介导分泌表达猪圆环病毒2型Cap蛋白的重组杆状病毒的构建

Construction of a Recombinant Baculovirus Secreted Expression Vector in Cap Protein of Porcine Circovirus Type 2

摘要: 应用杆状病毒表达系统，将猪圆环病毒2型 ORF2 基因插入供体质粒pFastBac^TMDual pH启动子控制下的多克隆位点，引入EGT信号肽取代 ORF2 原有核定位信号肽以实现在昆虫细胞中分泌型表达，并在C端融合6个组氨酸标签以便于后期的纯化.将构建质粒转化DH10Bac感受态细胞获得重组穿梭载体，提取重组Bacmid质粒.将阳性重组Bacmid质粒转染Sf9昆虫细胞，72 h收集培养上清液获得重组杆状病毒.间接免疫荧光试验和Western-blot表明，该重组杆状病毒可以在昆虫细胞实现猪圆环病毒2型Cap蛋白的分泌表达.

Abstract: The objective of this study was to utilize baculovirus expression vector system to express capsid(Cap)protein （in secreted form）of porcine circovirus 2.The gene sequences encoding Cap protein fused with a C-terminal 6 Histidine tag were cloned into the baculovirus pFastBac^TMDual vector under the control of pH promoter. The authentic signal peptide of porcine circovirus type 2 ORF2 was substituted with the ecdysteroid UDP-glucosyltransferase (EGT) signal peptide. Plasmid was transformed into Escherichia coli DH10Bac competent cells to obtain the recombinant shuttle vector Bacmid. The Bacmid was transfected into Sf9 cells to produce the recombinant baculovirus. Indirect immunofluorescent assay and Western-blot indicated that the recombinant baculovirus could express Cap protein in infected Sf9 cells.