A protein, self-replicating mRNA molecule and a rabies virus saRNA vaccine

Abstract

The application provides a protein, a self-replicating mRNA molecule and a rabies virus saRNA vaccine. The amino acid sequence of the protein is shown as SEQ ID NO: 5, the mRNA molecule comprises coding region RNA of the amino acid sequence, preferably, the nucleotide sequence of the mRNA molecule is shown as SEQ ID NO: 6, the vaccine is a lipid nanoparticle comprising the mRNA molecule shown as SEQ ID NO: 6, and additionally, the application also discloses a preparation method of the rabies virus saRNA vaccine, which comprises the steps of saRNA molecule design, supercoiled plasmid preparation, preparation of the supercoiled plasmid into a linear plasmid template, preparation of a saRNA stock solution, LNP-saRNA preparation and the like. On the basis of scientifically designing a target gene molecule, the rabies virus saRNA vaccine obtained by the application has the advantages of low use dosage, long duration of immune protection, an injection dosage of 5 mu even lower, LNP-saRNA secondary injection immunization, and a duration of immune protection of more than 126 days. The vaccine has good protection effect, and the NIH titer is as high as 217.4 IU / mL or higher, which is much higher than the reference vaccine of 11.4 IU / mL.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medical molecular biology, and in particular to a protein, a self-replicating mRNA molecule and a rabies virus saRNA vaccine. BACKGROUND

[0002] Rabies is an acute infectious disease caused by rabies virus (RABV). Rabies virus belongs to the Rhabdoviridae family and is a single-stranded RNA virus. It is mainly transmitted through the saliva of infected animals, usually through bites, licking open wounds or mucous membranes. Rabies virus is neurotropic, and the virus replicates at the wound site and enters the muscle ganglion, eventually reaching the central nervous system. The incubation period varies from a few days to several years, and when clinical symptoms appear, the virus has already spread widely in the central nervous system.

[0003] Therefore, timely prevention is crucial to reduce the risk of disease, and the development of rabies vaccine is the key to preventing rabies. In 1882, French scientist Louis Pasteur first successfully invented a human rabies vaccine, followed by early animal nerve tissue vaccines, avian embryo vaccines, and crude cell culture vaccines. The development has now reached the current technology of primary hamster kidney cells, chicken embryo cells, human diploid cells, and Vero cell culture purified vaccines. However, the production process of these vaccines is both time-consuming and expensive, and requires multiple vaccinations (four / five needles) to achieve protection.

[0004] saRNA vaccine is a new type of vaccine technology that uses RNA molecules encoding viral surface proteins to stimulate the body's immune response and has the ability to self-replicate for a long time in recipient cells, producing more RNA molecules and increasing the expression of target proteins. Compared with purified protein vaccines or viral vaccines, saRNA vaccines have the advantages of rapid development, low production cost, and easy mass production.

[0005] The present application is directed to the rabies virus G protein antigen and uses saRNA technology to provide a saRNA vaccine encoding the rabies virus G protein. Through cell experiments and animal experiments, it is confirmed that the saRNA vaccine designed by the inventors can normally express and continuously and efficiently produce antibodies against the rabies virus G protein, with broad market prospects. SUMMARY

[0006] The present application aims to overcome the above-mentioned defects of the prior art, take advantage of saRNA vaccine technology, and provide a protein, a self-replicating mRNA molecule, and a rabies virus saRNA vaccine with long protection time and good protection effect.

[0007] To achieve the above object, the present application first provides a protein, whose amino acid sequence is shown as SEQ ID NO: 5.

[0008] The present application also provides an mRNA molecule, wherein the mRNA sequence comprises the RNA of the coding region of the amino acid sequence.

[0009] Preferably, the nucleotide sequence of the mRNA molecule is shown as SEQ ID NO: 6.

[0010] The present application also provides the use of the mRNA molecule in the preparation of a rabies virus medicine, wherein the use is to prepare a composition comprising an immunologically effective amount of the construct of the mRNA molecule.

[0011] The present application also provides a rabies virus saRNA vaccine, characterized in that the vaccine is a lipid nanoparticle comprising the mRNA molecule shown as SEQ ID NO: 6.

