CN108743934A - Prevent Porcine epidemic diarrhea virus vaccine using recombination vesicular stomatitis virus structure - Google Patents

Prevent Porcine epidemic diarrhea virus vaccine using recombination vesicular stomatitis virus structure Download PDF

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CN108743934A
CN108743934A CN201810412129.1A CN201810412129A CN108743934A CN 108743934 A CN108743934 A CN 108743934A CN 201810412129 A CN201810412129 A CN 201810412129A CN 108743934 A CN108743934 A CN 108743934A
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pedv
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epidemic diarrhea
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孙涛
方心葵
柯勇
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Shanghai Jiaotong University
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Abstract

The invention discloses build to prevent Porcine epidemic diarrhea virus (PEDV) vaccine using recombination vesicular stomatitis virus (VSV)；Including：By the VSV (VSV for the height reduction that three different aminoacids sites of stromatin (Matrix, M) mutateMT) that expresses PEDV2012 Shanghai strain protective antigens S proteins to be prepared prevents the new generation vaccine of PEDV.The recombinant virus replicates titre and is close to 10 in vitro9TCID50/ml；In pig body; the new generation vaccine can efficiently induce neutralization protection antibody; simultaneously; has the characteristics of quickly activation animal body immune protective efficiency in view of by the recombinant vaccine of platform construction of VSV, the contingent immunization vaccine for effectively preventing Porcine epidemic diarrhea virus can be become by expressing the VSV vaccines of PEDV protective antigens.

Description

Prevent Porcine epidemic diarrhea virus vaccine using recombination vesicular stomatitis virus structure
Technical field
The present invention relates to a kind of built using recombination vesicular stomatitis virus (VSV) to prevent Porcine epidemic diarrhea virus epidemic disease Seedling.
Background technology
Pig epidemic diarrhea (porcine epidemic diarrhea, PED) is by Porcine epidemic diarrhea virus With diarrhea, vomiting, dehydration and to the cause of suckling pig height caused by (porcine epidemic diarrhea virus, PEDV) Dead rate is a kind of high degree in contact enteric infectious disease (Pensaert et al., 1978) of main feature, and PEDV is a kind of animal Coronavirus, which can cause each age group pig to infect, but piglet incidence is usually 100%, and sow is 15%~90%, In, the virus is to newborn piglet lethality up to since 50% or more, 2011 year, and PEDV is in a wide range of popular to cultivation of China Industry causes very big economic loss.The course of disease caused by PEDV is anxious, and spread speed is fast, therefore is directed to the pig in epidemic-stricken area and surrounding area in time Group especially newborn piglet and sow to be produced carry out urgent immunization campaign, are allowed to quickly generate immune protective efficiency, for controlling epidemic disease Feelings are spread, and it is extremely crucial to reduce the piglet death rate.Although the extensive use at home of PEDV vaccines, the various regions at home PEDV The prevalence on pig farm is still relatively universal.
Prevent commercialized vaccine used in PEDV at present to have the following disadvantages：
1, it is mainly inactivated vaccine, need to be repeatedly inoculated with animal, formation immune protective efficiency is slower, is not suitable for when epidemic situation occurs Emergency vaccination is used；
2, inactivated vaccine another disadvantage is that host can not effectively be excited to generate specific CTL and mucosal immune response.Profit It is one of the method for overcoming these difficult with PEDV attenuated live vaccines, but since in natural conditions weight can occur for coronavirus gene Group, therefore, it is difficult to ensure that its safety used, American-European countries are forbidden to use attenuated live vaccine and prevent PEDV；
3, the preparation of current PEDV is mainly obtained by Vero cell injuring models, replicates titre relatively low (about 106- 107TCID50/ ml), a certain amount of pancreatin, which need to be added, in cell culture fluid could maintain viral amplification in vitro, this is to a certain extent Limit the extensive preparation of the virus.
In view of existing vaccine there are the problem of, be badly in need of studying novel PEDV vaccines.
