人AMPKα2基因pEGFP-N1-AMPKα2表达载体的构建和鉴定

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(AMPKα2) 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 lipofectanine into the SH-SY5Y 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-SY5Y ceils.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-SY5Y cells transfacted with pEGFP-N1-AMPKα2, which laid a basis for its application in the research of cell injury induced by local anesthetic.