高表达Delta4增加Notch基因的表达  

Delta4 overexpression increases Notch gene expression

作　　者：马道新[1] 纪春岩[1] 赵建强[2] 郭农建[2] 张茂宏[1] 于书彦[3] 

机构地区：[1]山东大学齐鲁医院血液学研究室 [2]济南市中心医院 [3]山东大学生理学研究所,济南250012

出　　处：《基础医学与临床》2004年第6期651-656,共6页Basic and Clinical Medicine

基　　金：山东省医药卫生资助项目 (2 0 0 1CA1CJA1) ;山东省科技发展计划资助项目 (2 0 0 1BB1CJB1)

摘　　要：Notch信号传导系统在介导造血细胞增殖与分化过程中具有重要作用。通过分子克隆技术成功构建含标记基因FLAG的Notch配基Delta4的高表达载体pTracer CMV Delta4 FLAG ,瞬时转染COS7细胞 ,4 8h后收获细胞并制备融合蛋白 ,SDS PAGE凝胶电泳和Western blot证实后 ,将其稳定转染CHO细胞 ,Western blot鉴定并筛选高表达Delta4的Delta4 CHO1 4及Delta4 CHO1 5细胞株 ,利用Luciferase分析方法进行Delta4功能性研究。研究结果发现Delta4对Notch1及Notch2均具有信号功能活性 ,且对后者的活性功能水平大于前者 ,说明Delta4为Notch1及Notch2的配基。与其他配基功能比较 :对Notch1,Delta4的功能活性高于Delta1及Jagged1,但略低于Jagged2 ;对Notch2 ,Delta4的功能活性低于Delta1、Jagged1及Jagged2 ,但亦具有较强的活性水平。Notch system plays an important role in the proliferation and differentiation of hematoplastic cells. The Delta4 highly-expressing vector pTracer.CMV.Delta4.FLAG was successfully constructed and transfected into COS7 cells transiently. The cells were and produced fusion protein 48 hours after transduction. Delta4 protein band was proved by SDS-PAGE and Western-blot. Then stably transfected into CHO cells and the high-expressing Delta4-CHO clones were set up and screened using Western-blot. The signal activity of Delta4 was found with Luciferase analysis. It was found that Delta4 had a signal activity to both a Notch1 and Notch2, and it is stronger to Notch2 via Luciferase analysis. To Notch1, Delta4 was significantly weaker than Jagged2, but stronger than Delta1 and Jagged1. To Notch2, Delta4 had a strong signaling activity, but weaker than Delta1、Jagged1 and Jagged2.

关 键 词：高表达 NOTCH基因 功能活性 配基 WESTERN-BLOT NOTCH信号 分子克隆技术 COS7细胞 CHO细胞 凝胶电泳 

分 类 号：Q78[生物学—分子生物学]