【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HindⅢ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。更多还原

【Abstract】 Objective To 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.Methods The 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.Results The 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.Conclusion The recombinant eukaryotic expression vector pEGFP-N1-PRMT2 will lay the foundation for further studying the role of PRMT2 in breast cancer and endocrine treatment.更多还原