Expression of ORF2Gene of Porcine Circovirus Type 2in Sf9Insect Cells

The ORF2gene of porcine circovirus type 2(PCV2) including complete open reading frame(ORF) was amplified by PCR,and cloned into the pFastBac1 TM vector of Bac-to-Bac baculovirus expression systems to construct the recombinant transfer vector pFast-ORF2.The transfer vector was transformed into DH10Bac competent cell which contained the BmNPV bacmid.A recombinant shuttle vector which contained ORF2gene was constructed by site-specific transposition and the colonies containing recombinant bacmid were collected by white selection.The cultured Sf9cells were transfected with recombinant bacmid DNA mediating with cellfectin reagent and the pure recombinant baculovirus Bacmid-ORF2was obtained.After transfecting this transformant into Sf9cells,Cap protein expression in Sf9cells was detected by indirect immunofluorescent assay and Western blotting.The experimental results showed that the recombinant baculovirus was obtained and the Cap protein with a molecular weight of 31ku was expressed in Sf9insect cells.The recombinant protein could be recognized by PCV2positive serum.This would lay a foundation for developing PCV2subunit vaccine and diagnosis method.