mRNA in-vitro expression plasmid, construction method therefor and use thereof

Abstract

Provided are a nucleic acid molecule and a preparation method therefor and a use thereof. The nucleic acid molecule solves the problem of Poly A loss / shortening, improving the stability of Poly A, achieving homogeneity of a Poly A sequence in a plasmid during production, also improving the stability of the nucleic acid molecule, reducing the recombination probability of a plasmid during amplification, and facilitating industrial production of mRNA products.

Description

An mRNA in vitro expression plasmid, construction method and application thereof Technical Field

[0001] The present invention belongs to the field of genetic engineering and specifically relates to a method for constructing an in vitro mRNA expression plasmid capable of improving plasmid stability and its application. Background Art

[0002] Since the outbreak of the COVID-19 pandemic in late 2019, mRNA vaccines have emerged and, thanks to media coverage, have gradually become widely known. mRNA technology offers numerous advantages: First, mRNA does not integrate into the genome, thus avoiding concerns about insertional mutagenesis. More importantly, unlike relatively stable DNA, mRNA degrades after performing its function, without causing any toxic or side effects. mRNA can be manufactured in a cell-free manner and rapidly and economically mass-produced using in vitro transcription techniques. Its short development cycle allows for rapid, cost-effective, and efficient production. Furthermore, a single mRNA can encode multiple antigens, proteins, or peptides, making the process highly adaptable and theoretically capable of expressing any protein.

[0003] The mRNA COVID-19 vaccine has proven the applicability of the mRNA technology platform in the vaccine field. Beyond COVID-19 vaccines, mRNA technology can also be widely used in infectious diseases, tumors, and protein replacement therapy.

[0004] For mRNA technology, a good production process begins with the design of a safe, stable, and efficient plasmid vector. Whether from a production process perspective, a therapeutic perspective, or a regulatory perspective, most non-essential sequences in plasmid DNA should be removed to minimize molecular weight for easier genetic manipulation. Large plasmids impose a significant metabolic burden on the host bacteria, reducing the cellular resources available for plasmid replication and leading to decreased plasmid DNA yield. Furthermore, in industrial production, plasmids must maintain a high degree of stability during continuous passage to ensure widespread application and stability of mRNA technology.

[0005] There is a problem in the production process of mRNA plasmid technology, that is, the PolyA sequence of the plasmid is easily lost during the bacterial fermentation culture process, and the uniformity of the produced plasmid PolyA sequence cannot be achieved, which does not meet the GMP-level production process and drug regulatory requirements. Moreover, the PolyA sequence is also a very critical element for mRNA stability and translation expression efficiency. Therefore, how to solve the problem of shortening the PolyA sequence in plasmid fermentation production is also the direction that R&D personnel have been working hard on. The current solutions in the industry include: using a segmented polyA tail, represented by the technical solution disclosed in BioNTech's patent US10717982B2, and the technology disclosed in Ethris' patent WO2020074642A1; in addition, other companies use shorter polyA sequences to achieve the purpose of stabilizing the plasmid.

[0006] The above methods are all aimed at improving polyA itself. The present invention conducts further research on plasmids suitable for RNA technology, breaking through the improvement methods only targeting polyA. It considers solving the problem of polyA loss from the perspective of the entire plasmid without affecting plasmid replication and yield, thereby improving plasmid integrity and stability. Summary of the Invention

[0007] The present invention provides a nucleic acid molecule comprising, in the 5'→3' transcription direction, 1) a first nucleic acid fragment, the first nucleic acid fragment comprising transcribable nucleotides or nucleotides for introducing a transcribable nucleotide fragment; 2) a second nucleic acid fragment, the second nucleic acid fragment comprising a nucleic acid sequence encoding a polyadenylation signal; and 3) a third nucleic acid fragment, the third nucleic acid fragment comprising a nucleic acid sequence encoding a transcription termination signal; the third nucleic acid fragment being located downstream of a plasmid replication initiation site (ori).

[0008] Specifically, the third nucleic acid fragment is located 5-300bp downstream of the plasmid replication initiation site (ori), preferably 10-300bp, 30-300bp, 50-300bp, 80-300bp, 100-300bp, 120-300bp, 130-300bp, 150-300bp, 200-300bp, 250-300bp, 5-50bp, preferably 5bp, 6bp, 7bp, 8bp, 9bp, 10bp, 11bp, 12bp, 13bp, 14bp, 15bp, 16bp, 17bp, 18b, 19bp, 20bp, 25b, 30bp, 35bp, 40b, 45bp, 50bp.

