CN108743934B - Vaccine for preventing porcine epidemic diarrhea virus constructed by recombinant vesicular stomatitis virus - Google Patents

Vaccine for preventing porcine epidemic diarrhea virus constructed by recombinant vesicular stomatitis virus Download PDF

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CN108743934B
CN108743934B CN201810412129.1A CN201810412129A CN108743934B CN 108743934 B CN108743934 B CN 108743934B CN 201810412129 A CN201810412129 A CN 201810412129A CN 108743934 B CN108743934 B CN 108743934B
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孙涛
方心葵
柯勇
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Abstract

The invention discloses a vaccine for preventing Porcine Epidemic Diarrhea Virus (PEDV) constructed by utilizing recombinant Vesicular Stomatitis Virus (VSV); the method comprises the following steps: highly attenuated VSV (VSV) mutated at three different amino acid positions by means of Matrix protein (Matrix, M)MT) And (3) expressing a protective antigen S protein of the Shanghai strain PEDV2012 to prepare the obtained novel vaccine for preventing the PEDV. The in vitro replication titer of the recombinant virus can be close to 109TCID50Per ml; in a pig body, the novel vaccine can efficiently induce and neutralize protective antibodies, and meanwhile, in view of the characteristic that a recombinant vaccine constructed by taking VSV as a platform has the capability of quickly activating the immune protective force of an animal body, the VSV vaccine expressing the PEDV protective antigen can become an emergency immune vaccine for effectively preventing porcine epidemic diarrhea viruses.

Description

Vaccine for preventing porcine epidemic diarrhea virus constructed by recombinant vesicular stomatitis virus
Technical Field
The invention relates to a vaccine for preventing porcine epidemic diarrhea virus, which is constructed by utilizing recombinant Vesicular Stomatitis Virus (VSV).
Background
Porcine Epidemic Diarrheia (PED) is a highly-contact enteric infectious disease (Pensaert et al, 1978) which is mainly characterized by diarrhea, vomiting, dehydration and high lethality to suckling piglets and is caused by Porcine Epidemic Diarrhea Virus (PEDV), the PEDV is an animal coronavirus which can cause infection of pigs of various ages, but the morbidity of piglets is usually 100%, and the morbidity of sows is 15% -90%, wherein the lethality of the virus to newborn piglets can reach more than 50%, and the extensive epidemic of PEDV in China causes great economic loss to the breeding industry since 2011. PEDV causes a critical course of disease and has a high propagation speed, so that emergency preventive vaccination is timely carried out on swinery, particularly newborn piglets and sows to be born in an epidemic area and peripheral areas, so that the immunization can be rapidly generated, and the method is very critical for controlling epidemic spread and reducing the death rate of the piglets. Although PEDV vaccines have been widely used domestically, the prevalence of PEDV in pig farms across the country remains relatively prevalent.
The commercial vaccines currently used to prevent PEDV suffer from the following disadvantages:
1. the vaccine is mainly an inactivated vaccine, and needs to be inoculated to animals for many times, so that the formed immune protection is slow, and the vaccine is not suitable for emergency inoculation when epidemic situations occur;
2. another disadvantage of inactivated vaccines is the inability to efficiently elicit specific CTL and mucosal immune responses in the host. One of the methods for overcoming the difficulties is to use live attenuated PEDV vaccine, but the coronavirus gene can be recombined under natural conditions, so that the use safety of the coronavirus gene is difficult to ensure, and European and American countries forbid the use of live attenuated vaccine to prevent PEDV;
3. currently, PEDV is mainly prepared by culturing Vero cells in vitro, and has low replication titer (about 10)6-107TCID50Per ml), the cell culture fluid needs to be added with a certain amount of pancreatin to maintain the virus amplification in vitro, which limits the large-scale preparation of the virus to a certain extent.
In view of the problems with existing vaccines, there is a great need to develop new PEDV vaccines.
Recombinant Vesicular Stomatitis Virus (VSV) has unique advantages as a vaccine vector and has been well applied. The expression of foreign antigen by means of genetic engineering attenuated virus vector is an effective way to develop novel vaccine, in which the recombinant Vesicular Stomatitis Virus (VSV) has made a breakthrough in the development and application of human emergency vaccine, and recent reports show that Ebola virus emergency vaccine prepared by using the recombinant Vesicular Stomatitis Virus (VSV) as vector can excite effective immune protection in the body of primate within 3-5 days, and the immunized animal can withstand the attack of lethal dose virus, and the vaccine will complete human body experiment and be put into clinical application.