Recombinate vesicular stomatitis virus (VSV) has unique advantage as vaccine carrier, is applied very well.By Genetic engineering attenuated viral vectors expression exogenous antigen is a kind of effective means of development of new vaccine, wherein recombinating vesiculovirus mouth Scorching virus (VSV) above makes a breakthrough in the emergent vaccine development of people and applying, and report recently shows vesiculovirus using recombination Stomatovirus (VSV) is the emergent vaccine of Ebola viruses prepared by carrier, can effectively be exempted from excitation Primate body in 3-5 days Epidemic disease protection, immune animal can withstand the attack of lethal dose virus, which is near completion human experimentation, and input clinic is answered With.
VSV includes as the major advantage of vaccine carrier：
1, many researchs confirm, as vaccine carrier, VSV can become expression exogenous antigen albumen, such as HIV, Ebola The efficient platform of viral (Ebola) etc.；
2, VSV replicative cycles are short (replicating a generation within about 8 hours), are easy to cultivate, BHK21 cells can be highly dense used in amplification virus Degree suspends culture, this is industrialized for the recombinant vaccine that is prepared by platform of VSV, large-scale production provide it is advantageous Advantage；
3, VSV recombinant vaccines are in addition to can effectively excite human body to generate comprehensive immune response, including cellullar immunologic response, body Except liquid immune response and mucosal immune response, also have the advantages that quickly to excite immune response, such as：It is prepared by carrier of VSV Ebola viruses meet an urgent need vaccine, have proven to can in inoculation Primate 3-5 days excitation immune protective efficiency.
In view of the plurality of advantages that VSV has as vaccine carrier, the application is quasi- to be directed to piglet and adult using its development The PEDV contingent immunization vaccines of pig.
Invention content
It is an object of the invention to be directed to current PEDV vaccine developments and apply existing problems, a kind of utilization is provided It recombinates vesicular stomatitis virus and builds Porcine epidemic diarrhea virus vaccine.
The purpose of the present invention is achieved through the following technical solutions：
The present invention relates to a kind of Porcine epidemic diarrhea virus vaccine, the vaccine includes by PEDV protective antigen genes S Or protective polypeptide S1 is cloned into the genome of the recombination vesicular stomatitis virus of M albumen mutation in S protein, and using instead What formula genetic technique was built can be used for preventing the recombinant virus of PEDV.
Preferably, the protective antigen gene S is cloned from the places the PEDV strain PEDV/SH/ of District of Shanghai in 2012 2012.The Strain is one of the representative strain of new hair PEDV.
Preferably, PEDV/SH/2012S gene orders are as shown in SEQ ID N0.1.
Preferably, the recombination vesicular stomatitis virus that the M albumen mutates include the 51st, M albumen, the 221st, The recombination vesicular stomatitis virus mutant of one or three mutation in 226th amino acids site.
Preferably, the recombination vesicular stomatitis virus that the M albumen mutates is recombination vesicular stomatitis virus VSVMT。
Preferably, the recombination vesicular stomatitis virus VSVMTIt is that three different aminoacids sites of matrix protein occur to dash forward The recombinant virus of change；The 51st methionine of the gene protein M is knocked, and 221 amino acids-valine mutation is benzene Alanine, 226 amino acids-glycine mutation are arginine.
Preferably, the vaccine is while preventing the bivalent vaccine of PEDV and VSV infection.
The invention further relates to a kind of construction method of Porcine epidemic diarrhea virus vaccine of the present invention, the method packets Include following steps：
S1, PEDV S and S1 genes are obtained using RT-PCR, genetic fragment is cloned into pVSV with XhoI and NheI double digestionsMT Plasmid multiple cloning sites, obtain pVSVMT- S and pVSVMT- S1 recombinant plasmids；
S2, with expression T7The poxvirus vTF7-3 of RNA polymerase infects BHK-21 cells；It includes plasmid pBS- to prepare simultaneously N, pBS-P, pBS-L and pVSVMT- S or pVSVMTThe plasmid transfection mixed liquor of-S1；
S3, after carrying out cell cotransfection to plasmid, poxvirus is filtered off, the BHK-21 that filtrate is added to exponential phase is thin In born of the same parents, cytopathy is observed；If lesion occurs in cell, cell conditioned medium is collected, carries out viruses indentification using RT-PCR, correctly Then carried out with Vero cells it is Plaque-purified, after VSV Identification of recombinant baculovirus the Porcine epidemic diarrhea virus vaccine.
