hSesn3基因真核表达载体的构建及在骨肉瘤细胞中的蛋白表达和定位  

Construction of eukaryotic expression plasmid of human Sesn3 gene and expression and localization of fusion protein in osteosarcoma cells

作　　者：沈涛[1] 郭洲洋 李妍[2] 巴根[1] 陈之光[1] 郭然[1] 杨蕾[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17期2699-2702,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:通过构建人Sesn3真核表达载体同时证实融合蛋白在细胞中的表达及定位。方法:提取Hela工具细胞的mRNA,反转录至c DNA。进一步通过PCR来扩增人Sesn3基因的c DNA全长片段,并将其亚克隆于p EGFP-C1真核表达载体中,进而将构建的重组质粒进行酶切及测序鉴定,转染到MG-63骨肉瘤细胞中,获取细胞总蛋白,通过Western blot方法检测表达。进一步利用激光共聚焦扫描显微镜方法观察MG-63细胞内p EGFP-h Sesn3的定位,最后利用免疫沉淀方法纯化人源h Sesn3蛋白。结果:h Sesn3基因c DNA全长片段克隆于真核表达载体p EGFP-C1中,酶切鉴定片段为1 410 bp,并成功测序。Western blot检测到了GFP-h Sesn3融合蛋白表达,分子量约为79 k Da。在MG-63细胞中p EGFP-h Sesn3主要定位于细胞质,进一步成功纯化了h Sesn3蛋白。结论:成功构建了h Sesn3基因c DNA全长的真核表达载体,在MG-63细胞中p EGFP-h Sesn3蛋白主要定位于细胞质,最终成功纯化了h Sesn3蛋白。Objective To construct the expression plasmid of human Sestrin 3 （ hSesn3 ） gene and identify expres-sion and localization of the fusion protein in osteosarcoma MG -63 cells. Methods： Total mRNA was extracted from Hela cells,and the cDNA was reversely transcripted. The hSesn3 coding sequence was amplified by polymerase chain reaction （PCR） and sub - cloned into pEGFP - Cl empty vector. After the PCR fragment was identified by double re-striction enzymes digestion and sequenced,the plasmid was transiently transfected into MG -63 osteosarcoma cells.The expression of the recombinant plasmid in MG -63 cells was detected by Western blot assay. The localization of pEGFP - hSesn3 in MG -63 cells was observed with laser scanning confocal microscopy. The hSesn3 protein was pu-rified by immunoprecipitation assay. Results ： hSesn3 was successfully constructed into the pEGFP - Cl expression plasmid. The length of the fragment identified by double restriction enzymes digestion was 1 410 bp. The molecular weight of pEGFP - hSesn3 fusion protein was 79 kDa. It is detected by Western blot assay. The pEGFP - hSesn3 fu-sion protein was mostly localized in the cytoplasm of MG -63 cells. Conclusion ： The recombinant hSesn3 plasmid was successfully cloned into eukaryotic expression vector. The GFP - hSesn3 fusion protein was identified and pulled down by GFP antibody. The pEGFP - hSesn3 fusion protein was majorly localized in the cytoplasm of MG -63 cells.

关 键 词：hSesn3 WESTERN BLOT 绿色荧光蛋白 质粒构建 

分 类 号：R73-3[医药卫生—肿瘤] R738.7[医药卫生—临床医学]