Isolation And Identification Of Porcine Circovirus Type 2 Isolated In HeBei Province And Cloning And Expression Of ORF2 Gene

Porcine circovirus type 2 (PCV2) is the main causative agent of Post-weaning Multisystemic Wasting Syndrome (PMWS) and the other related diseases. Immunodeficiency is a well-known consequence of PCV2 infection, PCV2 is a immunosupressive virus and induce other agents inderect infection, it can bring a great loss to our pig industry. It is often been ingored because of its sub-clinical infection, so the study of the epidemiology and molecular biology of virus should be furture investigated. The ORF2 gene of PCV2 is the main region encoding the structural protein, which has nature immunity and conservative gene sequence, so it is often been used in diagosis.In this study, a pair of primers which were specific to PCV2 were desigend, that were used to amplify a segment of 656 bp from the DNA extracted from the 17 collected samples suspected PMWS by PCR test. 16 samples were positive for PCV2, It was approved by EcoR I enzyme digested. Three PCV2 positive samples from hoggeries A,B,C were abraded,sedimented by centrfugation,filtered and inoculated to porcine kidney (PK15) cells. Extract viral DNA in PK15 cells after 6 passages infected PCV2 as template, and design a pair of primers to amplify the complete PCV2 genome, then which were successfully cloned into pMD19-T vetor, it was comfirmed by PCR, sequence analysis and restrict enzyme identification. Based on the analysis results of molecular software, we found the 3 isolates were all 1767 bp in size, and which shared 98.4%～99.6% homology with eachother. Phylogenetic analysis confirmed the 3 isolates with other PCV2 strains recored in GeneBank have no pertinence in geograpfy.ORF2 gene was amplyfied by PCR from the extracted DNA of isolate HBa. It was proved by PCR and restrict enzyme identification. According to the ORF2 gene nucleotide and amino acid sequence ananlysis: The N end have a lot of rare codes, which influnce the expression; the antigen index located in the C end, So the ORF2 gene was modified by PCR using another pair of primers, a fragment of 579 bp of ORF2 gene was obtained and cloned into pET-32a expression vector. The recombinent pET-ORF2 plasmid was transformed into BL21(DE3)competent cell of E.coli and induced by IPTG SDS-PAGE analysis showed that the recombinent protein expressed was 40 kDa in size, Western-bloting analysis showed the expressed protein can react with polyclonal antibody against PCV2.