靶向HBx小干扰RNA表达载体构建及其功能探讨  

The Construction of expression vector of the short interfering RNA targeting HBV X gene and its function exploration

作　　者：高国生[1] 翁彭剑[1] 纪礽斌[1] 李德周[1] 李永燕[1] 李红山[1] 丁世雄[1] 

机构地区：[1]浙江省宁波市第二医院,浙江宁波315010

出　　处：《中国卫生检验杂志》2011年第7期1682-1684,共3页Chinese Journal of Health Laboratory Technology

基　　金：宁波市自然科学基金项目资助(2010A610056)

摘　　要：目的:设计和构建乙型肝炎病毒(HBV)X基因(HBx)特异性的小干扰RNA(siRNA)体内表达载体,筛选抑制X基因表达的有效siRNA,并初步探讨其对HBV复制功能的影响。方法:以X基因为目的基因,以产生siRNA质粒载体pSilencerTM-U6为表达模板,细胞内转录合成3条siRNA,并构建携带荧光素酶报告基因的重组质粒载体plucF-HBx。将重组质粒载体plucF-HBx与产生siRNA的质粒pSilencerTM-U6共转染HepG2细胞,检测荧光素酶活性以筛选出抑制荧光素酶表达的有效siRNA,逆转录聚合酶链反应检测HBx mRNA的表达,进一步证实siRNA对HBx表达的抑制效果,然后将siRNA转染HepG2.2.15,酶联免疫吸附法和荧光定量PCR检测siRNA对HBV复制的影响。结果:合成的3条siRNA中有1条抑制荧光素酶表达,抑制效率为76.3%,并能特异性抑制HBV的复制。结论:成功构建并筛选到针对X基因表达的有效siRNA质粒,为HBV的基因治疗奠定基础。Objective：To design and construct the expression vector that can express the short interfering RNA（siRNA） against hepatitis B virus X gene,screen the effective target sequence of siRNA and explore the function of siRNA on HBV replication.Methods： The three siRNA against HBx gene were transcript synthesized intracelluarly by expressed templates of plasmid vector pSilencer^TM2-U6,and the HBx gene was inserted into the reporter gene in order to construct the recombinant plasmid vector plucF-HBx.The recombinant plasmid and pSilencer^TM-U6,which could produce siRNA,were co-transfected into HepG2 cells and screened out the effective siRNA that inhibits the expression of luciferase,the expression of HBx mRNA was detected by reverse transcription polymerase chain reaction（RT-PCR） to further identify the inhibiting function of siRNA on HBx expression,then siRNA were transfected into HepG2.2.15 to analyze the effect of siRNA on HBV replication.Results： One siRNA of the three synthesized siRNA displayed inhibitory effects on the luciferase expression with the inhibitory rate being 76.3%,and could specially inhibit the replication of HBV.Conclusion： Effective siRNA plasmids against HBx gene were constructed and screened out successfully to creat a favourable condition for gene therapy of HBV.

关 键 词：RNA干扰 乙型肝炎病毒 SIRNA 表达载体 基因治疗 

分 类 号：R373.21[医药卫生—病原生物学]