A modified mRNA vaccine against tetanus and its preparation method

Abstract

The present invention relates to the field of biomedical technologies, and more specifically to a modified mRNA vaccine against tetanus and its preparation method. A modified mRNA vaccine against tetanus, characterized in that: the vaccine comprises: (1) mRNA for expressing a tetanus toxin immunogen, the immunogen being one of an Hc protein, an Hc-TRX protein, and an Hn+Hc protein; (2) a vaccine carrier, the vaccine carrier being a liposome nanoparticle that can be utilized by the mRNA. The main advantages of the present invention are: (1) for the first time, the C-terminal domain of the heavy chain of tetanus toxin (Hc), the fusion protein of the C-terminal domain of the heavy chain and thioredoxin (TRX-Hc), and the complete heavy chain of tetanus toxin (Hn+Hc) are selected as antigens to develop a modified mRNA vaccine; (2) the three developed tetanus toxin mRNA vaccines can all effectively induce specific antibody responses.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and specifically to a modified mRNA vaccine against tetanus and a preparation method thereof. Background Art

[0002] Clostridium tetani, abbreviated as C. tetani, is the pathogen of tetanus and is usually present in soil, saliva, dust, feces, and intestines. C. tetani enters the human body through wounds on the skin, and the exotoxins produced cause symptoms such as muscle spasms, fever, sweating, headache, etc., and some cases can lead to death. In 2017, approximately 38,000 people died globally from C. tetani infections, and nearly half (49%) of the tetanus death cases occurred in children under 5 years old.

[0003] C. tetani is a common Gram-positive bacterium, and its spores have extremely strong resistance to temperature and can exist in the soil for a long time. The C. tetani genome consists of a 2799 kb long circular chromosome and has 2368 open reading frames. C. tetani infects the body and produces tetanus spasm toxin (tetanus toxin) (TeNT) and tetanolysin. Tetanus toxin is an important cause of tetanus disease, a 151 kDa protein, which consists of three domains: an N-terminal light chain domain (L, 50 kDa) and a heavy chain (H, 100 kDa); the heavy chain H includes an internal translocation domain (Hn, 50 kDa) and a C-terminal receptor-binding heavy chain domain (Hc, 50 kDa), and the light chain and the heavy chain are connected by a disulfide bond. The light chain L is a zinc metalloprotease that can block the release of inhibitory neurotransmitters, and the inhibition of neurotransmitter release leads to muscle spasm symptoms in infected individuals. The heavy chain H is responsible for binding the toxin to the axonal membrane and has two functional domains: fragment B (Hn domain) and fragment C (Hc domain). The Hn domain delivers the light chain into the neuron cell with the help of a transporter, blocking the release of inhibitory neurotransmitters, resulting in muscle spasms, difficulty breathing, and even death. The Hc domain is responsible for the binding of the toxin to the target cell receptors (ganglioside receptors and synaptic protein receptors), is a non-toxic region that binds to the axonal membrane, and at the same time retains various properties such as the binding of the intact toxin to gangliosides.

[0004] Compared with passive immunization and drug treatment, active immunization vaccine products are an ideal way to combat Clostridium tetani infection. With the in-depth understanding of the pathogenic mechanism of Clostridium tetani and the continuous innovation and improvement of vaccine technology, great progress has been made in the research and development of tetanus vaccines in recent years, and a new generation of safe and highly effective vaccines is just around the corner. The research history of tetanus vaccines is relatively long, and there are various forms of existing vaccine platforms, such as toxoid vaccines, subunit protein vaccines, nucleic acid vaccines, and bacterial or viral vector vaccines, etc. Current tetanus vaccines are generally prepared based on inactivated full-length tetanus toxin, with relatively large side effects. Despite the widespread vaccination globally, tetanus remains an important cause of death, especially neonatal tetanus in developing countries has not been completely improved.

[0005] The traditional method of preparing tetanus toxoid is relatively dangerous. The spores formed by Clostridium tetani pollute the environment, and the exotoxin it produces also has strong neurotoxicity. Therefore, it is imperative to develop new tetanus vaccines. An ideal tetanus vaccine should have a long-lasting and safe antitoxin protection effect against tetanus toxin, and at the same time meet the requirements of convenient and rapid preparation and low production cost, suitable for mass vaccination, especially in economically backward countries and regions where tetanus is prevalent. From the current development status of the tetanus vaccine field, with the gradual exploration of the process of Clostridium tetani invading the body and the toxin pathogenic mechanism, and the continuous development of new vaccine platforms, future tetanus vaccines will undoubtedly gradually solve many existing problems of current vaccines that cannot be ignored.

[0006] In summary, there is an urgent need in this field to develop new and highly effective tetanus vaccines that can induce protective antibodies against tetanus toxin. Summary of the Invention

[0007] In order to overcome the deficiencies of the prior art, the present invention provides a modified tetanus mRNA vaccine and its preparation method, which not only has the characteristics of high safety, strong immunogenicity, and easy production, but also has technological advancement.

[0008] To achieve the above object, a modified tetanus mRNA vaccine is designed, which is characterized in that: the vaccine includes:

[0009] (1) mRNA for expressing tetanus toxin immunogen, and the immunogen is one of Hc protein, Hc-TRX protein, and Hn + Hc protein;

[0010] (2) Vaccine carrier, and the vaccine carrier is a liposome nanoparticle that can be utilized by mRNA.

[0011] The described Hc protein is the C-terminal domain of tetanus toxin heavy chain; the Hc-TRX protein is a fusion protein of the C-terminal domain of tetanus toxin heavy chain and thioredoxin; the Hn+Hc protein is the complete heavy chain of tetanus toxin.

[0012] The described optimized immunogenic mRNA sequences are as shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3.

[0013] The described optimized immunogenic protein amino acid sequences are as shown in SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6.

[0014] The described mRNA sequence has the following structure: A1 - A2 - A3 - A4 - A5 - A6:

[0015] A1 is a 5' cap structure, preferably a Cap1 structure;

[0016] A2 is a 5' UTR element;

[0017] A3 is a signal peptide coding sequence, preferably an IgE signal peptide coding sequence;

[0018] A4 is a tetanus toxin antigen coding sequence, where the tetanus toxin antigen is selected from the Hc protein, the Hc-TRX protein, and the Hn+Hc protein;

[0019] A5 is a 3' UTR element;

[0020] A6 is a poly(A) tail structure, preferably 101 As.

