LXRα-shRNA真核表达质粒的构建及抑制LXRα表达的有效序列筛选  

Construction of eukaryotic expression plasmid of LXRα and screening of effective sequences to inhibit LXRα expression

作　　者：张琴[1] 彭俊[2] 沈薇[1] 

机构地区：[1]重庆医科大学附属第二医院消化内科,重庆400010 [2]凉山州第一人民医院

出　　处：《中国老年学杂志》2012年第8期1632-1635,共4页Chinese Journal of Gerontology

基　　金：重庆市自然科学基金计划项目(CSTC;2008BB5404)

摘　　要：目的构建和筛选对肝X受体α(LXRα)mRNA和蛋白有抑制作用的LXRα质粒。方法从NCBI网站获得人的LXRαcDNA序列,根据RNA干扰设计原则,设计三条理论上最佳的siRNA序列,相应的双链DNA插入pGenesil-1.1载体中,经酶切和测序鉴定符合设计要求后,用转染试剂PolyJetTM将三条质粒转染至HepG2.2.15细胞中并观察转染效果。然后用RT-PCR和Western印迹方法分析各组LXRα的表达,筛选出干扰效率最佳的质粒。结果三个质粒经测序分析与设计的序列完全一致,酶切凝胶电泳证实质粒构建成功。三个质粒均能抑制LXRαmRNA和蛋白的表达,与shRNA-LXRα2和shRNA-LXRα3质粒比较shRNA-LXRα1质粒表达更低。结论成功构建了LXRαRNAi表达载体,且三条质粒均能高效抑制转染细胞中LXRα的表达,其中第一条质粒shRNA-LXRα1干扰效率最好,从而为脂肪肝的治疗提供了新的方法和材料。Objective To construct and screen the plasmid of LXRαgene that can inhibit LXRα mRNA and protein expression.Methods Human LXRα cDNA sequence was obtained from NCBI website.Three small interfering RNA sequences were selected through online design software according to RNAi design principles.The corresponding double-stranded DNA was connected with pGenesil-1.1 plasmid vector,namely shRNA-LXRα1,shRNA-LXRα2 and shRNA-LXRα3.Digestion and DNA sequencing confirmed the cDNA sequence and the orientation of the insert.Three plasmids were transfected into HepG2.2.15 cells with PolyJetTM-reagent respectively to assess the transfection efficiency under fluorescence microscope and to determine the expression of LXRα gene,with RT-PCR and Western blot.Results It was designed successfully of three plasmids demonstrated by digestion and DNA sequencing analysis.Through PolyJetTM reagent,shRNA-LXRα plasmid was transfected into HepG2.2.15 cells successfully.Target mRNA and protein was demonstrated by RT-PCR and Western blot,which displayed that three plasmids could effectively inhibit the expression of LXRα and the first shRNA-LXRα had the most effect.Conclusions Eukaryotic expression recombinant shRNA-LXRα could be constructed successfully.All of the plasmids could inhibit LXRα mRNA and protein expression,especially the first one.It will be helpful for the therapy of fatty liver disease.

关 键 词：肝X受体Α RNA干扰 质粒 构建 

分 类 号：R394.3[医药卫生—医学遗传学]