引用本文:刘正淑1,李 凯2,邹程程2,王 森2,盛艳蕊2,汤 华2.HBx-shRNA重组腺病毒的构建及功能鉴定[J].重庆医科大学学报,2013,(12):1425~1428
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
DOI:
中文关键词:  重组腺病毒  乙型肝炎病毒X蛋白  shRNA  肝细胞肝癌
英文关键词:recombinant adenovirus  HBx  shRNA  hepatocellular carcinoma
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刘正淑1,李 凯2,邹程程2,王 森2,盛艳蕊2,汤 华2 1. 重庆医科大学附属第一医院健康体检部重庆 4000162. 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 
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中文摘要:
      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。
英文摘要:
      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.
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