Major advantages of VSV as a vaccine vector include:
1. many studies have demonstrated that VSV, as a vaccine vector, can be a highly efficient platform for the expression of foreign antigen proteins, such as HIV, Ebola virus (Ebola), and the like;
2. VSV replication cycle is short (about 8 hours for one generation), easy to culture, BHK21 cell used for virus amplification can be suspension cultured in high density, which provides unique advantage for the industrial and large-scale production of recombinant vaccine prepared by using VSV as platform;
3. the VSV recombinant vaccine can effectively stimulate the body to generate comprehensive immune response, including cellular immune response, humoral immune response and mucosal immune response, and has the advantage of rapidly stimulating the immune response, such as: ebola virus emergency vaccine prepared by using VSV as a vector is proved to stimulate immune protection within 3-5 days after the primate is inoculated.
In view of the many advantages of VSV as a vaccine vector, this application contemplates the development of PEDV emergency vaccines against piglets and adult pigs using VSV.
Disclosure of Invention
The invention aims to solve the problems in the conventional PEDV vaccine development and application, and provides a porcine epidemic diarrhea virus vaccine constructed by using a recombinant vesicular stomatitis virus.
The purpose of the invention is realized by the following technical scheme:
the invention relates to a porcine epidemic diarrhea virus vaccine, which comprises the steps of cloning PEDV protective antigen gene S or protective polypeptide S1 on S protein to the genome of recombinant vesicular stomatitis virus with M protein mutation, and constructing the obtained recombinant virus for preventing PEDV by using a trans-genetic technology.
Preferably, the protective antigen gene S is cloned from the endemic strain PEDV of PEDV in shanghai region in 2012. This strain is one of the representative strains of the new PEDV.
Preferably, the sequence of the PEDV/SH/2012S gene is shown as SEQ ID N0.1.
Preferably, the recombinant vesicular stomatitis virus with M protein mutation comprises a mutant of the recombinant vesicular stomatitis virus with M protein mutation at one or three of amino acid positions 51, 221 and 226.
Preferably, the recombinant vesicular stomatitis virus with M protein mutation is recombinant vesicular stomatitisToxic VSVMT。
Preferably, the recombinant vesicular stomatitis virus VSVMTIs a recombinant virus with matrix protein M mutated at three different amino acid sites; the 51 th methionine of the gene protein M is knocked out, the 221 th amino acid-valine is mutated into phenylalanine, and the 226 th amino acid-glycine is mutated into arginine.
Preferably, the vaccine is a bivalent vaccine that prevents both PEDV and VSV infection.
The invention also relates to a construction method of the porcine epidemic diarrhea virus vaccine, which comprises the following steps:
s1, obtaining PEDV S and S1 genes by RT-PCR, and cloning gene fragments to pVSV by XhoI and NheI double enzyme digestionMTPlasmid multiple cloning site to obtain pVSVMT-S and pVSVMT-S1 recombinant plasmid;
s2, expression T7The poxvirus vTF7-3 of RNA polymerase infects BHK-21 cells; simultaneously preparing plasmids pBS-N, pBS-P, pBS-L and pVSVMT-S or pVSVMTPlasmid transfection mixture of S1;
s3, after cell cotransfection is carried out on the plasmid, poxvirus is filtered out, the filtrate is added into BHK-21 cells in a logarithmic growth phase, and cytopathic effect is observed; and if the cells are diseased, collecting cell supernatants, performing virus identification by using RT-PCR, if the cells are correct, performing plaque purification by using Vero cells, and performing VSV recombinant virus identification to obtain the porcine epidemic diarrhea virus vaccine.
Preferably, the primer pair for amplifying the S gene of the PEDV/SH/2012 virus strain in the RT-PCR is shown as sequences SEQ ID NO.2 and SEQ ID NO. 3.
Preferably, the primer pair for amplifying the PEDV/SH/2012 virus strain S1 gene in the RT-PCR is shown as sequences SEQ ID NO.4 and SEQ ID NO. 5.
The invention also relates to application of the porcine epidemic diarrhea virus vaccine in emergency immunity of newborn piglets.
