核酶细胞内抗丙型肝炎病毒作用的研究

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.%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'-Don-coding region(5'-NCR)and C region,were synthesized.Eukaryotic expression vectors,pSV2-gpt.CD-SRa,containing either HCRZNC or HCRZC were constructed and transfected into MT-2 cells,which had been infected by HCV.Quantitative RT-PCR and hybridization methods were used to determine the effect of inhibition of HCV by ribozymes. Results HCRZNC and HCRZC suppressed the replication of HCV by 54.7%and 62.1%,respectively.Further-more,when the two ribozymes were cotransfected into cells,they suppressed replication by 78.8%.Conclusion Two specific antisense ribozymes have strong inhibitory effects On the replication of HCV in cultured cells,and have better effect when used together.