Preparation and application of rabies mRNA vaccine

Abstract

According to the optimized RVG mRNA provided by the invention, a rabies virus CTN-1 strain G protein (RVG) coding region is optimized, and the optimization comprises the following steps: adjusting codon preference during expression in a human body, adjusting the use frequency of common codons, improving the GC content of the sequence and the like, so that the obtained nucleotide sequence transcribed mRNA is more stable in structure, more stable in expression, and more stable in expression. And the translation efficiency of the target protein in mammals and human bodies is higher. And mixing the vaccine vector with the optimized RVG mRNA to obtain the rabies virus nucleic acid vaccine. The method is simple and easy to operate, the process difficulty of vaccine production is reduced, large-scale production can be realized, the batch difference is easy to control, and the production cost is greatly reduced. Enough protection effect can be achieved by using an extremely small dosage, the production cost is reduced, and the rabies vaccine is superior to the existing rabies vaccine in the aspect of effectiveness.

Description

Technical Field

[0001] The present invention relates to the field of nucleic acid vaccines, and in particular to the preparation and application of an mRNA rabies vaccine. Background Art

[0002] Rabies is a fatal neurological disease caused by infection with the rabies virus (RV). Once the patient is infected and enters the attack phase, the mortality rate is nearly 100%. The disease has always been the subject of large-scale public health intervention measures. There is still no effective treatment, and it is currently mainly prevented by vaccination. Most existing rabies vaccines require multiple doses to achieve a high neutralizing titer, and the production cost is high. Therefore, the development of innovative vaccines is the only way to reduce rabies to an acceptable rate. At present, many preclinical and clinical studies are underway, including new vaccines, adjuvants, and injection methods. mRNA vaccines have the advantages of short R&D and production cycles, easy mass production, high production capacity, and low safety risks. Compared with traditional rabies vaccines, mRNA vaccines have significant advantages.

[0003] Currently, the rabies vaccine on the market is mainly an inactivated rabies virus vaccine. The production cost of this vaccine is high. The main reason is that the rabies virus produced has a low titer and requires multiple injections to achieve satisfactory protective efficacy. The amount of effective antigen required in each milliliter of vaccine is high, so a higher titer of rabies virus needs to be produced.

[0004] The pathogen of rabies, RABV, is a negative-strand RNA virus of the genus Lyssavirus, belonging to the family Rhabdoviridae, encoding five structural proteins: nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and RNA-guided RNA polymerase (L). Among them, the trimeric G protein interacts with the matrix protein on its viral outer membrane. It is the only protein exposed on the surface of the rhabdovirus envelope and the only ligand of the cell receptor. Only G protein can stimulate the body to produce neutralizing antibodies. Subunit vaccines only contain the required immunogenicity, that is, glycoproteins are extracted from the whole rabies virus, expressed in prokaryotic or eukaryotic cells, and gene products-protein or polypeptide immune bodies are formed to induce animal immunity to RABV. It is a safer rabies vaccine, but how to choose a suitable expression system and purification process of the expression product is a technical difficulty in the research and development and production process.

[0005] The rabies virus vaccines currently available on the market usually have many disadvantages in their application, especially: 1) The preparation process is complicated and requires the use of cell culture methods to produce viruses, which poses certain safety risks. 2) High quality control and process amplification requirements, poor control will cause product quality accidents. 3) The effective antigen amount produced by the virus is low, and in order to ensure the effect, the virus titer must be increased, which increases the production cost.

[0006] In view of this, the present invention is proposed, wherein the mRNA rabies vaccine only requires one immunization to produce 100% protection efficacy. Summary of the invention

[0007] The first purpose of the present invention is to provide an optimized RVG mRNA to alleviate the technical problems of low antigen activity, poor translation efficiency and stability required to be improved in the prior art nucleic acid vaccines.

[0008] The second object of the present invention is to provide the application of the above-mentioned optimized RVG mRNA.

[0009] The third purpose of the present invention is to provide a rabies virus nucleic acid vaccine to alleviate the problems of low rabies vaccine virus titer, small effective antigen amount and high production cost in the prior art.

[0010] The fourth object of the present invention is to provide a method for preparing the above-mentioned rabies virus nucleic acid vaccine, providing a method with simple operation and high yield.