[0021] The described signal peptide coding sequence includes those from tissue-type plasminogen activator (tPA), from serum immunoglobulin heavy chain (IgE), and from immunoglobulin kappa gene (IgK).

[0022] The described immunogenic mRNA sequence is a modified RNA, and the described modified RNA includes N1-methyl-pseudouridine, N6-methyladenosine, pseudouridine, and 5-methylcytidine.

[0023] The described liposome nanoparticles include PEG-modified cationic lipids, neutral lipids, cationic lipids, cholesterol, or cholesterol analogs.

[0024] The average particle size of the described liposome nanoparticles is in the range of 10 - 500 nm, preferably in the range of 50 - 120 nm.

[0025] The mass ratio of the described liposome nanoparticles to the optimized immunogenic mRNA is 10 - 30:1.

[0026] The cationic lipids described above include Dlin-MC3-DMA, DODMA, DODAP, ALC-0315, and SM102.

[0027] The cholesterol analogs described above include campesterol, stigmastanol, ergosterol, and lanosterol.

[0028] A method for preparing a modified tetanus mRNA vaccine, the specific process is as follows:

[0029] S1, Codon optimization of the antigen gene sequence using human as the host, synthesizing the corresponding antigen expression gene, and obtaining three tetanus toxin antigen expression genes: Hc protein, Hc-TRX protein, and Hn+Hc protein;

[0030] S2, Cloning the three tetanus toxin antigen expression genes into the plasmid vector 5′UTR-IgE-3’UTR-pok12 respectively to obtain three corresponding recombinant plasmids;

[0031] S3, Prepare three kinds of mRNA;

[0032] S4, Dissolve the four lipids D-Lin-MC3-DMA, DSPC, cholesterol, and 14:0PEG2000 PE with ethanol respectively;

[0033] S5, Mix them in a certain molar ratio to obtain a lipid mixture, where D-Lin-MC3-DMA:DSPC:cholesterol:14:0PEG2000 PE = 50:10:38.5:1.5;

[0034] S6, Add the lipid mixture and the citrate buffer solution dissolved with mRNA to the two inlet channels of the NanoAssemblr TM machine respectively, collect the mRNA-LNP mixture, and obtain the vaccine.

[0035] The specific process for preparing the three kinds of mRNA in step S3:

[0036] S31, Use PCR to amplify the target fragment as the in vitro transcription template, use the in vitro transcription kit of NEB company for in vitro transcription, and completely replace UTP with modified nucleotides at the same time;

[0037] S32, After the reaction, use DNase to digest to remove the DNA in the reaction;

[0038] S33, Use the method of LiCL precipitation of RNA to remove impurities such as proteins and salts in the RNA;

[0039] S34. Use the Cap1 capping kit and the tailing kit from NEB to perform capping and tailing respectively to obtain the corresponding mRNA.

[0040] The modified nucleotide is N1-methylpseudouridine triphosphate.

[0041] The capping kit is the mRNA Cap 2′-O-Methyltransferase and Vaccinia Capping System kit; the tailing kit is the E. coli Poly(A) Polymerase.

[0042] Use of a modified mRNA vaccine against tetanus, characterized in that:

[0043] (1) Prevent tetanus;

[0044] (2) Induce a specific immune response against tetanus toxin in the administered subject.

[0045] The main advantages of the present invention are: (1) For the first time, the C-terminal domain of the heavy chain (Hc) of tetanus toxin, the fusion protein of the C-terminal domain of the heavy chain and thioredoxin (TRX-Hc), and the complete heavy chain of tetanus toxin (Hn + Hc) are selected as antigens to develop a modified mRNA vaccine; (2) The three developed tetanus toxin mRNA vaccines can effectively induce a specific antibody response. Description of the Drawings

[0046] Figure 1 It is a diagram for identifying the recombinant plasmid of tetanus toxin antigen. As shown in the figure, the gel electrophoresis diagrams of the PCR products are Hc, TRX-Hc, and Hn + Hc from left to right.

[0047] Figure 2 It is a curve graph for analyzing the particle size of the tetanus toxin mRNA vaccine.

[0048] Figure 3 It is a diagram showing the binding antibody levels induced by the tetanus toxin Hc-mRNA, TRX-Hc-mRNA, and Hn + Hc-mRNA vaccines in mice. Detailed Embodiments

[0049] The present invention will be further described below with reference to the drawings.

[0050] I. Experimental Method:

[0051] 1. Construct a recombinant plasmid:

[0052] The present invention designs an mRNA vaccine based on the tetanus toxin sequence. First, the antigen gene sequences (Hc, TRX-Hc, and Hn+Hc) optimized for codons with human as the host are amplified; then, the amplified target gene fragments are inserted into the plasmid vector 5’UTR-IgE-3’UTR-pok12 using a homologous recombination kit to obtain the corresponding recombinant plasmids pok12-Hc, pok12-TRX-Hc, and pok12-Hn+Hc; finally, identification is performed by PCR and sequencing.

[0053] 2. Preparation of tetanus toxin antigen mRNA:

[0054] First, the target fragment is amplified by PCR as an in vitro transcription template; then, an in vitro transcription kit from NEB is used for in vitro transcription, and at the same time, natural nucleotides are completely replaced with modified nucleotides; finally, capping and tailing are respectively performed using the Cap1 capping kit and the tailing kit from NEB to obtain the corresponding mRNA.

[0055] 3. Preparation of tetanus toxin mRNA vaccine:

[0056] First, dissolve these four lipids (D-Lin-MC3-DMA, DSPC, cholesterol, and 14:0 PEG2000 PE) with ethanol respectively and mix them in a certain molar ratio (D-Lin-MC3-DMA:DSPC:cholesterol:14:0 PEG2000 PE = 50:10:38.5:1.5); then add the lipid mixture and the citrate buffer solution dissolved with mRNA into the two inlet channels of the NanoAssemblr TM machine, collect the mRNA-LNP mixture, and the collected mRNA-LNP is used for animal immunization after ultrafiltration and filtration.

[0057] 4. Detection of the properties and encapsulation rate of LNP-mRNA:

[0058] The encapsulation rate of mRNA is detected using the Quant-it RiboGreen RNA assay kit. Take two PCR tubes: Tube A and Tube B. Add TAE without denaturant and mRNA-LNP to Tube A, and add TAE containing 1% Triton-X100 and mRNA-LNP to Tube B; incubate Tube A and Tube B at room temperature for 10 min; add the solutions in the above two PCR tubes into a 96-well plate respectively, and add the TAE solution containing RiboGreen dye. Place the 96-well plate in a multifunctional microplate reader and select the fluorescence value (excitation wavelength 485 nm, emission wavelength 530 nm) option to detect the encapsulation rate.