[0009] Specifically, the nucleic acid sequence encoding the transcription termination signal is a terminator; preferably, the terminator sequence is 5-100 bp in length; preferably, the terminator sequence is 5-90 bp, 5-80 bp, 5-70 bp, 5-60 bp, 5-50 bp, 5-40 bp, 5-30 bp, or 5-20 bp in length; the terminator is preferably one or more of rrnB T1, ECK120029600, ECK120033737, L3S2P21, ECK120033736, ECK120010818, ECK120015440, Bba B0062, ECK120010799, ECK120010876, ECK120015170, ECK120010869, or Bba B0010. Specifically, the transcribable nucleotide segment includes a nucleotide segment encoding a peptide or protein.

[0010] Specifically, the nucleotide sequence for introducing a transcribable nucleotide fragment includes a multiple cloning site or a homologous recombination site.

[0011] Specifically, the polyadenylation signal comprises at least 60 consecutive adenine nucleotides.

[0012] The polyadenylation signal is segmented, including at least two 20 bp continuous adenine nucleotide segments.

[0013] Specifically, the template plasmid further includes a 5' cap structure, a 5' non-coding region and a 3' non-coding region.

[0014] The present invention further provides a DNA plasmid comprising any one of the nucleic acid molecules described above.

[0015] The present invention further provides a method for preparing a DNA plasmid, which comprises transforming the DNA plasmid into competent Escherichia coli cells, applying an appropriate amount of the plasmid onto a resistant LB medium plate for culture, picking a monoclonal colony, sequencing it, obtaining a positive bacterial solution with correct sequencing, and then conducting large-scale culture.

[0016] The present invention further provides a method for producing a nucleic acid molecule, comprising propagating the template plasmid as described above in a host cell and propagating the host cell, wherein the host cell comprises bacteria or fungi, preferably Escherichia coli;

[0017] The present invention further provides a method for obtaining RNA, comprising amplifying a nucleic acid molecule according to the above method, and using the nucleic acid molecule as a template to transcribe into RNA in vitro.

[0018] The present invention further provides a method for obtaining a peptide or protein, comprising obtaining mRNA encoding the peptide or protein according to the above method, and translating the mRNA into peptide or protein amino acid fragments.

[0019] The present invention further provides a system for producing eukaryotic translatable mRNA, comprising a vector or plasmid of the nucleic acid molecule as described above; and a host cell.

[0020] The present invention further provides a composition comprising the RNA, peptide or protein obtained according to the above method.

[0021] Specifically, the composition is characterized in that it includes one or more of pharmaceutically acceptable excipients, carriers, buffers, protective agents, stabilizers, surfactants, osmotic pressure regulators, adjuvants, preservatives, and inactivators.

[0022] Specifically, the buffer of the present invention includes one or more of HEPES, HIS, TRIS, PB, succinic acid, and citric acid; the protective agent includes one or more of gelatin, ethanol, diethylaminotetraacetic acid (EDTA), disodium diethylaminotetraacetic acid (EDTA-2Na), and magnesium chloride; the stabilizer includes one or more of sucrose, mannitol, fucose, and maltose; the surfactant includes one or more of Tween, Span, and glycerol; the osmotic pressure regulator includes sodium chloride or is omitted; and the auxiliary material components include one or more of mannitol, sucrose, sodium chloride, magnesium chloride, HEPES, polysorbate 80, and glycerol.

[0023] The composition can be prepared as a mucosal immune preparation, a humoral immune preparation, a cellular immune preparation, or a skin immune preparation;

[0024] The composition can be prepared into liquid dosage form, solid dosage form, semisolid dosage form, gas dosage form, and inhalation dosage form;

[0025] The composition can be administered by intravenous injection, intramuscular injection, subcutaneous injection, oral administration, buccal administration, sublingual administration, rectal administration, respiratory tract administration, or transdermal administration. Respiratory tract administration can be inhaled through the mouth, through the nose, or after atomization by an atomizer.

[0026] The present invention further provides uses of the nucleic acid molecule or composition, including use in medicine, for delivering a transgene into a host cell, for eliciting a primary immune response in an animal to treat or prevent at least one disease, for boosting an immune response in an animal, and for inducing an immune response in an animal that breaks tolerance to self-antigens. Such immune responses include vaccines for cancer prevention, therapeutic tumor vaccines, and vaccines for preventing viral, bacterial, and fungal infections.

