人AMPKα2基因pEGFP-N1-AMPKα2表达载体的构建和鉴定  

Construction and identification of pEGFP-N1-AMPKα2 expression vector targeting at human AMPKα2

作　　者：卢俊[1] 徐世元[1] 崔睿[1] 张庆国[1] 雷洪伊[1] 

机构地区：[1]南方医科大学珠江医院麻醉科,广东广州510282

出　　处：《基础医学与临床》2011年第2期128-133,共6页Basic and Clinical Medicine

基　　金：国家自然科学基金(30972843)

摘　　要：目的构建表达人单磷酸腺苷激活的蛋白激酶催化亚单位α2(AMPKα2)基因的pEGFP-N1-AMPKα2载体,转染SH-SY5Y细胞系,观测其上调AMPKα2基因的效果。方法 PCR法扩增AMPKα2基因片段,克隆到pEGFP-N1质粒载体上。对重组质粒进行DNA序列测定和酶切分析。用质脂体将重组质粒pEGFP-N1-AMPKα2转染到SH-SY5Y细胞系,经G418筛选阳性克隆后,用RT-PCR和Western blot法检测AMPKα2的mRNA和蛋白表达。流式细胞仪检测转染重组质粒细胞内ROS变化。结果经DNA测序和酶切鉴定表明,pEGFP-N1-AMPKα2表达载体构建成功。重组质粒pEGFP-N1-AMPKα2转染SH-SY5Y细胞系后,细胞中AMPKα2蛋白表达量明显增高。SH-SY5Y细胞转染pEGFP-N1-AMPKα2后,ROS含量增多。结论成功构建了人AMPKα2基因的pEGFP-N1-AMPKα2表达载体。pEGFP-N1-AMPKα2能有效上调AMPKα2基因在SH-SY5Y细胞系中的表达,为将来应用其研究AMPK在局麻药致细胞损伤中的作用奠定了基础。Objective To construct pEGFP-N1-AMPKα2 expression vector and to observe its potential up-regulation effect on human AMP-activated protein kinase α2（AMPKa2） gene in the SH-SY5Y cell line. Methods Human AMPKα2 gene fragment was amplified and cloned into the pEGFP-N1-AMPKα2 vector. The recombinant vector was confirmed by DNA sequencing and enzyme digestion analysis. The recombinant vector was transfected by lipofectamine into the SH-SYSY cells. After the screening by G418, the expression levels of AMPKα2 mRNA and protein were detected by RT-PCR and Western blot. ROS was measured by flow cytometry. Results The expression vector pEGFP-N1-AMPKα2 was successfully constructed. The vector pEGFP-N1-AMPKα2 can up-regulate protein expression of AMPKα2 effectively after transfection in the SH-SYSY cells. ROS increased in cells transfected with pEGFP-N1-AMPKα2. Conclusion pEGFP-N1-AMPKα2 expression vector was successfully constructed. The protein expression of AMPKα2 gene was effectively up-regulated in SH-SYSY cells transfacted with pEGFP-N1- AMPKα2, which laid a basis for its application in the research of cell injury induced by local anesthetic.

关 键 词：局麻药 活性氧 单磷酸腺苷激活的蛋白激酶 

分 类 号：Q789[生物学—分子生物学]