Preferably, in RT-PCR expand PEDV/SH/2012 Strain S genes primer pair such as sequence SEQ ID NO.2, Shown in SEQ ID NO.3.
Preferably, in RT-PCR expand PEDV/SH/2012 Strain S1 genes primer pair such as sequence SEQ ID NO.4, Shown in SEQ ID NO.5.
The invention further relates to a kind of Porcine epidemic diarrhea virus vaccine of the present invention newborn piglet contingent immunization In application.
Compared with prior art, the present invention has the advantages that：
1, protective polypeptide S1 in PEDV protective antigen genes S or S protein is cloned into VSV by the present inventionMT, the epidemic disease of structure Seedling includes various T and B cell epitope, gives full play to the function of VSV vaccine carriers；It is important that the PEDVS expressed with VSV Albumen remains the transmembrane region and intracellular section functional domain of albumen, in this way, S protein will can be embedded in the cyst membrane of VSV, to structure Build out while preventing the bivalent vaccine of PEDV and VSV infection.
2, the course of disease caused by PEDV is anxious, and spread speed is fast, therefore especially first for the swinery of epidemic-stricken area and surrounding area in time Newborn pig and sow to be produced carry out urgent immunization campaign, are allowed to quickly generate immune protective efficiency in 3-4 days, for controlling epidemic situation Sprawling, the reduction piglet death rate is extremely crucial, compares traditional PEDV inactivated vaccines using the vaccine that VSV is carrier development and subtracts Virus live vaccine has unique advantage in this regard, as the VSV vaccines for expressing Ebola virus envelope protein development can be in Primate With the excitation immune protective efficiency that is exceedingly fast in human body.The present invention expresses PEDV protective antigens by carrier of VSV, can prepare quick excitation The new generation vaccine of animal organism immune protective efficiency is inoculated with for contingent immunization, this is for controlling epidemic situation, especially to reducing PEDV senses High mortality caused by piglet is contaminated, it is extremely crucial to reduce economic loss.
Description of the drawings
Upon reading the detailed description of non-limiting embodiments with reference to the following drawings, other feature of the invention, Objects and advantages will become more apparent upon：
Fig. 1 is the recombination vesicular stomatitis virus structural schematic diagram for expressing PEDV S proteins；Wherein, A is VSV virus structures Figure；B is the recombination vesicular stomatitis virus (VSV for expressing PEDV S proteinsMT- S) structure chart；
Fig. 2 be 2012 District of Shanghai prevalence strain (PEDV/SH/2012) S protein structural schematic diagram；In figure, S1： PEDVS1 polypeptides；S2：PEDVS2 polypeptides；
Fig. 3 is the structure schematic diagram for the recombination VSV plasmids for cloning PEDV S or S1 genes；In figure, N is nucleocapsid protein, P For phosphorylated protein, M be stromatin (matrix), G is envelope protein, L is viral RNA replicase, S is PEDV spike proteins Gene, S1 are spike protein S1 polypeptides；VSVMT：The VSV viruses of three site mutation of Matrix albumen；VSVMT-S：Expression The recombination VSV viruses of the Shanghai PEDV2012 strains protective antigens S protein；VSVMT-S1：Express the Shanghai PEDV2012 strains The VSV viruses of S1 polypeptides；XhoI and NheI is respectively restriction enzyme；
Fig. 4 is to express the recombination VSV of PEDV2012 Shanghai strains protective antigens S proteins or S1 polypeptides viral to draw Rescue flow chart；
Fig. 5 is VSVMTThe identification schematic diagram of-S viruses；Wherein, A is the Western-blotting detections of PEDV rehabilitation serum As a result, 1 in figure：PEDV totivirus；2：VSVMT-S；3：VSVMT；4：Bacillus coli expression PEDV N proteins；5：Virus is not connect BHK21 cell supernatants compare；B is VSV rehabilitation serum Western-blotting testing results, 1 in figure：PEDV totivirus； 2：VSVMT-S；3：VSVMT；4：Bacillus coli expression PEDVN albumen；
Fig. 6 is VSVMTThe Western-blotting of-S1 viruses identifies schematic diagram；1 in figure：Bacillus coli expression PEDV N Albumen；2-6 swimming lanes：VSVMT- S1 infects BHK21 cells 0,4,8,12 and cell conditioned medium for 24 hours；
Fig. 7 is VSVMTThe determination schematic diagram of-S duplication characteristics.
