| hFAK基因重组质粒的构建及蛋白表达 |
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| 引用本文： | 李晓东,郭冰玉,朱戈,李丰.hFAK基因重组质粒的构建及蛋白表达[J].中国医科大学学报,2011,40(7):584-586,591. |
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| 作者姓名： | 李晓东 郭冰玉 朱戈 李丰 |
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| 作者单位： | 中国医科大学基础医学院细胞生物学教研室,教育部医学细胞生物学重点实验室,沈阳110001 |
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| 基金项目： | 国家自然科学基金资助项目(31000627,30771128,90813038) |
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| 摘 要： | 目的构建hFAK原核、真核表达载体，证实融合蛋白在原核细胞的诱导表达以及在胃癌细胞内的表达、定位。方法
提取人胃癌细胞SGC-7901 的总mRNA 并进行反转。以反转录的cDNA 为模板PCR 扩增hFAK全长编码基因，分别克隆至
pCDNA3.1-Flag 以及pGEX-4T-2 表达载体中。原核重组质粒鉴定后转入BL21 细胞中并经过诱导表达及纯化，真核表达质粒转
入胃癌SGC-7901 细胞中，分别利用Western blot和激光共焦扫描显微技术检测重组质粒的表达以及在胃癌细胞中的定位。结
果hFAK全长基因序列克隆到原核、真核表达载体中，酶切鉴定片段为3 200 bp。原核诱导出GST-hFAK 并进行纯化；
Western blot 检测到真核转染的Flag-hFAK表达，条带为130 kD，免疫荧光显示蛋白定位于细胞质，并在细胞膜上有定位。结论
成功构建了hFAK原核、真核表达载体，并验证了其表达
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| 关 键 词： | hFAK 蛋白质印迹 融合蛋白 免疫荧光 胃癌 |
Construction of hFAK Gene-recombinant Plasmids and its Protein Expression |
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| LI Xiao-dong
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GUO Bing-yu
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ZHU Ge
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LI Feng.Construction of hFAK Gene-recombinant Plasmids and its Protein Expression[J].Journal of China Medical University,2011,40(7):584-586,591. |
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| Authors: | LI Xiao-dong GUO Bing-yu ZHU Ge LI Feng |
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| Affiliation: | LI Xiao-dong,GUO Bing-yu,ZHU Ge,LI Feng (Department of Cell Biology,Key Laboratory of Medical Cell Biology of Ministry of Education,College of Basic Medical Sciences,China Medical University,Shenyang 110001,China) |
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| Abstract: | Objective To construct the recombinant plasmids of human hFAK gene and identify its protein expression.Methods Total RNA was extracted from human gastric cancer SGC-7901 cells.The hFAK coding sequence was amplified by polymerase chain reaction(PCR)method and cloned into pCDNA3.1/Flag and pGEX-4T-2 vectors respectively.IPTG-induced GST-hFAK protein expression in BL21 cells and protein purification were identified by SDS-PAGE.The expression and localization of the pCDNA3.1/Flag-hFAK recombinant plasmid in SGC... |
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| Keywords: | hFAK Western blot fusion protein immunofluorescence gastric cancer |
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