Rabies virus glycoprotein-based mRNA vaccine as well as preparation method and application thereof

Abstract

The invention relates to a rabies virus glycoprotein-based mRNA vaccine as well as a preparation method and application thereof. The mRNA vaccine comprises: (1) an immunogen mRNA for expressing rabies virus glycoprotein, wherein the rabies virus glycoprotein is G protein, IG protein, 3G protein, 3IG protein or 3IG [delta] GC protein; and (2) a vaccine vector, wherein the vaccine vector is liposome nano-particles which can be utilized by mRNA (messenger Ribonucleic Acid). The invention also provides a preparation method of the vaccine and application of the vaccine in prevention of rabies virus infection. According to the invention, G proteins and IG proteins of rabies virus strains CTN strain, SAD strain and CVS strain are taken as immunogen basis for the first time and are optimized and combined, and the rabies virus mRNA vaccine with immunogen being G protein, IG protein, 3G protein, 3IG protein or 3IG delta GC protein and vaccine vector being liposome nanoparticles is developed.

Description

Technical Field

[0001] The present invention relates to an mRNA vaccine based on rabies virus glycoprotein and a preparation method and application thereof, belonging to the technical field of biomedicine. Background Art

[0002] Rabies is a zoonotic disease transmitted by the rabies virus (RABV). Humans are infected mainly because of bites or scratches from infected dogs. The mortality rate after onset is as high as 100%, making it one of the infectious diseases with the highest mortality rate. Every year, 59,000 to 61,000 people die from rabies in more than 150 countries around the world. The direct and indirect economic losses caused by rabies are as high as nearly US$8.6 billion (about RMB 54.4 billion) each year, which has brought a great burden to human public health.

[0003] The rabies vaccine currently on the market is an inactivated rabies virus vaccine, which has a high production cost. The main reason is that the rabies virus produced has a low titer, while 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] Rabies virus (RV) is a type of enveloped virus belonging to the genus Lyssavirus of the family Rhabdoviridae. It contains a 12kb single-stranded negative-sense RNA genome that encodes five structural proteins: nucleoprotein (N), phosphoprotein (P), matrix protein (M), surface glycoprotein (G) and RNA polymerase (L). Rabies virus particles are bullet-shaped, with a trimer structure of G protein distributed on the surface. N, P and L proteins bind to the genomic RNA and participate in RNA replication and translation. G protein is a 65KDa envelope protein with antigenic determinants that induce cellular immunity and humoral immunity, and is a molecular target for inducing neutralizing antibodies. Monoclonal antibodies and vaccines targeting G protein can provide effective protection for the host and are widely used in the development of nucleic acid vaccines.

[0005] However, the rabies vaccines available on the market have the following problems: 1) The preparation process is complicated and requires the use of cell culture to produce the virus, which poses certain safety risks. 2) There are high requirements for quality control and process amplification during the production process. If it cannot be well controlled, it will cause product quality accidents. 3) The effective antigen amount produced by the virus is low. In order to ensure the effect, the virus titer must be increased, which increases the production cost. Summary of the invention

[0006] In view of the shortcomings of the prior art, the present invention provides an mRNA vaccine based on rabies virus glycoprotein and its preparation method and application. The vaccine not only has the advantages of high safety and effectiveness, but also reaches a level of sufficient protection efficiency with a very small dose.

[0007] The technical solution of the present invention is as follows: A modified mRNA vaccine based on rabies virus glycoprotein, comprising: (1) Immunogen mRNA for expressing rabies virus glycoprotein, wherein the rabies virus glycoprotein is G protein, IG protein, 3G protein, 3IG protein or 3IG ΔGC protein; (2) Vaccine vectors, wherein the vaccine vectors are liposome nanoparticles that can be utilized by mRNA.

[0008] Preferably according to the present invention, the rabies virus glycoprotein is selected from the following rabies virus strains: CTN strain, SAD strain and CVS strain.

