串联重复核定位信号的绿色荧光蛋白融合载体的构建与原核表达  

Construction and Prokaryotic Expression of Green Fluorescent Protein Fusion Vector of Tandem Nuclear Localization Signals

作　　者：邓鹏[1] 陈腾祥[1] 龚小卫[1] 

机构地区：[1]南方医科大学病理生理学教研室广东省蛋白质组学重点实验室,广东广州510515

出　　处：《贵阳医学院学报》2008年第4期340-343,共4页Journal of Guiyang Medical College

基　　金：国家自然科学基金项目(No.30700291)

摘　　要：目的:构建绿色荧光蛋白-串联重复核定位信号融合蛋白的表达载体(His-EGFP-3xNLS),并在原核细胞中进行表达与纯化。方法:采用PCR方法从原先克隆在pEGFP-C1载体中的3xNLS与EGFP编码序列扩增出来,使用常规酶切和连接方法将其重组至pET-14b载体中,对阳性克隆进行酶切、PCR和测序鉴定。转化BL21(DE3)大肠杆菌株,用IPTG诱导融合蛋白表达,并利用Ni-NTA亲和层析纯化该融合蛋白。结果:构建的His-EGFP-3xNLS融合蛋白表达载体是正确的,该载体可在大肠杆菌内高效表达,用Ni-NTA纯化获得了相对分子质量约36 kD的融合蛋白。结论:成功构建了His-EGFP-3xNLS融合蛋白表达载体,并在原核细胞中获得了高效表达和纯化,为进一步研究NLS介导信号分子入核提供了实验材料。Objective： To construct the expression vector of His-EGFP-3xNLS fusion protein and obtain its expression and purification in E. coli. Methods： 3xNLS and EGFP sequence was amplified by PCR from pEGFP-C1-3xNLS vector and cloned into pET-14b vector following the routine procedures. After identification by enzyme digestion, PCR and sequencing, the positive clones were transformed into BL21 （DE3） competent ceils, and the expression of His-EGFP-3xNLS fusion protein was induced with IPTG and further purified by Ni-NTA affinity chromatography. Results： The constructed His-EG- FP-3xNLS fusion protein vector highly expressed in E. coli. With Ni-NTA affinity chromatography, a purified His fusion protein with relative molecular mass of approximately 36 kD was obtained. Conclu- sion： The expression vector of His-EGFP-3xNLS fusion protein is constructed, expressed and purified under non-denaturing conditions, which may significantly facilitate future study of the mechanism by which NLS mediate the nuclear translocation of certain signal molecules.

关 键 词：核定位信号 载体蛋白质类 基因表达 蛋白纯化 

分 类 号：Q782[生物学—分子生物学]