骨肉瘤细胞中Sestrin2的表达和定位

目的:构建新型人源真核表达载体3*Flag-hSesn2并同时检测其在骨肉瘤细胞中的表达及定位.方法:以GST-hSesn2作为模板,利用聚合酶链反应扩增目的基因hSesn2的cDNA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-hSesn2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:hSesn2基因cDNA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-hSesn2融合蛋白表达,分子量约为55kDa.3*Flag-hSesn2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hSesn2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-hSesn2融合蛋白的表达,最终成功纯化hSesn2蛋白.其中3*Flag-hSesn2蛋白主要定位在细胞质和核周.

Expression and localization of Sestrin2 in osteosarcoma cells

Objective:To construct the expression plasmid of human Sestrin2 (hSesn2)gene and identify the ex-pression and localization of hSesn2 in osteosarcoma MG - 63 cells. Methods:Human hSesn2 coding sequence was ob-tained by polymerase chain reaction (PCR)amplification and cloned into 3*Flag tag expression plasmid. After the target cDNA 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 - hSesn2 in MG - 63 cells was observed with laser scanning confocal microscopy. The hSesn2 protein was purified by immunoprecipitation assay. Results:Human Sesn2 plasmid containing 3*Flag tag was successfully constructed. The fragment length was identified as 1443bp by restriction enzyme digestion. The protein level of 3*Flag - hSesn2 fusion protein was detected by Western blot as a molecular weight of 55kDa and pulled down by Flag antibody. The localization of recombinant protein is in the cytoplasm and perinucleus of MG - 63 cells. Conclusion:The hSesn2 recombinant plasmid was successfully constructed. The expression of 3*Flag - hSesn2 fu-sion protein was identified and pulled down by Flag antibody,and it was localized in cytoplasm and perinucleus of MG- 63 cells.