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

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Western blot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Western blot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。

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

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.