[0027] The beneficial effects of the present invention include:

[0028] First, the stability of Poly A is improved by the transcription termination signal to solve the problem of Poly A loss / shortening.

[0029] Secondly, the stability of the template plasmid is improved through the transcription termination signal, which reduces the probability of plasmid recombination during amplification and further improves the safety of plasmid application.

[0030] Thirdly, by screening the transcription termination signal, the optimal length range and optimal insertion position are obtained, within which the replication and yield of the plasmid or vector are not affected, and the expression of the target gene is not affected.

[0031] Fourthly, through the transcription termination signal, RNA molecules, peptides or proteins with higher purity and that meet GMP-level production processes and drug regulatory requirements can be obtained.

[0032] The term "vector" is used herein in its general sense as known to those skilled in the art and includes any intermediate carrier of a nucleic acid, for example enabling the nucleic acid to be introduced into a prokaryotic and / or eukaryotic host cell or, where appropriate, integrated into a genome, where the vector is preferably replicated and / or expressed.

[0033] The term "pharmaceutically acceptable carrier" refers to an inactive substance formulated with a pharmaceutically active substance to meet or improve the requirements of the active substance in terms of dosage, adsorption, solubility or pharmacokinetics.

[0034] The term "plasmid" generally relates to a construct of extrachromosomal genetic material, usually a circular DNA duplex, which can replicate independently of the chromosomal DNA.

[0035] The term "host cell" refers to any cell that can be transformed or transfected with exogenous nucleic acid.

[0036] The term "host cell" includes prokaryotes (e.g., E. coli) or eukaryotic cells (e.g., yeast cells and insect cells). Mammalian cells are particularly preferred, such as cells from humans, mice, hamsters, pigs, goats, and primates. Cells can be derived from a variety of tissue types and include primary cells and cell lines.

[0037] The terms "peptide" and "protein" include substances containing not only amino acid components but also non-amino acid components such as sugar and phosphate structures, and also include substances containing bonds such as ester bonds, thioether bonds or disulfide bonds.

[0038] The term "nucleotide fragment encoding a peptide or protein" refers to a nucleotide fragment that, if present in an appropriate environment, preferably in a cell, can direct the translation of the assembly of amino acids to produce a peptide or protein.

[0039] The term "immune response" relates to the immune system's response, for example, to an immunogenic organism, such as a bacterium or virus, a cell, or a substance. The term "immune response" encompasses both innate and adaptive immune responses. Preferably, the immune response involves activation of immune cells, induction of cytokine biosynthesis, and / or antibody production. Preferably, the immune response includes the steps of activation of antigen-presenting cells, such as dendritic cells and / or macrophages, presentation of antigens or fragments thereof by the antigen-presenting cells, and activation of cytotoxic T cells as a result of this presentation.

[0040] The nucleic acid molecules of the present invention, and plasmids or vectors containing the nucleic acid molecules, can be used to express recombinant proteins in cells for transcription and expression. More specifically, when producing recombinant proteins, the expression vectors of the present invention can be used to transcribe recombinant nucleic acids and express recombinant proteins in cell-based systems. For example, recombinant antibodies, hormones, cytokines, enzymes, etc. can be prepared.

[0041] The nucleic acid molecules, plasmids or vectors containing the nucleic acid molecules of the present invention can be used, for example, for transient expression of genes. Possible application areas are RNA-based vaccines, which are transfected into cells in vitro or administered directly to transiently express functional proteins in vivo or in vitro.

[0042] The nucleic acid molecules of the present invention, plasmids or vectors containing the nucleic acid molecules, can be used in gene therapy applications. Thus, the nucleic acid molecules of the present invention can be gene therapy vectors and used to express transgenes. Any nucleic acid (DNA / RNA)-based vector system (e.g., plasmids, adenovirus, poxvirus vectors, influenza virus vectors, alphavirus vectors, etc.) can be used. These vectors can be used to transfect cells in vitro, such as lymphocytes or dendritic cells, or in vivo by direct administration.