[0011] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0012] An optimized RVG mRNA, wherein the coding nucleotide sequence of the optimized RVG mRNA is as shown in SEQ ID NO.1.

[0013] Further, the nucleotide sequence for transcribing the optimized RVG mRNA also includes the following elements: at least one of a 5′-cap structure, a 5′-UTR, a 3′-polyadenylic acid sequence, and a 3′-UTR;

[0014] Preferably, the 5′-UTR is 10-200 nucleotides, more preferably 15-100 nucleotides;

[0015] Preferably, the 5′-UTR is a 5′-UTR sequence or a KOZAK sequence;

[0016] Preferably, the KOZAK sequence is shown in SEQ ID NO.2:

[0017] Preferably, the ACTBB 5′-UTR sequence is shown in SEQ ID NO.3:

[0018]

[0019] Preferably, the 3′-poly(A) sequence is preferably 60-120 A, more preferably 80-110 A, and further preferably 100 A;

[0020] Preferably, the 3′-UTR comprises a β-globin 3′-UTR sequence or a hemoglobin 3-UTR sequence;

[0021] Preferably, the 3′-UTR sequence is shown in SEQ ID NO.4:

[0022]

[0023] Preferably, the sequence of the RVG mRNA is shown as SEQ ID NO.5.

[0024] Preferably, the sequence of the RVG mRNA is as shown in SEQ ID NO.6.

[0025] The above-mentioned optimized RVG mRNA is used in the preparation of rabies virus nucleic acid vaccine.

[0026] A rabies virus nucleic acid vaccine, comprising a vaccine vector and the optimized RVG mRNA provided by the present invention, further comprising a cationic liposome, a cationic protein, a cationic polymer or a cationic lipid nanoparticle, preferably a cationic liposome or a cationic lipid nanoparticle;

[0027] Preferably, the molar ratio of the ionizable nitrogen of the cationic lipid to the phosphorus of the mRNA phosphate backbone (i.e., NP ratio, N / P) is 3-7, preferably 4-6, more preferably 5-6;

[0028] Further, the vaccine carrier is a cationic lipid nanoparticle, which comprises, by mole percentage, 20-70% cationic lipid, 20-50% structural lipid, 5-20% auxiliary lipid and 1-5% surfactant, wherein the molar content of cationic lipid, structural lipid, auxiliary lipid and surfactant is 100% in total;

[0029] Preferably, the protonatable cationic lipid comprises SM-102, ALC-0315, DOTPA, Dlin-MC3-DMA, Dlin-KC2-DMA, DODMA or DIinDMA, preferably Dlin-MC3-DMA;

[0030] Preferably, the structural lipid comprises cholesterol, cholesterol ester, steroid hormone, steroid vitamin or bile acid, preferably cholesterol;

[0031] Preferably, the helper lipid comprises DSPC, DOPE, DOPC, DSPE or DOPS, preferably DSPC;

[0032] Preferably, the surfactant comprises PEG-DMG, PEG-DSPE or ALC-0519, preferably PEG-DMG;

[0033] Preferably, the administration of the rabies virus nucleic acid vaccine includes intravenous injection, intramuscular injection or intradermal injection, preferably intramuscular injection.

[0034] The method for preparing the rabies virus nucleic acid vaccine comprises mixing the vaccine vector and the optimized RVG mRNA provided by the present invention to obtain the rabies virus nucleic acid vaccine.

[0035] Furthermore, the vaccine carrier is the cationic lipid nanoparticles in the above-mentioned rabies virus nucleic acid vaccine, and the preparation method comprises:

[0036] (a) dissolving the cationic lipid, structural lipid, auxiliary lipid and surfactant in an organic solution according to a formula ratio to obtain an organic phase;

[0037] (b) dissolving the optimized RVG mRNA in a sodium citrate or sodium acetate solution at pH 3-4 to obtain an aqueous phase;

[0038] (c) mixing the organic phase obtained in (a) and the aqueous phase obtained in (b) to obtain a mixed solution, replacing excess organic solution and excess acidic salt solution in the mixed solution with a buffer solution to obtain a rabies virus nucleic acid vaccine;

[0039] Preferably, the organic solvent comprises anhydrous ethanol, tetrahydrofuran, acetone, isopropanol or DMSO, preferably anhydrous ethanol;

[0040] Preferably, the total concentration of cationic lipids, structural lipids, auxiliary lipids and surfactants in the organic phase is 10-35 mg / ml;

[0041] Preferably, the concentration of optimized RVG mRNA in the aqueous phase is 0.1-0.3 mg / ml;

[0042] Preferably, the volume ratio of the organic phase to the aqueous phase is 1:2-4;

[0043] Preferably, replacing the solvent in the mixed solution with a buffer solution comprises: replacing the mixed solution with the buffer solution by 30-80 times.

