3*Flag-hSesn1重组质粒的构建及其在骨肉瘤细胞中的表达

摘要

Objective:To construct the expression plasmid of human sorbin and SH3 domain containing 1(hSesn1)gene and identify the expression and localization of hSesn1 in MG-63 osteosarcoma cells.Methods:GST-hSesn1 was used as a template,human SESN1 coding sequence was amplified by polymerase chain reaction(PCR)amplification and cloned into 3*Flag empty vector.After the cloning target was identified by double enzyme digestion and sequenced,the plasmid was transiently transfected into MG-63 osteosarcoma cells.The expression of the recombinant protein in MG-63 cells was detected by Western blot assay.The localization of 3*Flag-hSesn1 in MG-63 cells was observed by laser confocal scanning microscopy.The hSesn1 protein was purified by immunoprecipitation assay.Results:hSesn1 gene was successfully constructed into the 3*Flag expressing plasmid.The expression of 3*Flag-hSesn1 fusion protein was detected by Western blot and pulled down by anti-flag antibody.The localization of fusion protein is at the cytoplasm and near nuclear membrane of MG-63 cells.Conclusion:The recombinant hSesn1 plasmid was successfully cloned into eukaryotic expressing vector,and the expression of 3*Flag-hSesn1 fusion protein was pulled down by anti-flag antibody and majorly expressed at the cytoplasm and partly nuclear.%目的:构建3*Flag-hSesn1真核表达载体并检测在骨肉瘤细胞内的表达及定位.方法:以GST-hSesn1为模板,利用聚合酶链反应扩增hSesn1基因cDNA全长,并将其克隆至3*Flag标签的真核表达载体中.将构建的重组载体进行双酶切和测序鉴定,并转染到MG-63骨肉瘤细胞中,提取总蛋白进行Western blot检测.进一步利用共聚焦激光显微镜观察3*Flag-hSesn1在MG-63细胞中的定位,使用免疫沉淀的方法纯化人源Sesn1蛋白.结果:hSesn1基因的cDNA全长成功构建到3*Flag标签的真核表达载体中,Western blot检测到了含有Flag标签的人源Sesn1融合蛋白表达,且在MG-63骨肉瘤细胞中主要定位于细胞质和核周,并成功纯化人源Sesn1蛋白.结论:成功构建了3*Flag-hSesn1真核表达质粒,同时鉴定了其融合蛋白的表达,并纯化人源Sesn1蛋白.3*Flag-hSesn1蛋白主要定位在细胞质,部分定位于细胞核周.