[0012] Preferably, the rabies virus saRNA vaccine is prepared by a method comprising the following steps: 1) saRNA molecule design

[0013] The amino acid sequence shown as SEQ ID NO: 5 is selected, and then the nucleotide sequence shown as SEQ ID NO: 6 is designed;

[0014] 2) Plasmid preparation

[0015] ① Use a DNA synthesizer to synthesize a nucleic acid fragment according to the sequence of SEQ ID NO: 6, linearize the vector using a restriction endonuclease, and use a DNA ligase to connect the synthesized linear DNA with the linearized plasmid;

[0016] ② Add the recombinant plasmid ligation product obtained in step ① to competent cells DH5α for heat shock treatment;

[0017] ③ Culture the competent cells treated in step ② with liquid LB medium, and then spread the cells on solid LB medium for further culture;

[0018] ④ Select well-grown single colonies, culture with a synthetic medium, extract plasmids from a small amount of bacterial solution, and perform SalI and XbaI enzyme digestion identification;

[0019] ⑤ Culture the bacterial solution with correct identification results, centrifuge, and harvest the bacterial slurry;

[0020] ⑥ Prepare a supercoiled plasmid solution from the bacterial slurry;

[0021] 3) Prepare the supercoiled plasmid into a linear plasmid template;

[0022] 4) Preparation of saRNA stock solution and preparation of LNP-saRNA preparation

[0023] The preparation method of the saRNA stock solution is to perform an in vitro transcription reaction on a linear plasmid template, and then obtain the saRNA stock solution through incubation, centrifugation, precipitation dissolution with enzyme-free water, affinity chromatography column purification, purification using a TFF system, and filtration;

[0024] The preparation steps of the LNP-saRNA preparation are: ① dilute the saRNA stock solution with a citrate buffer to obtain a saRNA working solution; ② mix the organic phase with the saRNA working solution at a volume ratio of 1:2-4 to prepare nanoparticle LNP; wherein the molar concentration ratio of cation DLin-MC3-DMA: DSPC: CHO-HP: DMP-PEG2000 in the organic phase is 40-60:5-10:30-60:2-5, and the total molar concentration of the organic phase is controlled within the range of 15-20 mmol / L; ③ dilute and centrifugally concentrate the nanoparticle LNP with 1xPBS, repeat once, filter, and obtain the LNP-saRNA preparation, i.e., the rabies virus saRNA vaccine.

[0025] The technical innovation points and beneficial effects of the present application are:

[0026] ① Target gene molecular design

[0027] The present application selects the rabies virus G protein as the target antigen, and adds a double Q alpha sequence (QPRFAAA) at the end of the G protein molecular sequence to increase the stability and translation efficiency of the saRNA, and the target antigen is connected with the Q alpha sequence and the Q alpha sequence with a flexible Linker.

[0028] The present application designs the codon optimization of the designed rabies G protein sequence, selects the commonly used codons according to the codon usage bias of the host cells, and adjusts the GC content in the target gene codon sequence to increase the stability of the secondary structure of the saRNA molecule, so as to improve the expression level in the host cells.

[0029] ② Low dose, long duration of immune protection

[0030] The present application uses the saRNA vaccine technology, uses an injection dose of 5 µg or even lower, and the LNP-saRNA secondary injection immunization has a long duration of immune protection (more than 126 days), which is obviously superior to other traditional vaccines and other mRNA vaccines.

[0031] ③ High NIH titer

[0032] The optimized rabies G protein sequence designed in this invention, SEQ ID NO: 5, was tested for potency according to the method for determining the potency of human rabies vaccine in Part III, 3503 of the 2020 edition of the Pharmacopoeia of the People's Republic of China. The potency was >217.4 IU / mL, which is much higher than the 11.4 IU / mL of the reference vaccine. Attached Figure Description

[0033] Figure 1 Electrophoresis image of the plasmid SEQ ID NO.6 / SalⅠ+ XbaⅠ double digestion product;

[0034] Figure 2 This is a Western blotting image;

[0035] Figure 3 This is a graph showing the antibody detection results on day 17.

[0036] Figure 4 This is a graph showing the antibody detection results on day 21.

