作　　者： (曹开源）; (冯炼强）; (付涌水）; ;

机构地区： 中山大学

出　　处： 《中国病理生理杂志》 2000年第9期809-813,共5页

摘　　要： 目的 :构建丙型肝炎病毒 (HCV) 5′末端非编码区 (5′NCR)和结构蛋白编码基因序列逆转录病毒重组体 ,用于探索控制HCV感染的新途径和基因治疗。方法 :对多株HCV核酸序列进行同源性比较设计引物 ,逆转录聚合酶链式反应 (RT -PCR)扩增 5′NCR、C、E1和E2 /NS14个编码区共 5个片段 ,分别克隆。以连接聚合酶链反应 (PCR)将这 5个片段拼接为一连续的长 2 5 47nt的片段 ,含HCV完整的 5′NCR和全部的结构蛋白编码序列。将此序列插入pGEM -Zf(+ )载体 ,与逆转病毒pLNSX载体中 ,转化大肠杆菌DH5a、转化菌落经酶切、PCR和Southern杂交鉴定。结果 :通过RT -PCR和连接聚合酶链式反应 (PCR)扩增出 2 5 47nt含HCV完整的 5′NCR和全部的结构蛋白编码序列 ,将此序列与pGEM -Zf(+ )重组得重组体pHC2 5 47,与逆转录病毒载体pLNSX重组得pL HC。结论 :成功构建了HCV逆转病毒重组体 ,以利于HCV的胞内基因表达调控研究 。 AIM:To construct retrovirus recombinant HCV/ pLNSX(pLHC), which is used to investigate new ways to control HCV infection and gene therapy. METHODS and RESULTS: Primers with their sequences homologous to multiple strains of HCV had been designed, synthesized and used in RT-PCR to amplify 5 fragments from 4 regions,5'NCR,C,E1 and E2/NS1.The products had been cloned, restrictively cut aside, and overlappingly amplified to ligate a consecutive sequence of 2547 bp in length that contains the whole HCV 5'NCR and all the structural protein coding regions. The sequence was then inserted into the vector pGEM-3Zf (+) yielding a recombinant pHC2547, and into the retrovirus pLNSX yielding a pLHC .Both plasmid DNA were analyzed by PCR, Southern blot, and enzymatic cut. CONCLUSION: Retrovirus recombinant HCV/pLNSX (pLHC) constructed successfully is useful to study both the regulation of intracellular HCV gene expression and the pathogenesis of HCV infection, as well as the molecular foundation of related transgene animals and gene therapy.

关 键 词： 克隆 丙型肝炎 结构蛋白 编码基因

领　　域： [医药卫生—内科学] [医药卫生—临床医学]