Sequence Analysis Of ORF2 Gene Of PCV-2 Strains And The Expression Of Proteine In Insect Cells

Porcine circovirus is a member of circoviridae.According to virulence and genomic porcine circovirus contains two types genome:PCV1 and PCV2.PCV1 has no pathogenicity and PCV2 is relation to many dieases and closed associated with postweaning multisystemic wasting syndrome.PCV2 has two open reading frames . ORF2-coding product is the major structural protein. This protein shares about 68% identity between two types virus; however, no cross-reaction of antigenicity is observed between them. Therefore, the ORF2 gene can be used to differentiate PCV1 and PCV2.1.Using a pair of specific primers designed according to published ORF2 gene sequence of PCV2,and the ORF2 gene was amplified from the suspected PMWS sample by PCR .The amplified fragment swere cloned into the pMD18-T vectors and the positive recombinant plasmids were designated PMD18T- ORF2.The results indicated that it share 91.5%to 99.4% homology at their nucleotide level.there has only 91.5% nucleotide identity with harbin isolates and share most high sequence identity with zhejiang isolate 99.4%.The data also indicate that there have genetic differences from different geographical regions.2.To produce the recombinant baculovirus transfer plasmid pFast-ORF2 , the ORF2 gene of porcine circovirus type 2 (PCV2) was subcloned into baculovirus transfer vector using Bac-to-Bac baculovirus expression system. E. coli DH10Bac containing baculovirus shutter vector and helper vector was transformed with recombinant plasmid pFast-ORF2. Within E. coli DH10Bac,the ORF2 gene was transposed into the bacmid. The colonies of E. coli containing recombinant bacmid (Bac-ORF2) were collected by blue and white selection. The Bac-ORF2 was transfected into sf9 cells to yield AcNPV carrying the PCV2 ORF2 gene , referred to as Ac. ORF2. Expression of the ORF2 gene of PCV2 was confirmed by indirect immunofluorescent assay,SDS-PAGE and Western-blotting. The expressed ORF2 gene product had a molecular mass of 28kD and could be recognized by the positive serum of PCV2.The results indicated the ORF2 gene was properly expressed in sf9 cell.