Recombinant plasmid based on Getah virus replicon, vesicle vaccine, preparation method and application

Abstract

The invention discloses a recombinant plasmid based on a Getah virus replicon, a vesicle vaccine, a preparation method and an application, and belongs to the technical field of bioengineering. The method comprises the following steps: by taking codon-optimized Getah virus non-structural protein as a skeleton, introducing a reporter gene eGEP-T2A-VSVG, and constructing a pGETV-eGFP-VSVG recombinant plasmid; further synthesizing a rabies virus RABVG protein, and replacing the VSVG protein with the rabies virus RABVG protein to construct a pGETV-eGFP-RABVG recombinant plasmid. The two obtained recombinant plasmids capable of stably passing can stably express a large amount of VSVG protein and RABVG protein under the action of a Geitai virus replication subsystem and a subgene promoter and the assistance of T2A, infectious and self-replicating VSVG virus-like vesicles and RABVG virus-like vesicles are formed, and the recombinant plasmids can be used for preparing novel vesicle vaccines and can be used for preparing VSVG virus-like vesicles and RABVG virus-like vesicles. The important technical support is provided for the prevention and control of the rabies virus.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and in particular to a recombinant plasmid, a vesicle vaccine based on a Getah virus replicon, a preparation method and an application thereof. Background Art

[0002] When an alphavirus RNA replicon expresses vesicular stomatitis virus glycoprotein (VSVG) as the only structural protein, self-replicating and infectious virus-like vesicles (VLVs) will be produced, which can be modified to express foreign antigens to trigger a protective immune response and can overcome the problems of instability and poor in vivo delivery effect faced by nucleic acid vaccines. The alphavirus replicon can be used as a self-amplifying RNA (self-amplification mRNA, saRNA) backbone. Currently, saRNA vaccines are mainly constructed using the replicon backbone sequences of alphavirus Venezuelan equine encephalitis virus (VEEV) and Semliki Forest virus (SFV).

[0003] Getah virus (GETV), belonging to the family Togaviridae and the genus Alphavirus, GETV is a spherical particle containing single-stranded positive-strand RNA. It has a diameter of about 70 nm, has an envelope and spikes, and a buoyant density of 1.22 g / cm 3 , it has poor resistance, is sensitive to acids (below pH 5.0) and alkalis (above pH 10.0), and cannot resist treatment with high concentrations (above 0.25%) of trypsin. Although it is sensitive to temperatures above 50 °C, it can survive for 3 months at 10 °C and 6 months at 4 °C. Getah virus is a newly emerging and re-emerging zoonotic animal infectious pathogen of an important pathogen. The first 2 / 3 part of the 5'-end of the alphavirus genome encodes non-structural proteins, which is called the non-structural region. This region encodes a total of 4 non-structural proteins (nspl-nsp4), while the latter 1 / 3 near the 3'-end is called the structural region, which encodes several structural proteins. These structural proteins are translated from subgenomic mRNA, which includes capsid protein (C), membrane glycoprotein E1, B2, E3, and 6K protein, etc.

[0004] GETV can cause diseases in horses. Diseased horses show symptoms such as elevated body temperature, skin rashes and swelling, and generally recover within 1 week, showing a benign course. The diagnosis of diseased horses can be made by isolating and identifying the virus from the blood in the early stage of the disease, and corresponding complement-fixing antibodies, neutralizing antibodies and hemagglutination-inhibiting antibodies can also be detected in the sera of diseased horses. The symptoms of diseased pigs are similar to those of diseased horses, manifested as many sick piglets after farrowing. After infection, listlessness, loss of appetite, whole body tremors, tongue tremors, hind limb paralysis, red body surface, etc. can appear 24 hours after infection, and a dying state appears 2-3 days later. By collecting diseased tissues such as the brain, lungs, kidneys and intestines, the virus was isolated using ESK cells, identified as GETV by serology, and cytopathic effect (CPE) could appear in ESK cells. Artificially infected pigs also showed the same cases as naturally infected pigs, thus confirming the infection and pathogenicity of the virus to pigs.

