丙型肝炎病毒核心基因靶向性M1 GS核酶的构建及鉴定

Construction and identification of an M1GS ribozyme targeting the core gene of hepatitis C virus

目的构建一种具有丙型肝炎病毒(hepatitis C virus,HCV)靶向性的M1GS核酶,为进一步研究引导序列(guide sequence,GS)引导核酶P在RNA水平阻断HCV核心基因(core gene,C)的表达作前期基础。方法选择HCV基因组中序列相对保守且在病毒复制过程中起关键作用的核心基因作为靶基因,针对该基因mRNA中的潜在切割位点(第52位),设计相应的引导序列及桥序列(bridge sequence),以PCR扩增M1GS,将其插入pGEM-3Z克隆载体;重组的克隆载体经EcoRⅠ和HindⅢ双酶切、测序及体外转录鉴定。结果 M1GS-HCV/C52克隆载体经双酶切、琼脂糖凝胶电泳证明插入片段与M1RNA片段大小相符;体外转录产生的M1GS-HCV/C52核酶在体外切割实验中将HCV核心基因RNA切割成2个片段,片段大小符合预期。结论成功构建出具有体外靶向切割活性的HCV核心基因靶向性M1GS核酶。

Objective A M1GS ribozyme targeting hepatitis C virus core gene is constructed for investigating the cleavage effect of RNase P lead by guide sequence. Method HCV core gene is aimed for gene targeting due to its relatively conservative sequence and the key role in viral replication. A corresponding sequence- specific M1GS ribozyme is constructed based on the catalytic subunit （M1 RNA） of E. coli RNase P and the potential targeting sites （No. 52） in HCV mRNA. Result M1GS prokaryotic expression recombitnant vector was identified and confirmed by PCR, partial nucleotide sequencing and in vitro transcription. Conclusion A potential M1 GS ribozyme targeting hepatitis C virus core gene which possessing transcription activity is successfully constructed.