[0059] 5. Immunization of experimental animals and serum collection:

[0060] Six- to eight-week-old female Balb / c mice were immunized by intramuscular injection. A total of two immunizations were performed, with a two-week interval between each. Serum was collected one week after the last immunization to detect the immune response.

[0061] 6. Detection of binding antibody levels using ELISA:

[0062] The coated antigen was inactivated tetanus toxin. 400-fold diluted tetanus toxin was added to each well of a 96-well ELISA plate and incubated overnight at 4°C; ELISA was blocked with PBST solution containing 5% skim milk powder and placed at 37°C for 2 h; the inactivated serum was diluted in a 3-fold gradient, the supernatant in the ELISA plate was discarded, the diluted serum was added to the ELISA plate and placed at 37°C for 1 h; the supernatant was discarded, the plate was washed 3 times with 1×PBST, HRP-conjugated anti-mouse IgG antibody was added and placed at 37°C for 1 h; the supernatant was discarded, the plate was washed 3 times with 1×PBST, TMB substrate was added for color development for 2 min, and then the color development reaction was terminated with 2 M HCl. The OD450 value corresponding to each well was read using an ELISA reader, the data was statistically analyzed, and the endpoint dilution of each serum was calculated.

[0063] II. Experimental results:

[0064] 1. Construction and identification of recombinant plasmids:

[0065] Design of tetanus toxin mRNA vaccine: Tetanus toxin contains two chains - the heavy chain and the light chain. The heavy chain includes the N-terminal (Hn) and the C-terminal (Hc). Hc mediates the binding of tetanus toxin to target cells and is an antigen that is widely used and can induce neutralizing antibodies. The three antigens selected in the present invention are respectively: (1) the C-terminal Hc of the tetanus toxin heavy chain; (2) the fusion protein of the C-terminal (Hc) of the tetanus toxin heavy chain and human thioredoxin (TRX), TRX-Hc, and TRX can increase the secretion of the Hc protein; (3) the entire N-terminal and C-terminal (Hn + Hc) of the tetanus toxin heavy chain, and Hn is beneficial to the formation of the natural conformation of Hc and is beneficial to inducing a high-titer antibody response. The codons of the antigen gene sequences were optimized with humans as the host, and the corresponding antigen expression genes were synthesized, named TeNT-Hc, TeNT-Hc-TRX, and TeNT-Hn + Hc respectively. The synthesized antigen sequences were inserted into the plasmid vector 5’UTR-IgE-3’UTR-pok12 using a homologous recombination kit to obtain the corresponding recombinant plasmids.

[0066] As Figure 1As shown, the gene expression sequences of three antigens were successfully inserted into a plasmid with mRNA translation elements to obtain recombinant plasmids. The target bands were amplified by PCR method, which were consistent with the expected size. The product sizes of Hc, TRX-Hc, and Hn+Hc were 1578 bp, 1965 bp, and 2769 bp, respectively.

[0067] Preparation of tetanus toxin mRNA: First, the successfully amplified and sequenced plasmid was used to prepare a linearized DNA template; then, the DNA was in vitro transcribed into RNA using an in vitro transcription kit; finally, the mRNA was prepared using a capping and tailing kit.

[0068] 2. Detection of the particle size of liposome nanoparticles:

[0069] Four lipids, namely cationic lipid (D-Lin-MC3-DMA), neutral lipid (DSPC), cholesterol, and PEG lipid (14:0 PEG2000 PE), were selected as the basic components of LNP. The four lipids in LNP were assembled in a certain molar ratio (D-Lin-MC3-DMA:DSPC:Cholesterol:PEG2000 PE 2000 = 50:10:38.5:1.5). Tetanus toxin mRNA and nuclease-free water were encapsulated with LNP and named V1 (TeNT-Hc), V2 (TeNT-Hc-TRX), V3 (TeNT-Hn+Hc), and V4 (Empty-LNP). The successfully prepared mRNA-LNP needed to go through steps such as dialysis, concentration, and filtration before immunizing mice, and the particle size distribution of LNP was detected by a Malvern nanoparticle size analyzer (Malvern Zetasizer Nano ZS). As Figure 2 shown, the average particle size of mRNA-LNP was between 50 - 80 nm; the average particle sizes of V1 (TeNT-Hc), V2 (TeNT-Hc-TRX), V3 (TeNT-Hn+Hc), and V4 (Empty-LNP) were 78 nm, 80 nm, 85 nm, and 52 nm, respectively.

[0070] 3. Immune responses induced by tetanus toxin TRX-TTC and HN vaccines:

[0071] Tetanus toxin mRNA vaccine induced binding antibodies: As Figure 3 shown in A, each mouse was immunized with 10 μg of the vaccine, and a total of two doses were given. Serum was collected one week after the last immunization to detect the antibody response induced by the vaccine. The collected serum was serially diluted, and ELISA plates were coated with tetanus toxin antigen. The binding degree of antibodies in the serum to different antigen components and the virus was detected by ELSIA experiment. The experimental results showed ( Figure 3B), after the serum was diluted 400-fold, the OD450 values of the three immunization groups of V1 (Hc), V2 (TRX-Hc), and V3 (Hn+Hc) were around 2.8, 2.5, and 3.0 respectively. All three immunogen groups induced strong IgG antibody levels, and no tetanus toxin-specific antibody response was detected in the negative control group V4 (empty LNP). Among them, the binding degree of the binding antibody to tetanus toxin in the sera of the V1 (Hc) and V3 (Hn+Hc) immunization groups was significantly stronger than that of the V2 (TRX-Hc) group. In addition, the three immunization groups still had binding strength even when the serum dilution was diluted to hundreds of thousands of times, and the end-point dilution factors were 409,600, 102,400, and 537,031( Figure 3 C).

[0072] In summary, all three tetanus toxin mRNA vaccines can induce the production of specific antibodies. The mRNA vaccine against tetanus toxin was successfully prepared, indicating that this type of vaccine has good immunogenicity and can induce a high-intensity specific antibody response.