[0005] Veterinary vaccines based on alphavirus vectors can produce good immune effects in animals. Alphavirus vector vaccines are mainly modified based on attenuated or non-pathogenic strains of SFV and VEEV. Due to the replication characteristics of the virus, the carried antigen genes can be further amplified, thereby enhancing the expression level of antigens. The double-stranded RNA structure formed during the amplification process can bind to pattern recognition receptors and activate dendritic cells. Therefore, such vaccines are natural adjuvants themselves, and alphavirus vectors can also be used as delivery vectors for antibodies to provide passive immune protection. Based on the presence of the alphavirus RNA polymerase gene on the alphavirus replicon vector, it can mimic the replication of the virus. Therefore, the carried antigen genes are superior to subunit vaccines, inactivated vaccines and traditional vaccines in terms of expression level, duration and immune effect. The present invention uses GETV as a replicon as an expression platform to achieve two expression forms of enveloped virus VLVs and non-enveloped virus saRNA vaccines, providing a reference for the application of alphavirus as an antigen delivery vector in veterinary vaccines and for the research and development of new veterinary vaccines. Summary of the Invention

[0006] The object of the present invention is to provide a recombinant plasmid, vesicle vaccine, preparation method and application based on the Getah virus replicon to solve the problems existing in the above-mentioned prior art. By constructing a stably expressed pGETV replicon vector, constructing a saRNA vaccine backbone and VSVG virus-like vesicles, it can be used as a vaccine platform to provide a platform for the research and development of new vaccines.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides a recombinant plasmid pGETV-eGFP-VSVG, which is obtained by homologous recombination of a codon-optimized Getah virus non-structural protein as a backbone and a reporter gene eGFP-T2A-VSVG. Among them, the coding gene sequence of the codon-optimized Getah virus non-structural protein is shown in SEQ ID NO.1, and the nucleotide sequence of eGFP-T2A-VSVG is shown in SEQ ID NO.2.

[0009] The present invention also provides a preparation method of the recombinant plasmid pGETV-eGFP-VSVG, comprising the following steps:

[0010] Synthesize the coding gene of the codon-optimized Getah virus non-structural protein;

[0011] Connect the coding gene with an expression vector, and after enzymatic digestion, perform homologous recombination with the reporter gene eGFP-T2A-VSVG to obtain the recombinant plasmid pGETV-eGFP-VSVG;

[0012] Among them, the coding gene sequence of the codon-optimized Getah virus non-structural protein is shown in SEQ ID NO.1, and the nucleotide sequence of eGFP-T2A-VSVG is shown in SEQ ID NO.2.

[0013] The present invention also provides a recombinant plasmid pGETV-eGFP-RABVG, and the synthesis method of the pGETV-eGFP-RABVG is any one of the following:

[0014] (1) It is obtained by homologous recombination of a codon-optimized Getah virus non-structural protein as a backbone and the RABVG gene;

[0015] (2) It is obtained by replacing the VSVG gene in the pGETV-eGFP-VSVG described in claim 1 with the RABVG gene;

[0016] Among them, the nucleotide sequence of the RABVG gene is shown in SEQ ID NO.3.

[0017] The present invention also provides a preparation method of the recombinant plasmid pGETV-eGFP-RABVG, comprising the following steps:

[0018] Recombine the RABVG gene with an expression vector to construct a recombinant vector;

[0019] After enzymatic digestion of the recombinant vector and the recombinant plasmid pGETV-eGFP-VSVG respectively, perform homologous recombination to obtain the recombinant plasmid pGETV-eGFP-RABVG;

[0020] Among them, the nucleotide sequence of the RABVG gene is shown in SEQ ID NO.3.

[0021] The present invention also provides the use of the recombinant plasmid pGETV-eGFP-VSVG or the recombinant plasmid pGETV-eGFP-VSVG in the preparation of a Getah virus-like vesicle vaccine.

[0022] The present invention also provides the use of the recombinant plasmid pGETV-eGFP-VSVG or the recombinant plasmid pGETV-eGFP-VSVG in the preparation of an saRNA vaccine.

