目的 针对禽流感病毒(AIV)复制酶PB2亚基的基因构建具有靶向切割作用的M1GS核酶,为新型抗AIV反义药物的开发奠定基础.方法 基于大肠埃希菌的核糖核酸酶P(RNase P),根据AIV PB2基因的序列特征,设计一小段与之互补的引导序列GS,通过PCR技术将其共价连接至大肠埃希菌RNase P催化性亚基(M1 RNA)的3'末端,以构建靶向性M1GS核酶.通过体外切割试验测定M1GS核酶的活性.结果 构建了M1GS-T436、M1GS-C341两种M1GS核酶.体外切割实验证实,核酶M1GS-T436可对靶RNA片段产生特异性切割;核酶M1GS-C341虽可切割靶RNA片段,但可能属于非特异性切割.结论 成功构建并筛选出一种具有显著体外切割活性的M1GS核酶(M1GS-T436),为今后进一步研究其胞内及体内抗病毒活性奠定了基础.%Objective To construct a MIGS ribozyme that targeting to replicase PB2 gene of avian influenza virus ( AIV) , and provide a reference for development of the novel anti-AIV strategy. Methods Based on the ribonuclease P ( RNase P) derived from Escherichia coli, a guide sequence ( GS) complementary with PB2 gene sequence was designed, and covalently linked to the 3′end of catalytic subunit of RNase P ( M1 RNA) by PCR. Then the activity of this targeting M1GS ribozyme was measured by the in vitro cleavage assay. Results Two kinds of M1GS ribozymes, namely M1GS-C341 and M1GS-T436 , were constructed. The ribozyme M1GS-T436 specifically cleaved the target RNA, however the ribozyme M1GS-C341 showed the nonspecific cleavage activity. Conclusion A potential M1GS ribozyme ( M1GS-T436 ) , that possess the specific cleavage activity, has been successfully constructed, which may lay the foundation for further study of its intracellular and in vivo anti-viral activity.
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