Compared with the prior art, the invention has the following beneficial effects:
1. the invention protects PEDV againstCloning of protective polypeptide S1 on Progen S or S protein into VSVMTThe constructed vaccine contains various T and B cell epitopes, and the functions of the VSV vaccine vector are fully exerted; importantly, the PEDVS protein thus expressed with VSV retains the transmembrane and intracellular segment domains of the protein, so that the S protein will be embedded in the envelope of VSV, thereby creating a bivalent vaccine that prevents both PEDV and VSV infection.
2. PEDV causes the course of disease to be urgent, the propagation speed is fast, therefore in time carry on the urgent prevention inoculation to the swinery of epidemic area and peripheral area, especially newborn piglet and awaiting delivery sow, make it produce the immune protective power rapidly within 3-4 days, it is extremely crucial to control the epidemic situation to spread, reduce the piglet mortality, utilize VSV to have unique advantage in this respect compared with traditional PEDV inactivated vaccine and attenuated live vaccine for the vaccine that the vector was developed, for example, vaccine that VSV expresses Ebola virus envelope protein can stimulate the immune protective power very fast in primate and human body. According to the invention, the VSV is used as a vector to express the PEDV protective antigen, so that a novel vaccine capable of rapidly stimulating the immune protective power of animal bodies can be prepared for emergency immunization, which is very critical for controlling epidemic situation, especially for reducing the high mortality rate caused by PEDV infected piglets and reducing economic loss.
Drawings
Other features, objects and advantages of the invention will become more apparent upon reading of the detailed description of non-limiting embodiments with reference to the following drawings:
FIG. 1 is a schematic representation of the structure of a recombinant vesicular stomatitis virus expressing the PEDV S protein; wherein A is a VSV virus structure diagram; b is a recombinant Vesicular Stomatitis Virus (VSV) expressing the PEDV S proteinMT-S) structural diagram;
FIG. 2 is a schematic diagram of the S protein structure of an epidemic strain (PEDV/SH/2012) in Shanghai region in 2012; in the figure, S1: a PEDVS1 polypeptide; s2: a PEDVS2 polypeptide;
FIG. 3 is a schematic diagram showing the construction of a recombinant VSV plasmid for cloning the PEDV S or S1 gene; in the figure, N is nucleocapsid protein, P is phosphorylated protein, M is matrix protein (matrix), G is envelope protein, L is viral RNA replicase, S is PEDV spike protein gene, S1 is spike protein S1 polypeptide; VSVMT: VSV virus with a Matrix protein three-site mutation; VSVMT-S: a recombinant VSV virus expressing a protective antigen S protein of a PEDV2012 shanghai strain; VSVMT-S1: VSV virus expressing PEDV2012 shanghai square strain S1 polypeptide; XhoI and NheI are restriction endonucleases respectively;
fig. 4 is a flowchart of a rescue scheme for a recombinant VSV virus expressing the protective antigen S protein or S1 polypeptide of PEDV2012 shanghai strain;
FIG. 5 shows VSVMT-identification scheme of S virus; wherein A is a Western-blotting detection result of PEDV recovery serum, and 1: PEDV holovirus; 2: VSVMT-S；3：VSVMT(ii) a 4: expressing PEDV N protein by escherichia coli; 5: control of cell supernatant of non-viral BHK 21; b is a detection result of Western-blotting of VSV recovery serum, wherein 1: PEDV holovirus; 2: VSVMT-S；3：VSVMT(ii) a 4: expressing PEDVN protein by escherichia coli;
FIG. 6 shows VSVMTA Western-blotting identification schematic diagram of the S1 virus; in the figure 1: expressing PEDV N protein by escherichia coli; lanes 2-6: VSVMTS1 cell supernatants from BHK21 cells infected at 0, 4, 8, 12 and 24 h;
FIG. 7 shows VSVMT-determination of the S copy characteristics.
Detailed Description
The present invention will be described in detail with reference to examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that it would be apparent to those skilled in the art that several modifications and improvements can be made without departing from the inventive concept. All falling within the scope of the present invention.
The key points of the scheme of the invention are as follows:
1. vaccine vector (VSV) used in the present inventionMT) The invention is successfully constructed for the first time by the applicant, and repeated experiments show that the virus vector realizes weakening and has good application prospect.