[0009] Preferably, according to the present invention, the immunogen mRNA is RABV-G, RABV-IG, RABV-3G, RABV-3IG, RABV-3IG designed according to the rabies virus glycoprotein. ΔGC ; The nucleotide sequence of RABV-G is shown in SEQ ID NO.1; the nucleotide sequence of RABV-IG is shown in SEQ ID NO.2; the nucleotide sequence of RABV-3G is shown in SEQ ID NO.3; the nucleotide sequence of RABV-3IG is shown in SEQ ID NO.4; the nucleotide sequence of RABV-3IG is shown in SEQ ID NO.5. ΔGC The nucleotide sequence is shown in SEQ ID NO.5.

[0010] The structure of the immunogen mRNA is: A1-A2-A3-A4-A5-A6; Wherein, A1 is a 5' cap structure; A2 is a 5' UTR element; A3 is an IgG signal peptide coding sequence; A4 is a coding gene sequence of rabies virus glycoprotein, wherein the rabies virus glycoprotein is G protein, IG protein, 3G protein, 3IG protein or 3IG ΔGC protein; the A5 is a 3'UTR element; and the A6 is a poly (A) tail structure.

[0011] Preferably, according to the present invention, the G protein is the full-length glycoprotein of the CTN strain, and the amino acid sequence is shown in SEQ ID NO.6; the IG protein is the full-length glycoprotein of the CTN strain with the signal peptide sequence replaced, and the amino acid sequence is shown in SEQ ID NO.7; the 3G protein is the glycoprotein of the CTN strain, SAD strain, and CVS strain according to the combination of CTN: SAD: CVS = 1: 1: 1, and the amino acid sequence is shown in SEQ ID NO.8; the 3IG protein is the glycoprotein of the CTN, SAD, and CVS strains with the signal peptide sequence replaced according to the combination of CTN: SAD: CVS = 1: 1: 1, and the amino acid sequence is shown in SEQ ID NO.9; ΔGC The protein is a protein obtained by optimizing the amino acid sites of the 3IG protein, and the amino acid sequence is shown in SEQ ID NO.10.

[0012] Preferably according to the present invention, the liposome nanoparticles include ionizable cationic lipids, neutral phospholipids, cholesterol, and PEG-modified lipids; and the particle size of the liposome nanoparticles is 100-150 nm.

[0013] Preferably according to the present invention, the mass ratio of the liposome nanoparticles to the immunogen mRNA is 23:1.

[0014] The method for preparing the modified mRNA vaccine based on rabies virus glycoprotein comprises the following steps: S1, G protein, IG protein, 3G protein, 3IG protein, 3IG ΔGC The amino acid sequence of the protein was codon optimized and the corresponding rabies virus glycoprotein coding gene was synthesized to obtain G protein, IG protein, 3G protein, 3IG protein, 3IG ΔGC Proteins A total of five rabies virus glycoprotein expression genes; S2. The five rabies virus immunogen expression genes were cloned into the plasmid vector pVAX1-BsaI-SpeI-SapI-BspMI-free-Terminator-T7 deleted (C9863TRUG0-34) to obtain five corresponding recombinant plasmids; S3, preparation of five immunogen mRNAs; S4, transfect five immunogen mRNAs into 293T cells respectively; S5. Western-Blot was used to detect protein expression. After confirming that the immunogen mRNA can be expressed normally, the immunogen mRNA was dissolved in a citrate buffer to obtain a citrate buffer containing the immunogen mRNA; S6. Dissolve the ionizable cationic lipid SM-102, the neutral phospholipid DSPC, cholesterol and the PEG-modified lipid DMG-PEG2000 respectively using ethanol; then mix them in a molar ratio of SM-102: DSPC: cholesterol: DMG-PEG2000 = 50:10:38.5:1.5 to obtain a lipid mixture; S7. The lipid mixture is mixed evenly with the citrate buffer containing the immunogen mRNA to obtain an RNA-LNP mixture. After ultrafiltration and filtration, a modified mRNA vaccine based on rabies virus glycoprotein is obtained.

[0015] Preferably, according to the present invention, in step S3, the immunogen mRNA is prepared according to the following method: (1) The recombinant plasmid obtained in step S2 is subjected to restriction enzyme linearization treatment, and then the restriction enzyme linearized plasmid is used as an in vitro transcription template. An in vitro transcription kit is used with ATP, GTP, CTP, and N1-Me-pUTP as substrates. The DNA sequence downstream of the plasmid promoter after restriction enzyme linearization is transcribed in vitro by T7 RNA polymerase, and UTP is completely replaced with the modified nucleotide N1-Me-pUTP; (2) After the reaction is completed, use DNase digestion to remove the DNA in the reaction; (3) Purify mRNA using magnetic beads to remove proteins and salts from mRNA; (4) Use the cap analog CAP5011 to perform chemical one-step capping to obtain the corresponding immunogen mRNA.