[0073] Sequence information:

[0074] SEQ ID NO Description SEQ ID NO.1 Nucleotide sequence of optimized immunogenic mRNA-Hc protein SEQ ID NO.2 Nucleotide sequence of optimized immunogenic mRNA-Hc-TRX protein SEQ ID NO.3 Nucleotide sequence of optimized immunogenic mRNA-Hn+Hc protein SEQ ID NO.4 Amino acid sequence of optimized immunogenic mRNA-Hc protein SEQ ID NO.5 Amino acid sequence of optimized immunogenic mRNA-Hc-TRX protein SEQ ID NO.6 Amino acid sequence of optimized immunogenic mRNA-Hn+Hc protein

[0075]

[0076]

[0077]

[0078]

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[0080]

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[0085] Sequence Listing <110> Shanghai Sailun Biotechnology Co., Ltd. <120> A Modified mRNA Vaccine Against Tetanus Toxin and Its Preparation Method <130> Sailun Invention - Party <160> 6 <170> SIPO Sequence Listing 1.0 <210> 1 <211> 1578 <212> DNA <213> Nucleotide sequence of optimized immunogenic mRNA-Hc protein (2 Ambystoma laterale x Ambystoma jeffersonianum) <400> 1 aaataagaga gaaaagaaga gtaagaagaa atataagagc caccatggac tggacctgga 60 ttctgttcct cgtggccgcc gctactcgtg tgcactctaa gaacctcgac tgctgggtgg 120 acaacgagga ggacatcgac gtgatcctca agaagtccac catcctgaac ctcgacatca 180 acaacgacat catctctgac atctctggct tcaactccag cgtgatcacc taccccgacg 240 cccagctcgt gcccggcatc aacggcaagg ccatccacct cgtgaacaac gagtctagcg 300 aggtgatcgt gcacaaggct atggacatcg agtacaacga catgttcaac aacttcaccg 360 tgtctttctg gctcagggtg cctaaggtga gcgcctccca cctggagcag tacggcacca 420 acgagtactc tatcatctct agcatgaaga agcacagcct gtctatcggc tctggctggt 480 ccgtgagcct gaagggcaac aacctgatct ggaccctgaa ggactccgcc ggcgaggtga 540 ggcagatcac cttccgggac ctgcccgaca agttcaacgc ctacctcgcc aacaagtggg 600 tgttcatcac catcaccaac gacagactgt ctagcgccaa cctgtacatc aacggcgtgc 660 tcatgggctc cgccgagatc accggcctcg gcgccatccg ggaggacaac aacatcaccc 720 tcaagctgga cagatgcaac aacaacaacc agtacgtgtc tatcgacaag ttcagaatct 780 tctgcaaggc cctgaaccct aaggagatcg agaagctgta caccagctac ctgtctatca 840 ccttcctcag agacttctgg ggcaaccctc tccgctacga caccgagtac tacctgatcc 900 ccgtggccag cagcagcaag gacgtgcagc tgaagaacat caccgactac atgtacctga 960 ccaacgcccc ttcttacacc aacggcaagc tgaacatcta ctaccgcaga ctgtacaacg 1020 gcctcaagtt catcatcaag agatacaccc ctaacaacga gatcgactct ttcgtgaagt 1080 ctggcgactt catcaagctg tacgtgtctt acaacaacaa cgagcacatc gtgggctacc 1140 ctaaggacgg caacgccttc aacaacctcg acagaatcct cagagtgggc tacaacgccc 1200 ccggcatccc tctctacaag aagatggagg ccgtgaagct cagggacctt aaaacctact 1260 ccgtgcagct gaagctgtac gacgacaaga acgccagcct gggcctcgtg ggcacccaca 1320 acggccagat cggcaacgac cctaacagag acatcctgat cgcctctaac tggtacttca 1380 accacctgaa ggacaagatc ctcggctgcg actggtactt cgtgcctacc gacgagggct 1440 ggaccaacga ccatcaccat caccatcact gataataggc tggagcctcg gtggccatgc 1500 ttcttgcccc ttgggcctcc ccccagcccc tcctcccctt cctgcacccg tacccccgtg 1560 gtctttgaat aaagtctg 1578 <210> 2 <211> 1578 <212> DNA <213> Nucleotide sequence of optimized immunogenic mRNA-Hc-TRX protein (2 Ambystoma laterale x Ambystoma jeffersonianum) <400> 2 aaataagaga gaaaagaaga gtaagaagaa atataagagc caccatggac tggacctgga 60 ttctgttcct cgtggccgcc gctactcgtg tgcactctaa gaacctcgac tgctgggtgg 120 acaacgagga ggacatcgac gtgatcctca agaagtccac catcctgaac ctcgacatca 180 acaacgacat catctctgac atctctggct tcaactccag cgtgatcacc taccccgacg 240 cccagctcgt gcccggcatc aacggcaagg ccatccacct cgtgaacaac gagtctagcg 300 aggtgatcgt gcacaaggct atggacatcg agtacaacga catgttcaac aacttcaccg 360 tgtctttctg gctcagggtg cctaaggtga gcgcctccca cctggagcag tacggcacca 420 acgagtactc tatcatctct agcatgaaga agcacagcct gtctatcggc tctggctggt 480 ccgtgagcct gaagggcaac aacctgatct ggaccctgaa ggactccgcc ggcgaggtga 540 ggcagatcac cttccgggac ctgcccgaca agttcaacgc ctacctcgcc aacaagtggg 600 tgttcatcac catcaccaac gacagactgt ctagcgccaa cctgtacatc aacggcgtgc 660 tcatgggctc cgccgagatc accggcctcg gcgccatccg ggaggacaac aacatcaccc 720 tcaagctgga cagatgcaac aacaacaacc agtacgtgtc tatcgacaag ttcagaatct 780 tctgcaaggc cctgaaccct aaggagatcg agaagctgta caccagctac ctgtctatca 840 ccttcctcag agacttctgg ggcaaccctc tccgctacga caccgagtac tacctgatcc 900 ccgtggccag cagcagcaag gacgtgcagc tgaagaacat caccgactac atgtacctga 960 ccaacgcccc ttcttacacc aacggcaagc tgaacatcta ctaccgcaga ctgtacaacg 1020 gcctcaagtt catcatcaag agatacaccc