Isolation And Identification Of Porcine Circovirus Type 2 From Xinjiang And Prodaryotic Expression Of ORF2

In this study, some samples were collected from (contain lymph nodes, spleens, lungs and livers and so on) pigs with PMWS in Changji.The samples were frozen in-20℃and homogenized.The inocula composed of homogenized tissues were centrifuged and filtered to eliminate bacterial contaminant.The monolayer of PK15 cells free of PCV1 and PCV2 were inoculated with the inocula, and then incubated at 37℃for 48h in 5%CO2 atmosphere.The specific primers were designed, then the presence of PCV2 was tested by PCR from PK15 cell.The PCR products were digested with appropriate restriction enzymes and PCR-positive cell were identified by immunofluorescence.Results showed that the tissues infected the PCV2 viruses.The specific primers were designed and synthesized on the basis of the published sequence of PCV2 and the complete genomes were amplified by PCR from viral DNA of the PK15 cell.The purified PCR products were cloned into pMD18-T simple vector for sequencing.The identification of PCR and digested enzymes and sequencing results showed that the two isolates were obtained, and were named XJPCV2-a and XJPCV2-b.The phylogenetic analysis showed that the two PCV2 isolates were closely related to each other, shared 99.3% nucleotide sequence identities and displayed 95.8%-99.4% nucleotide homology and 75.9%-76.1% nucleotide homology with other PCV1 isolates in GenBank.Phylogenetic analysis confirmed the two isolates with other PCV2 strains have no pertinence in geograpfy.The specific primers were designed to amplify the ORF2 gene about 654 bp from the extracted DNA of isolate XJPCV2-a.According to the ORF2 gene nucleotide and amino acid analysis:The N end have a lot of rare codes, which influnce the expression.Main antigen eptiod of capsid protine located in the C end, therefore, the ORF2 gene was modified by enzymed digestion.The fragment of 393 bp of ORF2 gene and were cloned into pGEX-6P-1 vector.The recombinant plasmid (pGEX-6P-dORF2) was identified by PCR and digested enzymes and sequencing, then the postive recombinant plasmid (pGEX-6P-dORF2) was transformed into E.coli BL21.The fusion protein was expressed under the induction of IPTG and identification by SDS-PAGE and western blotting.Results showed that the fusion protein identical with the predicted molecular weight of 40.7 kDa, and that the protein could be recognized by monoantibodies.These research results would provide a basis for further study on the diagnosis technology of PCV2, clinic diagnosis and the devolopment of vaccine.