By utilizing genetic engineering technology, we construct a novel recombinant vesicular propertyStomatitis Virus (VSV)MT) The Matrix protein (Matrix, M) of the virus has three different amino acid site mutations, including that the 51 st methionine of the Matrix protein is knocked out, the 221 th valine of the Matrix protein is mutated into phenylalanine, and the 226 th glycine of the Matrix protein is mutated into arginine. The in vivo and in vitro biological property experiments of the pig show that the pathogenicity of the virus is obviously reduced, and the virus can effectively stimulate the organism to generate protective immune response reaction and has good application prospect. In addition, by inserting a multiple cloning site (XhoI and NheI) between the G and L proteins of the VSV genome, as shown in FIG. 1, exogenous protein genes, especially large fragment genes, can be cloned and expressed. The existing research results show that the vaccine developed by using attenuated VSV capable of expressing exogenous antigen as vector can fully and quickly stimulate animal immune response, especially cytotoxic T cell response (CTL) and mucosal immunity, and has unique significance for virus which is difficult to propagate in vitro and has high pathogenicity.
2. Cloning the PEDV protective antigen gene S and the protective polypeptide S1 on the S protein into VSVMTAnd a safe and effective novel vaccine for preventing PEDV is constructed by using the vector.
Spike protein (S) is an important structural protein of PEDV, responsible for virus recognition of host cell receptors, and can induce neutralization of protective antibodies; the S1 polypeptide is a subunit of the S protein and contains a protective epitope of the S protein. Therefore, in the application, the two antigens are cloned to the VSV genome respectively, and the novel strategy enables the constructed vaccine to comprise various T and B cell epitopes and fully exert the functions of the VSV vaccine vector; importantly, the PEDVS protein thus expressed with VSV retains the transmembrane and intracellular segment domains of the protein, so that the S protein will be embedded in the envelope of VSV, thereby creating a bivalent vaccine that prevents both PEDV and VSV infection, a technical effect that one of skill in the art would have had unexpected, as shown in fig. 1. As can be seen in FIG. 1B, both the PEDVS protein and the VSV G protein will be embedded in the envelope of this recombinant virus.
3. Protective antigen gene S for constructing vaccine is cloned from a PEDV local strain (PEDV/SH/2012) in Shanghai region in 2012, and the strain has better representativeness
The subject group clones spike protein gene S (Genbank accession number JX110659) from epidemic strain PEDWSH/2012 in Shanghai region in 2012 at the earlier stage, and carries out systematic evolution analysis by comparing related sequences of the epidemic strains in various regions in recent years, and the result shows that the PEDV/SH/2012 protein amino acid sequence is highly homologous with PEDV strain sequences reported in 2012 in China and 2013 in North America (as shown in figure 2), and is far away from the genetic relationship with vaccine strain CV777 widely used at present. The determination of the sequence of the S gene by the applicant shows that the encoded S protein comprises 1386 amino acids (aa) and the S1 polypeptide is 789 amino acids, as shown in figure 2, the whole-length sequence (from the start codon to the stop codon) of the PEDV/SH/2012S gene has 4161 bases, and is shown in SEQ ID NO. 1.
4. The application of the invention to the emergency immunization of newborn piglets and adult pigs is another innovation point of the invention.
PEDV is mainly lethal to suckling piglets within 2 weeks, most of commercial PEDV vaccines used domestically are inactivated vaccines, and are used for immunization and vaccination of adult pigs, no proper PEDV vaccine exists for newborn piglets at present, in addition, because the inactivated vaccines need repeated vaccination for many times to generate effective immune response, the time of weeks or even months is usually needed, the emergency immunization of piglets and pregnant sows is not facilitated when epidemic outbreaks occur, and the PEDV protective antigen is expressed by taking VSV as a carrier, so that a novel vaccine for rapidly exciting the animal organism immune protection can be prepared, and the vaccine is used for the emergency immunization, is favorable for controlling the epidemic situation and reducing the piglet mortality.
In addition, VSV is easy to culture, and particularly BHK21 cells used for amplifying the virus can be subjected to high-density suspension culture, which provides unique advantages for industrial and large-scale production of recombinant vaccines prepared by taking VSV as a platform.
Examples
The embodiment relates to a method for constructing a porcine epidemic diarrhea virus vaccine by utilizing recombinant vesicular stomatitis virus; the method specifically comprises the following steps:
1.1 cloning of protective antigen S1 or S protein of a 2012 epidemic virus strain in the Shanghai region of PEDV into the VSV viral genome
VSVMTThe 51 th methionine of the matrix protein is knocked out, the 221 th amino acid-valine is mutated into phenylalanine, and the 226 th amino acid-glycine is mutated into arginine.