[0037] Figure 5 This is a graph showing the antibody detection results on day 28.

[0038] Figure 6 This is a graph showing the antibody detection results on day 35.

[0039] Figure 7 This is a graph showing the antibody detection results on day 66.

[0040] Figure 8 The image shows the results of cytokine (IFN-γ) secretion detection on day 44.

[0041] Figure 9 This is a graph showing the CD25 / CD69 test results on day 52. Detailed Implementation

[0042] To better understand the present invention, embodiments are described in detail below with reference to the accompanying drawings. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0043] Unless otherwise specified, the technical means used in this embodiment are conventional technical means in the field.

[0044] In the following embodiments, saRNA (Self-amplifying RNA) refers to a specially designed synthetic RNA.

[0045] RNA molecules have the property of self-replication within cells; LNP-saRNA refers to drugs formed by encapsulating saRNA with LNPs (Lipid Nanoparticles).

[0046] The overall technical solution of the present application is as follows:

[0047] 1. Molecular design

[0048] 1) 5' end signal peptide design

[0049] To ensure the secretion of the antigen, a signal peptide sequence is added at the 5' end of the molecule, preferably a sequence as shown in SEQ ID NO: 1: MIPQALLFVPLLVFPLCFG

[0050] 2) Molecular design of target gene protein

[0051] ① Selection of target antigen

[0052] Rabies virus glycoprotein (G protein) is the only envelope protein, responsible for binding to cell receptors, mediating viral invasion, determining the pathogenicity and tissue tropism of the virus. G protein is the main antigen of rabies virus, which can effectively stimulate the proliferation of specific T helper cells (Th) and cytotoxic T lymphocytes (CTL), and induce the body to produce protective neutralizing antibodies. The G protein sequence (GenBank: ACR39382.1) is preferably used as a target antigen in the application, and the sequence is shown as SEQ ID NO: 2: KFPIYTIPDKLGPWSPIDIHHLSCPNNLVVEDEGCTNLSGFSYMELKVGYISAIKVNGFTCTGVVTEAETYTNFVGYVTTTFKRKHFRPTPDACRSAYNWKMAGDPRYEESLHNPYPDYHWLRTVKTTKESVVIISPSVADLDPYDKSLHSRVFPRGKCSGITVSSAYCSTNHDYTIWMPENPRLGTSCDIFTNSRGKRASKGSKTCGFVDERGLYKSLKGACKLKLCGVLGLRLMDGTWVAIQTSNETKWCPPDQLVNLHDFHSDEIEHLVVEELVKKREECLDALESIMTTKSVSFRRLSHLRKLVPGFGKAYTIFNKTLMEADAHYKSVRTWNEIIPSKGCLRVGGRCHPHVNGVFFNGIILGPDGHVLIPEMQSSLLQQHMELLESSVIPLMHPLADPSTVFKDGDEVEDFVEVHLPDVHKQVSGVDLGLPNWGKDVLMGAGVLTALMLMIFLMTCCRRTNRAESIQHSLGETGRKVSVTSQSGRVISSWESYKSGGETKL

[0053] ②Double Q alpha sequence design

[0054] The Qa sequence is a short peptide sequence consisting of 7 amino acids, which can increase the stability of mRNA and greatly increase the yield of target protein when it is added to the coding region of the target gene. In order to promote the expression of target antigen in vivo, the mRNA stability and translation efficiency are increased by adding double Qa sequences (QPRFAAA) at the end of the target antigen sequence, and the target antigen is connected with Qa sequence, Qa sequence with Qa sequence by flexible linker (GGGSGGGS). Preferably, the design optimizes the double Qa sequence as shown in SEQ ID NO: 3: GGGSGGGSQPRFAAAGGGSGGGSQPRFAAA

[0055] 3) Optimization design of stop codon

[0056] In order to achieve precise control of protein translation, the synthesis of protein can be terminated by adding a stop codon, so as to produce the desired protein variant or avoid the production of unnecessary proteins. The stop codon sequence is designed as SEQ ID NO: 4: TAGTAATGATGA

