Cloning And Sequence Analysis Of The ORF2Gene Of PCV2and Its Expression In Eukaryotic System

The porcine circoviruses (PCV) are members of the genus Circovirus, family Circoviridae, which are the smallest non-enveloped, single-stranded, circular DNA viruses. Two types of PCV have been characterized, and subsequently named PCV1and PCV2. PCV1is a persistent contaminant of the porcine kidney cell lines (PK-15), it has never been associated with naturally occurring disease and experimental inoculation of pigs did not result in clinical disease, but antibody can be detected in heathy swine herds. In contrast, PCV2is identified as virulent porcine pathogen. PCV2infection has been associated with post-weaning multisystemic wasting syndrome (PMWS), porcine dermatitis and nephropathy syndrome (PDNS), porcine respiratory disease complex and reproduction disorders. PCV2has two primary open reading frames. ORF2-coding product is the major structural protein, also is primary immunogen. This frame shares about68%nucleotide identity between two types viruses, however, no cross-reaction of antigenicity is observed between them. Therefore, ORF2protein has important significance in epidemiologic diagnosis and vaccine research. And it has become a research hot topic.1. According to the sequence of a PCV2strain isolated from a pig with clinical PMWS and XSC strain (GenBank accession No:DQ104422), a pair of specific primers was respectively designed to amplify the ORF2gene of PCV2(SD-11strain), and cloned into pMD18-T simple vector. After sequence analysis, the homology comparison were conducted with the ORF2sequence of ten PCV2strains deposited in GenBank and the phylogenetic tree was drawn. The results suggested that ORF2gene of the isolated PCV2(SD-11) strain exhibited a low nucleotide homology compared with those of the other ten strains, homology of nucleotide sequence and amino acid sequence between the isolated strain and those ten strains is90.2%-93.2%and87.6%-94%, respectively. ORF2gene of the isolated PCV2(SD-11) was classified into an independent cluster, suggesting ORF2gene of the isolated PCV2had far genetic relationship compared with other ten strains. XSC strain exhibited a high nucleotide homology compared with that of C7155strain (GenBank accession No:FJ905463.1) of Korea and09GDLB strain (GenBank accession No:HQ395027.1) of China, homology of nucleotide sequence and amino acid sequence was more than98.7%, and ORF2gene of XSC strain was classified into an small cluster with that of C7155strain of Korea, suggesting the two strains were revelant with the import of the breed.The complete genome of SD-11and XSC strains, nucleotides of ORF1and ORF2genes were respectively compared with the different genotypes of PCV (PCV1, PCV2a, PCV2b, PCVl/2a, PCV2c) strain, the results suggested that SD-11strain had the highest homology with PCV2a (GenBank accession No:AJ223185), speculating SD-11strain for PCV2a genotype; XSC strain had the highest homology with PCV2b (GenBank accession No:AY909004) strain, speculating XSC strain for PCV2b genotype.2. One pair of specific primers was respectively designed according to ORF2gene sequence of PCV2b (XSC strain) and PCV2a strain (isolated from clinical PMWS pig), and the ORF2gene was amplified by PCR. To construct the recombinant baculovirus transfer plasmid pFast-ORF2a and pFast-ORF2b, the ORF2genes of PCV2a and PCV2b were respectively subcloned into baculovirus transfer vector (pFastBacTM1. E. coli DH1OBac containing baculovirus shutter vector was transformed with recombinant plasmid pFast-ORF2a and pFast-ORF2b. Within E. coli DH10Bac, the ORF2gene was respectively transposed into the bacmid. The colonies of E. coli containing recombinant bacmid (reBacmid-ORF2) were collected by blue and white selection. The reBacmid-ORF2a and reBacmid-ORF2b were respectively transfected into sf9cells to yield recombinant virus (reBacmid-Cap) carrying ORF2gene of the PCV2a and PCV2b. mRNA of ORF2gene of PCV2a and PCV2b was detected by RT-PCR in cells carrying recombinant virus. Expression of the ORF2genes of PCV2was confirmed by indirect immunofluorescent assay, SDS-PAGE and Western-blotting. The expression products of ORF2genes had a molecular mass of about30kD and could be recognized by the anti-serum against PCV2. The results indicated that the ORF2gene expressed properly in sf9cells. Three methods for titering recombinant baculovirus including the limited dilution method, the indirect immunofluorecent assay and the plaque assay were evaluated to develop a suitable method to titer the recombinant baculovirus. The results indicated that the limited dilution method and the indirect immunofluorecent assay were stable and exhibited a good repeatability. However, the plaque assay represented high false positive and negative rates and a poor repeatability.