| 钟警，陈亚军，杨靖，曹仁贤，文格波.PRMT2真核表达载体构建及在乳腺癌MCF7细胞的亚细胞定位.[J].中南医学科学杂志.,2012,40(6):550-554. |
| PRMT2真核表达载体构建及在乳腺癌MCF7细胞的亚细胞定位 |
| Construction of PRMT2 Gene Eukaryotic Expression Vector and Subcellular Localization in Breast Cancer MCF7 Cells |
| 投稿时间：2012-05-02 |
| DOI： |
| 中文关键词: 乳腺癌 蛋白质精氨酸N-甲基转移酶 真核表达载体 亚细胞定位 |
| 英文关键词:breast cancer protein-arginine N-methyltransferase eukaryotic expression vector subcellular localization |
| 基金项目:国家自然科学基金资助项目（30670993）和湖南省高等学校科学研究项目（07C627）. |
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| 中文摘要: |
| 目的构建携带人蛋白质精氨酸甲基转移酶2(PRMT2)基因的真核表达载体pEGFP-N1-PRMT2，观察其转染乳腺癌MCF7细胞后融合蛋白在细胞中的亚细胞定位，为进一步研究PRMT2基因在乳腺癌中的作用奠定实验基础。方法以本实验室保存的pGEM-T-PRMT2载体为模板进行聚合酶链反应（PCR）特异扩增PRMT2基因片段，将扩增片段经Hind Ⅲ和SalⅠ双酶切后克隆到pEGFP-N1载体，酶切与测序鉴定阳性克隆；激光共聚焦显微镜观察融合蛋白在乳腺癌MCF7细胞中的定位。结果成功构建了pEGFP-N1-PRMT2重组真核表达载体，融合蛋白主要聚集成颗粒状分布于除核仁以外的核浆中，胞质中少量弥散分布。结论pEGFP-N1-PRMT2重组真核表达载体的构建为进一步研究PRMT2基因在乳腺癌及其内分泌治疗中的作用奠定实验基础。 |
| 英文摘要: |
| ObjectiveTo construct a eukaryotic expression vector pEGFP-N1-PRMT2 that can express the human protein arginine methyltransferase 2 (PRMT2) and observe the subcellular localization of GFP-fusion protein in the transfected breast cancer MCF7 cells under laser confocal scanning microscope in order to study the role of PRMT2 in breast cancer.MethodsThe PRMT2 gene was amplified from a vector pGEM-T-PRMT2，digested by Hind Ⅲ and Sal Ⅰ and then cloned into eukaryotic expression plasmid pEGFP-N1 which was also digested by Hind Ⅲ and Sal Ⅰ.The recombinant plasmid of pEGFP-N1-PRMT2 was identified by enzyme digestion and sequence analysis.The subcellular localization of GFP protein arginine methyltransferase 2 fusion protein in breast cancer MCF7 cells were observed under laser scanning microscope.ResultsThe construction of recombinant eukaryotic expression vector pEGFP-N1-PRMT2 was successfully constructed.The result of laser confocal miccroscope scanning showed that the GFP-PRMT2 fusion protein expression were predominantly localized to the nuclear compartment excluding the nucleoli，and a weak fluorescence was detected in the cytosol.ConclusionThe recombinant eukaryotic expression vector pEGFP-N1-PRMT2 will lay the foundation for further studying the role of PRMT2 in breast cancer and endocrine treatment. |
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