[0016] Preferably, according to the present invention, in step S4, the specific method of transfecting the immunogen mRNA into 293T cells is as follows: 1) Resuscitate 293T cells and inoculate the cells into a 6-well plate when the cells reach 90-100% density; 2) After 12 hours, when the cells grow to 90% density, the culture medium is replaced with serum-free culture medium and the mRNA is transfected into 293T cells using the transfection kit Lipofectamine; 3) Culture cells with 10% FBS DMEM medium and harvest cells 24 hours after transfection; 4) The levels of mRNA expression proteins were analyzed by Western-blot staining, and untransfected cells were used as negative controls.

[0017] Preferably, according to the present invention, in step S5, the specific method of Western-Blot detection of protein expression is as follows: a. 24 hours after transfection, cells were collected, 1× SDS-PAGE loading buffer was added, and the samples were denatured in a 100°C metal bath for 10 min; b. Add the heat-denatured sample to the prepared SDS-PAGE gel and perform electrophoresis at 120V for 80 minutes; c. After electrophoresis, transfer the membrane at 100V for 1 hour, use 5% milk as blocking solution for 1 hour at room temperature, wash three times with TBST eluent, use anti-G protein antibody as primary antibody (1:3000), shake slowly at room temperature for 2 hours, and wash three times with TBST eluent; use horseradish peroxidase (HRP)-labeled goat anti-rabbit 1gG (1:2000) as secondary antibody, shake slowly at room temperature for 1 hour, wash three times with TBST eluent, add substrate staining solution, and take pictures of the membrane after the reaction is terminated.

[0018] Beneficial effects: 1. The present invention is the first to use the G protein and IG protein of the rabies virus strains CTN, SAD and CVS as the basis of immunogens, optimize and combine them, and develop immunogens that are G protein, IG protein, 3G protein, 3IG protein or 3IG ΔGC Protein, rabies virus mRNA vaccine with liposome nanoparticles as the vaccine carrier.

[0019] 2. The present invention uses 3IG protein or 3IG ΔGC Protein-based mRNA vaccine RV-3IG and RV-3IG ΔGC Can effectively induce efficient neutralizing antibody response. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is the protein expression level of 5 immunogen mRNAs in HEK-293T cells; In the figure, the Western-Blot bands from left to right are RABV-G, RABV-IG, RABV-3G, RABV-3IG, RABV-3IG ΔGC .

[0021] Figure 2 The results of particle size analysis of modified mRNA vaccines based on rabies virus glycoprotein; In the figure, A is RV1 (G), B is RV2 (IG), C is RV3 (3G), D is RV4 (3IG), E is RV5 (3G ΔGC ).

[0022] Figure 3 Results are shown for the neutralizing antibody levels induced in mice by a modified mRNA vaccine based on rabies virus glycoprotein.

[0023] Figure 4 The results are shown for the neutralizing antibody levels induced in mice after immunization with different doses of modified mRNA vaccine based on rabies virus glycoprotein. DETAILED DESCRIPTION

[0024] The technical solution of the present invention is further described below in conjunction with the embodiments, but the protection scope of the present invention is not limited thereto. The reagents and materials involved in the embodiments are all common commercially available products unless otherwise specified.

[0025] In vitro transcription kit is available from Thermo Fisher Scientific.

[0026] The cap analog CAP5011 is available from Jiangsu Shenji Biotechnology Co., Ltd.

[0027] Quant-it RiboGreen RNA assay kit is available from Thermo Fisher Scientific.

[0028] FITC-labeled anti-rabies virus nucleoprotein antibody is available from Fujirebio.

[0029] Plasmid vector pVAX1-BsaI-SpeI-SapI-BspMI-free-Terminator-T7 deleted (C9863TRUG0-34), GenScript Biotech Co., Ltd.