[0023] The present invention also provides the use of the recombinant plasmid pGETV-eGFP-VSVG or the recombinant plasmid pGETV-eGFP-VSVG in the preparation of a drug for preventing and treating rabies virus infection.

[0024] The present invention also provides a Getah virus-like vesicle vaccine, which comprises the recombinant plasmid pGETV-eGFP-VSVG or the recombinant plasmid pGETV-eGFP-RABVG.

[0025] The present invention also provides an saRNA vaccine, which comprises the recombinant plasmid pGETV-eGFP-VSVG or the recombinant plasmid pGETV-eGFP-RABVG.

[0026] The present invention also provides a drug for preventing and treating rabies virus infection, which comprises the recombinant plasmid pGETV-eGFP-VSVG or the recombinant plasmid pGETV-eGFP-RABVG.

[0027] The present invention discloses the following technical effects:

[0028] (1) For the first time, the present invention obtained stably passaged pGETV-eGFP-VSVG and pGETV-eGFP-RABVG by replacing the structural proteins of GETV with VSVG and RABVG and using its non-structural proteins as firmware, providing a basis for exploring alphavirus-vesicular stomatitis virus vaccines with infectivity and self-replication ability.

[0029] (2) The VSVG and RABVG virus-like vesicles obtained in the present invention can achieve a large amount of proliferation in BHK-21 in 48 h, showing strong infectivity. The VSVG virus-like vesicles can be stably passaged with a titer of up to 10 6 . The RABVG virus-like vesicles can express a high concentration and relatively high purity of RABVG protein in BHK-21 in 72 h, and a large amount of RABVG can be expressed in a short time, and the titer can reach 10 6.125 at the 3rd generation.

[0030] (3) The present invention designs a codon-optimized GETV replicon vector to obtain a saRNA vaccine backbone. After entering cells at a low dose, it has the ability to self-replicate and amplify, providing longer-lasting immune protection with fewer side effects, and can provide a new expression platform for saRNA vaccines.

[0031] (4) The pGETV-eGFP-RABVG virus-like vesicles constructed in the present invention achieve stable and large-scale expression of the RABVG protein under the action of the Geta virus replicon and the subgene promoter based on the pGETV-eGFP-VSVG virus-like vesicle platform, thus providing important technical support for the prevention and control of rabies virus and offering a new idea for preparing virus-like vesicle vaccines using the Geta virus replicon as the backbone; it can also serve as the backbone of self-replicating RNA vaccines and lay a foundation for the development of self-replicating RNA vaccines.

[0032] (5) The alphavirus-vesicular stomatitis virus vaccines pGETV-eGFP-VSVG and pGETV-eGFP-RABVG obtained using the present invention can overcome the problems of instability and poor in vivo delivery effect faced by nucleic acid vaccines, with relatively low production costs, providing a potential vaccine for the prevention of RABV and a new vaccine platform for all enveloped viruses. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0034] Figure 1 It is the overall technical roadmap for constructing pGETV-eGFP-VSVG and -RABVG of the present invention;

[0035] Figure 2 It is the genomic structure diagram of pGETV-GFP-VSVG (A) and pGETV-GFP-RABVG (B);

[0036] Figure 3 It is the double digestion verification diagram of GETV-eGFP-VSVG; M: 15000bp DNA molecular standard; 1: GETV-eGFP-VSVG;

[0037] Figure 4 It is the diagram of pGETV-GFP-VSVG infecting BHK-21 cells; A: Fluorescence diagram at 24 h after transfection; B: Fluorescence diagram at 48 h after transfection; C: Fluorescence diagram at 72 h after transfection; D: Negative diagram of BHK-21 cells;

[0038] Figure 5 Scanning electron micrograph of GETV-eGFP-VSVG;

[0039] Figure 6 Coomassie brilliant blue graph of GETV-eGFP-VSVG; M: 10-200kD protein molecular standard; 1: pGETV-eGFP-VSVG; 2: blank control;

[0040] Figure 7 Single enzyme digestion verification graph of GETV-eGFP-RABVG; M: 5000bp DNA molecular standard; 1: GETV-eGFP-RABVG;

