中文摘要：

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。

英文摘要：

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.