[0030] Example 1. Preparation of modified mRNA vaccine based on rabies virus glycoprotein 1. Cells and viruses: HEK-293T cells and BHK21 cells were cultured in DMEM medium (GIBCO) containing 10% fetal bovine serum. The selected rabies virus strains were CVS-11 and CVS-24.

[0031] 2. Construction of recombinant plasmid: The present invention designs mRNA vaccines based on the immunogenic sequence of rabies virus strains, as follows: First, human hosts were used to test the rabies virus glycoprotein G protein, IG protein, 3G protein, 3IG protein, and 3IG ΔGC The amino acid sequence of the protein was codon optimized, and GenScript Biotech Co., Ltd. synthesized the immunogens G protein, IG protein, 3G protein, 3IG protein, and 3IG protein based on the optimized amino acid sequence. ΔGC The gene encoding the rabies virus glycoprotein.

[0032] Then, according to the gene recombination method of the prior art, the above-mentioned G protein, IG protein, 3G protein, 3IG protein, 3IGΔGC The rabies virus glycoprotein encoding genes of the proteins were cloned into the plasmid vector pVAX1-BsaI-SpeI-SapI-BspMI-free-Terminator-T7 deleted (C9863TRUG0-34) to obtain five corresponding recombinant plasmids.

[0033] Among them, the G protein is the full-length glycoprotein of the CTN strain, and the amino acid sequence is shown in SEQ ID NO.6; the IG protein is the full-length glycoprotein of the CTN strain with the signal peptide sequence replaced, and the amino acid sequence is shown in SEQ ID NO.7; the 3G protein is a combination of the full-length glycoproteins of the CTN strain, the SAD strain, and the CVS strain, and the amino acid sequence is shown in SEQ ID NO.8; the 3IG protein is a combination of the full-length glycoproteins of the CTN, SAD, and CVS strains with the signal peptide sequence replaced, and the amino acid sequence is shown in SEQ ID NO.9; ΔGC The protein is a protein obtained by optimizing amino acid sites of 3IG protein, and the amino acid sequence is shown in SEQ ID NO.10.

[0034] 3. Preparation of mRNA The recombinant plasmid obtained in step 2 was subjected to enzyme linearization treatment, and then the enzyme linearized plasmid was used as an in vitro transcription template, and an in vitro transcription kit was used with ATP, GTP, CTP, and N1-Me-pUTP as substrates. The DNA sequence downstream of the plasmid promoter after enzyme linearization was transcribed in vitro by T7 RNA polymerase, and UTP was completely replaced with the modified nucleotide N1-Me-pUTP; after the reaction, DNA was digested to remove the DNA in the reaction; and the mRNA was purified by magnetic beads to remove impurities such as proteins and salts in RNA. Finally, the cap analog CAP5011 was used for chemical one-step capping to prepare the corresponding immunogen mRNA.

[0035] In this step, five immunogen mRNAs were prepared, namely RABV-G, RABV-IG, RABV-3G, RABV-3IG, and RABV-3IG. ΔGC The nucleotide sequence of the RABV-G is shown in SEQ ID NO.1; the nucleotide sequence of the RABV-IG is shown in SEQ ID NO.2; the nucleotide sequence of the RABV-3G is shown in SEQ ID NO.3; the nucleotide sequence of the RABV-3IG is shown in SEQ ID NO.4; the RABV-3IG ΔGC The nucleotide sequence is shown in SEQ ID NO.5.

[0036] The structure of the resulting immunogen mRNA sequence was: A1-A2-A3-A4-A5-A6; Wherein, A1 is a 5' cap structure; A2 is a 5' UTR element; A3 is an IgG signal peptide coding sequence; A4 is a coding gene sequence of rabies virus glycoprotein, wherein the rabies virus glycoprotein is G protein, IG protein, 3G protein, 3IG protein or 3IG ΔGC protein; the A5 is a 3'UTR element; and the A6 is a poly (A) tail structure.

[0037] 4. Transfect HEK-293T cells to express proteins: HEK-293T cells were revived and inoculated into 6-well plates when they reached 90% to 100% density. After 12 hours, the cells grew to 90% density and the culture medium was replaced with serum-free medium. The five immunogen mRNAs (RABV-G, RABV-IG, RABV-3G, RABV-3IG, and RABV-3IG) prepared in step 3 were transfected into the culture medium using the commercial transfection kit Lipofectamine. ΔGC ) were transfected into HEK-293T cells, and the cells were cultured with 10% FBS DMEM medium. The cells were harvested 24 hours after transfection. Untransfected HEK-293T cells were used as negative control (Mock group).

