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 ,更为探索HCV分子致病机制和转基因动物及基因治疗提供可靠的物质基础 OBJECTIVE: To construct a recombinant retrovirus containing 5 ’untranslated region (5’NCR) and structural protein coding sequence of Hepatitis C virus (HCV), and explore new ways and gene therapy for HCV infection control. Methods: Primers were designed for homology comparison of multiple HCV nucleic acid sequences. A total of 5 fragments of 14 coding regions of 5’NCR, C, E1 and E2 / NS were amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) clone. The 5 fragments were spliced ​​into a continuous 2547-nt fragment by polymerase chain reaction (PCR), containing the complete 5’NCR of HCV and the entire structural protein coding sequence. The sequence was inserted into the pGEM-Zf (+) vector, and the retrovirus vector pLNSX was transformed into E. coli DH5a. The transformed colonies were identified by restriction enzyme digestion, PCR and Southern blotting. RESULTS: A total of 2547 nt HCV-containing 5’NCR and all structural protein coding sequences were amplified by RT-PCR and linked polymerase chain reaction (PCR). This sequence was recombined with pGEM-Zf (+) PHC2 5 47, pL HC was recombined with the retroviral vector pLNSX. Conclusion: HCV retrovirus recombinant was successfully constructed to facilitate the regulation of intracellular gene expression in HCV, and provide a reliable material basis for further exploration of molecular pathogenesis of HCV and transgenic animals and gene therapy