| 3*Flag-hPAK4重组质粒的构建及其在COS7细胞中的表达与定位 |
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| 引用本文： | 张红艳,王春玉,李彦姝,王迪,李丰.3*Flag-hPAK4重组质粒的构建及其在COS7细胞中的表达与定位[J].东南大学学报(医学版),2011,30(6):869-873. |
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| 作者姓名： | 张红艳 王春玉 李彦姝 王迪 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,细胞生物学卫生部重点实验室,辽宁,沈阳,110001 |
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| 基金项目： | 国家自然科学基金资助项目(30900752); 国家自然基金重大研究计划(90813038); 博士点基金(20102104110016); 辽宁省教育厅创新团队项目(2008T195) |
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| 摘 要： | 目的:构建3*Flag-hPAK4真核表达质粒,证实融合蛋白在细胞内的表达与定位,并纯化PAK4蛋白。方法:提取人乳腺癌细胞MCF-7 mRNA,反转录为cDNA。PCR扩增hPAK4全长编码基因,亚克隆至含有3*Flag标签的真核表达载体中。将构建的重组质粒测序并转染到非洲绿猴肾成纤维细胞COS7中,提取细胞蛋白进行Western blotting检测,同时利用共聚焦激光扫描显微镜观察3*Flag-hPAK4在COS7细胞内定位。使用免疫沉淀的方法纯化PAK4蛋白。结果:hPAK4全长基因序列克隆到了真核表达载体3*Flag中,酶切鉴定片段为1 800 bp。Western blotting检测到了融合蛋白3*Flag-hPAK4的表达,分子质量约为68 kDa,3*Flag-hPAK4在COS7细胞中表达定位在细胞浆中,成功纯化了PAK4蛋白。结论:成功地构建了3*Flag-hPAK4真核表达质粒,同时鉴定了3*Flag-hPAK4融合蛋白的表达,并纯化了PAK4蛋白。3*Flag-hPAK4蛋白主要定位在细胞浆中。
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| 关 键 词： | 人P21活化激酶4 蛋白质印记 免疫沉淀 |
Construction of 3*Flag-PAK4 recombinant plasmid and its protein expression and localization in COS7 cell line |
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| ZHANG Hong-yan,WANG Chun-yu,LI Yan-shu,WANG Di,LI Feng.Construction of 3*Flag-PAK4 recombinant plasmid and its protein expression and localization in COS7 cell line[J].Journal of Southeast Univ: Medical Sci Ed,2011,30(6):869-873. |
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| Authors: | ZHANG Hong-yan WANG Chun-yu LI Yan-shu WANG Di LI Feng |
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| Institution: | ZHANG Hong-yan,WANG Chun-yu,LI Yan-shu,WANG Di,LI Feng(Department of Cell Biology,The Key Laboratory of Cell Biology of Ministry of Health,College of Basic Medical Sciences,China Medical University,Shenyang 110001,China) |
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| Abstract: | Objective: To construct the expression plasmid of 3*Flag-hPAK4 and identify its recombinant protein expression and localization,and purify the Pak4 protein.Methods: Total RNA was extracted from human breast cancer MCF-7 cells.The hPAK4 coding sequence was amplified by polymerase chain reaction(PCR) method and subcloned into 3*Flag vector.After the target region was sequenced,the plasmid was transfected into COS7 cell line.The expression of the recombinant plasmid in COS7 cells was proved by Western blotting... |
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| Keywords: | hPAK4 Western blotting immunoprecipitate |
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