[0044] Compared with the prior art, the present invention has the following beneficial effects:

[0045] The present invention provides an optimized RVG mRNA, and the nucleotide sequence of the transcribed RVG mRNA is shown in SEQ ID NO.1. The inventor optimizes the coding region of the G protein (RVG) of the rabies virus CTN-1 strain, and the sequence optimization includes: adjusting the codon preference when expressed in the human body, adjusting the usage frequency of common codons, increasing the GC content of the sequence, etc., to obtain the nucleotide sequence of the optimized RVG mRNA sequence shown in SEQ ID NO.1. The nucleotide sequence makes the transcribed mRNA structure more stable, and the target protein translation efficiency in mammals and humans is higher.

[0046] The rabies virus nucleic acid vaccine provided by the present invention includes a vaccine vector and an optimized RVG mRNA. The nucleic acid vaccine delivers the optimized RVG mRNA synthesized by in vitro transcription into the body to produce rabies virus glycoprotein (i.e., antigen protein), thereby achieving sufficient protection effect using an extremely small dose, reducing production costs, and being superior to existing rabies vaccine technologies in terms of effectiveness.

[0047] The present invention provides a method for preparing the above-mentioned rabies virus nucleic acid vaccine, wherein the vaccine vector and the optimized RVG mRNA are mixed to obtain the rabies virus nucleic acid vaccine. The method is simple and easy to operate, reduces the process difficulty of vaccine production, can be produced on a large scale, and the batch difference is easy to control, which greatly reduces the production cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 a. Western blot image showing the expression of linear and circular RVG mRNA in HEK293 cells;

[0049] Figure 1 b. A bar graph showing the data analysis of the expression levels of linear and circular RVG mRNA transfected HEK293 cells;

[0050] Figure 2 .The expression intensity of Fluc encapsulated by different LNP preparations in mice;

[0051] Figure 3 a. Schematic diagram of animal experiment scheme;

[0052] Figure 3 b. is a statistical chart of mouse weight;

[0053] Figure 3 c. is a statistical graph of neutralizing antibody data of mice 14 days after administration;

[0054] Figure 3 d. Statistical chart of vaccine protection rate;

[0055] Figure 4a. The results of neutralizing antibody titers at 7D after mice were immunized with different doses of linear mRNA vaccines;

[0056] Figure 4 b. The results of 14D neutralizing antibody titers after mice were immunized with different doses of linear mRNA vaccines;

[0057] Figure 4 c. Statistical graph of weight changes of mice at different doses after immunization;

[0058] Figure 4 d. is a statistical chart of the weight changes of mice after challenge;

[0059] Figure 4 e. Protection rate after immunization with different doses of linear mRNA vaccine;

[0060] Figure 5 a. Statistical graph of the survival of mice after exposure and vaccination;

[0061] Figure 5 b. Neutralizing antibodies 3D / 5D after mice were vaccinated;

[0062] Figure 5 c. is a statistical chart of mouse weight;

[0063] Figure 5 d. 3D / 5D positive rate after mice were vaccinated with mRNA vaccine. DETAILED DESCRIPTION

[0064] The embodiments of the present invention will be described in detail below in conjunction with examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. In the examples, if no specific conditions are specified, the conditions are carried out according to normal conditions or manufacturer recommendations. Unless otherwise indicated, the professional and scientific terms used herein are the same as those familiar to those skilled in the art. In addition, any method or material similar or equivalent to the described content may also be applied to the present invention.

[0065] An optimized RVG mRNA transcription-optimized RVG mRNA encoding nucleic acid sequence is as SEQ ID NO.1.

[0066]

[0067]

[0068] Example 1 RVG mRNA preparation

[0069] The kit used for in vitro transcription was Thermo scientific's Transcript Aid T7 High Yield Transcription Kit (Cat. No.: #K0441), and the reaction system is shown in Table 1.

