反向重复序列法构建神经病靶标酯酶RNA干涉表达质粒  

Construction of RNA interference expression plasmid of human neuropathy target esterase by cloning reverse repeats into plasmid DNA

作　　者：常平安[1] 伍一军[2] 

机构地区：[1]重庆邮电学院生物信息学院,重庆400065 [2]中国科学院动物研究所

出　　处：《毒理学杂志》2007年第1期4-8,共5页Journal of Toxicology

基　　金：国家自然科学基金资助项目(30600329);重庆市自然科学基金资助项目(CSTC2005BB5072)

摘　　要：目的构建基于通用真核表达载体的人神经病靶标酯酶双链RNA的稳定表达载体。方法在正义和反义引物两端分别加上EcoRⅠ和BamHⅠ的识别位点扩增得到神经病靶标酯酶活力域序列,构建含有目标基因的倒置重复序列的双链RNA表达载体pcDNA.NTE.dsRNA,酶切分析鉴定重组载体。脂质体法转染到Hela细胞中瞬时表达重组载体,酶活力测定其对细胞内神经病靶标酯酶活力的影响。结果成功构建了NTE双链RNA稳定表达载体pcDNA.NTE.dsRNA,表达该载体细胞内NTE活力明显下降至对照细胞的20%左右。结论采用反向重复序列法将靶标基因的编码序列正反向插入到通用真核细胞表达载体中,是构建哺乳细胞基因双链RNA稳定表达载体的有效方法。Objective To construct stable RNA interference expression vector of human neurepathy target esterase（NTE） on the universal eukaryotic expression vecter.Methods Two specific restriction endonuclearase sites, EcoR Ⅰ and BamH Ⅰ , were added to the sense and antisense primer to amplify the sequence of the NTE active domain, and then to generate double strand RNA expressing plasmids, pcDNA. NTE. dsRNA, containing inverted DNA repeats of the target gene, which was identified with digestiong of restriction endonuclearase. The construct was tansfected into Hela cell by liposome methods and the effect was detected by NTE activity assaying. Results The stable expressing vector of NTE double strand RNA, pcDNA. NTE. dsRNA, was successfully constructed and its transient expressing reduced the NTE activity to less 20% of the control cells. Conclusion Introducing plasmid DNA encoding inverted repeats of a target sequence on the universal eukaryotic expression vecter is an effective methond to construct double strand RNA expressing vector in mammalin cells.

关 键 词：神经病靶标酯酶 RNA干涉 表达载体 反向重复序列 

分 类 号：R994.6[医药卫生—毒理学]