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

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 pro－tein，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-HBx-shRNA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。

Abstract:

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 PmeⅠ，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 PacⅠand 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.