Primers for amplifying the S gene and the S1 gene were designed according to the PEDV/SH/2012S gene, as shown in table 1; then obtaining PEDV S and S1 genes by RT-PCR, and cloning the gene fragment to pVSV by XhoI and NheI double enzyme digestionMTPlasmid multiple cloning site to obtain pVSVMT-S and pVSVMT-S1 recombinant plasmid (the construction scheme of the recombinant VSV plasmid is shown in FIG. 3).
TABLE 1 primers for amplification of the S gene and S1 gene of the PEDV/SH/2012 virus strains
Figure BDA0001647974190000061
1.2 rescue of recombinant VSV viruses expressing the protective antigen S protein or S1 polypeptide of PEDV2012 Shanghai strain
VSV rescue was performed as described by Schnell et al, scheme 4. By expression of T7BHK-21 cells were infected with poxvirus vTF7-3 of RNA polymerase (MOI 5), incubated for 1h, and a plasmid transfection mixture was prepared, including plasmids pBS-N, pBS-P, pBS-L and pVSVMT-S or pVSVMTS1, cell cotransfection of the plasmid was carried out according to the transfection method provided by Lipofectamine2000 of Invitrogen. And after 48 hours, sucking the supernatant, filtering the poxvirus by using a filter membrane with the diameter of 0.22 mu m, adding the filtrate into BHK-21 cells in a logarithmic growth phase, observing cytopathic effect, collecting the cell supernatant if the cells are diseased, identifying the viruses by using RT-PCR (reverse transcription-polymerase chain reaction), and performing plaque purification by using Vero cells if the cells are correct.
1.3 identification of VSV recombinant viruses
1.3.1 VSVMT-S and VSVMTWestern-blotting identification of-S1 virus
Inoculating the VSV virus after plaque purification to BHK-21 cells for 24 hours, collecting cell supernatant, centrifuging at 10000rpm for 10min,collecting the supernatant, performing ultracentrifugation on the supernatant for 1.5h at 30000rpm by using a Hitachi ultracentrifuge, collecting precipitates, resuspending viruses by using PBS, treating the viruses by using a protein loading buffer solution, performing 10% SDS-PAGE gel electrophoresis, transferring the proteins to a PVDF membrane, sealing the PVDF membrane by using 5% skimmed milk powder for two hours, washing the PVDF membrane for three times by using PBST after sealing is completed, performing 10min each time, and performing primary incubation overnight by using PEDV infected pig recovery serum and PEDV infected pig recovery serum respectively at 4 ℃, wherein the PEDV infected pig recovery serum is diluted according to the working concentration of 1: 500; whereas the recovered serum from VSV-infected mice was diluted 1: 1000 working concentration. The primary antibody incubated PVDF membrane was washed three times with PBST for 10min each time, and incubated for 2h with HRP-labeled rabbit anti-porcine IgG as the secondary antibody at a dilution of 1: 20000. PBST was washed three times, and then developed with ECL kit, tabletted, and the target protein was detected. As shown in fig. 5 below. The result shows that the PEDV infected pig recovery serum can detect VSVMTS expressed PEDVS protein, as shown by arrow in FIG. 5A, and VSVMTThe virus did not express the S protein, and infection of mouse convalescent serum with VSV detected the respective structural proteins G, N/P and M protein of VSV, as shown in FIG. 5B. Similarly, in VSVMTEach structural protein of VSV was also detected, demonstrating that VSVMTThe S virus is constructed correctly, namely the recombinant VSV obtained by the trans-genetic technology is a novel virus with PEDV S protein embedded into VSV cyst membrane, and has not been reported before.
1.3.2 VSVMrWestern-blotting identification of-S1 virus
Inoculating the VSV virus after plaque purification to BHK-21 cells for 24 hours, collecting cell supernatant, and performing Western-blotting identification by the method described in section 1.3.1, wherein the primary antibody is a monoclonal antibody against 6 × His tag, and the working concentration is 1: 1000; the secondary antibody is HRP-labeled rabbit anti-mouse IgG, and the working concentration is 1: 20000. FIG. 6 shows VSVMTWestern-blotting identification of S1 virus; as can be seen from FIG. 6, VSVMTInfection of host cells with S1 virus results in efficient expression of PEDV S1 polypeptide, which when injected into animals will effectively induce neutralization of protective antibodies against PEDV.
