| hSesn1基因真核表达载体构建及蛋白的表达和定位 |
| 投稿时间：2016-11-24 修订日期：2016-12-30 点此下载全文 |
| 引用本文：沈涛,巴根,李妍,杨蕾,郭然,陈之光,郭洲洋,付勤.hSesn1基因真核表达载体构建及蛋白的表达和定位[J].医学研究杂志,2017,46(6):41-44 |
| DOI：
10.11969/j.issn.1673-548X.2017.06.011 |
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| 基金项目: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） |
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| 中文摘要: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cDNA。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pEGFP-C1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pEGFP-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80kDa。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。 |
| 中文关键词:hSesn1 Western blot 绿色荧光蛋白 质粒构建 |
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| Construction of Eukaryotic Plasmid of Human hSesn1 Gene and Expression and Localization of Fusion Protein |
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| Abstract:Objective To construct the expression plasmid of human Sestrin 1 (hSesn1) gene and identify expression and localization of fusion protein. Methods Total mRNA was extracted from Hela cells, and cDNA was synthesized via reverse transcription. The hSesn1 coding sequence was sucessfully amplified by polymerase chain reaction (PCR) and cloned into pEGFP-C1 expression plasmid. After the hSesn1 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-hSesn1 in COS-7 cells was observed with laser confocal microscopy. Results hSesn1 cDNA was successfully constructed into the pEGFP-C1 expression vector. The fragment length identified by double restriction enzymes digestion was 1479bp. The expression of pEGFP-hSesn1 fusion protein was detected by Western blot assay. Its molecular weight is 80kDa. The pEGFP-hSesn1 fusion protein was mostly localized at cytoplasm and perinuclear of COS-7 cells. Conclusion The recombinant pEGFP-hSesn1 plasmid was successfully constructed, and the pEGFP-hSesn1 fusion protein was mostly localized at cytoplasm and perinuclear of COS-7 cells. |
| keywords:hSesn1 Western blot GFP Plasmid construction |
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