[0043] The nucleic acid molecules, plasmids or vectors containing the nucleic acid molecules of the present invention can be used to transfect antigen-presenting cells and thus serve as a means of delivering and loading antigen-presenting cells with the antigen to be presented, which corresponds to or is derived from a peptide or protein RNA expressed from the antigen. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 Effect of transcription termination signal on plasmid copy number under non-resistance conditions

[0045] Figure 2 Effect of transcription termination signal on plasmid copy number under Kana resistance conditions

[0046] Figure 3 Effects of different lengths of transcription termination elements on plasmid copy number under non-resistance conditions

[0047] Figure 4 Effects of different lengths of transcription termination elements on plasmid copy number under Kana resistance conditions

[0048] Figure 5 Effects of inserting transcription termination elements at different positions on the stability of PolyA under non-resistant conditions

[0049] Figure 6 Effects of inserting transcription termination elements at different positions on PolyA stability under Kana resistance conditions

[0050] Figure 7 Effects of different lengths of transcription termination elements on PolyA stability under non-resistant conditions

[0051] Figure 8 Effects of different lengths of transcription termination elements on PolyA stability under Kana resistance conditions

[0052] Figure 9 Changes in plasmid copy number during passage of universal transcription vector under non-resistance conditions

[0053] Figure 10 Changes in plasmid copy number during passage of the universal transcription vector under Kana resistance conditions

[0054] Figure 11 Stability of PolyA during passage of universal transcription vector under non-resistance conditions

[0055] Figure 12 Stability of PolyA during the passage of the universal transcription vector under Kana resistance conditions DETAILED DESCRIPTION

[0056] Example 1 Effect of Inserting Transcription Termination Signals at Different Positions Downstream of Origin on Plasmid Copy Number

[0057] In order to explore the effect of inserting the transcription termination signal into different positions downstream of ori on the plasmid copy number, the present invention inserted the ECK120029600 or dA10 transcription termination sequence into different positions downstream of the transcription vector ori, and observed its effect on the copy number during plasmid passage. The specific design method is shown in Table 1.

[0058] Table 1 Effects of terminator sequence insertion into different positions downstream of ori on plasmid copy number

[0059] The transcription vector of the present invention comprises a 5'UTR sequence, a Kozak sequence, an antigen coding sequence, a 3'UTR sequence, a polyA tail sequence and a terminator sequence, wherein the sequences are synthesized into a pUC57-kana vector by Nanjing KingSher Biotechnology Co., Ltd., and then are sequentially connected to a universal transcription vector by homologous recombination. The present invention compares the effects of inserting ECK120029600 into 5bp, 25bp, 60bp, 90bp, 128bp, 160bp, 200bp, 300bp downstream of ori and inserting dA10 into the transcription vector ori downstream 5bp, 9bp, 15bp, 20bp position on the plasmid copy number. The constructed plasmid is transferred into Trans1-Blue Escherichia coli competent cells, an appropriate amount is applied to a Kana-resistant LB culture plate and cultured at 37°C for a certain period of time, a monoclonal colony is picked, sequenced, and the positive bacterial solution with correct sequencing is preserved. The frozen bacterial suspension was inoculated into kanamycin-resistant LB medium at a ratio of 1:100 and incubated in a 37°C constant-temperature shaker for 4-6 hours for activation. Subsequently, a certain amount was inoculated into fresh LB medium containing kanamycin resistance and fresh LB medium without resistance at a ratio of 1:1000 and incubated for 8-16 hours. After the incubation period, the bacterial suspension was removed and stored in two tubes of glycerol, each labeled as the first generation. An appropriate amount of the first-generation bacterial suspension was inoculated into kanamycin-resistant LB medium and fresh LB medium without resistance at a ratio of 1:1000. The second-generation bacterial suspension was harvested and stored as described above, and the same procedure was repeated until the 10th generation. Plasmid copy number was detected by qPCR using the first, fifth, and tenth generation bacterial suspensions. The results are shown in Figures 1 and 2. The results showed that inserting ECK120029600 at the position 128bp-300bp downstream of ori and inserting dA10 at the position 5-20bp downstream of ori had no significant effect on the copy number of the plasmid. It is preferred that ECK120029600 be inserted 128bp downstream of ori and dA10 be inserted 9bp downstream of ori.

[0060] Example 2 Effects of Different Lengths of Transcription Termination Elements on Plasmid Copy Number

[0061] In order to explore the effect of different lengths of transcription termination elements on the plasmid copy number, the present invention further selected ECK120010869, ECK120015170, L3S2P21, rrnB T1 or dA5, dA10, dA20 transcription termination sequences to be inserted downstream of the transcription vector ori to observe their effects on the plasmid copy number. The specific design methods are shown in Table 2.

[0062] Table 2. Effects of different lengths of transcription termination elements on plasmid copy number.