1.4 VSVMrDetermination of S copy characteristics
VSV was assayed in BHK21 cells using VSV-GFP as a controlMT-replication characteristics of S by a one-step growth curve test, i.e.: BHK21 cells were plated in 24-well plates at a cell density of 2X 105One cell/well, after overnight cell growth, each was inoculated with VSVMT-S and VSV-GFP in an amount of MOI 5, incubated for 1h and washed three times with PBS, and DMEM medium containing 10% fetal bovine serum was added, cell supernatants were collected at infection 4, 8, 12, 24 and 48h, respectively, and virus content was quantified using plaque assay performed on Vero cells to confirm virus replication characteristics, as shown in fig. 7. Specifically, BHK21 cells were plated in 24-well plates at a density of 2X 105One cell/well, after overnight cell growth, each was inoculated with VSVMT-S and VSV-GFP, at MOI ═ 5, and cell supernatants were collected at infection 4, 8, 12, 24 and 48h, respectively, and virus content was quantified using plaque experiments performed on Vero cells. As can be seen from FIG. 7, VSVMTThe amplification propagation curve of the S virus in the host cells shows a gradual increase, reaching its peak around 8 hours after inoculation of the cells and then a gradual decrease, notably VSV expressing the PEDV S protein geneMTThe maximum replication titer of S virus can be > 106pFU/mL, such a rapid incremental speed is of great benefit to future production.
The foregoing description of specific embodiments of the present invention has been presented. It is to be understood that the present invention is not limited to the specific embodiments described above, and that various changes and modifications may be made by one skilled in the art within the scope of the appended claims without departing from the spirit of the invention.

Claims (3)

1. The porcine epidemic diarrhea virus vaccine is characterized by comprising a recombinant vaccine for preventing PEDV, which is obtained by cloning PEDV protective antigen gene S or protective polypeptide S1 on S protein into genome of recombinant vesicular stomatitis virus with M protein mutated and constructing by using a trans-genetic technology; the protective antigen gene S is cloned from a PEDV local strain PEDV/SH/2012 Shanghai region in 2012; the recombinant vesicular stomatitis virus with M protein mutated is recombinant vesicular stomatitis virus VSVMT(ii) a The recombinant vaccine comprises T cell epitopes and B cell epitopes, and PEDVS protein expressed by VSV reserves the functional regions of a transmembrane region and an intracellular region of the protein, and S protein can be embedded into a capsule membrane of the VSV, so that a bivalent vaccine for preventing PEDV and VSV infection simultaneously is constructed;
the porcine epidemic diarrhea virus vaccine is constructed by a method comprising the following steps:
s1, obtaining PEDV S and S1 genes by RT-PCR, and cloning gene fragments to pVSV by XhoI and NheI double enzyme digestionMTPlasmid multiple cloning site to obtain pVSVMT-S and pVSVMT-S1 recombinant plasmid;
s2, expression T7The poxvirus vTF7-3 of RNA polymerase infects BHK-21 cells; simultaneously preparing plasmids pBS-N, pBS-P, pBS-L and pVSVMT-S or pVSVMTPlasmid transfection mixture of S1;
s3, after cell cotransfection is carried out on the plasmid, poxvirus is filtered out, the filtrate is added into BHK-21 cells in a logarithmic growth phase, and cytopathic effect is observed; if the cells are diseased, collecting cell supernatants, performing virus identification by using RT-PCR, if the cells are correct, performing plaque purification by using Vero cells, and performing VSV recombinant virus identification to obtain the porcine epidemic diarrhea virus vaccine;
the recombinant vesicular stomatitis virus with M protein mutated comprises a recombinant vesicular stomatitis virus mutant with M protein mutated at one or three of amino acid positions 51, 221 and 226;
the recombinant vesicular stomatitis virus VSVMTIs a recombinant virus with matrix protein M mutated at three different amino acid sites; the 51 th methionine of the gene protein M is knocked out, the 221 th amino acid-valine is mutated into phenylalanine, and the 226 th amino acid-glycine is mutated into arginine.
2. The porcine epidemic diarrhea virus vaccine of claim 1, wherein the primer pair for amplifying the S gene of the PEDV/SH/2012 virus strain in RT-PCR is represented by SEQ ID No.2 and SEQ ID No. 3.
3. The porcine epidemic diarrhea virus vaccine of claim 1, wherein the primer pair for amplifying the PEDV/SH/2012 strain S1 gene in RT-PCR is represented by SEQ ID No.4 and SEQ ID No. 5.
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