骨肉瘤细胞中Sestrin2的表达和定位  

Expression and localization of Sestrin2 in osteosarcoma cells

作　　者：沈涛[1] 陈之光[1] 李妍[2] 巴根[1] 郭然[1] 杨蕾[1] 郭洲洋 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11000

出　　处：《现代肿瘤医学》2017年第7期1011-1014,共4页Journal of Modern Oncology

基　　金：国家自然科学基金资助项目(编号:30800415;81372337)

摘　　要：目的:构建新型人源真核表达载体3*Flag-h Sesn2并同时检测其在骨肉瘤细胞中的表达及定位。方法:以GST-h Sesn2作为模板,利用聚合酶链反应扩增目的基因h Sesn2的c DNA全长,并将其克隆到含有3*Flag标签的真核表达载体中。进一步将构建的重组质粒酶切并测序分析鉴定,转染至MG-63骨肉瘤细胞中,提取细胞总蛋白后进行Western blot检测重组蛋白表达。此外利用荧光共聚焦激光扫描显微镜检测3*Flag-h Sesn2在MG-63细胞系中的定位,并最终利用免疫沉淀方法纯化人源Sesn2蛋白。结果:h Sesn2基因c DNA全长成功构建于含有3*Flag标签的真核表达载体中,利用Western blot检测3*Flag-h Sesn2融合蛋白表达,分子量约为55k Da。3*Flag-h Sesn2在MG-63骨肉瘤细胞系中主要定位于细胞质及核周,进一步成功纯化了h Sesn2蛋白。结论:成功的构建了含有3*Flag标签的人源Sesn2真核表达质粒,同时鉴定3*Flag-h Sesn2融合蛋白的表达,最终成功纯化h Sesn2蛋白。其中3*Flag-h Sesn2蛋白主要定位在细胞质和核周。Objective：To construct the expression plasmid of human Sestrin2（ h Sesn2） gene and identify the expression and localization of h Sesn2 in osteosarcoma MG- 63 cells. Methods：Human h Sesn2 coding sequence was obtained by polymerase chain reaction（ PCR） amplification and cloned into 3 ＊ Flag tag expression plasmid. After the target c DNA was identified by double enzymes digestion and sequencing,the plasmid was transiently transfected into osteosarcoma MG- 63 cell line. The expression of the recombinant plasmid in MG- 63 cells was detected by Western blot. The localization of 3＊ Flag- h Sesn2 in MG- 63 cells was observed with laser scanning confocal microscopy. The h Sesn2 protein was purified by immunoprecipitation assay. Results：Human Sesn2 plasmid containing 3＊ Flag tag was successfully constructed. The fragment length was identified as 1443 bp by restriction enzyme digestion. The protein level of 3＊ Flag- h Sesn2 fusion protein was detected by Western blot as a molecular weight of 55 k Da and pulled down by Flag antibody. The localization of recombinant protein is in the cytoplasm and perinucleus of MG- 63 cells.Conclusion：The h Sesn2 recombinant plasmid was successfully constructed. The expression of 3 ＊ Flag- h Sesn2 fusion protein was identified and pulled down by Flag antibody,and it was localized in cytoplasm and perinucleus of MG- 63 cells.

关 键 词：hSesn2 WESTERN BLOT 免疫沉淀 

分 类 号：R738.1[医药卫生—肿瘤]