[0041] Figure 8 Graph of pGETV-GFP-RABVG infecting BHK-21 cells; A: Fluorescence graph at 24 h after transfection; B: Fluorescence graph at 48 h after transfection; C: Fluorescence graph at 72 h after transfection; D: Negative graph of BHK-21 cells;

[0042] Figure 9 Coomassie brilliant blue graph of GETV-eGFP-RABVG; M: 10-180kD protein molecular standard; 1: pGETV-eGFP-RABVG; 2: blank control. Detailed implementation manners

[0043] The various exemplary implementation manners of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0044] It should be understood that the terms described in the present invention are only for describing particular implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0045] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0046] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the present invention's specification, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0047] Regarding the terms "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.

[0048] The optimized design of the GETV replicon in the present invention provides a backbone for obtaining saRNA vaccines. In the present invention, the GETV non-structural proteins were replaced with VSVG and RABVG by homologous recombination, and pGETV-eGFP-VSVG and pGETV-eGFP-RABVG that can be stably passaged were obtained, generating self-replicating and infectious virus-like vesicles VSVG VLVs and RABVG VLVs, achieving stable high-titer / high-level protein expression. The technical route of the present invention is as Figure 1 shown.

[0049] Example 1

[0050] (1) Construction of recombinant pGETV-eGFP-VSVG plasmid

[0051] According to the Getah virus genome sequence provided in NCBI, codon-optimized Getah virus 5'UTR, non-structural proteins nps1-4, 23S promoter, and 3'UTR were synthesized and named GETVrep. Using pUC57 as a vector, pUC57-GETVrep was synthesized and named pGETV. The nucleotide sequence of GETVrep is shown in SEQ ID NO.1; the reporter gene eGFP-T2A-VSVG was synthesized, and the nucleotide sequence is shown in SEQ ID NO.2. The specific steps are as follows:

[0052] Using the product obtained by digesting pGETV with Xho I as a vector and performing homologous recombination with eGFP-VSVG to obtain pGETV-eGFP-VSVG (as Figure 2 shown), and verifying with Xho I and Miu I digestion to meet the required conditions. pGETV-eGFP-VSVG (as Figure 2 shown) was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The double digestion result of pGETV-GFP-VSVG is as Figure 3 shown. The double digestion result combined with the sequencing result shows that eGFP-VSVG was successfully ligated to the vector.

[0053] GETVrep sequence (SEQ ID NO.1):

[0054]

[0055] eGFP-T2A-VSVG sequence (SEQ ID NO.2):

[0056]

[0057] (2) Rescue of pGETV-GFP-VSVG virus

[0058] According to the instructions of the mMESSAGE mMACHINE TM T7 transcription kit, after digesting the constructed plasmid with EcoR V, in vitro transcription was carried out; according to the instructions of Lipofectamine2000, the in vitro transcribed and recovered mRNA was transfected onto a 6-well plate of well-grown BHK-21 cells. The transfection dose was 5 μg, 300 μL of opli-MEM culture medium was replaced in the 6-well plate, and the transfected cell plate was placed in a 37 °C constant temperature incubator containing 5% CO2 for culture. After 6 h, 2 mL of MEM maintenance solution containing 2% fetal bovine serum was replaced, and fluorescence was observed at 24 h, 48 h, and 72 h.

[0059] The supernatant was collected 72 h after pGETV-GFP-VSVG infected BHK-21 cells, centrifuged and stored at -80 °C, as Figure 4 shown, the results showed that pGETV-GFP-VSVG produced self-replicating and infectious VSVG virus-like vesicles in BHK-21.

[0060] (3) Titration of pGETV-GFP-VSVG

[0061] BHK-21 cells were seeded in a 6-well plate. When the cell confluence reached 80%, BHK-21 cells were infected with pGETV-eGFP-VSVG at an MOI of 0.01, and 3 replicates were set. The supernatant was collected 72 h after infection, and the virus titer was measured to be 10 6 FFU / mL.