ctaacaacga gatcgactct ttcgtgaagt 1080 ctggcgactt catcaagctg tacgtgtctt acaacaacaa cgagcacatc gtgggctacc 1140 ctaaggacgg caacgccttc aacaacctcg acagaatcct cagagtgggc tacaacgccc 1200 ccggcatccc tctctacaag aagatggagg ccgtgaagct cagggacctt aaaacctact 1260 ccgtgcagct gaagctgtac gacgacaaga acgccagcct gggcctcgtg ggcacccaca 1320 acggccagat cggcaacgac cctaacagag acatcctgat cgcctctaac tggtacttca 1380 accacctgaa ggacaagatc ctcggctgcg actggtactt cgtgcctacc gacgagggct 1440 ggaccaacga ccatcaccat caccatcact gataataggc tggagcctcg gtggccatgc 1500 ttcttgcccc ttgggcctcc ccccagcccc tcctcccctt cctgcacccg tacccccgtg 1560 gtctttgaat aaagtctg 1578 <210> 3 <211> 1965 <212> DNA <213> Nucleotide sequence of optimized immunogenic mRNA-Hn+Hc protein (2 Ambystoma laterale x Ambystoma jeffersonianum) <400> 3 aaataagaga gaaaagaaga gtaagaagaa atataagagc caccatggac tggacctgga 60 ttctgttcct cgtggccgcc gctactcgtg tgcactctat gagcgacaag atcatccacc 120 tgaccgacga ctctttcgac accgacgtgc tcaaggccga cggcgccatc ctggtggact 180 tctgggccga gtggtgcggc ccttgcaaga tgatcgcccc tatcctcgac gagatcgccg 240 acgagtacca gggcaagctg accgtggcca agctgaacat cgaccagaac cccggcaccg 300 cccctaagta cggcatccgg ggcatcccca ccctgctgct gttcaagaac ggcgaggtgg 360 ccgccaccaa ggtgggcgcc ctgtctaagg gccagctcaa ggagttcctg gacgccaacc 420 tcgccggcgg cggcggctct ggcggcggcg gctctggcgg cggcggctct ggcggcggcg 480 gctctaagaa cctcgactgc tgggtggaca acgaggagga catcgacgtg atcctcaaga 540 agtccaccat cctgaacctc gacatcaaca acgacatcat ctctgacatc tctggcttca 600 actccagcgt gatcacctac cccgacgccc agctcgtgcc cggcatcaac ggcaaggcca 660 tccacctcgt gaacaacgag tctagcgagg tgatcgtgca caaggctatg gacatcgagt 720 acaacgacat gttcaacaac ttcaccgtgt ctttctggct cagggtgcct aaggtgagcg 780 cctcccacct ggagcagtac ggcaccaacg agtactctat catctctagc atgaagaagc 840 acagcctgtc tatcggctct ggctggtccg tgagcctgaa gggcaacaac ctgatctgga 900 ccctgaagga ctccgccggc gaggtgaggc agatcacctt ccgggacctg cccgacaagt 960 tcaacgccta cctcgccaac aagtgggtgt tcatcaccat caccaacgac agactgtcta 1020 gcgccaacct gtacatcaac ggcgtgctca tgggctccgc cgagatcacc ggcctcggcg 1080 ccatccggga ggacaacaac atcaccctca agctggacag atgcaacaac aacaaccagt 1140 acgtgtctat cgacaagttc agaatcttct gcaaggccct gaaccctaag gagatcgaga 1200 agctgtacac cagctacctg tctatcacct tcctcagaga cttctggggc aaccctctcc 1260 gctacgacac cgagtactac ctgatccccg tggccagcag cagcaaggac gtgcagctga 1320 agaacatcac cgactacatg tacctgacca acgccccttc ttacaccaac ggcaagctga 1380 acatctacta ccgcagactg tacaacggcc tcaagttcat catcaagaga tacaccccta 1440 acaacgagat cgactctttc gtgaagtctg gcgacttcat caagctgtac gtgtcttaca 1500 acaacaacga gcacatcgtg ggctacccta aggacggcaa cgccttcaac aacctcgaca 1560 gaatcctcag agtgggctac aacgcccccg gcatccctct ctacaagaag atggaggccg 1620 tgaagctcag ggaccttaaa acctactccg tgcagctgaa gctgtacgac gacaagaacg 1680 ccagcctggg cctcgtgggc acccacaacg gccagatcgg caacgaccct aacagagaca 1740 tcctgatcgc ctctaactgg tacttcaacc acctgaagga caagatcctc ggctgcgact 1800 ggtacttcgt gcctaccgac gagggctgga ccaacgacca tcaccatcac catcactgat 1860 aataggctgg agcctcggtg gccatgcttc ttgccccttg ggcctccccc cagcccctcc 1920 tccccttcct gcacccgtac ccccgtggtc tttgaataaa gtctg 1965 <210> 4 <211> 469 <212> PRT <213> Amino acid sequence of optimized immunogenic mRNA-Hc protein (2 Ambystoma laterale x Ambystomajeffersonianum) <400> 4 Met Asp Trp Thr Trp Ile Leu Phe Leu Val Ala Ala Ala Thr Arg Val 1 5 10 15 His Ser Lys Asn Leu Asp Cys Trp Val Asp Asn Glu Glu Asp Ile Asp 20 25 30 Val Ile Leu Lys Lys Ser Thr Ile Leu Asn Leu Asp Ile Asn Asn Asp 35 40 45 Ile Ile Ser Asp Ile Ser Gly Phe Asn Ser Ser Val Ile Thr Tyr Pro 50 55 60 Asp Ala Gln Leu Val Pro Gly Ile Asn Gly Lys Ala Ile His Leu Val 65 70 75 80 Asn Asn Glu Ser Ser Glu Val Ile Val His Lys Ala Met Asp Ile Glu 85 90 95 Tyr Asn Asp Met Phe Asn Asn Phe Thr Val Ser Phe Trp Leu Arg Val 100 105 110 Pro Lys Val Ser Ala Ser His Leu Glu Gln Tyr Gly Thr Asn Glu Tyr 115 120 125 Ser Ile Ile Ser Ser Met Lys Lys His Ser Leu Ser Ile Gly Ser Gly 130 135 140 Trp Ser Val Ser Leu Lys Gly Asn Asn Leu Ile Trp Thr Leu Lys Asp 145 150 155 160 Ser Ala Gly Glu Val Arg Gln Ile Thr Phe Arg Asp Leu Pro Asp Lys 165 170 175 Phe Asn Ala Tyr Leu Ala Asn Lys Trp Val Phe Ile Thr Ile Thr Asn 180 185 190 Asp Arg Leu Ser Ser Ala Asn Leu Tyr Ile Asn Gly Val Leu Met Gly 195 200 205 Ser Ala Glu Ile Thr Gly Leu Gly Ala