agent.

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