Construction Of Recombinant Attenuated Pseudorabies Virus Co-expressing Porcine Epidemic Diarrhea Virus And Group A Rotavirus And Partial Biological Characterization Analysis

Porcine epidemic diarrhea,porcine rotavirus disease and swine pseudorabies are three major infectious diseases that seriously harm the global swine industry.Porcine epidemic diarrhea virus?PEDV?and rotatovirus?PoRV?mainly cause diarrhea in piglets,and Porcine pseudorabies virus?PRV?causes reproductive failure in sows and acute neonatal deaths,accompanied by vomiting,diarrhea,tremor,movement disorders and other symptoms.The co-infection of the three viruses often leads to more harmful.Therefore,it is urgent and necessary to prevent these three diseases at the same time with a stable and effective vaccine.This project proposes to use the immunodominant antigen regions of the PoRV VP7 and PEDV S genes as target genes.Then,we construct the Recombinant Pseudorabies Virus Strain Coexpressing PEDV S and PoRV VP7 through the method of homologous recombination reaction that VP7.S take the place of PRV XJ strain,we researched its biological characteristics,culture characteristics,genetic stability,safety and so on.1 Construction of PRV eukaryotic transfer plasmid pEGFP-VP7.SIn this study,we selected the dominant antigenic epitopes of PEDV S protein and PoRV VP7 protein according to the existing literature.Combined with bioinformatics software analysis,we designed the primers S-a/S-b and VP7-a/VP7-b by using PEDV and PoRV sequences on NCBI,respectively.A suitable restriction enzyme site,protective base,and flexible peptide sequence are introduced 5?upstream and downstream of the primer.The VP7 and S genes were amplified by RT-PCR as candidate gene fragments of this study and cloned into pMD19-T?Simple?to construct pMD-VP7 and pMD-S cloning plasmids respectively,then Kpn I and BamH I double digestion reactions were carried out on the pMD-VP7 and pMD-S,the target fragments were recovered from the gel and transformed to DH5?after the ligation reaction.pMD-VP7.S was identified by PCR,enzyme digestion and sequence determination,successfully constructed recombinant plasmid pMD-VP7.S.The pEGFP-gI28k eukaryotic expression vector and the pMD-VP7.S recombinant plasmid were digested with Xho I and Sal I,respectively,and the target fragments were recovered from the gel and transformed to DH5?after the ligation reaction.pEGFP-VP7.S was identified by PCR,enzyme digestion and sequence determination,successfully constructed the PRV eukaryotic transfer plasmid pEGFP-VP7.S.2 Construction of recombinant pseudorabies virus PRV?CM?expressing PEDV S and PoRV VP7 geneThe PRV eukaryotic transfer plasmids pEGFP-VP7.S and PRV XJ strains were co-transfected into 293T cells,through the method of homologous recombination to constructed the PRV?CM?trivalent genetic engineering vaccine,and then identified by PCR,SDS-PAGE and Western-Blotting,indicating that PRV?CM?was successfully constructed.3 Study on Biological Characteristics of PRV?CM?of Pseudorabies Recombinant Virus StrainsThe pathogenic effect on BHK-21 cells observed that the insertion of the foreign gene VP7.S did not affect the proliferation of the recombinant virus.The titer of the TCID₅₀0 of PRV?CM?was slightly lower compared to the parental strain,and the one-step growth curve showed similar growth kinetics.The PRV?CM?was transmitted to the 20th passage in BHK-21 cells.The virus proliferation was similar in each passage.Safety tests showed that PRV?CM?is safe for whether pregnant sows or suckling pigs.