[0057] 4) Optimization of mRNA sequence and secondary structure

[0058] According to the target gene protein molecule sequence string design, preferably the amino acid sequence is shown in SEQ ID NO: 5: MIPQALLFVPLLVFPLCFGKFPIYTIPDKLGPWSPIDIHHLSCPNNLVVEDEGCTNLSGFSYMELKVGYISAIKVNGFTCTGVVTEAETYTNFVGYVTTTFKRKHFRPTPDACRSAYNWKMAGDPRYEESLHNPYPDYHWLRTVKTTKESVVIISPSVADLDPYDKSLHSRVFPRGKCSGITVSSAYCSTNHDYTIWMPENPRLGTSCDIFTNSRGKRASKGSKTCGFVDERGLYKSLKGACKLKLCGVLGLRLMDGTWVAIQTSNETKWCPPDQLVNLHDFHSDEIEHLVVEELVKKREECLDALESIMTTKSVSFRRLSHLRKLVPGFGKAYTIFNKTLMEADAHYKSVRTWNEIIPSKGCLRVGGRCHPHVNGVFFNGIILGPDGHVLIPEMQSSLLQQHMELLESSVIPLMHPLADPSTVFKDGDEVEDFVEVHLPDVHKQVSGVDLGLPNWGKDVLMGAGVLTALMLMIFLMTCCRRTNRAESIQHSLGETGRKVSVTSQSGRVISSWESYKSGGETKLGGGSGGGSQPRFAAAGGGSGGGSQPRFAAA

[0059] According to the codon usage bias of the host cell, the commonly used synonymous codons in the host cell are selected to improve the expression level of the gene. The stability of the mRNA secondary structure is closely related to the half-life of the mRNA and the amount of protein expression. Enhancing the stability of the mRNA secondary structure is beneficial to the expression of the target protein. High content of guanine and cytosine (G and C) can improve the stability and translation efficiency of mRNA. Adjusting the GC content in the codon sequence of the target gene can improve its expression level in the host cell. The average GC content of the optimized sequence is preferably 57%-61%; more preferably 58%-60%; more preferably 59%, and the sequence is SEQ ID NO: 6:

[0060]

[0061] 2. Drug preparation

[0062] 1) Seed bank construction

[0063] The sequence of SEQ ID NO.6 was cloned into a self-replicating vector using SalI / XbaI double digestion to construct a supercoiled plasmid. The supercoiled plasmid was then introduced into *E. coli* cells using a heat shock method to complete the construction of the research seed bank.

[0064] 2) Plasmid template preparation

[0065] ① A modified microbial strain containing a specific recombinant plasmid is introduced into a culture medium containing a specific antibiotic. The role of these antibiotics is to ensure that only the strain containing the recombinant plasmid can survive and reproduce. Subsequently, the strain is cultured in a bioreactor under suitable conditions, including but not limited to precise control of temperature, pH, and oxygen supply, to ensure efficient plasmid replication and quality.

[0066] ② Once the culture is complete, a series of physical and chemical methods are needed, such as centrifugation and cell precipitation.

[0067] The plasmids are collected through lysis and filtration. Then, they are further purified using a series of chromatographic techniques, including gel filtration, affinity chromatography, and ion exchange chromatography, to remove any impurities such as proteins, RNA, and endotoxins, thereby obtaining pure, supercoiled plasmids.

[0068] ③ The supercoiled plasmid was cleaved into a linear form using a specific enzyme. Then, the linear plasmid was purified again using chromatography.

[0069] 3) Preparation of saRNA stock solution

[0070] ① In vitro transcription (IVT) was used, with carefully purified linear plasmids as templates. RNA polymerase and essential nucleoside triphosphates (NTPs) were introduced during this process, and these components worked together to catalyze the synthesis of the target saRNA molecule in an in vitro environment.

[0071] ② The synthesized saRNA molecules then undergo a series of sophisticated purification steps, including ammonium sulfate precipitation, affinity chromatography, and ultrafiltration. These steps are designed to ensure that the obtained saRNA molecules are not only intact but also possess high purity and biological activity, meeting the high standards required for subsequent experiments or applications.

