pEGFP-MEK1/Q56P重组质粒的构建及融合基因在293T细胞中的表达  

Construction of Recombinant pEGFP- MEK1/Q56P and Expression of Fusion Gene in 293T Cell

作　　者：沈晗[1] 彭芳芳[1] 张百芳[1] 吴少波[1] 李宪奎[1] 杞少华[1] 武栋成[1] 

机构地区：[1]武汉大学医学院,湖北武汉430071

出　　处：《武汉大学学报（理学版）》2004年第6期751-755,共5页Journal of Wuhan University:Natural Science Edition

基　　金：国家自然科学基金资助项目(30170335)

摘　　要：构建第56位氨基酸发生突变的MEK1基因(MEK1/Q56P)与增强绿色荧光蛋白(EGFP)报道基因融合表达的真核重组质粒pEGFP MEK1/Q56P,经限制性酶切及测序鉴定后,将其导入293T细胞中,用荧光显微镜观察绿色荧光蛋白的表达,同时进行Westernblot检测。酶切鉴定及测序结果表明构建的pEGFP MEK1/Q56P与预期结果一致,荧光观察及Westernblot结果表明MEK1/Q56P和EGFP在293T细胞中能以融合蛋白的形式表达,且MEK1/Q56P能特异性活化ERK1/2.本研究成功构建了含有MEK1/Q56P的绿色荧光蛋白真核表达质粒,便于对Raf/MEK1/ERK1/2信号传导通路做进一步研究.A new plasmid pEGFP-MEK1/Q56P was constructed for mammalian cells transfection, which contains a constitutively active MEK1 mutant gene MEK1/Q56P and a green fluorescent protein gene EGFP. The MEK1/Q56P DNA fragment was obtained from pBabe-MEK1/Q56P with PCR technique. pEGFP-MEK1/Q56P was constructed by inserting MEK1/Q56P into pEGFP-C1 plasmid vector with routine molecular techniques. After structure identification by restriction analysis, pEGFP\|MEK1/Q56P plasmid was further transferred into 293T cells using calcium-phosphate mediated gene transfer. The expression of pEGFP-MEK1/Q56P was detected using fluoroscopy and western blot analysis. Restriction analysis showed that the structure of pEGFP-MEK1/Q56P plasmid was the same as anticipated. The transfected 293T cells emitted strong green fluorescence and the fusion gene EGFP-MEK1/Q56 could be expressed successfully. The levels of MEK1 and pp-ERK1/2 were strongly up-regulated in MEK1/Q56P-transfected 293T cells. It can be concluded that a new plasmid pEGFP-MEK1/Q56P was constructed as anticipated, in which EGFP gene can serve as a reporter gene reflecting the expression of MEK1/Q56P gene.

关 键 词：绿色荧光蛋白 质粒 表达 

分 类 号：Q782[生物学—分子生物学]