表达猪圆环病毒2型SH1分离株Cap蛋白的重组杆状病毒的构建与鉴定

Construction and identification of recombinant baculovirus expressing Cap protein of porcine circovirus type 2 SH1 isolate

通过PCR方法扩增完整的猪圆环病毒2型(PCV2)ORF2基因,随后将其克隆到Bac-to-Bac杆状病毒表达系统pFastBacTMHTB载体中,将筛选获得的阳性重组质粒pF-Cap转化至含有杆状病毒穿梭载体的DH10Bac感受态大肠杆菌中,经蓝白菌落筛选和PCR鉴定,获得重组表达载体rBac-Cap。在脂质体介导下转染处于对数生长期的Sf9昆虫细胞,获得重组杆状病毒rAc-Cap。Western-blot和间接免疫荧光试验结果表明,成功构建了表达PCV2Cap蛋白的重组杆状病毒,表达的蛋白具有良好的反应原性,这为进一步研究Cap蛋白的功能及免疫学特性奠定了基础。

The complete ORF2 gene was amplified from porcine circovirus type 2（PCV2） SH1 isolate by PCR and then cloned into the donor vector pFastBac TMHT B of Bac-to-Bac Baculovirus Expression System.After sequencing and analysis,the purified recombinant plasmid pF-Cap was transformed into Escherichia coli DH10Bac competent cells containing baculovirus shuttle vector.The white colonies were selected and the recombinant bacmid rBac-Cap was verified by PCR.Then the recombinant bacmid was transfected into Sf9 insect cells,the recombinant baculovirus rAc-Cap was constructed successfully,confirmed by Western-blot and indirect immunofluorescence assay with antibody against Cap protein.The recombinant Cap protein with good reactogenicity laid the foundation for further studies of the function and immuno-logic characteristics of the recombinant Cap protein.