丙型肝炎病毒RNA多聚酶在昆虫细胞中的表达

Expression of Hepatitis C Virus RNA Polymerase in Insect Cells

HCVNS5B基因片段克隆入BAC TO BACTM 重组杆状病毒表达系统的pFASTHTc载体质粒 ,转化DH10BACTM感受态细菌获得重组的Bacmid质粒 ,将重组Bacmid质粒转染Sf9细胞 ,获得的重组杆状病毒可表达目的蛋白。免疫印迹和体外活性检测表明 ,所表达蛋白为HCVNS5B蛋白 ,具有多聚酶活性。

A recombinant transposing vector pFASTHTc NS5B was constructed by inserting HCV NS5B gene into pFASTHTc vector of BAC TO BAC TM recombinant baculovirus expression system. The plasmid pFASTHTc NS5B was then transformed into DH10BAC TM competent cell. High molecular weight DNA, the recombinant bacmid, was prepared from the overnight cultures of selected E. coli DH10BAC TM colonies. By transfecting Sf9 cells with the recombinant bacmid the recombinant baculovirus from the superenatant was obtained, which could further infect Sf9 cells obtained and express the target protein. The expression of HCV NS5B was confirmed by Western blot and in vitro characterization showed that HCV NS5B possessed polymerase activity.