3*Flag-hPAK4重组质粒的构建及其在COS7细胞中的表达与定位

Construction of 3*Flag-PAK4 recombinant plasmid and its protein expression and localization in COS7 cell line

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。

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.The localization of 3＊Flag-hPAK4 in COS7 cells was observed by using laser scanning confocal microscopy.The hPak4 protein was purify by immunoprecipitate.Results： hPAK4 had been constructed into expressing vector 3＊Flag successfully.The length of the fragment was 1 800 bp,identified by restriction enzymes digestion.The expression of 3＊Flag-hPAK4 fusion protein was detected by Western blot,with a molecular weight 68 kDa,was pulled down by Flag antibody,its localization in the cytoplasm.Conclusion：The recombinant plasmid is successfully cloned into eukaryotic expressing vector,the expression of 3＊Flag-hPAK4 fusion protein is identified and pulled down by Flag antibody,it was expressed in cytoplasm.