[0038] 5. Western-blot detection of protein expression: Collect the 6 transfected cells obtained in step 3, add 1×SDS-PAGE loading buffer respectively, and denature the samples in a 100℃ metal bath for 10 minutes; then, add the heated denatured samples to the prepared SDS-PAGE gel, and electrophoresed at 120V for 80 minutes. After the electrophoresis, transfer the membrane at 100V for 1 hour; 5% milk was used as the blocking solution, blocked at room temperature for 1 hour, and washed three times with TBST elution solution; anti-G protein antibody was used as the primary antibody (1:3000), slowly shaken at room temperature for 2 hours, and washed three times with TBST elution solution; horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (1:2000) was used as the secondary antibody, slowly shaken at room temperature for 1 hour, and washed three times with TBST elution solution; add the substrate staining solution, and take pictures of the membrane after the reaction is terminated. The results are as follows Figure 1 shown.

[0039] Depend on Figure 1 It can be seen that the five immunogen mRNAs (RABV-G, RABV-IG, RABV-3G, RABV-3IG, RABV-3IG ΔGC ) were successfully expressed in HEK-293T cells, and the expression levels were relatively high.

[0040] 6. Liposome nanoparticles encapsulate mRNA: First, the five immunogen mRNAs (RABV-G, RABV-IG, RABV-3G, RABV-3IG, RABV-3IG) prepared in step 3 were ΔGC ) are respectively dissolved in citrate buffer to obtain citrate buffer containing immunogen mRNA.

[0041] Then, the ionizable cationic lipid SM-102, the neutral phospholipid DSPC, cholesterol and the PEG-modified lipid DMG-PEG2000 were dissolved in ethanol respectively, and mixed in a molar ratio of SM-102: DSPC: cholesterol: DMG-PEG2000 = 50:10:38.5:1.5 to obtain a lipid mixture.

[0042] Finally, the lipid mixture and the citrate buffer containing mRNA were added to the two inlet channels of Micro&Nano INano L at a mass ratio of 23:1, and the mRNA-LNP mixture was collected. After ultrafiltration and filtration, five modified mRNA vaccines based on rabies virus glycoprotein, RV1 (G), RV2 (IG), RV3 (3G), RV4 (3IG), RV5 (3G ΔGC ).

[0043] Example 2: Particle size determination of five modified mRNA vaccines based on rabies virus glycoprotein The particle size distribution of the five modified mRNA vaccines based on rabies virus glycoprotein prepared in Example 1 was measured by a Malvern Pagona Lab Zetasizer nanoparticle size potential instrument. The results are as follows: Figure 2 shown.

[0044] Depend on Figure 2 It is known that the five modified mRNA vaccines based on rabies virus glycoprotein, RV1 (G), RV2 (IG), RV3 (3G), RV4 (3IG), RV5 (3G ΔGC The average particle size of RV1 (G) is 133.8nm, the average particle size of RV2 (IG) is 133.5nm, the average particle size of RV3 (3G) is 123.5nm, the average particle size of RV4 (3IG) is 105.3nm, and the average particle size of RV5 (3G) is 123.5nm. ΔGC ) have an average particle size of 130.1 nm.

[0045] Example 3: Using fluorescent antibody virus neutralization assay to detect the neutralizing antibody levels induced by five modified mRNA vaccines based on rabies virus glycoprotein in mice 1. Five modified mRNA vaccines based on rabies virus glycoprotein and the existing rabies virus inactivated vaccine were injected into the hind limb muscles of KM mice at a dose of 2 μg / mouse. Serum was collected 21 days after immunization, and the rabies virus neutralizing antibody titer was detected by in vitro cell fluorescent antibody virus neutralization test (FAVN). The results are as follows: Figure 3 shown.

