人神经病靶标酯酶RNAi表达载体的构建及其对哺乳动物细胞中NTE表达的抑制

Construction of RNA interference expression vectors of human neuropathy target esterase and its inhibition for expression of NTE in mammalian cells

目的构建人神经病靶标酯酶(NTE)双链RNA的稳定表达载体。方法用Spe I和Xho I将pSUPER质粒中的H1 RNA聚合酶Ⅲ启动子及多克隆位点部分切下替换pcDNA3．1(+)载体中的巨嗜细胞病毒(CMV)启动子及其多克隆位点,构建了适合在哺乳动物细胞中表达具有干涉作用的小 RNA的载体pSUPER／neo,进而将针对NTE表达的双链DNA插入到BglⅡ和HindⅢ酶切的载体pSUPER ／neo中构建NTE的双链RNA表达载体pSUPER／neo-NTE,后者被转染到COS7和SH-SY5Y细胞中, 采用蛋白质杂交和酶活力测定的方法,检测其表达效率并验证载体构建的正确性。结果构建了适合在真核细胞用抗生素筛选的NTE双链RNA表达载体pSUPER／neo-NTE。蛋白质杂交检测显示,转染细胞能有效抑制外源性NTE的表达;酶活力测定则显示其能有效地抑制内源性NTE的表达。结论采用启动子替换的策略,成功构建了NTE双链RNA的表达载体并在哺乳动物细胞内有效抑制NTE 的表达。

Objective To construct the RNA interference expression vector for explession of human neuropathy target esterase （NTE） gene in mammalian ceils. Methods Spe I and Xho I-digested insert from pSU-PER, which comprised HI RNA polymerase Ⅲ promoter and the multiple cloning sites, were cloned into the compatible in the pcDNA3.1 （＋） to generate pSUPER/neo that could express small interfering RNA in mammalian ceils. The annealed oligos targeting the expression of NTE were ligated into pSUPER/neo vector digested with Bgl Ⅱ and Hind Ⅲ to generate pSUPER/neo-NTE, which was transfected into COS7 and SH-SY5Y ceils. The inhibitory effect of the expression of NTE was detected by western blot analysis and the enzyme activity assay. Results pSU- PER/neo-NTE could stably express double-stranded RNA of NTE. The expression of pSUPER/neo-NTE in COS7 and SH-SYSY ceils could efficiently inhibit the activity of NTE in the mammalian cells. Conclusion Stable eukaryotic expression vector of double-stranded RNA of NTE, pSUPER/neo-NTE, has been constructed successfully with promoter substitution strategy.