| 人PAK1重组蛋白质的表达及在人胃癌BGC-823细胞内定位 |
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| 引用本文： | 王桂玲,周颖,赵大林,李家滨,刘芙蓉,李丰.人PAK1重组蛋白质的表达及在人胃癌BGC-823细胞内定位[J].细胞生物学杂志,2007,29(1):86-90. |
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| 作者姓名： | 王桂玲 周颖 赵大林 李家滨 刘芙蓉 李丰 |
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| 作者单位： | 中国医科大学细胞生物学卫生部重点实验室 沈阳110001(王桂玲，周颖，李家滨，刘芙蓉，李丰)，沈阳体育学院生理学教研室 沈阳110001(赵大林) |
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| 基金项目： | 国家自然科学基金;高等学校博士学科点专项科研项目 |
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| 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。
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| 关 键 词： | 基因克隆 蛋白表达 转染 绿色荧光蛋白 |
| 修稿时间： | 2006-03-30 |
Expression of Recombinant Protein of Human PAK1 and Its Location in Cytoplasm of BGC-823 Cells |
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| Gui-Ling Wang,Ying Zhou,Da-Lin Zhao,Jia-Bin Li,Fu-Rong Liu,Feng Li.Expression of Recombinant Protein of Human PAK1 and Its Location in Cytoplasm of BGC-823 Cells[J].Chinese Journal of Cell Biology,2007,29(1):86-90. |
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| Authors: | Gui-Ling Wang Ying Zhou Da-Lin Zhao Jia-Bin Li Fu-Rong Liu Feng Li |
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| Institution: | Key Laboratory o f Cell Biology, Ministry of Public Health, China Medical University, Shenyang 110001, China 1.Department of Physiology, Shenyang Institute of Athletics, Shenyang 110001, China |
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| Abstract: | To detect p21-activated kinase1(PAK1)expression in prokaryocyte and its location in trans-fected 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 SDS-PAGE,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 cyto-plasm of BGC-823 cells transfected with pEGFP/PAK1.This study provides the basis for research on biological features of PAK1. |
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| Keywords: | PAK1 |
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