构建和鉴定PTEN基因的shRNA重组腺病毒表达载体  

Construction and identification of the recombinant adenovirus expressing the short hairpin RNA targeting phosphatase and tensin homolog deleted on chromosome ten gene

作　　者：韦永琼[1] 曾照芳[1] 陈力学[2] 

机构地区：[1]重庆医科大学医学检验系/临床检验诊断学教育部重点实验室 [2]重庆医科大学第一附属医院重庆市神经病学重点实验室,重庆400016

出　　处：《南方医科大学学报》2009年第12期2414-2417,2420,共5页Journal of Southern Medical University

基　　金：重庆市自然科学基金(2007BB5287)

摘　　要：目的构建PTEN特异性shRNA重组腺病毒表达载体,为应用基因沉默技术进行缺血性脑损伤的基因治疗奠定基础。方法将前期构建的PTENshRNA特异性真核表达载体pGenesil-1-shRNA的表达启动子U6连同shRNA亚克隆至穿梭质粒pAdTrack,酶切及DNA测序鉴定后,将含PTEN基因的重组穿梭质粒pAdTrack-U6-shRNA经PmeI线性化后转化入pAdEasy-1感受态细菌。pAd-U6-shRNA质粒经PacI线性化后转染293细胞,包装重组腺病毒Ad-U6-shRNA,并进行PCR鉴定、病毒滴度测定及Westernblotting检测受感染海马神经元细胞内PTEN蛋白的表达。结果证实pAdTrack-U6-shRNA及pAd-U6-shRNA质粒构建正确,收获病毒后PCR及DNA测序结果证明Ad-U6-shRNA包被成功,受腺病毒感染海马神经元细胞内PTEN蛋白表达明显降低。结论成功构建了PTEN基因的shRNA重组腺病毒载体Ad-U6-shRNA,为应用基因沉默技术研究PTEN对缺血性脑损伤后的神经保护作用奠定了基础。Objective To construct the recombinant adenovirus expression vector of a short hairpin RNA （shRNA） targeting phosphatase and tensin homolog deleted on chromosome ten （PTEN） gene for gene therapy of ischemic cerebral injury. Methods The U6 expression promoter and shRNA of pGenesil-1-shRNA, which was constructed and identified in our previous experiment, were subcloned to pAdTrack shuttle plasmid. The product pAdTrack-U6-shRNA was linearized by PmeI for homologous recombination with pAdEasy-1 in pAdEasy-1 competence bacteria. The positive clone was identified by enzyme digestion, PCR analysis and DNA sequence analysis. After linearization by PacI, the recombinant adenovirus DNA shuttle plasmid pAdEasy-U6-shRNA was transfected into 293 cells for packaging and amplification of Ad-U6-shRNA, which was further identified by PCR analysis and DNA sequence analysis. Western blotting was used to detect the expression of PTEN protein in the hippocampal neurons infected with the adenovirus. Results The pAdTrack-U6-shRNA and pAd-U6-shRNA plasmids had been successfully constructed as verified by PCR analysis, enzyme digestion and DNA sequence analysis. PCR analysis and DNA sequence analysis confirmed successful packaging of the recombinant adenovirus Ad-U6-shRNA in 293 cells. PTEN protein expression decreased significantly in the hippocampal neurons after infection by the recombinant virus. Conclusion We have successfully constructed the recombinant adenovirus Ad-U6-shRNA targeting PTEN gene, which provides a basis for investigating the role of PTEN in neuroprotection after cerebral ischemic injury using RNA interference.

关 键 词：PTEN基因 RNA干扰 腺病毒 表达载体 

分 类 号：R346[医药卫生—基础医学]