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

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.