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核酶细胞内抗丙型肝炎病毒作用的研究 |
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陈智 刘勇 刘克洲 Dennin RH 窦俊 Reinhard U
【摘要】目的 研究核酶抗丙型肝炎病毒(HCV)的有效切割位点并获得高效、特异、无毒、价廉的HCV特异性 反式核酶。方法 根据文献报道的序列设计、合成HCV 5′非结构(NC)区和核心(C)区 的特异性反式核 酶基因并分别克隆进入真核细胞表达载体pSV2-gpt.CD-SRα中，转染HCV感染的MT-2细胞 ，用定量逆转录-聚合酶链反应(RT-PCR)、核酸杂交等方法测定核酶对HCV的抑制作用。结果 所用的HCV 5′NC区核酶和C区核酶对HCV均有显著的抑制作用，抑制 率分别为54.7%和62.1%。两者联合作用时抑制率达78.8%。结论 HCV 5′NC区和C区特异性反式核酶在HCV感染的体外细胞培养模型中 对HCV复制有明显的抑制作用；双靶位核酶联合使用较单靶位核酶效果更好。
【关键词】核酶；肝炎病毒，丙型；MT-2细胞
An in vitro study of ribozyme against hepatitis C virus
CHEN Zhi LIU Yong LIU Kezhou et al.
（The Institute of Infectious Diseases, First Affiliated Hospital, Medic al College, Zhejiang University, Hangzhou 310003, China）
【Abstract】Objective To determine the cleavage sites of ribozymes against hepatitis C virus(HCV), and to obtain a highly effective, nontoxic and inexpensive antisense ribozyme specific for HCV. Methods Two effective ribozymes, targeted to HCV 5′-non-coding region(5′-NCR) and C region, were synthesized. Eukaryotic expression vectors, pSV2-gpt. CD-SRα, containing either HCRZ NC or HCRZC were constructed and transfected into MT-2 cells, which had been in fected by HCV. Quantitative RT-PCR and hybridization methods were used to deter mine the effect of inhibition of HCV by ribozymes. Results HCRZNC and HCRZC suppressed the replication of HCV by 54. 7% and 62.1%, respectively. Furthermore, when the two ribozymes were cotransfect ed into cells, they suppressed replication by 78.8%. Conclusion Two specific antisense ribozymes have strong inhibit ory effects on the replication of HCV in cultured cells, and have better effect when used together.
【Key words】Ribozyme； Hepatitis virus, type C; MT-2 cells
核酶(Ribozyme)是一类具有特异性剪切RNA活性的RNA分子。1990年Sarver等［1］ 首先报道在体外培养的细胞中用核酶成功抑制了人类免疫缺陷病毒(HIV)的复制，引起越 来越多的医学和生物 工作者的重视。目前核酶已开始应用于人体内治疗［2］。然而，核酶步入临床实践 前，还有许多问题有待于解决。例如：如何选择靶序列中最佳切割位点，如何获得高效、无 毒的特异性反式核酶，如何提高核酶进入靶细胞的效率并稳定发挥作用等。我们在已建立的 丙型肝炎病毒[1] [2] 下一页
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