乙型肝炎病毒多聚酶基因在重组杆状病毒系统中的表达

目的利用杆状病毒一昆虫细胞表达系统制备乙型肝炎病毒多聚酶重组蛋白。方法构建含有乙型肝炎病毒多聚酶基因的杆状病毒转移载体pFastbac Dual—polymerase，转化大肠杆菌DH10Bac进行转座，提取重组Bacmid转染昆虫细胞获得含有HBVpol基因的重组杆状病毒。用重组杆状病毒感染昆虫细胞进行蛋白表达，用SDS—PAGE电泳分析表达情况。结果将所得Bacmid行PCR鉴定，结果表明成功构建了的含乙型肝炎病毒多聚酶基因的重组杆状病毒，SDS-PAGE分析表明该病毒能在昆虫细胞中高效表达重组的多聚酶蛋白。结论利用杆状病毒表达系统，构建的重组杆状病毒在昆虫细胞中高效表达乙型肝炎病毒多聚酶，为进一步的乙型肝炎病毒多聚酶的体外功能研究奠定了基础。

Expression of hepatitis B virus polymerase in recombinant baculovirus

Objectives To produce the recombinant polymerase of the hepatitis B virus in insect ceils by Bac-to-Bac system. Methods The gene of HBV polymerase was amplified by PCR and cloned into pFastBac Dual to construct the recombinant plasmid pFastbac Dual polymerase. The recombinant plasmid was transformed into DH10Bac competent cells for transposition. After the recombinant bacmid was determined by PCR analysis , the isolated bacmid DNAs were transfected into Sf9 insect cells by using lipofectin and recombinant baculoviruses were harvested to infect the insect cells. The insect cells infected by recombinant baculovirus were determined by SDS-PAGE. Results The recombinant bacmid DNA was analyzed by PCR to verify the presence of the polymerase gene of the hepatitis B viru in the bacmid , It was comfirmed that recombinant baculovirus containing the HBV polymerase gene was constructed successfully. The recombi- nant hepatitis B virus polymerase expressed in cells infected by recombinant baculovirus was determined by SDS-PAGE, The hepatitis B virus polymerase was expressed in insect cells at high level. Conclusions Using the baculovirus expression system, recombinant hepatitis B virus polymerase was expressed in insect cells at high level, which would provide valuable reagent for analysis of various aspects of HBV replication in vitro. The results lay the foundation for the research in vitro on HBV polymerase biological characteristics.