[0063] The transcription vector of the present invention comprises a 5'UTR sequence, a Kozak sequence, a 3'UTR sequence, a polyA tail sequence and a terminator sequence, wherein the sequences are synthesized into the pUC57-kana vector by Nanjing KingSher Biotechnology Co., Ltd., and then they are sequentially connected to the universal transcription vector by homologous recombination. The constructed plasmid is transferred into Trans1-Blue Escherichia coli competent cells, and an appropriate amount is applied to a Kana-resistant LB culture medium plate and cultured at 37°C for a certain period of time. Monoclonal colonies are picked and sequenced, and the positive bacterial solution with correct sequencing is preserved. The bacterial solution is passaged according to the method described in Example 1 to obtain bacterial solutions of the 1st, 5th and 10th generations, and the plasmid copy number is detected by the qPCR method. The results are shown in Figures 3 and 4. The results show that the insertion of transcription termination elements of different lengths downstream of ori has no significant effect on the plasmid copy number during passage. Example 3 Effect of insertion of transcription termination elements at different positions on the stability of PolyA

[0064] In order to explore the effect of inserting the transcription termination signal into different positions downstream of ori on the stability of PolyA, the present invention inserted the ECK120029600 or dA10 transcription termination sequence into different positions downstream of the transcription vector ori, constructed a recombinant plasmid, and transformed the plasmid into Trans1-Blue Escherichia coli competent cells to obtain a positive clone with correct sequencing. Then, according to the above method, the bacterial solution of the 1st, 5th and 10th generations was extracted with plasmids and sent for sequencing to observe the stability of polyA during the passage process. The specific design method is shown in Table 3.

[0065] Table 3 Effects of terminator sequence insertion into different positions downstream of ori on the stability of PolyA

[0066] The results are shown in Figures 5 and 6. Compared with the control group, ECK120029600 inserted 128bp-300bp downstream of ori and dA10 inserted 5-20bp downstream of ori can significantly improve PolyA stability. Preferably, ECK120029600 is inserted 128bp downstream of ori, and preferably, dA10 is inserted 9bp downstream of ori.

[0067] Example 4 Effects of Transcription Termination Elements of Different Lengths on PolyA Stability

[0068] In order to explore the effect of transcription termination elements of different lengths on PolyA stability, the present invention further selected ECK120010869, ECK120015170, L3S2P21, rrnB T1 or dA5, dA10, dA20 transcription termination sequences to be inserted downstream of the transcription vector ori, respectively. According to the above method, plasmids were extracted from the bacterial solution of the 1st, 5th and 10th generations and sent for sequencing to observe their effects on PolyA stability. The specific design method is shown in Table 4.

[0069] Table 4. Effects of different lengths of transcription termination elements on PolyA stability

[0070] The results are shown in Figures 7 and 8 . The results show that compared with the control group, the insertion of transcription termination elements of different lengths downstream of ori can significantly improve the stability of PolyA, especially ECK120029600 and dA10-10.

[0071] Example 5 Determination of a universal high-stability plasmid vector

[0072] In order to further verify the versatility of the above-mentioned plasmid vector, the present invention inserted the target gene sequences derived from five different pathogens, including novel coronavirus (COVID-19), dengue virus (DENV), malaria, varicella zoster virus (VZV) and feline parvovirus (FPV), into the above-mentioned vectors containing ECK120029600 (128 bp downstream of Ori) and dA10 (9 bp downstream of Ori), respectively, and constructed them into expression vectors P001-P010, and observed the copy number content and the stability of continuous 100A during plasmid passage.

[0073] The results showed that ECK120029600 and dA10 were universally applicable to different target gene sequences, had no significant effect on plasmid copy number after insertion into plasmid vectors, and could ensure the polyA stability of each recombinant plasmid within 10 generations, with dA10 being preferred (as shown in Figures 9, 10, 11, and 12).

[0074] In summary, the present invention provides a universal high-stability plasmid vector, which has universal applicability and can solve the problem of plasmid instability caused by polyA sequences in the mRNA field.

Claims

1. A nucleic acid molecule, characterized in that In the 5'→3' transcription direction, it comprises 1) a first nucleic acid fragment, which includes transcribable nucleotides or nucleotides for introducing a transcribable nucleotide fragment; 2) a second nucleic acid fragment, which includes a nucleic acid sequence encoding a polyadenylation signal; and 3) a third nucleic acid fragment, which includes a nucleic acid sequence encoding a transcription termination signal; the third nucleic acid fragment is located downstream of the plasmid replication initiation site (ori).

