RNA干扰抑制土拨鼠肝炎病毒X基因表达

目的:研究土拨鼠肝炎病毒(WHV)序列特异性siRNA对WHV X基因表达的抑制作用。方法:以WHV全长质粒为模板PCR扩增WHV X基因,克隆到pXF3H载体构建WHX-HA融合蛋白真核表达质粒(pXF3H-WHx);合成siRNA的模板序列,克隆到siRNA表达质粒psiRNA构建shRNA表达质粒(psiWx1、psiWx2);质粒pXF3H-WHx与psiWx1、psiWx2共转染Hela细胞,Western blot检测WHV X蛋白的表达。结果:Hela细胞中WHVX蛋白的表达受siRNA的抑制,psiWx1表达的siRNA抑制90%以上的X蛋白表达,并呈剂量依赖性,但是WHV X基因靶位点中2个突变的核苷酸可使这种干扰作用消失。结论:质粒表达的siRNA序列特异性抑制WHV X蛋白的表达。

Inhibition of Woodchuck Hepatitis Virus X Gene Expression by RNA interference

Objective To investigate the inhibition on woodchuck hepatitis virus(WHV) X gene expression by RNA interference.Methods WHV X gene was amplified and restricted with EcoR I and Pst I,then inserted into the EcoR I-Pst I site of pXF3H vector,which finally generated a plasmid expressing WHX-HA fusion protein(pXF3H-WHx).Two plasmids(psiWx1 and psiWx2) expressing siRNA targeting against WHV X gene were constructed by directly inserting the synthesized siRNA templates to the Bgl Ⅱ and Hind Ⅲ sites of psiRNA vector.Hela cells were cotransfected with pXF3H-WHx and psiWx1 or psiWx2,and the expression level of WHx-HA fusion protein was investigated.Results WHX-HA fusion protein expression was inhibited over 90% with siRNA transcribed from psiWx1 dose-dependently,while two nucleotides mutation in the target sequence thoroughly abrogated the RNAi effect.Conclusion RNAi could inhibit the WHV X protein expression sequence-specifically and dose-dependently.