一种HCV靶向性M1GS核酶的构建及其体外活性

摘要

Objective To construct a targeting ribozyme (M1GS) which is specific to HCV genome. Methods According to the sequence of conservative region (5'UTR) of HCV genome, a guide sequence was designed and synthesized. And then by the PCR method, a kind of targeting ribozyme can be constructed by covalently linking the guide sequence to the 3'terminus of M1 RNA, the catalytic subunit of RNase P from Escherichia coli. Results The engineered ribozyme( M1GS-HCV/C67) is targeted to the 5'UTR of HCV genome, and can effectively cleave the substrate RNA segment in vitro. The cleavage is specific and the cleavage site is between 67 nt and 68 nt of the target region. Conclusion The M1GS-HCV/C67 would be a useful experimental material to further study its cleavage activity in vivo, and can be even used for evaluating its anti-viral effect in the animal model. It was believed that this study would markedly facilitate the research of a general gene targeting agent for anti-HCV applications, and layed the foundation for developing a new nucleic acid drug of anti-HCV therapy.%目的 构建对HCV基因组具有特异切割作用的新型靶向性核酶-M1GS.方法 针对HCV基因组的保守区(5'UTR)设计并合成一段引导序列,通过PCR扩增直接将该引导序列连接于大肠杆菌核糖核酸酶P的催化亚基(M1 RNA)的3'末端,从而构建一类靶向性M1GS核酶.结果 体外切割实验表明,所构建的核酶(MIGS-HCV/C67)对HCV 5'UTR具有明显的切割作用,切割的位点在靶序列67～68 nt之间,属于特异性切割.结论 构建了一种对HCV 5'UTR具有靶向切割活性的MIGS核酶,为胞内反义效应及动物模型内抗病毒效应的评价提供了实验材料,为新型抗HCV药物的研究奠定了基础.