【摘要】 目的构建乙型肝炎病毒(HBV)中国株及其拉米夫定耐药相关变异株的重组杆状病毒。方法将1.2倍HBV基因组(血清adr型、基因C型)连接至杆状病毒的转移载体pFastBacⅠ上,然后转化入DH10Bac菌株中,在细菌内的辅助质粒辅助作用下与杆状病毒基因组Bacmid发生位点特异性转座,形成含有HBV基因组的重组Bacmid,转染昆虫sf9细胞后,包装产生HBV重组杆状病毒vAcHBVc。采用连续聚合酶链反应(PCR)引入方法进行定点突变,构建YVDD变异株质粒,并按照上述方法构建HBV YVDD变异株重组杆状病毒vAcHBVYVDD,扩增并滴定。为便于观察重组杆状病毒的转染和扩增效率及病毒滴度测定,同时构建了含绿色荧光蛋白(GFP)的HBV重组杆状病毒vAcGFPHBVc。结果HBV野毒株和变异株重组杆状病毒均构建成功,通过免疫空斑及TCID50定量测定病毒滴度为106~108pfu/ml左右。结论应用Bac to Bac杆状病毒表达系统可有效快速地构建HBV C型重组杆状病毒,以用于HBV体外复制系统的建立,为HBV野毒株及变异株生物学特性的研究及抗病毒药物的筛选提供技术平台。更多还原

【Abstract】 Objective To construct Hepatitis B Virus (HBV) Chinese-strain wild type and Lamivudine-resistant variant recombinant baculovirus. Methods 1.2-unit length HBV construct (adr serotype, C genotype) was cloned into the multiple cloning region of the pFastBac Ⅰ vector and then transformed into DH10Bac, in which homologous recombination occurred between the pFastBac vector and the inside bacmid with the help of helper plasmid. So HBV recombinant bacmid was produced and transfected into sf9 cells to generate a Recombinant HBV baculovirus called vAcHBVc. The viral titers were detected by immune plaque assay and TCID 50. Lamivudine resistance mutation (YVDD) was introduced by site-directed mutagenesis using continuous PCR. HBV YVDD recombinant baculovirus (vAcHBVcYVDD) was generated and titered as above. Meanwhile, the recombinant HBV baculovirus containing GFP reporter was constructed for evaluating the efficacy of transfection and titration of virus. Results Recombinant wild and YMDD variant HBV baculovirus were successfully developed and the titer was between 10~6～10~8pfu/ml through plaque assay. Conclusion Different kinds of recombinant HBV baculovirus could be generated rapidly and efficiently with Bac to Bac expression system. The developed recombinant HBV baculovirus could be further used in the study of anti-viral resistances.更多还原