[0046] The specific method for detecting the rabies virus neutralizing antibody titer by the in vitro cell fluorescent antibody virus neutralization test (FAVN) is as follows: The inactivated sample serum and rabies standard serum (0.5 IU / mL) diluted 3 times in equal proportion were incubated with an equal amount of rabies virus CVS-11 at 37°C for 1 hour, and then 10,000 to 20,000 BHK21 cells were added to each well. After 48 hours of culture, the cell culture plate was removed, the culture supernatant was removed, and the cells were fixed with 80% acetone for fluorescent immunostaining. FITC-labeled anti-rabies virus nucleoprotein antibody was used for staining for 1 hour, the supernatant was removed, the plate was washed 3 times with PBS buffer, 100 μL PBS buffer was added, and fluorescent staining was performed under an inverted fluorescence microscope. More than one fluorescent cell in the culture well was recorded as "+", and cells without fluorescent signal were recorded as "-". The neutralizing antibody titer was calculated according to the Spearman-karber formula.

[0047] Depend on Figure 3 It is known that the five modified mRNA vaccines based on rabies virus glycoprotein, RV1 (G), RV2 (IG), RV3 (3G), RV4 (3IG), RV5 (3G ΔGC ) induced neutralizing antibodies 21 days after immunization in mice, with antibody titers of 0.5IU / ml, 1.13IU / ml, 2.59IU / ml, 5.92IU / ml and 23.38IU / ml, respectively. RV5 (3G ΔGC ) The antibody titer in the vaccine group was the highest.

[0048] 3. Modified mRNA vaccines based on rabies virus glycoprotein RV3 (3G), RV4 (3IG), RV5 (3G ΔGC ) Mice were immunized with a single injection of 0.2μg, 1μg, or 2μg. Serum was collected 21 days after immunization and the rabies virus neutralizing antibody titer was tested by in vitro cell fluorescent antibody virus neutralization test (FAVN). The results were as follows Figure 4 Wherein, the method for detecting the rabies virus neutralizing antibody titer by the in vitro cell fluorescent antibody virus neutralization test (FAVN) is the same as the above step 1.

[0049] Depend on Figure 4It can be seen that RV3 (3G), RV4 (3IG), RV5 (3G ΔGC ) were immunized with 0.2μg, 1μg, and 2μg doses of RV3 (3G), RV4 (3IG), and RV5 (3G ΔGC ) The neutralizing antibody titers of immunized mice 21 days later were 0.21IU / ml, 0.89IU / ml, and 5.29IU / ml, respectively. The 1μg dose of RV3 (3G), RV4 (3IG), and RV5 (3G ΔGC ) The neutralizing antibody titers of mice immunized 21 days later were 0.5IU / ml, 1.5IU / ml, and 10.25IU / ml, respectively. The 2μg dose of RV3 (3G), RV4 (3IG), and RV5 (3G ΔGC ) The neutralizing antibody titers of immunized mice 21 days later were 1.13U / ml, 5.29IU / ml, and 23.38IU / ml, respectively.

[0050] In summary, the five modified mRNA vaccines based on rabies virus glycoprotein can induce specific neutralizing antibodies, RV4 (3IG) and RV5 (3G ΔGC ) has a potency superior to existing veterinary vaccines, RV5 (3G ΔGC ) At a low dose of 0.2 μg, the potency is still higher than that of existing veterinary inactivated vaccines. The mRNA vaccine against rabies virus was successfully prepared, showing that this type of vaccine has good immunogenicity and a high level of neutralizing antibodies.

[0051] The above-described embodiments are only preferred specific implementation schemes of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A modified mRNA vaccine based on rabies virus glycoprotein, characterized in that: include: (1) Immunogen mRNA for expressing rabies virus glycoprotein, wherein the rabies virus glycoprotein is G protein, IG protein, 3G protein, 3IG protein or 3IG ΔGC protein; Among them, the immunogen mRNAs are RABV-G, RABV-IG, RABV-3G, RABV-3IG, RABV-3IG designed according to rabies virus glycoprotein. ΔGC ; The nucleotide sequence of the RABV-G is shown in SEQ ID NO.1; the nucleotide sequence of the RABV-IG is shown in SEQ ID NO.2; the nucleotide sequence of the RABV-3G is shown in SEQ ID NO.3; the nucleotide sequence of the RABV-3IG is shown in SEQ ID NO.4; the RABV-3IG ΔGC The nucleotide sequence is shown in SEQ ID NO.5; (2) Vaccine vectors, wherein the vaccine vectors are liposome nanoparticles that can be utilized by mRNA.

