2a型丙型肝炎病毒非结构基因5区(NS5)全长克隆的构建

Amplification and construction of the vector containing full-length nonstructure gene 5 of hepatitis C virus

目的 构建 2a型丙型肝炎病毒 (HCV)非结构基因 (NS) 5区全长克隆质粒。方法 用聚合酶链反应 (PCR)从HCVNS5A和NS5B质粒中扩增出NS5A和NS5BPCR片段 ,再采用融合PCR技术扩增出全长NS5的cDNA片段 ,克隆后RFLP和序列分析鉴定重组子。结果 融合PCR获得约 2 8kb全长NS5序列 ,同源性分析表明 :与HC J6的核苷酸同源性为 91 2 % ,推导的氨基酸同源性为 95 5 %。结论 利用融合PCR技术简单方便 ,利用该技术获得了 2a型HCVNS5全长序列 。

Objective To construct the recombinant of the nonstructure gene 5(NS5)of hepatitis C(HCV).Methods Two individual fragments were obtained by polymerase chain reaction (PCR) with recombinants of HCV NS5A and NS5B as template,which were used as templates and primers for over-lapping PCR;amplicon was cloned and confirmed by RFLP and sequencing.Results The recombinant contains full-length HCV NS5 gene,which is 58 7%,60 0%,91 2% and 2 3% homologous with HCV-1,HC-J6,HC-C2,HC-J8 respectively.Conclusion The recombinant plasmid containing full-length HCV NS5 gene was constructed successfully by over-lapping PCR.