hSesn1基因真核表达载体构建及蛋白的表达和定位  

Construction of Eukaryotic Plasmid of Human hSesn1 Gene and Expression and Localization of Fusion Protein

作　　者：沈涛[1] 巴根[1] 李妍[2] 杨蕾[1] 郭然[1] 陈之光[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,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,沈阳110001

出　　处：《医学研究杂志》2017年第6期41-44,共4页Journal of Medical Research

基　　金：国家自然科学基金资助项目(面上项目)(81372337);国家自然科学基金青年基金资助项目(31201053)

摘　　要：目的构建人源的Sesn1真核表达载体同时检测融合蛋白在COS-7细胞系内的表达和定位。方法提取工具细胞系HeLa的mRNA,进而反转录成为c DNA。聚合酶链反应方法扩增人源Sesn1基因的cDNA全长片段,将其克隆于pEGFPC1真核表达载体中。进一步将已构建好的重组表达载体进行双酶切以及测序鉴定,继而转染到工具细胞系COS-7中,进行Western blot方法检测。最后应用激光共聚焦扫描显微镜方法来观察pEGFP-hSesn1在COS-7细胞中的定位情况。结果hSesn1基因cDNA全长片段克隆至p EGFP-C1真核表达载体中,酶切鉴定片段大小为1479bp,测序证实成功。Western blot方法检测出GFP-hSesn1融合蛋白的成功表达,分子质量(Mr)约为80k Da。pEGFP-hSesn1在COS-7于细胞中主要定位在细胞质及核周。结论成功构建了pEGFP-hSesn1真核表达载体,进而检测到融合蛋白的表达,并且在COS-7细胞中主要定位于核周及细胞质。Objective To construct the expression plasmid of human Sestrin 1 （hSesnl） gene and identify expression and localiza- tion of fusion protein. Methods Total mRNA was extracted from Hela ceils, and cDNA was synthesized via reverse transcription. The hSesnl coding sequence was sucessfully amplified by polymerase chain reaction （PCR） and cloned into pEGFP- C1 expression plasmid. After the hSesnl gene was successfully identified by double enzymes digestion and sequencing, the plasmid was transiently transfected into COS -7 cells. The expression of the recombinant plasmid in COS - 7 cells was detected by Western blot assay. The localization of pEGFP - hSesnl in COS - 7 cells was observed with laser eonfocal microscopy. Results hSesnl eDNA was successfully constructed into the pEGFP - C1 expression vector. The fragment length identified by double restriction enzymes digestion was 1479bp. The expression of pEGFP - hSesnl fusion protein was detected by Western blot assay. Its molecular weight is 80kDa. The pEGFP - hSesnl fusion protein was mostly localized at cytoplasm and perinuclear of COS - 7 cells. Conclusion The recombinant pEGFP - hSesnl plasmid was success- fully constructed, and the pEGFP - hSesnl fusion protein was mostly localized at cytoplasm and perinuclear of COS - 7 cells.

关 键 词：hSesnl WESTERN blot绿色荧光蛋白 质粒构建 

分 类 号：Q291[生物学—细胞生物学]