3~* Flag-hSesn1重组质粒的构建及其在骨肉瘤细胞中的表达  

Construction and expression of 3~* Flag-hSesn1 plasmid in osteosarcoma cells

作　　者：沈涛[1] 郭然[1] 李妍[2] 巴根[1] 杨蕾[1] 郭洲洋 陈之光[1] 付勤[1] 

机构地区：[1]中国医科大学附属盛京医院骨科,辽宁沈阳110004 [2]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

出　　处：《现代肿瘤医学》2017年第9期1353-1356,共4页Journal of Modern Oncology

基　　金：国家自然科学基金资助项目(编号:31201053;81372337)

摘　　要：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GSThSesn1为模板,利用聚合酶链反应扩增hSesn1基因c DNA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-hS esn1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:hS esn1基因的cD NA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-hS esn1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-hS esn1蛋白主要定位在细胞质,部分定位于细胞核周。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.

关 键 词：hSesn1 WESTERN BLOT 免疫沉淀 

分 类 号：R738.1[医药卫生—肿瘤]