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

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cDNA.进一步通过PCR来扩增人Sesn3基因的cDNA全长片段,并将其亚克隆于pEGFP-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pEGFP-hSesn3的定位,最后利用免疫沉淀方法纯化人源hSesn3蛋白.结果:hSesn3基因cDNA全长片段克隆于真核表达载体pEGFP-C1中,酶切鉴定片段为1 410 bp,并成功测序.Western blot检测到了GFP-hSesn3融合蛋白表达,分子量约为79 kDa.在MG-63细胞中pEGFP-hSesn3主要定位于细胞质,进一步成功纯化了hSesn3蛋白.结论:成功构建了hSesn3基因cDNA全长的真核表达载体,在MG-63细胞中pEGFP-hSesn3蛋白主要定位于细胞质,最终成功纯化了hSesn3蛋白.%Objective:To construct the expression plasmid of human Sestrin 3 (hSesn3) gene and identify expression 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-C1 empty vector.After the PCR fragment was identified by double restriction 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 purified by immunoprecipitation assay.Results:hSesn3 was successfully constructed into the pEGFP-C1 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 fusion 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.