[0072] 4) Preparation of the formulation

[0073] ① Citric acid buffer solution is selected as the buffer medium of the stock solution to maintain the stability and pH balance of the solution. The lipid solution is prepared according to a specific mass ratio, in which the ratio of cationic lipid to other lipids is 1.1:1, to ensure the charge balance and stability of the lipid membrane. Protonatable cationic lipids are used to enhance the interaction between the lipid membrane and the drug molecules. The mixing flow rates of the oil phase and the water phase are controlled during the preparation process, which are set to 12 mL / min, 16 mL / min, 32 mL / min and 36 mL / min, respectively, to optimize the formation and size distribution of the lipid nanoparticles.

[0074] ② When the LNP structure is solidified, an appropriate amount of phosphate buffer solution is added to the mixed solution, which is 4 to 20 times the volume of the stock solution, to promote the solidification and stability of the lipid membrane. After ultrafiltration purification, the obtained lipid nanoparticles are the rabies saRNA vaccine.

[0075] 3. In vitro experiment

[0076] The saRNA stock solution is delivered into cells through in vitro transfection, and Western Blotting is used to detect whether the saRNA can translate the target protein in cells.

[0077] 4. Animal experiment proves that the saRNA drug of the application can effectively prevent rabies

[0078] BALB / c mice and Kunming mice are selected for the experiment to establish an animal model, and the effectiveness of the drug is determined by detecting the binding antibody, neutralizing antibody and NIH titer.

[0079] Example 1 saRNA molecule design

[0080] According to the sequence of the target gene protein molecule, the amino acid sequence shown in SEQ ID NO: 5 is designed, and the amino acid sequence is optimized. The optimization includes adjusting the codon bias for expression in different organisms, adjusting the frequency of commonly used codons, and adjusting the GC content in the sequence. The optimized nucleotide sequence is shown in SEQ ID NO: 6.

[0081] Example 2 Plasmid preparation

[0082] Step 1: Use a DNA synthesizer to synthesize a nucleic acid fragment according to the sequence of SEQ ID NO: 6, linearize the vector using a restriction endonuclease, and use a DNA ligase to connect the synthesized linear DNA with the linearized plasmid;

[0083] Step 2: Recover the enzyme digestion product obtained in Step 1, prepare a reaction system according to the molar ratio of ligation gene to vector of 3:1 to 5:1, add T4 DNA ligase and appropriate reaction buffer, mix well, and carry out the ligation reaction at room temperature for 30 to 60 minutes.

[0084] Step 3: Take 3-5 μL of the recombinant plasmid ligation product obtained in Step 2 and add it to 50 μL of thawed competent DH5α cells. After mixing, perform heat shock treatment at 42 °C for 30 to 60 seconds.

[0085] Step 4: Add the competent cells treated in step 3 to 500 μL of liquid LB medium and culture at 37°C and 200-220 rpm for about one hour. Then spread the cells on solid LB medium and continue to culture at 37°C for 16 to 18 hours.

[0086] Step 5: Select a single colony with good growth from the plate cultured in Step 4, inoculate it into synthetic medium, and culture it at 37 ℃ and 200-220 rpm for 4 to 6 hours. Then take a small amount of bacterial culture, extract plasmids using a plasmid extraction kit, and perform SalI and XbaI restriction enzyme digestion identification.

[0087] Step 6: The bacterial culture with correct enzyme digestion identification results in step 5 is scaled up and cultured at 37℃ and 200-220 rpm for 14 to 16 hours. After that, it is centrifuged at 4000 rpm for 5 to 10 minutes to harvest the bacterial sludge.

[0088] Step 7: Lyse the bacterial sludge obtained in Step 6. First, add lysis buffer I and suspend the bacteria thoroughly. The volume ratio of bacterial sludge to lysis buffer I is 4:6 to 6:250. After the bacteria are fully suspended, add lysis buffer II and gently invert to mix and achieve bacterial lysis. Then, add lysis buffer III and immediately gently invert to mix and neutralize. Incubate at room temperature for 5 to 10 minutes. The volume ratio of lysis buffer I, lysis buffer II, and lysis buffer III is 5:5:7. Centrifuge the lysed mixture at 12,000 rpm for 5 to 10 minutes. Add the supernatant to a DNA adsorption column and centrifuge at 12,000 rpm for 1 minute to remove waste liquid. Then, add washing buffer to the adsorption column and centrifuge at 12,000 rpm for 1 minute to remove waste liquid. Finally, add elution buffer, incubate at 37 °C for 2 to 5 minutes, centrifuge at 12,000 rpm for 1 to 2 minutes, and collect the elution buffer to obtain the supercoiled plasmid solution.

