p38调节活化蛋白激酶绿色荧光蛋白融合载体的构建及表达  

Construction and expression of green fluorescent protein fusion vector of p38 regulated/activated protein kinase

作　　者：彭毅[1] 龚小卫[1] 邓鹏[1] 秦清和[1] 姜勇[1] 

机构地区：[1]南方医科大学病理生理教研室,广东广州510515

出　　处：《第一军医大学学报》2005年第3期285-288,共4页Journal of First Military Medical University

基　　金：国家杰出青年科学基金(39925014);国家自然科学基金重点项目(30030060)~~

摘　　要：目的构建在哺乳动物细胞中表达的PRAK绿色荧光蛋白融合表达载体。方法将克隆在pET-14b上的PRAK亚克隆到绿色荧光蛋白载体pEGFP-C2上,随后转染Hela细胞,并在荧光显微镜下观察。结果重组质粒经酶切、PCR和测序鉴定正确无误,并在Hela细胞中得到高量表达。融合蛋白发出的绿色荧光表明EGFP-PRAK主要分布在细胞核中。结论成功构建了PRAK绿色荧光蛋白融合载体,该载体能在哺乳动物细胞中进行表达,为研究PRAK的细胞内定位和移位提供了一个重要的工具。Objective To construct the expression vector of p38 regulated/activated protein kinase (PRAK) fused to green fluorescent protein which is capable of expression in mammalian cells. Methods PRAK with His-tag in pET-14b expression vector was subcloned into the green fluorescent protein vector pEGFP-C2. The recombinant vector was then transfected into HeLa cells, followed by observation of the cells with fluorescent microscope. Results Identification by enzyme digestion, PCR and sequencing confirmed successful construction of the recombinant vector, which was highly expressed in HeLa cells. Green fluorescence of the fusion protein EGFP-PRAK was observed mainly in the cell nuclei. Conclusion The expression vector of PRAK fused to green fluorescent protein is successfully constructed and expressed in mammalian cells, which may facilitate the study of intracellular localization and translocation of PRAK.

关 键 词：p38调节活化蛋白激酶(PRAK) 绿色荧光蛋白(GFP) 载体构建 基因表达 

分 类 号：Q78[生物学—分子生物学]