[0070] Table 1. IVT reaction system

[0071] DPEC Water Up to 20ul A / C / GTP, Psedo UTP, clean cap 2ul 5x Reaction Buffer 4ul Enzyme Mix 2ul Template lug Total 20ul

[0072] After mixing, react at 37℃ for 30min. Add 2ul DNase I to the transcription product after the reaction is completed and digest it at 37℃ for 20min; add an equal volume of LiCl Precipitation Solution 22uL and mix thoroughly, cool at -20℃ for 1h to terminate and precipitate RNA. Centrifuge at 4℃ for 15min at the highest speed to precipitate RNA and remove the supernatant. Wash the precipitate with 1mL of 70% ethanol (prepared with DEPC water), centrifuge again, remove the supernatant, dry the precipitate, and add appropriate solution to dissolve RNA as needed, and store mRNA at -70℃.

[0073] Example 2 Sequence Optimization of RVG mRNA

[0074] The sequence optimization scheme of RVG mRNA in this example is shown in Table 2, and is as follows:

[0075] Table 2. Sequence information of RVG mRNA

[0076]

[0077]

[0078] The final RVG-1 mRNA coding sequence was:

[0079]

[0080]

[0081] The final RVG-3 mRNA coding sequence was:

[0082]

[0083]

[0084]

[0085] The key elements of the RVG-1 mRNA sequence are the use of different 5′UTRs, the open reading frame nucleotides encoding the G protein of the CTN-1 strain (SEQ ID NO.1 shows the optimized sequence), 3′UTR and polyA sequences (refer to the CureVac rabies virus mRNA vaccine patent (CN105517569A). The coding region (CDS) of RVG-3 mRNA is consistent with that of RVG-1 mRNA, and the optimized sequences are used in the 5′UTR, 3′UTR, and polyA parts. The coding region of RVG-2 mRNA is consistent with that of RVG-1 mRNA, and a circular RNA method is used. The protein sequences finally translated from these three ORFs are consistent, which is the G protein of the CTN-1 strain.

[0086] Example 3 Detection of RVG mRNA antigen expression in vitro

[0087] The RVG-1 mRNA, RVG-2 mRNA and RVG-3 mRNA prepared by the above in vitro transcription process were transfected into HEK293 cells respectively. HEK293 cells in the logarithmic growth phase were taken and the cell suspension concentration was adjusted to 5×10 5Cells / ml, 1 ml per well was inoculated in a 6-well plate, placed in a 37°C incubator containing 5% CO2 to allow the cells to adhere to the wall, and cultured overnight. During transfection, 4 μg mRNA and 8 μl of transfection reagent Lipofectamine 2000 (Thermo Fisher Scientific) were diluted in 250 μl serum-free culture medium (Opti-MEM) and mixed, incubated at room temperature for 5 minutes, then the above mRNA solution was mixed with lipofectamine 2000 solution, left to stand at room temperature for 20 minutes, 500 μl of oligonucleic acid-liposome complex was added to the cell culture plate, and 500 μl serum-free culture medium (Opti-MEM) was added to the 6-well plate to make the final transfection system 1 ml. 24 hours after transfection, HEK293 cells were lysed with M-PER Mammalian Protein Extraction Reagent (Thermo) for protein extraction, and the obtained protein was used for Western Blot detection. Specifically, the total protein concentration was determined by the BRADFORD method; the total protein concentration of each group was adjusted with 1X PBS; and the gel was run in the Tianneng VE-180 vertical electrophoresis tank. After the electrophoresis, the membrane was transferred using the Tianneng VE-186 transfer electrophoresis tank. After the transfer, the transfer membrane was blocked with Blocking Buffer for 2 hours. The diluted primary antibody was added and incubated at 4°C overnight. Washed 3 times with 1X PBST. The diluted secondary antibody was added and incubated at room temperature for 1 hour. Washed 3 times with 1X PBST. The ECL substrate was used for chemiluminescence detection, and the grayscale analysis was quantified after exposure by the Tanon 4200 fully automatic chemiluminescence image analysis system and using the Gel-Pro analyzer software.

[0088] The results are as follows Figure 1 As shown, from Figure 1 The results show that the G protein expression in the RVG-2mRNA and RVG-3mRNA transfected cell groups is higher than that in the RVG-1mRNA transfected group. This shows that different 5UTRs and a perfect CleanCap GAG promoter can increase the G protein expression. The G protein content in the RVG-2mRNA transfected cells is higher than that in the RVG-3mRNA transfected group. This shows that circular RNA can increase the G protein expression.

