HBx-shRNA重组腺病毒的构建及功能鉴定  

Construction of recombinant adenovirus HBx-shRNA and identification of its inhibition effect

作　　者：刘正淑[1] 李凯[2] 邹程程[2] 王森[2] 盛艳蕊[2] 汤华[2] 

机构地区：[1]重庆医科大学附属第一医院健康体检部,重庆400016 [2]重庆医科大学教育部感染性疾病分子生物学重点实验室,重庆400016

出　　处：《重庆医科大学学报》2013年第12期1425-1428,共4页Journal of Chongqing Medical University

基　　金：重庆市渝中区软科学研究资助项目(编号:20110213);国家临床重点专科护理建设资助项目(编号:财社【2010】305号)

摘　　要：目的:构建携带绿色荧光蛋白(green fluorescence protein,GFP)标签的乙型肝炎病毒X蛋白(hepatitis B virus X protein,HBx)特异性shRNA重组腺病毒,并对其干扰效果进行鉴定。方法:将前期构建的真核表达载体pGenesil-1-HBx-shRNA和pGenesil-1-Scramble sequence的表达启动子U6连同shRNA亚克隆到穿梭质粒pAdTrack-CMV,构建pAdTrack-CMV-U6-HBxshRNA和对照pAdTrack-CMV-U6-Scramble sequence穿梭质粒;酶切及DNA测序鉴定后,经PmeⅠ线性化后转化入感受态AdEasier细菌,获得重组腺病毒质粒pAd-U6-HBx-shRNA和pAd-U6-Scramble sequence,PacⅠ酶切后转染AD293细胞获得重组腺病毒Ad-U6-HBx-shRNA和Ad-U6-Scramble sequence;扩增病毒,测定滴度;以RT-PCR和real-time PCR鉴定重组腺病毒Ad-U6-HBx-shRNA对HBx的干扰效果。结果:酶切鉴定得到阳性pAd-U6-HBx-shRNA和pAd-U6-Scramble sequence重组质粒;转染到AD293细胞并包装成功;RT-PCR以及real-time PCR表明,重组腺病毒Ad-U6-HBx-shRNA携带的干扰片段能够明显干扰HBx表达(P=0.01)。结论:成功构建了HBx特异性shRNA重组腺病毒Ad-U6-HBx-shRNA,它能抑制HepG2.2.15细胞中的HBx的表达,为研究HBx作为肝癌基因治疗作用的一个靶点以及机制奠定基础。Objective：To construct and identify the recombinant adenovirus vector of a short hairpin RNA（shRNA） targeting HBx. Methods：U6 expression promoter and shRNA of pGenesil-1-HBx-shRNA and pGenesil-1-Scramble sequence,which was construct- ed and identified in our previous experiment,were subcloned to pAdTrack-CMV shuttle plasmid, and further identified by enzyme di- gestion and DNA sequence analysis. Recombinant shuttle plasmids were restrictedly digested with Pme I ,and subsequently trans- formed into competent AdEasier E.coli for homologous recombinant to obtain the adenovirus plasmids pAd-U6-HBx-shRNA and pAd-U6-Scramble sequence（control）. Recombinants were digested with Pac I and finally transfected into AD293 for packaging. Re- combinant adenoviruses Ad-U6-HBx-shRNA and Ad-U6-Scramble sequence were amplified in AD293 and the titers were deter- mined. Expression of HBx gene was identified by RT-PCR and real-time PCR. Results：Recombinant adenovirus plasmid was cor- rectly constructed. Real-time PCR proved that the expression of HBx was reduced by Ad-U6-HBx-shRNA（P=0.01 ）. Conclusions： Recombinant adenovirus vector Ad-U6-HBx-shRNA is correctly constructed,and it could inhibit HBx expression in HepG2.2.15 cells. This recombinant adenovirus vector provides a useful tool for further study of HBx function.

关 键 词：重组腺病毒 乙型肝炎病毒X蛋白 SHRNA 肝细胞肝癌 

分 类 号：R373.2[医药卫生—病原生物学]