Studies On Molecular Epidemiology Of Porcine Circovirus Type2and Expression Of Viral Capsid Protein In Insect Cells

Porcine circovirus (PCV) is a small, non-enveloped virus with a single-strand circular DNAgenome of1.76kb, which makes it one of the smallest known animal viruses. Based on the antigenicityand sequence analyses of PCV isolates, they can be further divided into two genotypes Porcinecircovirus type1(PCV1) and Porcine circovirus type2(PCV2). PCV1was initially detected as acontaminant of porcine kidney-15cells(PK-15) in1974by Tischer, but it does not induce a disease inswine. Unlike PCV1, PCV2is the etiological agent of postweaning multisystemic wasting syndrome(PMWS), a new emerging and multi-factorial disease in swine which was first recognized in Canada in1991by Ellis. Since then, this scourge has devastated almost every pig-producing area of the world.PCV2may be primary causative pathogen of other diseases such as abortion and mortalitysyndrome(SAMS), sow porcine dermatitis and nephropathy syndrome(PDNS), porcine respiratorydisease complex(PRDC), necrotizing pneumonia(PNP) and congenital tremor(CT), etc.To investigate the molecular epidemiological features and dynamic trends of PCV2,452sampleswere collected from diseased pigs and clinically healthy pigs in12provinces during2008-2011anddetected by PCR. The results showed that354samples were positive (78.3%), from which the ORF2sequences of31PCV2isolates were amplified and sequenced, phylogenetics of the ORF2wereanalyzed by molecular biological software. All the31isolates belonged to sub-genotype PCV2b, amongthem,21belonged to sub-group PCV1A/1B,10belonged to sub-group PCV1C. A comparison of thededuced amino acid residues indicated that PCV2sub-groups have their specific mutation sites. At genelevel, PCV2sub-group1A/1B was dominant PCV2strain since2008in China. However, the statusmight change in future because of the general increasing tendency of PCV2sub-group1C strain.We re-designed and synthesized a full-length PCV1A/1B ORF2gene by optimizing condons biasfor insect cells. The capsid gene and codon optimized capsid gene were both cloned into the multiplecloning site of the pFastBacTMⅠvector of the Bac-to-Bac expression system under the control of PPHpromotor. The recombinant plasmids rBacmid-cap and rBacmid-opt-cap were transfomed into DH10Bacfor bule/white plaque and antibotics selection. The positive recombinant bacmids were transfected toSf9cells in logarithmic growth phase for harvesting recombinant Baculovirus. The expression protein inSf9which were infected by recombinant Baculovirus rvBac-cap and rvBac-opt-cap were identified byIFA and western blot. The optimal conditions for amplification of the recombinant virus and expressionlevel were obtained by changing the M.O.I and the harvest time. The results showed that, compared towide type, the expression level of optimized gene was increased. The recombinant protein wasself-assembled into PCV2-like particles(PCV2-VLPs) with a diameter of17nm. The optimal conditionsfor amplifying the recombinant virus were that the recombinant baculovirus was inoculated at M.O.I of0.05and the supernatant was harvested in96h. The optimal conditions for expressing the recombinantprotein were that the recombinant baculovirus was inoculated at M.O.I of5.0and the protein wasexpressed in48h. The efficient expression and formation of VLPs could be an important basis forfurther developing a PCV2-VLPs vaccine.