人PAK1重组蛋白质的表达及在人胃癌BGC-823细胞内定位  

Expression of Recombinant Protein of Human PAK1 and Its Location in Cytoplasm of BGC-823 Cells

作　　者：王桂玲[1] 周颖[1] 赵大林[2] 李家滨[1] 刘芙蓉[1] 李丰[1] 

机构地区：[1]中国医科大学细胞生物学卫生部重点实验室,沈阳110001 [2]沈阳体育学院生理学教研室,沈阳110001

出　　处：《细胞生物学杂志》2007年第1期86-90,共5页Chinese Journal of Cell Biology

基　　金：国家自然科学基金(No.30370736;No.30570966);教育部博士点基金(No.20050159023)资助项目~~

摘　　要：为检测人PAK1在原核细胞中的表达情况及在真核细胞中的定位,利用RT-PCR技术获得PAK1目的基因,构建GST融合蛋白原核表达载体pGEX-5X-1/PAK1,IPTG诱导后进行SDS-PAGE及Western印迹检测,并用体外激酶实验测定其生物学活性;同时,构建带绿色荧光蛋白(GFP)标签的真核表达载体pEGFP/PAK1,利用脂质体法转入人胃癌BGC-823细胞系中,在共焦激光扫描显微镜下观察癌细胞中绿色荧光蛋白的表达。结果表明,pGEX-5X-1/PAK1载体能在大肠杆菌BL-21中正确表达GST-PAK1融合蛋白,大小约为94kDa,具有生物活性;PAK1绿色荧光蛋白定位于细胞浆。上述研究为进一步探索PAK1的生物学特性及信号转导通路打下了基础。To detect p21-activated kinasel （PAK1） expression in prokaryocyte and its location in transfected eukaryotic cells, we obtained PAK1 gene by RT-PCR and cloned the coding sequences of PAK1 into the procaryotic expression vector pGEX-5X-1.The expressed proteins were induced by IPTG and identified by SDSPAGE, Western blot and kinase assay in vitro. At the same time, GFP-tagged eukaryotic expression vector pEGFP/ PAK1 was constructed and then transfected into human gastric cancer cell line BGC-823 with liposome, and the expression of GFP was observed by confocal laser scanning microscopy, pGEX-5X-1/PAK1 could express GST-PAK1 fusion protein with 94kDa. Under microscopy, the expression of GFP-tagged PAK1 was observed in cytoplasm of BGC-823 cells transfected with pEGFP/PAK1. This study provides the basis for research on biological features of PAK1.

关 键 词：PAK1 基因克隆 蛋白表达 转染 绿色荧光蛋白 

分 类 号：R735.2[医药卫生—肿瘤]