伪狂犬病病毒gE基因在昆虫细胞中的表达

Expression of pseudorabies virus gE gene in insect cells

为获得具有天然活性的伪狂犬病病毒(PRV)gE蛋白,建立PRV gE抗体快速检测方法,将PRV gE基因克隆至p Fast Bac/HBM-TOPO载体,并转化含有杆状病毒穿梭质粒的DH10Bac E.coli,通过三重抗性和蓝白斑筛选,得到含有PRV gE基因的重组Bacmid,将其转染Sf9昆虫细胞,获得重组杆状病毒。然后分别用间接免疫荧光、SDS-PAGE和Western-blot对表达蛋白进行鉴定和分析,结果表明,gE蛋白在杆状病毒表达系统中得到成功表达,并且具有良好的反应原性,这为建立PRV gE抗体快速检测方法提供了良好的抗原。

In order to acquire naturally active gE protein of pseudorabies virus（PRV） used for establishing a rapid antibody detection method, PRV gE gene was cloned into p Fast Bac/HBM-TOPO vector,and transformed into DH10 Bac E.coli containing baculovirus shuttle plasmid. Recombinant bacmid with PRV gE gene was gained by three antibiotics and blue/white selection. Then the recombinant bacmid was transfected into Sf9 insect cells, and recombinant baculovirus was obtained. The gE protein was expressed in Sf9 cell infected with recombinant baculovirus,which was identified by indirect immunofluorescence, SDS-PAGE and Western-blot. The results showed that gE protein has been successfully expressed through baculovirus expression system, and demonstrates good reactionogenicity, which provides a good antigen for establishing a rapid PRV gE antibody detection method.