Specific implementation mode
The following describes the present invention in detail with reference to examples.Following embodiment will be helpful to those skilled in the art The present invention is further understood, but the invention is not limited in any way.It should be pointed out that those skilled in the art For, without departing from the inventive concept of the premise, it can also make certain adjustments and improvements.These belong to the guarantor of the present invention Protect range.
The main points of the present invention program are as follows：
1. vaccine carrier (VSV used in the present inventionMT) it is that success is built by patent application people for the first time, by testing repeatedly Show that it realizes reduction, is a viral vectors for having good application prospect.
Using technique for gene engineering, we construct one plant of novel recombination vesicular stomatitis virus (VSVMT), the virus base There are three different aminoacids site mutations, including the 51st methionine of stromatin to be knocked for matter albumen (Matrix, M), 221st valine mutation is phenylalanine, and the 226th glycine mutation is arginine.In pig body, exobiology characteristic test Show that the pathogenicity significantly reduces, and human body can effectively be excited to generate protective immune response reaction, has good Application prospect.In addition, by inserting a multiple cloning sites (XhoI and NheI) between G the and L albumen of VSV genomes, As shown in Figure 1, can be used to clone and express the gene of exogenous protein gene especially large fragment.Existing result of study table It is bright, by can express the attenuation VSV of exogenous antigen as carrier development vaccine can comprehensively, rapidly excite animal immune (CTL) and mucosal immunity are reacted in response, especially cytotoxic T cell, this is difficult to vitro propagation, highly pathogenic for those For virus, more there is unique meaning.
2. protective polypeptide S1 in PEDV protective antigen genes S and S protein is cloned into VSVMT, utilize the vector construction Safely effectively prevent PEDV new generation vaccines.
Spike protein (S) is the important feature albumen of PEDV, is responsible for virus identification host cell receptor, can induce to neutralize and protect Shield property antibody；S1 polypeptides are a subunits of S protein, contain the protective epitope of S protein.Therefore, in this Shen Please in, the two antigens are cloned into VSV genomes by us respectively, and new strategy will so that the vaccine of structure includes various T and B thin Born of the same parents' epitope gives full play to the function of VSV vaccine carriers；It is important that remaining albumen with the VSV PEDVS albumen expressed Transmembrane region and intracellular section functional domain, in this way, S protein will can be embedded in the cyst membrane of VSV, to construct while prevent PEDV With the bivalent vaccine of VSV infection, belong to the unexpected technique effect of those skilled in the art, such as Fig. 1.By Figure 1B it is found that this By the G-protein of embedded PEDVS albumen and VSV simultaneously on the cyst membrane of a recombinant virus.