[0062] (4) Characterization of pGETV-GFP-VSVG virus-like vesicles

[0063] The pGETV-eGFP-VSVG sample was prepared with PEG8000. The sample was taken and thawed at 4 °C, centrifuged at 8000 r / min for 5 min at 4 °C, and the supernatant was taken. 20 μL of the sample was added dropwise to the activated 200-mesh copper grid, allowed to stand for 5 min, and observed and photographed under a transmission electron microscope with an operating voltage of 80 kv, as Figure 5 shown, typical virus-like vesicle structures could be observed.

[0064] (5) Coomassie brilliant blue detection of the protein composition of pGETV-GFP-VSVG

[0065] Inoculate BHK-21 cells into each well of a 6-well plate. When the cell confluence reaches 80%, infect the BHK-21 cells with pGETV-eGFP-VSVG at an MOI of 0.01. After 24 h, take the supernatant of the BHK-21 cells infected with pGETV-eGFP-VSVG, add 5×SDS loading buffer, boil it, and then perform SDS-PAGE gel electrophoresis. After the electrophoresis, perform Coomassie brilliant blue staining for 30 min, and decolorize with the decolorizing solution until the bands are clear. The results are shown in Figure 6 。

[0066] Example 2

[0067] (1) Construction of recombinant pGETV-eGFP-RABVG plasmid

[0068] According to the rabies virus RABVG genome sequence provided in NCBI, synthesize pUC57-RABVG using pUC57 as the vector, and use premier 5.0 to design primers to amplify the left and right arms for Overlap PCR respectively. Add an Mlu I restriction site to the 5' end of the upstream primer of the left arm, and introduce an Mlu I restriction site to the 5' end of the downstream primer of the right arm. The primer sequences are as follows:

[0069] GETV-RABVF (SEQ ID NO.4): 5'-AGAATCCTGGCCCACTCGAGGCCACCACGCGTGCCACCATGGTACCCCAAGCCC-3';

[0070] GETV-RABVR (SEQ ID NO.5): 5'-TATGTCAAGCCTCCCGGTCAACGCGTTTAGAGCCTTGTCTCACCTCCTGACTTGTGG-3'.

[0071] Using PUC57-RABVG as the template, the fragment RABVG (shown as SEQ ID NO.3) amplified with GETV-RABVF / GETV-RABVR as the target gene to be introduced, and the fragment obtained by digesting pGETV-eGFP-VSVG with Mlu I as the vector. Insert the fragment RABVG into the vector through homologous recombination to obtain pGETV-GFP-RABVG. Verify it with single digestion by Miu I to meet the required situation, and send pGETV-GFP-RABVG to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0072] The single digestion result of pGETV-GFP-RABVG is as Figure 7 shown, and combined with the sequencing results, it shows that RABVG has been successfully inserted into the vector.

[0073] RABV sequence (as shown in SEQ ID NO.3):

[0074]

[0075] (2) Rescue of pGETV-GFP-RABVG virus

[0076] According to the instructions of the mMESSAGE mMACHINE TM T7 Transcription Kit, after digesting the constructed plasmid with EcoR V, in vitro transcription was carried out; according to the instructions of Lipofectamine2000, the in vitro transcribed and recovered mRNA was transfected onto a 6-well plate of well-grown BHK-21 cells. The transfection dose was 5 μg. The 300 μL opli-MEM culture medium in the 6-well plate was replaced, and the transfected cell plate was placed in a constant temperature incubator at 37 °C with 5% CO2 for culture. After 6 h, 2 mL of MEM maintenance solution containing 2% fetal bovine serum was replaced, and fluorescence was observed at 24 h, 48 h, and 72 h.

[0077] As Figure 8 shown, the supernatant was collected at 72 h, centrifuged and stored at -80 °C. The results showed that pGETV-GFP-RABVG produced self-replicating and infectious RABVG virus-like vesicles in BHK-21.

[0078] (3) Titration of pGETV-GFP-RABVG

[0079] BHK-21 cells were inoculated in a 6-well plate. When the cell confluence reached 80%, BHK-21 cells were infected with pGETV-eGFP-RABV at an MOI of 0.01, and 3 replicates were set. The supernatant was collected 72 h after infection, and the virus titer was measured to be 10 6.125 FFU / mL.