[0089] Example 4 Preparation method of rabies virus nucleic acid vaccine

[0090] The mRNA prepared by in vitro transcription was dissolved in a sodium citrate buffer at pH 4 and the concentration was adjusted to 0.2 mg / ml to obtain an aqueous phase.

[0091] According to the formula SM-102 or Stama 1-4 (Xingrui Pharmaceutical (Suzhou) Co., Ltd.), SM-102, cholesterol, DSPC / DOPE and PEG-DMG are dissolved in anhydrous ethanol in proportion, and the total lipid concentration is adjusted to 10-35 mg / ml to obtain an organic phase. The organic phase and the aqueous phase are mixed at a volume ratio of 1:3 using a microfluidic device with a mixing flow rate of 12.0 ml / min. The obtained mixed solution is immediately diluted with PBS solution, and tangential flow filtration (TFF) is used to remove ethanol and excess citrate buffer components in the solution. The solution is concentrated to an mRNA concentration of about 0.1 mg / ml to obtain lipid nanoparticles encapsulating mRNA. Lipid nanoparticles encapsulating Fluc-mRNA of different prescriptions were injected intramuscularly into mice. The specific prescription information is shown in Table 3. The fluorescence value of the mouse was observed by in vivo imaging ( Figure 2 ), select the better Stama-3 formula to encapsulate the corresponding RVG-mRNA, which is the RVG-mRNA rabies virus nucleic acid vaccine.

[0092] Table 3. LNP prescription table (the content in the table is molar percentage)

[0093]

[0094] Example 5 Determination of neutralizing antibody titer in mice

[0095] The mRNA selected was RVG-2 mRNA and RVG-3 mRNA in Example 3 for the Balb / C mouse immunization experiment, and the vaccine carrier selected was cationic lipid nanoparticles prepared using the Stama-3 formula in Example 4. The commercial vaccine was an inactivated whole virus vaccine (Intervet) as a positive control PC (≥10 6.3 FAID 50 / ml, the administration volume was 100ul / mouse), and each mouse was injected with a dosage of 5ug, and the injection volume was 100ul / mouse. The negative control was an equal volume of saline injection. The rabies virus neutralizing antibody titer of the mouse serum was detected 14 days after the above vaccine preparations were administered once, and the mice were challenged with rabies virus (100 times LD50 RABV CVS-24) by intracerebral injection 14 days after administration. The weight of the mice and the survival rate of the mice were monitored 21 days after the challenge. Figure 3 As shown, from Figure 3 It can be seen from the results that the neutralizing antibody titer induced by a single immunization of RVG-mRNA vaccine is much higher than that of commercially available rabies vaccines, indicating that the technology of the present invention is superior to the existing technology.

[0096] Example 6 Dose-dependent humoral immune response

[0097] The RVG-3 mRNA was encapsulated in the Stama-3 formula of Example 5 for the Balb / C mouse immunization experiment. The dose gradient was set as: 0.5ug, 5ug, 50ug, and the injection volume was 100ul / mouse. The commercial vaccine was an inactivated whole virus vaccine (Intervet) as a positive control PC (≥10 6.3 FAID 50 / ml, the administration volume was 100ul / mouse). The negative control was an equal volume of saline injection. The rabies virus neutralizing antibody titer of the mouse serum was detected 14 days after the above vaccine preparations were administered once, and the mice were challenged with rabies virus (100 times LD50 RABV CVS-24) by intracerebral injection 17 days after administration. The weight and survival rate of the mice were monitored 39 days after the challenge. The results are as follows Figure 4 As shown: At the same time, refer to the Pharmacopoeia (2020) General Chapter 1141 Inspection Method to monitor the abnormal toxicity of the preparation. The results are as follows Figure 4 As shown:

[0098] from Figure 4 The results show that the neutralizing antibody titer induced by a single administration of 0.5ug of RVG-mRNA vaccine is equivalent to that of commercially available rabies vaccines, and the protection rate of mice after challenge is comparable to that of commercially available rabies vaccines. The neutralizing antibody titer induced by a single administration of 5ug and 50ug of RVG-mRNA vaccine is much higher than that of commercially available rabies vaccines, and all mice vaccinated are protected against lethal rabies virus attack infection without weight loss. Abnormal toxicity showed no significant difference in the weight of mice after immunization.