3. building the protective antigen gene S used in vaccine to clone from the places the PEDV strain (PEDV/ of District of Shanghai in 2012 SH/2012), which has preferable representative
Seminar's early period of the present invention cloned its spike protein from District of Shanghai prevalence strain PEDWSH/2012 in 2012 Gene S (Genbank accession number JX110659), by the related sequence for comparing various regions prevalence strain in recent years Row, have carried out phylogenetic analysis, the results showed that, PEDV/SH/2012 S proteins amino acid sequence with domestic 2012 since with And the PEDV strain sequence very high homologies (as shown in Figure 2) that North America is reported for 2013, and with now widely used vaccine virus Then affiliation is farther out by strain CV777.The S protein of its coding, which includes 1386, to be shown to the measurement of the S gene orders through applicant Amino acid (amino acid, aa), and S1 polypeptides are 789 amino acid, see Fig. 2, PEDV/SH/2012 S full length gene sequences (from initiation codon to terminator codon), totally 4161 bases, specific as shown in SEQ ID NO.1.
4. the contingent immunization applied to newborn piglet and Adult Pig is another innovative point of the present invention.
Nursery-age pig in main lethal 2 week old of PEDV, the commercialization PEDV vaccines that the country uses are mostly inactivated vaccine, For Adult Pig Vaccination and Immunoprophylaxis, to newborn piglet, there are no suitable PEDV vaccines at present, further, since inactivated vaccine needs Effective immune response can be generated by being repeatedly inoculated with repeatedly, this usually requires the time of several weeks or even several months, is unfavorable in epidemic disease The urgent immunity of piglet and farrowing sow is inoculated with when feelings are broken out, and PEDV protective antigens is expressed using VSV as carrier, can be prepared The quickly new generation vaccine of excitation animal organism immune protective efficiency, is inoculated with for contingent immunization, is conducive to control epidemic situation, reduces piglet The death rate.
In addition, VSV is easy to cultivate, especially expand BHK21 cells used in the virus can high density suspension culture, this be with VSV is industrialized for recombinant vaccine prepared by platform, large-scale production provides advantageous advantage.
Embodiment
The present embodiment is related to a kind of utilize and recombinates vesicular stomatitis virus structure Porcine epidemic diarrhea virus vaccine；Specific packet Include following steps：
The protective antigens S1 of the District of Shanghai PEDV circulating virus strains in 2012 or S protein are cloned into VSV virus bases by 1.1 Because of group
VSVMT51st methionine of stromatin is knocked, and 221 amino acids-valine mutation is phenylpropyl alcohol ammonia Acid, 226 amino acids-glycine mutation are arginine.
The primer of amplification S genes and S1 genes is designed according to PEDV/SH/2012 S genes, as shown in table 1；Recycle RT- PCR obtains PEDV S and S1 genes, and genetic fragment is cloned into pVSV with XhoI and NheI double digestionsMTPlasmid multiple cloning sites, obtain To pVSVMT- S and pVSVMT- S1 recombinant plasmids (the structure schematic diagram of recombination VSV plasmids is as shown in Figure 3).
Table 1. expands the primer of PEDV/SH/2012 Strain S genes and S1 genes
The redemption of 1.2 expression PEDV2012 Shanghai strains protective antigens S proteins or the recombination VSV viruses of S1 polypeptides
VSV rescue of virus is carried out by the methods such as Schnell, flow is shown in Fig. 4.With expression T7The acne disease of RNA polymerase Malicious vTF7-3 infects BHK-21 cells, and inoculum concentration MOI=5 is incubated 1h, while preparing plasmid transfection mixed liquor, including Plasmid pBS-N, pBS-P, pBS-L and pVSVMT- S or pVSVMT- S1, by the Lipofectamine2000 of Invitrogen companies The transfection method provided carries out cell cotransfection to plasmid.Supernatant is drawn after 48h, and poxvirus is filtered off with 0.22 μm of filter membrane, Filtrate is added in the BHK-21 cells of exponential phase, observation cytopathy collects cell if lesion occurs in cell Supernatant carries out viruses indentification using RT-PCR, is correctly then carried out with Vero cells Plaque-purified.
