The Isolation Of Swinepox Virus&Construction Of Recombinant Swinepox Virus Expressing Capsid Protein Of Porcine Circovirus Type 2

Swinepox is a swine infectious disease caused by Swinepox Virus(SWPV),and is characterized by pox and scabby formed in skin.The vast majority of infected pigs can recover themselves.SWPV has a large genome(146kb),contain multiple replication non-essential areas,can insert the large fragment of exogenous gene,strict host specificity and other characteristics make it particularly suitable as a live vaccine vector.To establish a technology platform of recombinant swinepox virus vaccine vector,in this study,a strain isolated from pig in Jiangxi province as the parental virus,constructed using homologous recombination expression of porcine circovirus type 2 recombinant swinepox virus capsid protein.1.Swine pox virus isolation and identification Virus isolated from suspected swine fattening pigs skin pox scab in a pig farm in Nanchang,Jiangxi province,one strain of swine pox virus isolated,and named by JX-20G strain.References in GenBank SWP(AF410153.1),design SPV 027-029,SPV043-044,SPV063,SPV071 4 pairs of primers,with JX-20G strain DNA as a template,after PCR amplification was approximately 1688bp,1560bp,960bp,975bp 4 specific target band.Got the SPV 027,SPV 028,SPV 043,SPV 044,SPV 063(TK),SPV 071(P35)6 complete genes.DNAstar software using a sequence of six nucleotide and amino acid sequences of the gene JX-20G strains analyzed with reference strains(AF410153.1)and by the isolation and identification of the experimental JX-08 strains for homology.The results showed that:(1)six genes JX-20G with reference strains(AF410153.1)homology between 97.1%?99.6%;Isolation and identification of the laboratory strain of JX-08 homology between 98.2%?99.6%;(2)the amino acid sequence homology with the reference strain(AF410153.1)analysis of genetic evolution in 97.3%to 100%homology,with the JX-08 98.8%to 100%.Display JX-20G with the reference drug strains(AF410153.1),JX-08 closest relationship,but significant differences exist with other pox viruses.2.Cloning and gene sequence analysis of Porcine circovirus type 2 A strain of swine PCV2 isolated from Jiangxi Nanchang area,through a whole genome sequencing,the strain of the whole genome is 1767bp,ORF2 total length is 705bp.Comparison of the amino acid sequence homology with 13 strains PCV2 and 1 strain PCV1 that GenBank included,found that with 13 PCV2 genomic nucleotide sequence homology between 93.9%?99.0%,Only 76.2%with 1 PCV1.So this PCV2 strains named JX-CZH.3.Construction of TK deletion recombinant swinepox virus expressing PCV capsid protein In order to explore the potential of swine pox virus as a PCV2 vaccine vector,in this study,Vaccinai virus(VV)P11,P28,P7.5 as exogenous gene promoter,JX-20G TK gene as an insertion site for a foreign gene EGFP as selection marker,JX-CZH ORF2 gene as exogenous gene,constructed two transfer vector pSWE11-28C andpSWE11-7.5C.By homologous recombination technology,obtained two recombinant virus rSWPV-E11-28C and rSWPV-E11-7.5C.The results showed that:(1)pSW-E11 have been infected with strains of the virus JX-20G PK15 cells were transfected,three days later,it can be observed lager green fluorescent cell under fluorescence microscope,After 7 round purified and multiplied,cells all can observed fluoresce in cells bottle.(2)Recombinant viruses were identified by PCR,genetic stability analysis,Western blotting analysis and electron microscopy.Western blotting analysis showed that two recombinant virus expressed VP60 protein,sizes were about 60 ku,can react with inmmuned His-Cap mice serum.The recombinant protein had good reactogenicity.Electron microscopy showed that the shape and structure of two recombinant virus consistent with the parental virus with no change.In this study,a SWPV strain isolated from Jiangxi province,on this basis,the use of homologous recombination construct two strains of PCV2 capsid protein expression of recombinant porcine poxvirus,recombinant virus infected PK-15 cells can express the PCV2 capsid protein,and the protein has a good reactogenicity,the future of the laboratory for further study of recombinant porcine poxvirus vaccine vectors based foundation.