[0089] Supercoiled plasmid samples were stored at -80℃; plasmid identification chromatograms are shown below. Figure 1 As shown.

[0090] Example 3: Preparation of linear plasmid template

[0091] The supercoiled plasmid prepared in Example 2 was incubated at 50℃ for 1-2 hours with Xbal or BspQl enzyme system to obtain linearized plasmid. Then, 0.5 times volume of magnetic beads was added to the remaining enzyme digestion product and mixed well. After incubation at room temperature for 10-15 minutes, the mixture was placed on a magnetic stand until the solution was clear, and the supernatant was removed. The magnetic beads were rinsed with freshly prepared 80% ethanol solution, and dried after removing the supernatant. Finally, an appropriate volume of nuclease-free water was added and mixed well, and the supernatant was removed to obtain the purified linear plasmid solution.

[0092] The linear template sample was stored at -80℃.

[0093] Example 4: Preparation of saRNA stock solution

[0094] First step: After mixing the linear plasmid template, NTPs (nucleotide triphosphates), Clean Cap, buffer, and other substrates, T7 RNA polymerase was added, and the mixture was incubated at 37℃ in a PCR instrument for in vitro transcription reaction (IVT). After 1-3 hours of reaction, DNase I was added to terminate the reaction.

[0095] Second step: Buffer A: 20 mM Tris-HCl, pH=7.0; Buffer B: 20 mM Tris-HCl, 3.5M ammonium sulfate, pH=7.0. The solution obtained after the reaction in step one was mixed with 64 uL buffer A and 86 uL buffer B, and the sample was incubated at 20℃ for 1 hour. Then, the mixture was centrifuged at 12000 rpm for 10 min at 20℃ to separate the supernatant and the precipitate, and 200 uL nuclease-free water was added to dissolve the precipitate.

[0096] Third step: Purification was performed by affinity chromatography column chromatography, and Oligo dT was selected as the chromatography filler. Before purification, the solution obtained in step two was diluted 15-30 times using the equilibration solution, and the linear flow rate was 6 cm / hour-20 cm / hour. The equilibration solution had a mass ratio of NaCl: EDTA: Tris HCl = 9.35: 0.067: 0.32. After the washing step, pure water was used for elution to collect the elution peak.

[0097] Third step: Purification was performed using a TFF system, and the replacement solution was pure water. The transmembrane pressure was 5-20 psi, the shear rate was 1000-4000 / sec, the washing was 8-15 times, and the concentration was concentrated to the target concentration. The obtained sample was filtered with 0.2 μm to obtain the saRNA stock solution.

[0098] The saRNA stock solution is stored at -80°C.

[0099] Example 5: Preparation of LNP-saRNA formulation

[0100] First step: The saRNA stock solution prepared in Example 4 is thawed in a cold water bath. Then, the saRNA stock solution is diluted using citrate buffer to achieve a concentration of about 0.1-0.5 mg / ml, so as to obtain a saRNA working solution;

[0101] Second step: The saRNA is encapsulated in lipids, wherein the molar concentration ratio of the cationic DLin-MC3-DMA: DSPC: CHO-HP: DMP-PEG2000 in the lipid phase is 40-60: 5-10: 30-60: 2-5, and the total molar concentration of the lipid phase is controlled in the range of 15-20 mmol / L. The organic phase is mixed with the aqueous phase (saRNA working solution) at a volume ratio of 1:2-4, and nanoparticles LNP are prepared by a microfluidic device;

[0102] Third step: The harvested nanoparticles LNP is diluted with 5-10 times volume of 1xPBS. Using a 100KD ultrafiltration centrifuge tube, centrifugal concentration is carried out at 4°C, 1500 g, until the expected concentration is achieved;

[0103] Fourth step: Continue to dilute with 5-10 times volume of 1xPBS, and centrifugal concentration is carried out again until the expected concentration volume is achieved. Finally, filtration is carried out through a 0.2 μm membrane to obtain the LNP-saRNA formulation;

[0104] The LNP-saRNA formulation is stored at -20°C.