1.3 VSV Identification of recombinant baculovirus
1.3.1 VSVMT- S and VSVMTThe Western-blotting of-S1 viruses is identified
After the VSV virus inoculation BHK-21 cells 24 hours after Plaque-purified, collect cell conditioned medium and Centrifuge 10min under the rotating speed of 10000rpm, collect supernatant, then by supernatant with Hitachi ultracentrifuges 30000rpm exceed the speed limit from Heart 1.5h collects precipitation, and virus is resuspended with PBS, albumen sample-loading buffer is used in combination to handle, then carries out 10%SDS-PAGE gels Electrophoresis, then by protein delivery to pvdf membrane, close two hours with 5% skimmed milk power, after the completion of closing, three are washed with PBST Secondary, then each 10min uses PEDV infected pigs rehabilitation serum and PEDV infected pigs rehabilitation serum as primary antibody respectively at 4 DEG C It is incubated overnight, wherein PEDV infected pigs rehabilitation serum is diluted by 1: 500 working concentration；And VSV infecting mouse rehabilitation serum is with 1: 1000 working concentrations dilute.Primary antibody is incubated pvdf membrane and is washed three times with PBST, each 10min, then marks rabbit-anti pig IgG with HRP Incubation 2h, dilution 1: 20000 are carried out as secondary antibody.After PBST washings three times, developed the color with ECL kits, tabletting, inspection Survey destination protein.It is illustrated in fig. 5 shown below.As a result show that PEDV infected pigs rehabilitation serum can detect VSVMTThe PEDVS eggs of-S expression In vain, as shown in Fig. 5 A arrows, and VSVMTIt is viral then do not express S protein, it then can detect VSV with VSV infecting mouse rehabilitations serum Each structural proteins G, N/P and M albumen, as shown in Figure 5 B.Equally, in VSVMTAlso each structural proteins of VSV are can detect, this card VSV is illustratedMT- S virus formulations are correct, i.e., the present invention is that PEDV S proteins are embedding using the recombination VSV that trans- genetic technique is obtained The new virus for entering VSV cyst membranes, before this without report.
1.3.2 VSVMrThe Western-blotting of-S1 viruses is identified
After the VSV virus inoculation BHK-21 cells 24 hours after Plaque-purified, cell conditioned medium is collected, such as the portions 1.3.1 The content the method divided carries out Western-blotting identifications, and primary antibody is the monoclonal antibody of anti-6 × His labels, work A concentration of 1: 1000；Secondary antibody is that HRP marks rabbit anti-mouse igg, working concentration 1: 20000.Fig. 6 is VSVMT- S1 viruses Western-blotting is identified；It will be appreciated from fig. 6 that VSVMT- S1 viruses infect host cell can be more with effective expression PEDV S1 Peptide can effectively induce in PEDV and protection antibody after such virus injection animal.
1.4 VSVMrThe determination of-S duplication characteristics
It is control with VSV-GFP, VSV is measured in BHK21 cellsMTThe method of the duplication characteristic of-S, use is step life Long curve detection, i.e.,：BHK21 cells are laid in 24 orifice plates, cell density is 2 × 105A/hole, after cells grew overnight, point VSV is not inoculated with itMT- S and VSV-GFP, inoculum concentration MOI=5 are washed three times after being incubated 1h with PBS, and are added and are contained 10% tire ox blood Clear DMEM culture mediums are infecting 4,8,12,24 and 48h collection cell conditioned mediums respectively, are utilizing the plaque that Vero cells carry out real The amount of assaying viral level, to clear virus replication characteristic, as shown in Figure 7.Specially BHK21 cells are laid in 24 orifice plates, Density is 2 × 105A/hole after cells grew overnight, is inoculated with VSV respectivelyMT- S and VSV-GFP, inoculum concentration MOI=5, respectively Cell conditioned medium is collected infecting 4,8,12,24 and 48h, the plaque assay carried out using Vero cells quantifies viral level.By Fig. 7 It is found that VSVMTThe amplification reproduction curve of-S viruses in host cell shows a process gradually risen, in inoculating cell It reaches its peak value within 8 hours or so afterwards, then gradually declines, it is notable that the VSV of expression PEDV S protein genesMT- S diseases The maximum duplication titre of poison can > 106PFU/mL, such quick value-added speed have larger benifit for production from now on.