2. The modified mRNA vaccine based on rabies virus glycoprotein according to claim 1, characterized in that The rabies virus glycoprotein is selected from the following rabies virus strains: CTN strain, SAD strain and CVS strain.

3. The modified mRNA vaccine based on rabies virus glycoprotein according to claim 1, characterized in that: The G protein is the full-length glycoprotein of the CTN strain, and the amino acid sequence is shown in SEQ ID NO.6; The IG protein is the full-length glycoprotein of the CTN strain with the signal peptide sequence replaced, and the amino acid sequence is shown in SEQ ID NO.7; the 3G protein is the combination of the glycoproteins of the CTN strain, the SAD strain, and the CVS strain according to CTN: SAD: CVS = 1:1:1, and the amino acid sequence is shown in SEQ ID NO.8; The 3IG protein is a glycoprotein of the CTN, SAD, and CVS strains with a signal peptide sequence replaced according to the combination of CTN: SAD: CVS = 1:1:1, and the amino acid sequence is shown in SEQ ID NO.9; 3IG ΔGC The protein is a protein obtained by optimizing the amino acid sites of the 3IG protein, and the amino acid sequence is shown in SEQ ID NO.

10.

4. The modified mRNA vaccine based on rabies virus glycoprotein according to claim 1, characterized in that: The liposome nanoparticles include ionizable cationic lipids, neutral phospholipids, cholesterol, and PEG-modified lipids; the particle size of the liposome nanoparticles is 100-150 nm; and the mass ratio of the liposome nanoparticles to the immunogen mRNA is 23:

1.

5. The method for preparing the modified mRNA vaccine based on rabies virus glycoprotein according to claim 1, characterized in that: The steps include: S1, G protein, IG protein, 3G protein, 3IG protein, 3IG ΔGC The amino acid sequence of the protein was codon optimized and the corresponding rabies virus glycoprotein coding gene was synthesized to obtain G protein, IG protein, 3G protein, 3IG protein, 3IG ΔGC Proteins A total of five rabies virus glycoprotein expression genes; S2. The five rabies virus immunogen expression genes were cloned into the plasmid vector pVAX1-BsaI-SpeI-SapI-BspMI-free-Terminator-T7 deleted (C9863TRUG0-34) to obtain five corresponding recombinant plasmids; S3, preparation of five immunogen mRNAs; S4, transfect five immunogen mRNAs into 293T cells respectively; S5. Western-Blot was used to detect protein expression. After confirming that the immunogen mRNA can be expressed normally, the immunogen mRNA was dissolved in a citrate buffer to obtain a citrate buffer containing the immunogen mRNA; S6. Dissolve the ionizable cationic lipid SM-102, the neutral phospholipid DSPC, cholesterol and the PEG-modified lipid DMG-PEG2000 respectively using ethanol; then mix them in a molar ratio of SM-102: DSPC: cholesterol: DMG-PEG2000 = 50:10:38.5:1.5 to obtain a lipid mixture; S7. The lipid mixture is mixed evenly with the citrate buffer containing the immunogen mRNA to obtain an RNA-LNP mixture. After ultrafiltration and filtration, a modified mRNA vaccine based on rabies virus glycoprotein is obtained.

6. The method for preparing a modified mRNA vaccine based on rabies virus glycoprotein according to claim 5, characterized in that: In step S3, the immunogen mRNA is prepared as follows: (1) The recombinant plasmid obtained in step S2 is subjected to restriction enzyme linearization treatment, and then the restriction enzyme linearized plasmid is used as an in vitro transcription template. An in vitro transcription kit is used with ATP, GTP, CTP, and N1-Me-pUTP as substrates. The DNA sequence downstream of the plasmid promoter after restriction enzyme linearization is transcribed in vitro by T7 RNA polymerase, and UTP is completely replaced with the modified nucleotide N1-Me-pUTP; (2) After the reaction is completed, use DNase digestion to remove the DNA in the reaction; (3) Purify mRNA using magnetic beads to remove proteins and salts from mRNA; (4) Use the cap analog CAP5011 to perform chemical one-step capping to obtain the corresponding immunogen mRNA.

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