2. The nucleic acid molecule according to claim 1, wherein The third nucleic acid fragment is located 5-300bp downstream of the plasmid replication initiation site (ori), preferably 10-300bp, 30-300bp, 50-300bp, 80-300bp, 100-300bp, 120-300bp, 130-300bp, 150-300bp, 200-300bp, 250-300bp, 5-50bp, preferably 5bp, 6bp, 7bp, 8bp, 9bp, 10bp, 11bp, 12bp, 13bp, 14bp, 15bp, 16bp, 17bp, 18b, 19bp, 20bp, 25b, 30bp, 35bp, 40b, 45bp, 50bp.

3. The nucleic acid molecule according to any one of claims 1 or 2, wherein The nucleic acid sequence encoding the transcription termination signal is a terminator; preferably, the length of the terminator sequence is 5-100bp; preferably, the length is 5-90bp, 5-80bp, 5-70bp, 5-60bp, 5-50bp, 5-40bp, 5-30bp, 5-20bp; the terminator is preferably one or more of rrnB T1, ECK120029600, ECK120033737, L3S2P21, ECK120033736, ECK120010818, ECK120015440, Bba B0062, ECK120010799, ECK120010876, ECK120015170, ECK120010869 or Bba B0010.

4. The nucleic acid molecule according to any one of claims 1 to 3, wherein The nucleic acid fragment encoding the transcription termination signal is 5-20 bp of continuous adenylate, preferably 5 bp-10 bp, 5-15 bp, 10-20 bp, or 15-20 bp.

5. The nucleic acid molecule according to any one of claims 1 to 4, wherein The transcribable nucleotide fragments include nucleotide fragments encoding peptides or proteins.

6. The nucleic acid molecule according to any one of claims 1 to 5, wherein The nucleotides for introducing transcribable nucleotide fragments include a multiple cloning site or a homologous recombination site.

7. The nucleic acid molecule according to any one of claims 1 to 6, wherein The polyadenylation signal comprises at least 60 consecutive adenine nucleotides.

8. The polyadenylation signal according to claim 7, wherein The polyadenylation signal is segmented, including at least two 20 bp continuous adenine nucleotide segments.

9. The nucleic acid molecule according to any one of claims 1 to 8, characterized in that The nucleic acid molecule further comprises a 5' cap structure, a 5' non-coding region and a 3' non-coding region.

10. The nucleic acid molecule according to any one of claims 1 to 9, wherein The nucleic acid molecule is an expression vector or a plasmid.

11. A DNA plasmid, characterized in that The DNA plasmid comprises the nucleic acid molecule of any one of claims 1-9.

12. A method for preparing a DNA plasmid, characterized in that: The DNA plasmid of claim 11 is transformed into competent E. coli cells, and an appropriate amount is applied to a resistant LB medium plate for culture, and a monoclonal colony is picked and sequenced to obtain a positive bacterial solution with correct sequencing, and then large-scale culture is carried out.

13. A method for amplifying a nucleic acid molecule, characterized in that: comprising propagating the nucleic acid molecule of claims 1 to 9 in a host cell and propagating the host cell, the host cell comprising bacteria or fungi, preferably Escherichia coli; 14. A method for obtaining RNA, characterized in that: The method comprises amplifying a nucleic acid molecule according to the method of claim 13, and using the nucleic acid molecule as a template to transcribe into RNA in vitro.

15. A method for obtaining a peptide or protein, characterized in that: The method comprises obtaining mRNA encoding a peptide or protein according to the method of claim 14, and translating the mRNA into peptide or protein amino acid fragments.

16. A system for producing eukaryotic translatable mRNA, characterized in that The invention comprises 1) a vector or plasmid containing the nucleic acid molecule according to claim 1-15; 2) a host cell.

17. A composition, characterized in that Comprising RNA, peptide or protein obtained according to the method of claim 14 or 15.

18. The composition according to claim 17, characterized in that The composition comprises one or more of pharmaceutically acceptable excipients, carriers, buffers, protective agents, stabilizers, surfactants, osmotic pressure regulators, adjuvants, preservatives, and inactivators.

19. Use of the nucleic acid molecule according to any one of claims 1 to 10, the system according to claim 16, or the composition according to any one of claims 17 to 18, characterized in that: The invention also includes use in medicine, for delivering transgenes into host cells, for eliciting a primary immune response in an animal to treat or prevent at least one disease, for enhancing an immune response in an animal, and for inducing an immune response in an animal to break tolerance to self-antigens.

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