Specific embodiments of the present invention are described above.It is to be appreciated that the invention is not limited in above-mentioned Particular implementation, those skilled in the art can make various deformations or amendments within the scope of the claims, this not shadow Ring the substantive content of the present invention.

Claims (10)

1. a kind of Porcine epidemic diarrhea virus vaccine, which is characterized in that the vaccine include by PEDV protective antigen genes S or Protective polypeptide S1 is cloned into the genome of the recombination vesicular stomatitis virus of M albumen mutation in S protein, utilizes trans- something lost Teach skill the recombinant vaccine for preventing PEDV that art is built.
2. Porcine epidemic diarrhea virus vaccine according to claim 1, which is characterized in that the protective antigen gene S It clones from the places the PEDV strain PEDV/SH/2012 of District of Shanghai in 2012.
3. Porcine epidemic diarrhea virus vaccine according to claim 2, which is characterized in that PEDV/SH/2012 S gene sequences Row are as shown in SEQ ID NO.1.
4. Porcine epidemic diarrhea virus vaccine according to claim 1, which is characterized in that the M albumen mutated Recombination vesicular stomatitis virus includes the 51st, M albumen, the 221st, one or three generation in the 226th amino acids site The recombination vesicular stomatitis virus mutant of mutation.
5. Porcine epidemic diarrhea virus vaccine according to claim 1, which is characterized in that the M albumen mutated It is recombination vesicular stomatitis virus VSV to recombinate vesicular stomatitis virusMT；The recombination vesicular stomatitis virus VSVMTIt is matrix The recombinant virus that the tri- different aminoacids sites albumen M mutate；The 51st methionine of the gene protein M is struck It removes, 221 amino acids-valine mutation is phenylalanine, and 226 amino acids-glycine mutation is arginine.
6. Porcine epidemic diarrhea virus vaccine according to claim 1, which is characterized in that the vaccine is while preventing The bivalent vaccine of PEDV and VSV infection.
7. a kind of construction method of such as Porcine epidemic diarrhea virus vaccine according to any one of claims 1 to 6, feature exist In described method includes following steps：
S1, PEDV S and S1 genes are obtained using RT-PCR, genetic fragment is cloned into pVSV with XhoI and NheI double digestionsMTPlasmid Multiple cloning sites obtain pVSVMT- S and pVSVMT- S1 recombinant plasmids；
S2, with expression T7The poxvirus vTF7-3 of RNA polymerase infects BHK-21 cells；Simultaneously prepare include plasmid pBS-N, PBS-P, pBS-L and pVSVMT- S or pVSVMTThe plasmid transfection mixed liquor of-S1；
S3, after carrying out cell cotransfection to plasmid, poxvirus is filtered off, filtrate is added to the BHK-21 cells of exponential phase In, observe cytopathy；If lesion occurs in cell, cell conditioned medium is collected, carries out viruses indentification using RT-PCR, correctly then Carried out with Vero cells it is Plaque-purified, after VSV Identification of recombinant baculovirus the Porcine epidemic diarrhea virus vaccine.
8. the construction method of Porcine epidemic diarrhea virus vaccine according to claim 7, which is characterized in that expand in RT-PCR Increase the primer pair of PEDV/SH/2012 Strain S genes as shown in sequence SEQ ID NO.2, SEQ ID NO.3.
9. the construction method of Porcine epidemic diarrhea virus vaccine according to claim 7, which is characterized in that expand in RT-PCR Increase the primer pair of PEDV/SH/2012 Strain S1 genes as shown in sequence SEQ ID NO.4, SEQ ID NO.5.
10. a kind of as Porcine epidemic diarrhea virus vaccine according to any one of claims 1 to 6 is exempted from the emergent of newborn piglet Application in epidemic disease.
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