小鼠mdm2的表达及活性鉴定

Expression and activity analysis of mouse mdm2

mdm2(murine double minute 2,鼠双微粒体-2)是抑制p53(肿瘤抑制因子)活性的重要分子之一,它们之间相互作用对细胞的生存具有重要功能。克隆得到小鼠Mus musculus mdm2 cDNA,构建其真核表达载体p3xFLAG-CMV-7.1-mdm2,Western blotting验证其在真核细胞中的表达大约为60 kDa的蛋白表达产物,间接免疫荧光技术检测mdm2的表达和亚细胞定位,mdm2主要在细胞核内表达。利用p53蛋白泛素化试验和p53报告基因活性检测试验,验证了FLAG-mdm2的活性,构建了mdm2调节p53泛素化和抑制p53活性的细胞模型,为研究p53活性调控、mdm2与p53相互作用等提供了基础和研究手段。

The mdm2（murine double minute 2） is one of the factors inhibiting p53 activity,and their interaction is important to hold cell survival.In this paper,mouse mdm2 gene was cloned from mouse 3T3 cell strain,and the recombinant plasmid p3xFLAG-CMV-7.1-mdm2 was constructed and confirmed by restriction enzyme digestion and DNA sequencing.Western blotting analysis and indirect immune-fluorescent analysis showed that FLAG-mdm2 expressed in p3xFLAG-CMV-7.1-mdm2-transfected H1299 cells,which was 60 kDa in size and dominantly localized in nucleus.The activity of FLAG-mdm2 protein was confirmed by p53 ubiquitination assay and luciferase assay.The result showed that mdm2 cell model inhibiting p53 activity is established.Our results would be useful for studying the mdm2 biological fuction in regulating p53 activity and interaction between mdm2 and p53.