Isolation And Identification Of Porcine Circovirus Type1in Yangzhou And Its Surrounding Areas And Development Of PCV1Monoclonal Antibody

Initially found in PK-15cell lines as a kind of contaminant, the porcine circovirus (PCV) was a member of Circovirus belonged to Circoviridae, with the size of about17nm, non-enveloped, including a closed circular DNA genome. The PCV is classified into two kinds of genotype, the PCV1and PCV2. The PCV2is the pathogenic one which has caused huge economic losses to the global pig industry. Although the PCV1is not considered to be pathogenic, it can produce the serum antibody and contaminate the cells of porcine origin. As a result, it is necessary for us to put more emphasis on the research of the PCV1.The PCV has two large open reading frames, the ORF1and the ORF2. The ORF1codes the virus replication associated protein, while the ORF2codes the viral capsid protein. As the main structural protein of the virus, the viral capsid protein exhibits great difference between the PCV1and the PCV2, which can stimulate the host to produce specific antibodies. In view of the above, it is of great significance to do research on the PCV1capsid protein.1. The isolation and identification of the porcine circovirus type1The tissue or blood samples collected from the slaughter houses and pig farms in Yangzhou and its surrounding areas were detected for PCV1by PCR, and five PCV1strains were isolated from these samples. Through sequencing and sequential analysis, it is found that among these5strains, the full genome length was1759bp in3isolates, the other two were1758bp and1760bp, respectively. The length of the ORF2was all702bp. Phylogenic analysis showed that5strains were in the same branch of the Phylogenic tree and the genetic distance was close. Whole genomic sequence was99.0-99.9%homology, ORF2gene was99.0-99.9%identical among these isolates. Epidemiological investigations suggested that whole genome size of PCV1is often1759bp, and1758bp or1760bp is uncommon. We found that PCV1habored three kinds of genomic lengths in Yangzhou and its surrounding areas, which was helpful in the research of PCV1prevalence, genetic variation and genetic evolution.2. Expression of porcine circovirus type1ORF2gene in eukaryotic and prokaryotic system and development of PCV1monoclonal antibodyBoth prokaryotic expression system and eukaryotic baculovirus expression system were employed for PCV1ORF2gene expression. First, the PCV1ORF2gene without signal peptide gene was inserted into the prokaryotic expression vector PET-32a and the whole PCV1ORF2gene was cloned into the baculovirus transfer vector pFastBacTM1, respectively. And the former was transformed into competent cells of E. coli BL21(DE3) and the latter was transformed into competent cells of E. coli DH10Bac. Recombinant protein PCV1-His-Cap was produced by the recombinant plasmid pET-PCV1-ORF2induced by IPTG in BL21(DE3) in the form of inclusion body. The soluble protein PCV1-His-Cap was obtaind by the urea-denatured and-refolded and purified by Ni column and immunized6-8weeks old BALB/c mice after confirmed by SDS-PAGE and Western-Blot. By the hybridoma technique, monoclonal antibody (mAb)1C9against PCV1Cap protein with ELISA (enzyme-linked immunosorbent assay) titer of1:51200was obtained. The transposition took place between the recombinant plasmid pFastBacTM1-PCV1-ORF2and the shuttle vector Bacmid in DH10Bac, the recombinant bacmid Bacmid-PCV1-ORF2was picked out based on blue-white screening. There was significant cytopathic effect after72hours when Bacmid-PCV1-ORF2was liposome-mediated transfected into Sf9insect cells, Transfected cells gave rise to the first generation of recombinant baculovirus reBacmid-PCV1-ORF2. The supernatant of cultured cells was concentrated by Vivaflow200and was used to immunize8-week-old BALB/c mice, based on the hybridoma technique, mAb13D4with IFA (indirect immunofluorescence assay) titer of1:1200against PCV1Cap protein. Using IFA,13D4MAb reacted with all PCV1strains stored in the laboratory while not reacted with PCV2or chimeric PCV1-2strains. The MAbs mentioned above might be a potential tool for detection of PCV1contaminated in porcine cells used in tissue culture.