中文摘要：

本文构建了甲基化 CpG 结合结构域（MBD）蛋白的原核表达质粒,并表达了 GST-MBD融合蛋白,采用GST柱对该融合蛋白进行纯化。体外 pull-down 实验发现,GST-MBD融合蛋白能够与小鼠胚胎成纤维细胞MEF中 oct4基因的启动子 DNA 结合,验证了该蛋白的体外生物活性。利用相同的实验方法,发现 GST-MBD融合蛋白能够与 MEF 细胞中inhbb基因的启动子区结合,而不能与小鼠胚胎干细胞 R1中的inhbb基因的启动子区 DNA 结合,同时采用半定量PCR方法检测到inhbb基因在R1细胞中高表达,而在MEF细胞中不表达,表明inhbb基因受到DNA甲基化的调控。

英文摘要：

The prokaryotic expression plasmid of methylated CpG binding domain （MBD）protein was constructed.GST-MBD fusion protein was expressed in E.coliBL21（DE3）and purified by GST beads. Pull-down experiments invitro showed that GST-MBD fusion protein could bind the promoter DNA of oct4 gene in mouse embryonic fibroblast （MEF）cells,confirming the biological activity of GST-MBD protein.Same experiments revealed that GST-MBD protein could bind the promoter DNA of inhbb gene in MEF cells,not in R1 cells.Expression of inhbb gene was observed in R1 cells,but not in MEF cells by semi-quantitive PCR.These results indicate that inhbb gene is regulated by DNA methylation.