[0099] Example 7 Post-exposure immune protection

[0100] The Balb / C mouse immunization experiment was performed using the Stama-3 formula in Example 5 and SM102 encapsulated RVG-3 mRNA. 50 times LD50 CVS-24 rabies virus was injected into the left hind limb muscle of the mouse. Six hours after the virus injection, the above vaccine preparation was injected into the right hind limb muscle of the mouse. The negative control was an equal volume of saline injection. The mouse serum rabies virus neutralizing antibody titer was detected 3 days and 5 days after administration. The mouse body weight and mouse survival rate after administration were then monitored. The results are shown in Figure 2. Figure 5 As shown:

[0101] from Figure 5It can be seen from the results that a single dose of the Stama-3 formula and the SM102 commercial formula encapsulated RVG-3mRNA vaccine can induce high neutralizing antibodies 5 days after administration, and is sufficiently higher than the WHO standard of 0.5IU / mL, indicating that the above vaccines can quickly induce mice to produce neutralizing antibodies. The protection rate of mice vaccinated with the Starna-3 formula vaccine after exposure to rabies virus was 60%, and the protection rate of mice vaccinated with the SM102 formula vaccine after exposure was 40%. However, all mice in the negative control group died of rabies within 14 days. The above data show that the Stama-3 formula vaccine is superior to the SM102 commercial formula vaccine and has a better protective effect on mice after exposure.

[0102] The present invention illustrates the process method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned process steps, that is, it does not mean that the present invention must rely on the above-mentioned process steps to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of the raw materials selected by the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. An RVG mRNA, characterized in that The coding sequence of the RVG mRNA is shown in SEQ ID NO.

1.

2. An optimized RVG mRNA, characterized in that The optimized RVG mRNA comprises the following elements from 5' to 3' end: a 5'-cap structure, a 5'-UTR, the coding sequence of the RVG mRNA according to claim 1, a 3'-UTR and a 3'-polyadenylic acid sequence.

3. The optimized RVG mRNA according to claim 2, characterized in that The 5'-UTR has a length of 10-200 nucleotides, preferably 15-100 nucleotides.

4. The optimized RVG mRNA according to claim 3, characterized in that The 5'-UTR is an ACTBB 5'-UTR sequence or a KOZAK sequence.

5. The optimized RVG mRNA according to claim 2, characterized in that The 3'-poly(A) sequence preferably has 60-120 A's.

6. The optimized RVG mRNA according to claim 2, characterized in that The 3'-UTR includes a β-globin 3'-UTR sequence or a hemoglobin HBA2 3'-UTR sequence.

7. Use of the RVG mRNA according to any one of claims 1 to 6 in the preparation of rabies virus nucleic acid vaccine.

8. A rabies virus nucleic acid vaccine, characterized in that: The rabies virus nucleic acid vaccine comprises a vaccine vector and the RVG mRNA according to any one of claims 1-6.

9. The rabies virus nucleic acid vaccine according to claim 8, characterized in that: The vaccine carrier includes one or more of cationic liposomes, cationic proteins, cationic polymers or cationic lipid nanoparticles.

10. A method for preparing a rabies virus nucleic acid vaccine, characterized in that: The vaccine vector is mixed with the RVG mRNA described in any one of claims 1 to 6 to obtain a rabies virus nucleic acid vaccine.

11. The method according to claim 10, characterized in that The steps include: 1) dissolving a protonable cationic lipid, a structural lipid, an auxiliary lipid and a surfactant in an organic solution to obtain an organic phase; 2) dissolving the RVG mRNA according to any one of claims 1 to 6 in a sodium citrate or sodium acetate solution at pH 3-4 to obtain an aqueous phase; 3) The obtained organic phase and aqueous phase are mixed to obtain a mixed solution, and the organic solution and excess acidic salt solution in the mixed solution are replaced with a buffer solution to obtain a rabies virus nucleic acid vaccine.

12. The method according to claim 11, characterized in that The concentration of RVG mRNA in the aqueous phase of step 2) is 0.1-0.4 mg / ml.

13. The method according to claim 12, characterized in that In the step 3), the volume ratio of the organic phase to the aqueous phase is 1:2-4.

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