Ile Arg Glu Asp Asn Asn Ile 210 215 220 Thr Leu Lys Leu Asp Arg Cys Asn Asn Asn Asn Gln Tyr Val Ser Ile 225 230 235 240 Asp Lys Phe Arg Ile Phe Cys Lys Ala Leu Asn Pro Lys Glu Ile Glu 245 250 255 Lys Leu Tyr Thr Ser Tyr Leu Ser Ile Thr Phe Leu Arg Asp Phe Trp 260 265 270 Gly Asn Pro Leu Arg Tyr Asp Thr Glu Tyr Tyr Leu Ile Pro Val Ala 275 280 285 Ser Ser Ser Lys Asp Val Gln Leu Lys Asn Ile Thr Asp Tyr Met Tyr 290 295 300 Leu Thr Asn Ala Pro Ser Tyr Thr Asn Gly Lys Leu Asn Ile Tyr Tyr 305 310 315 320 Arg Arg Leu Tyr Asn Gly Leu Lys Phe Ile Ile Lys Arg Tyr Thr Pro 325 330 335 Asn Asn Glu Ile Asp Ser Phe Val Lys Ser Gly Asp Phe Ile Lys Leu 340 345 350 Tyr Val Ser Tyr Asn Asn Asn Glu His Ile Val Gly Tyr Pro Lys Asp 355 360 365 Gly Asn Ala Phe Asn Asn Leu Asp Arg Ile Leu Arg Val Gly Tyr Asn 370 375 380 Ala Pro Gly Ile Pro Leu Tyr Lys Lys Met Glu Ala Val Lys Leu Arg 385 390 395 400 Asp Leu Lys Thr Tyr Ser Val Gln Leu Lys Leu Tyr Asp Asp Lys Asn 405 410 415 Ala Ser Leu Gly Leu Val Gly Thr His Asn Gly Gln Ile Gly Asn Asp 420 425 430 Pro Asn Arg Asp Ile Leu Ile Ala Ser Asn Trp Tyr Phe Asn His Leu 435 440 445 Lys Asp Lys Ile Leu Gly Cys Asp Trp Tyr Phe Val Pro Thr Asp Glu 450 455 460 Gly Trp Thr Asn Asp 465 <210> 5 <211> 598 <212> PRT <213> Amino acid sequence of the optimized immunogenic mRNA-Hc-TRX protein (2 Ambystoma laterale x Ambystoma jeffersonianum) <400> 5 Met Asp Trp Thr Trp Ile Leu Phe Leu Val Ala Ala Ala Thr Arg Val 1 5 10 15 His Ser Met Ser Asp Lys Ile Ile His Leu Thr Asp Asp Ser Phe Asp 20 25 30 Thr Asp Val Leu Lys Ala Asp Gly Ala Ile Leu Val Asp Phe Trp Ala 35 40 45 Glu Trp Cys Gly Pro Cys Lys Met Ile Ala Pro Ile Leu Asp Glu Ile 50 55 60 Ala Asp Glu Tyr Gln Gly Lys Leu Thr Val Ala Lys Leu Asn Ile Asp 65 70 75 80 Gln Asn Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile Pro Thr 85 90 95 Leu Leu Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val Gly Ala 100 105 110 Leu Ser Lys Gly Gln Leu Lys Glu Phe Leu Asp Ala Asn Leu Ala Gly 115 120 125 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 130 135 140 Gly Gly Ser Lys Asn Leu Asp Cys Trp Val Asp Asn Glu Glu Asp Ile 145 150 155 160 Asp Val Ile Leu Lys Lys Ser Thr Ile Leu Asn Leu Asp Ile Asn Asn 165 170 175 Asp Ile Ile Ser Asp Ile Ser Gly Phe Asn Ser Ser Val Ile Thr Tyr 180 185 190 Pro Asp Ala Gln Leu Val Pro Gly Ile Asn Gly Lys Ala Ile His Leu 195 200 205 Val Asn Asn Glu Ser Ser Glu Val Ile Val His Lys Ala Met Asp Ile 210 215 220 Glu Tyr Asn Asp Met Phe Asn Asn Phe Thr Val Ser Phe Trp Leu Arg 225 230 235 240 Val Pro Lys Val Ser Ala Ser His Leu Glu Gln Tyr Gly Thr Asn Glu 245 250 255 Tyr Ser Ile Ile Ser Ser Met Lys Lys His Ser Leu Ser Ile Gly Ser 260 265 270 Gly Trp Ser Val Ser Leu Lys Gly Asn Asn Leu Ile Trp Thr Leu Lys 275 280 285 Asp Ser Ala Gly Glu Val Arg Gln Ile Thr Phe Arg Asp Leu Pro Asp 290 295 300 Lys Phe Asn Ala Tyr Leu Ala Asn Lys Trp Val Phe Ile Thr Ile Thr 305 310 315 320 Asn Asp Arg Leu Ser Ser Ala Asn Leu Tyr Ile Asn Gly Val Leu Met 325 330 335 Gly Ser Ala Glu Ile Thr Gly Leu Gly Ala Ile Arg Glu Asp Asn Asn 340 345 350 Ile Thr Leu Lys Leu Asp Arg Cys Asn Asn Asn Asn Gln Tyr Val Ser 355 360 365 Ile Asp Lys Phe Arg Ile Phe Cys Lys Ala Leu Asn Pro Lys Glu Ile 370 375 380 Glu Lys Leu Tyr Thr Ser Tyr Leu Ser Ile Thr Phe Leu Arg Asp Phe 385 390 395 400 Trp Gly Asn Pro Leu Arg Tyr Asp Thr Glu Tyr Tyr Leu Ile Pro Val 405 410 415 Ala Ser Ser Ser Lys Asp Val Gln Leu Lys Asn Ile Thr Asp Tyr Met 420 425 430 Tyr Leu Thr Asn Ala Pro Ser Tyr Thr Asn Gly Lys Leu Asn Ile Tyr 435 440 445 Tyr Arg Arg Leu Tyr Asn Gly Leu Lys Phe Ile Ile Lys Arg Tyr Thr 450 455 460 Pro Asn Asn Glu Ile Asp Ser Phe Val Lys Ser Gly Asp Phe Ile Lys 465 470 475 480 Leu Tyr Val Ser Tyr Asn Asn Asn Glu His Ile Val Gly Tyr Pro Lys 485 490 495 Asp Gly Asn Ala Phe Asn Asn Leu Asp Arg Ile Leu Arg Val Gly Tyr 500 505 510 Asn Ala Pro Gly Ile Pro Leu Tyr Lys Lys Met Glu Ala Val Lys Leu 515 520 525 Arg Asp Leu Lys Thr Tyr Ser Val Gln Leu Lys Leu Tyr Asp Asp Lys 530 535 540 Asn Ala Ser Leu Gly Leu Val Gly Thr His Asn Gly Gln Ile Gly Asn 545 550 555 560 Asp Pro Asn Arg Asp Ile Leu Ile Ala Ser Asn Trp Tyr Phe Asn His 565 570 575 Leu Lys Asp Lys Ile Leu Gly Cys Asp Trp Tyr Phe Val Pro Thr Asp 580 585 590 Glu Gly Trp Thr Asn Asp 595 <210> 6 <211> 866 <212> PRT <213> Amino