[0105] Example 6: In vitro experiment of saRNA drug

[0106] First step: HEK293 cells are cultured on cell well plates until the confluence of each well reaches 80%-90%;

[0107] Second step: 2-5 μg of the saRNA stock solution prepared in Example 3 and the LNP-saRNA formulation prepared in Example 4 are taken respectively, diluted to 150 μL using Opti-MEM (serum-free medium), and then mixed with 150 μL of the transformed reagent Lipofectamine™ MessengerMAX™ Reagent diluted with Opti-MEM, and allowed to stand for 5 min. The solution after standing is added to the cell well plates, and placed in a carbon dioxide incubator for culture for 24 hours;

[0108] Third step: Take out the cell hole plate, observe the cell state under the microscope, remove the culture solution, and then wash it once with 1xPBS after ice bath, add 200 μL RIPA lysis buffer (add 1% PMSF to the lysis buffer) to each hole, and place it on ice for 5-10 min for lysis, collect the lysis buffer and vortex, and centrifuge the collected lysis buffer at 4 ℃, 17000 g for 15-30 min, collect the supernatant and store it at -20 ℃;

[0109] Fourth step: Western Blotting is used to detect protein expression, and the detection results are as shown in Figure 2

[0110] Example 7: saRNA drug in vivo experiment

[0111] First step: Select Balb / c mice as experimental animals, and randomly divide the experimental mice into 4 groups. The blank control group is given LNP solution, and the other three groups are given LNP-saRNA preparations prepared in Example 5. The dosages are 5 μg / dose, 2.5 μg / dose and 1 μg / dose, respectively. Each group is given 2 doses, and the interval between the two doses is one week.

[0112] Second step: Take the mouse serum on the 17th, 21st, 28th, 35th and 66th day after administration. ELISA method is used to detect mouse serum IgG binding antibody, and the results are as shown in Figures 3-7 . The detection results show that the binding antibody titer gradually increases from 1:3200 on the 17th day to 1:1024000 on the 28th day, and then remains unchanged. The value indicated by the Cutoff line is the preset threshold value for distinguishing the positive and negative results of the detection results. The results higher than the value indicated by the Cutoff line are positive, and the results lower than the value indicated by the Cutoff line are negative.

[0113] Third step: Take the spleen cells of the mice on the 44th day and use the ELISPOT method to detect IFN-γ secretion. Prepare mouse cell suspension from the spleen cells, add polypeptide library for stimulation, make the spleen cells secrete IFN-γ, add avidin-enzyme complex, combine with biotin, add color developing substrate, and form visible spots. The results are as shown in Figure 8 . After long-term stimulation of saRNA translated antigens, the cellular immune system has been completely activated, and there is no obvious trend change in the IFN-γ detection results between different administration groups.

[0114] Fourth step: Take the spleen of the mice on the 52nd day to check T cell activation. After polypeptide stimulation, the spleen cells are stained with CD25 / CD69 dye, and then the FACs method is used to analyze the T cell activation. The detection results are shown in Figure 9 . The data show that saRNA translated antigens stimulate the body to induce cellular immune response after administration. ​

[0115] Fifth step: Take the serum of the 126th day mouse to detect neutralizing antibodies. According to the RFFIT rapid fluorescent focus inhibition test method, the rabies virus standard challenge strain CVS-11 is used as the challenge virus to neutralize the neutralizing antibodies of the rabies virus in the serum to be tested, and then BHK-21 cells are used to detect the remaining virus, and the results are detected by the fluorescent antibody test. The results are as follows in Table 1. All test results are much higher than the standard ≥0.5 IU / ml (WHO), indicating that the mice after vaccination still have very high immune protection at D126.