[0080] (4) Detection of the protein composition of pGETV-GFP-RABVG by Coomassie Brilliant Blue

[0081] BHK-21 cells were inoculated in each well of a 6-well plate. When the cell confluence reached 80%, BHK-21 cells were infected with pGETV-eGFP-RABVG at an MOI of 0.01. The supernatant of BHK-21 cells infected with pGETV-eGFP-RABV was taken at 24 h, added with 5×SDS loading buffer, boiled, and then subjected to SDS-PAGE gel electrophoresis. After the electrophoresis was completed, Coomassie Brilliant Blue staining was carried out for 30 min, and decolorization was performed with decolorizing solution until the bands were clear. The results were as Figure 9 shown, and the results showed that the purified target protein could be obtained.

[0082] The above-described embodiments are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A recombinant plasmid pGETV-eGFP-VSVG, characterized in that, The pGETV-eGFP-VSVG is obtained by homologous recombination of a codon-optimized Getah virus non-structural protein as a backbone and the reporter gene eGFP-T2A-VSVG. Among them, the coding gene sequence of the codon-optimized Getah virus non-structural protein is shown in SEQ ID NO.1, and the nucleotide sequence of eGFP-T2A-VSVG is shown in SEQ ID NO.

2.

2. A method for preparing a recombinant plasmid pGETV-eGFP-VSVG, characterized in that, It includes the following steps: Synthesize the coding gene of the codon-optimized Getah virus non-structural protein; Connect the coding gene with an expression vector, and after enzyme digestion, perform homologous recombination with the reporter gene eGFP-T2A-VSVG to obtain the recombinant plasmid pGETV-eGFP-VSVG; Among them, the coding gene sequence of the codon-optimized Getah virus non-structural protein is shown in SEQ ID NO.1, and the nucleotide sequence of eGFP-T2A-VSVG is shown in SEQ ID NO.

2.

3. A recombinant plasmid pGETV-eGFP-RABVG, characterized in that, The synthesis method of the pGETV-eGFP-RABVG is any one of the following: (1) It is obtained by homologous recombination of a codon-optimized Getah virus non-structural protein as a backbone and the RABVG gene; (2) It is obtained by replacing the VSVG gene in the pGETV-eGFP-VSVG described in claim 1 with the RABVG gene; Among them, the nucleotide sequence of the RABVG gene is shown in SEQ ID NO.

3.

4. A method for preparing a recombinant plasmid pGETV-eGFP-RABVG, characterized in that, It includes the following steps: Recombine the RABVG gene with an expression vector to construct a recombinant vector; After enzyme digestion of the recombinant vector and the recombinant plasmid pGETV-eGFP-VSVG described in claim 1 respectively, perform homologous recombination to obtain the recombinant plasmid pGETV-eGFP-RABVG; Among them, the nucleotide sequence of the RABVG gene is shown in SEQ ID NO.

3.

5. The application of the recombinant plasmid pGETV-eGFP-VSVG described in claim 1 or the recombinant plasmid pGETV-eGFP-VSVG described in claim 3 in the preparation of a Getah virus-like vesicle vaccine.

6. The application of the recombinant plasmid pGETV-eGFP-VSVG described in claim 1 or the recombinant plasmid pGETV-eGFP-VSVG described in claim 3 in the preparation of an saRNA vaccine.

7. The application of the recombinant plasmid pGETV-eGFP-VSVG described in claim 1 or the recombinant plasmid pGETV-eGFP-VSVG described in claim 3 in the preparation of a drug for preventing and treating rabies virus infection.

8. A Getah virus-like vesicle vaccine, characterized in that, It contains the recombinant plasmid pGETV-eGFP-VSVG described in claim 1 or the recombinant plasmid pGETV-eGFP-RABVG described in claim 3.

9. A saRNA vaccine, characterized in that, It contains the recombinant plasmid pGETV-eGFP-VSVG described in claim 1 or the recombinant plasmid pGETV-eGFP-RABVG described in claim 3.

10. A drug for preventing and treating rabies virus infection, characterized in that, It contains the recombinant plasmid pGETV-eGFP-VSVG described in claim 1 or the recombinant plasmid pGETV-eGFP-RABVG described in claim 3.

Images