表面展示猪圆环病毒2型衣壳蛋白的重组杆状病毒的构建与鉴定

通过PCR方法扩增猪圆环病毒2型(porcine circovirus 2,PCV2)核定位序列部分缺失的ORF2基因(pdCap),将其克隆至Bac-to-Bac杆状病毒表达系统的pBACsurf-1质粒中,得到重组质粒pBACsurf-1-pdCap。再将含有pdCap和gp64的融合基因克隆到杆状病毒并转移到质粒pFastBac^TM Dual中,得到重组质粒pFastBac-pdCap。然后将该重组质粒转化至DH10Bac感受态细胞中,经蓝白斑筛选和PCR鉴定,得到重组穿梭质粒Bacmid-pdCap。在脂质体介导下转染Sf9昆虫细胞,获得重组杆状病毒BV-pdCap。间接免疫荧光和Western blotting试验结果表明,该重组杆状病毒成功表达了具有良好反应原性的pdCap蛋白。

Construction and Identification of Recombinant Baculovirus Surface Displaying the Cap Protein of Porcine Circovirus Type 2

The Cap gene partially deleted nuclear localization signal sequence(pdCap) of porcine circovirus 2(PCV2)was cloned into pBACsurf-1 between the upstream gp64 signal sequence and downstresm gp64 mature domain to construct the recombinant plasmid pBACsurf-1-pdCap.The fusion gene containing pdCap and gp64 was subcloned into pFastBac^TM Dual to construct the recombinant plasmid pFastBac-pdCap.And then the plasmid was transformed into E.coli DH10Bac competent cells to obtain the recombinant shuttle vector Bacmid-pdCap.Eventually,the recombinant Bacmid-pdCap was transfected into Sf9 cells to produce the recombinant baculovirus BV-pdCap mediating by Lipofectamine^TM2000.Indirect immunofluorescent and Western blotting assay indicated that the recombinant baculovirus BV-pdCap could express pdCap protein with significant antigencity.