人神经病变靶标酯酶RNA干扰表达载体的构建及其对哺乳动物细胞中NTE表达的抑制作用

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

作者信息

Chang Ping-an, Chen Rui, Li Wei, Wu Yi-jun

机构信息

Laboratory of Molecular Toxicology, State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, China.

出版信息

Zhonghua Lao Dong Wei Sheng Zhi Ye Bing Za Zhi. 2006 Jan;24(1):27-30.

PMID:16600090

Abstract

OBJECTIVE

To construct the RNA interference expression vector for expression of human neuropathy target esterase (NTE) gene in mammalian cells.

METHODS

Spe I and Xho I-digested insert from pSUPER, which comprised H1 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 cells. 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 cells. The inhibitory effect of the expression of NTE was detected by western blot analysis and the enzyme activity assay.

RESULTS

pSUPER/neo-NTE could stably express double-stranded RNA of NTE. The expression of pSUPER/neo-NTE in COS7 and SH-SY5Y cells 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.

摘要

目的

构建用于在哺乳动物细胞中表达人神经病靶酯酶（NTE）基因的RNA干扰表达载体。

方法

将经Spe I和Xho I酶切的pSUPER插入片段（包含H1 RNA聚合酶III启动子和多克隆位点）克隆到pcDNA3.1(+)的相应位点，构建能在哺乳动物细胞中表达小干扰RNA的pSUPER/neo。将退火后的靶向NTE表达的寡核苷酸连接到经Bgl II和Hind III酶切的pSUPER/neo载体上，构建pSUPER/neo-NTE，并将其转染到COS7和SH-SY5Y细胞中。通过蛋白质免疫印迹分析和酶活性测定检测NTE表达的抑制效果。