[0116] Table 1. D126 serum neutralizing antibody detection data

[0117]

[0118] Example 8: LNP-saRNA formulation titer experiment

[0119] The drug product is prepared according to Example 5, with a specification of 50 μg / mL / dose, batch number: F2402202. The titer is detected according to the "Pharmacopoeia of the People's Republic of China" 2020 edition three 3503 human rabies vaccine titer determination method. The detection results are shown in Table 2. The detection results show that the vaccine of the inventor has a very high titer (>217.4 IU / mL), which is much higher than the standard (each human dose is 0.5 mL or 1.0 mL, and the titer of rabies vaccine should not be less than 2.5 IU).

[0120] Table 2. Detection data of mouse challenge experiment

[0121]

[0122] The human rabies vaccine of the present application not only meets the minimum titer requirement of the pharmacopoeia, but also has much higher actual neutralizing antibody and titer than the standard, showing the high efficiency of the vaccine. This high efficiency means that the vaccine has very high protection in preventing rabies, and can provide more reliable immune protection for the vaccinated. In addition, high vaccine titer may also mean that the vaccination dose can be reduced in actual application, thereby improving the accessibility and economy of the vaccine. Therefore, the vaccine of the present application performs well in safety, effectiveness and practical application value, and has important clinical significance and market potential.

Claims

1. A protein having the amino acid sequence shown in SEQ ID NO:

5.

2. A self-replicating mRNA molecule comprising a coding region RNA sequence of the amino acid sequence as described in claim 1.

3. The mRNA molecule as described in claim 2, characterized in that: The nucleotide sequence of the mRNA molecule is shown in SEQ ID NO:

6.

4. Use of the mRNA molecule of claim 2 or 3 in the preparation of a rabies virus prevention drug, wherein the use is for the preparation of a composition comprising an immunologically effective amount of a construct of the mRNA molecule of claim 2 or 3.

5. A rabies virus saRNA vaccine, characterized in that: The vaccine is a lipid nanoparticle containing the mRNA molecule as described in claim 3.

6. The rabies virus saRNA vaccine as described in claim 5, characterized in that... It is prepared by a method including the following steps: 1) saRNA molecular design Select the amino acid sequence shown in SEQ ID NO:5, and then design the nucleotide sequence shown in SEQ ID NO:6; 2) Plasmid preparation ① The nucleic acid fragment was synthesized according to the sequence of SEQ ID NO:6 using a DNA synthesizer, the vector was linearized using restriction endonuclease, and the synthesized linear DNA was ligated with the linearized plasmid using DNA ligase; ② The recombinant plasmid ligation product obtained in step ① was added to competent DH5α cells for heat shock treatment; ③ Culture the competent cells treated in step ② in liquid LB medium, and then spread the cells on solid LB medium for further culture; ④ Select well-grown single colonies, culture them in synthetic medium, extract plasmids from a small amount of bacterial solution, and identify them by SalI and XbaI enzyme digestion. ⑤ The bacterial culture with correct identification results is scaled up, centrifuged, and the bacterial sludge is harvested; ⑥ Prepare supercoiled plasmid solution using fungal sludge; 3) Prepare linear plasmid templates from supercoiled plasmids; 4) Preparation of saRNA stock solution and LNP-saRNA formulation; The method for preparing the saRNA stock solution is to perform an in vitro transcription reaction on a linear plasmid template, and then obtain the saRNA stock solution by incubation, centrifugation, dissolving the precipitate in enzyme-free water, purification by affinity chromatography, purification using a TFF system, and filtration. The preparation steps of the LNP-saRNA formulation are as follows: ① Dilute the saRNA stock solution with citrate buffer to obtain the saRNA working solution; ② Mix the organic phase with the saRNA working solution at a volume ratio of 1:2-4 to prepare LNP nanoparticles; wherein the molar concentration ratio of the cations DLin-MC3-DMA:DSPC:CHO-HP:DMP-PEG2000 in the organic phase is 40-60:5-10:30-60:2-5, and the total molar concentration of the organic phase is controlled within the range of 15-20 mmol / L; ③ Dilute the LNP nanoparticles with 1×PBS, centrifuge and concentrate, repeat once, filter, and obtain the LNP-saRNA formulation, which is the rabies virus saRNA vaccine.

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