acid sequence of optimized immunogenic mRNA-Hn+Hc protein (2 Ambystoma laterale x Ambystoma jeffersonianum) <400> 6 Met Asp Trp Thr Trp Ile Leu Phe Leu Val Ala Ala Ala Thr Arg Val 1 5 10 15 His Ser Cys Ile Lys Ile Lys Asn Glu Asp Leu Thr Phe Ile Ala Glu 20 25 30 Lys Asn Ser Phe Ser Glu Glu Pro Phe Gln Asp Glu Ile Val Ser Tyr 35 40 45 Asn Thr Lys Asn Lys Pro Leu Asn Phe Asn Tyr Ser Leu Asp Lys Ile 50 55 60 Ile Val Asp Tyr Asn Leu Gln Ser Lys Ile Thr Leu Pro Asn Asp Arg 65 70 75 80 Thr Thr Pro Val Thr Lys Gly Ile Pro Tyr Ala Pro Glu Tyr Lys Ser 85 90 95 Asn Ala Ala Ser Thr Ile Glu Ile His Asn Ile Asp Asp Asn Thr Ile 100 105 110 Tyr Gln Tyr Leu Tyr Ala Gln Lys Ser Pro Thr Thr Leu Gln Arg Ile 115 120 125 Thr Met Thr Asn Ser Val Asp Asp Ala Leu Ile Asn Ser Thr Lys Ile 130 135 140 Tyr Ser Tyr Phe Pro Ser Val Ile Ser Lys Val Asn Gln Gly Ala Gln 145 150 155 160 Gly Ile Leu Phe Leu Gln Trp Val Arg Asp Ile Ile Asp Asp Phe Thr 165 170 175 Asn Glu Ser Ser Gln Lys Thr Thr Ile Asp Lys Ile Ser Asp Val Ser 180 185 190 Thr Ile Val Pro Tyr Ile Gly Pro Ala Leu Asn Ile Val Lys Gln Gly 195 200 205 Tyr Glu Gly Asn Phe Ile Gly Ala Leu Glu Thr Thr Gly Val Val Leu 210 215 220 Leu Leu Glu Tyr Ile Pro Glu Ile Thr Leu Pro Val Ile Ala Ala Leu 225 230 235 240 Ser Ile Ala Glu Ser Ser Thr Gln Lys Glu Lys Ile Ile Lys Thr Ile 245 250 255 Asp Asn Phe Leu Glu Lys Arg Tyr Glu Lys Trp Ile Glu Val Tyr Lys 260 265 270 Leu Val Lys Ala Lys Trp Leu Gly Thr Val Asn Thr Gln Phe Gln Lys 275 280 285 Arg Ser Tyr Gln Met Tyr Arg Ser Leu Glu Tyr Gln Val Asp Ala Ile 290 295 300 Lys Lys Ile Ile Asp Tyr Glu Tyr Lys Ile Tyr Ser Gly Pro Asp Lys 305 310 315 320 Glu Gln Ile Ala Asp Glu Ile Asn Asn Leu Lys Asn Lys Leu Glu Glu 325 330 335 Lys Ala Asn Lys Ala Met Ile Asn Ile Asn Ile Phe Met Arg Glu Ser 340 345 350 Ser Arg Ser Phe Leu Val Asn Gln Met Ile Asn Glu Ala Lys Lys Gln 355 360 365 Leu Leu Glu Phe Asp Thr Gln Ser Lys Asn Ile Leu Met Gln Tyr Ile 370 375 380 Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu Lys Lys Leu Glu 385 390 395 400 Ser Lys Ile Asn Lys Val Phe Ser Thr Pro Ile Pro Phe Ser Tyr Ala 405 410 415 Asn Leu Asp Cys Trp Val Asp Asn Glu Glu Asp Ile Asp Val Ile Leu 420 425 430 Lys Lys Ser Thr Ile Leu Asn Leu Asp Ile Asn Asn Asp Ile Ile Ser 435 440 445 Asp Ile Ser Gly Phe Asn Ser Ser Val Ile Thr Tyr Pro Asp Ala Gln 450 455 460 Leu Val Pro Gly Ile Asn Gly Lys Ala Ile His Leu Val Asn Asn Glu 465 470 475 480 Ser Ser Glu Val Ile Val His Lys Ala Met Asp Ile Glu Tyr Asn Asp 485 490 495 Met Phe Asn Asn Phe Thr Val Ser Phe Trp Leu Arg Val Pro Lys Val 500 505 510 Ser Ala Ser His Leu Glu Gln Tyr Gly Thr Asn Glu Tyr Ser Ile Ile 515 520 525 Ser Ser Met Lys Lys His Ser Leu Ser Ile Gly Ser Gly Trp Ser Val 530 535 540 Ser Leu Lys Gly Asn Asn Leu Ile Trp Thr Leu Lys Asp Ser Ala Gly 545 550 555 560 Glu Val Arg Gln Ile Thr Phe Arg Asp Leu Pro Asp Lys Phe Asn Ala 565 570 575 Tyr Leu Ala Asn Lys Trp Val Phe Ile Thr Ile Thr Asn Asp Arg Leu 580 585 590 Ser Ser Ala Asn Leu Tyr Ile Asn Gly Val Leu Met Gly Ser Ala Glu 595 600 605 Ile Thr Gly Leu Gly Ala Ile Arg Glu Asp Asn Asn Ile Thr Leu Lys 610 615 620 Leu Asp Arg Cys Asn Asn Asn Asn Gln Tyr Val Ser Ile Asp Lys Phe 625 630 635 640 Arg Ile Phe Cys Lys Ala Leu Asn Pro Lys Glu Ile Glu Lys Leu Tyr 645 650 655 Thr Ser Tyr Leu Ser Ile Thr Phe Leu Arg Asp Phe Trp Gly Asn Pro 660 665 670 Leu Arg Tyr Asp Thr Glu Tyr Tyr Leu Ile Pro Val Ala Ser Ser Ser 675 680 685 Lys Asp Val Gln Leu Lys Asn Ile Thr Asp Tyr Met Tyr Leu Thr Asn 690 695 700 Ala Pro Ser Tyr Thr Asn Gly Lys Leu Asn Ile Tyr Tyr Arg Arg Leu 705 710 715 720 Tyr Asn Gly Leu Lys Phe Ile Ile Lys Arg Tyr Thr Pro Asn Asn Glu 725 730 735 Ile Asp Ser Phe Val Lys Ser Gly Asp Phe Ile Lys Leu Tyr Val Ser 740 745 750 Tyr Asn Asn Asn Glu His Ile Val Gly Tyr Pro Lys Asp Gly Asn Ala 755 760 765 Phe Asn Asn Leu Asp Arg Ile Leu Arg Val Gly Tyr Asn Ala Pro Gly 770 775 780 Ile Pro Leu Tyr Lys Lys Met Glu Ala Val Lys Leu Arg Asp Leu Lys 785 790 795 800 Thr Tyr Ser Val Gln Leu Lys Leu Tyr Asp Asp Lys Asn Ala Ser Leu 805 810 815 Gly Leu Val Gly Thr His Asn Gly Gln Ile Gly Asn Asp Pro Asn Arg 820 825 830 Asp Ile Leu Ile Ala Ser Asn Trp Tyr Phe Asn His Leu Lys Asp Lys 835 840 845 Ile Leu Gly Cys Asp Trp Tyr Phe Val Pro Thr Asp Glu Gly Trp Thr 850 855 860 Asn Asp 865

