摘要
目的进行丙型肝炎病毒(HCV)RNA干扰(RNA interference RNAi)作用研究。方法本实验选择HCV NS3基因片段作为靶序列,合成含有靶序列的DNA片段并克隆到pGCsi-U6/Neo/GFP/siNeGative载体中,构建可表达具有发夹结构双链siRNA的真核表达质粒pGCsi-U6/Neo/GFP/A,并转染Hela细胞,用HCV攻击该细胞,通过TCID50和HCV负链RT-PCR检测该载体对HCV感染细胞的保护效果。结果通过测序及荧光显微镜观察证明载体构建成功,实验组TCID50明显高于对照组,且RT-PCR结果显示pGCsi-U6/Neo/GFP/A转染组的HCV负链RNA表达量显著低于对照组。结论证实重组质粒表达产物能成功地使模型细胞中的HCV NS3基因沉默,并间接抑制了病毒的复制,本实验的成功无疑为HCV的治疗及蛋白功能的研究打下坚实的基础。
Objective To investigate the antiviral effect of HCV RNAi.Methods we constructed the siRNA expressing vector pGCsi-U6/Neo/A with U6 promoter and GFP marker targeting to HCV NS3 gene, then transfected it into Hela cells. The HCV was infected after different transfection time as 5 h,24 h,48 h and 72 h.Results Then the inhibition effects were tested by the TCID50 and the expressing level of viral RNA. Conclusion It is show that the expressing vector pGCsi-U6/Neo/A could inhibit the replication of HCV effectively. And the inhibition is strongest after 24 h transfection.
出处
《中国实验诊断学》
北大核心
2009年第6期723-726,共4页
Chinese Journal of Laboratory Diagnosis