Claims

1. A modified mRNA vaccine against tetanus, characterized in that: The vaccine includes: (1) mRNA for expressing tetanus toxin immunogen, and the immunogen is Hc protein; the Hc protein is the C-terminal domain of the heavy chain of tetanus toxin, and its immunogenic mRNA sequence is as shown in SEQ ID NO.1; (2) A vaccine carrier, and the vaccine carrier is a liposome nanoparticle that can be utilized by mRNA.

2. The modified mRNA vaccine against tetanus according to claim 1, characterized in that: The mRNA sequence has the following structure: A1 - A2 - A3 - A4 - A5 - A6: A1 is a 5' cap structure; A2 is a 5' UTR element; A3 is a signal peptide coding sequence; A4 is a tetanus toxin antigen coding sequence, and the tetanus toxin antigen selects Hc protein; A5 is a 3' UTR element; A6 is a poly(A) tail structure.

3. The modified mRNA vaccine against tetanus according to claim 2, characterized in that: The signal peptide coding sequence includes those from tissue-type plasminogen activator (tPA), from serum immunoglobulin heavy chain (IgE), and from immunoglobulin kappa gene (IgK).

4. The modified mRNA vaccine against tetanus according to claim 1, characterized in that: The immunogenic mRNA sequence is a modified RNA, and the modified RNA includes N1-methyl-pseudouridine, N6-methyladenosine, pseudouridine, 5-methylcytidine.

5. The modified mRNA vaccine against tetanus according to claim 1, characterized in that: The liposome nanoparticle includes neutral lipid, cationic lipid, cholesterol, campesterol, stigmastanol, ergosterol, lanosterol or PEG-modified lipid.

6. The modified mRNA vaccine against tetanus according to claim 1 or 5, characterized in that: The average particle size of the liposome nanoparticle is in the range of 10 - 500 nm.

7. The modified mRNA vaccine against tetanus according to claim 1 or 5, characterized in that: The mass ratio of the liposome nanoparticle to the immunogenic mRNA is 10 - 30:

1.

8. The modified mRNA vaccine against tetanus according to claim 5, characterized in that: The cationic lipid includes Dlin-MC3-DMA, DODMA, DODAP, ALC-0315, SM102.

9. A preparation method of the modified mRNA vaccine against tetanus according to claim 1, characterized in that: The specific process is as follows: S1, codon-optimize the antigen gene sequence with human as the host, synthesize the corresponding antigen expression gene, and obtain the Hc protein tetanus toxin antigen expression gene; S2, clone the tetanus toxin antigen expression gene into the plasmid vector 5´UTR-IgE-3’UTR-pok12 respectively to obtain the corresponding recombinant plasmid; S3, prepare mRNA; S4. Dissolve four kinds of lipids, namely D-Lin-MC3-DMA, DSPC, cholesterol, and 14:0 PEG2000 PE, separately with ethanol. S5. Mix them in a certain molar ratio to prepare a lipid mixture, where D-Lin-MC3-DMA:DSPC:cholesterol:14:0 PEG2000 PE = 50:10:38.5:1.

5. S6. Add the lipid mixture and the citrate buffer solution dissolved with mRNA into two inlet channels of the NanoAssemblr machine respectively, and collect the mRNA-LNP mixture to obtain the vaccine.

10. The preparation method of a modified mRNA vaccine against tetanus according to claim 9, characterized in that: The specific process of preparing mRNA in step S3: S31. Use PCR to amplify the target fragment as the in vitro transcription template, and use the in vitro transcription kit of NEB company for in vitro transcription, while completely replacing UTP with modified nucleotides. S32. After the reaction, use DNase to digest to remove DNA in the reaction. S33. Use the method of LiCL precipitation of RNA to remove proteins and salts in the RNA. S34. Use the Cap1 capping kit and the tailing kit of NEB company to perform capping and tailing respectively to obtain the corresponding mRNA.

11. The preparation method of a modified mRNA vaccine against tetanus according to claim 10, characterized in that: The modified nucleotide is N1-methylpseudouridine triphosphate.

12. The preparation method of a modified mRNA vaccine against tetanus according to claim 10, characterized in that: The capping kit is the mRNA Cap 2´-O-Methyltransferase and Vaccinia Capping System kit; The tailing kit is E. coli Poly(A) Polymerase.

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