NF-κB决定小鼠胎肝激酶-1基因启动子的基本活性  

NF-κB Determinates Basic Activity of Mouse Fetal Liver Kinase-1 Promoter

作　　者：郑丽端[1] 童强松[2] 杨渝珍[3] 侯晓华[4] 杨秀萍[1] 董继华[5] 

机构地区：[1]华中科技大学同济医学院附属协和医院病理科,武汉430022 [2]华中科技大学同济医学院附属协和医院外科,武汉430022 [3]华中科技大学同济医学院生物化学与分子生物学教研室,武汉430030 [4]华中科技大学同济医学院附属协和医院消化内科,武汉430022 [5]华中科技大学同济医学院附属协和医院中心实验室,武汉430022

出　　处：《中国生物化学与分子生物学报》2007年第8期625-630,共6页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金资助项目(No.30200284;No.30600278)~~

摘　　要：为克隆小鼠胎肝激酶-1(fetal liver kinase-1,FLK-1)基因上游启动子序列,并观察其不同截短片段在小鼠血管内皮细胞中的启动子活性,以小鼠全基因组为模板,通过PCR扩增方法获得-258^+299bp、-96^+299bp、-71^+299bp、-36^+299bp大小的FLK-1启动子片段,将其定向克隆入pGL3-Basic,构建荧光素酶报告基因载体,并制备NF-κB结合位点的突变或缺失体.在阳离子脂质体介导下,报告基因载体瞬时转染小鼠血管内皮细胞株SVEC4-10.结果发现,在小鼠血管内皮细胞中,各FLK-1启动子片段均有活性;-71^-36bp区存在FLK-1启动子的核心调控元件.针对该区域NF-κB结合位点进行突变或缺失,能导致启动子活性显著降低;凝胶电泳迁移率实验表明该区段能结合转录因子NF-κB.结果提示,成功克隆了在血管内皮细胞中具有活性的FLK-1上游启动子序列,NF-κB是决定其基本活性的重要转录因子,为进一步研究FLK-1基因的转录调控机制奠定了基础.To clone the upstream promoter sequence of mouse fetal liver kinase-1 （FLK-1） and investigate the activities of its truncated segments in mouse vascular endothelial cells, four segments of FLK-1 promoter, -258- ＋299 bp, -96- ＋299 bp, -71- ＋299 bp and -36- ＋299 bp, were obtained by PCR amplification from mouse genomic DNA and were subcloned into pGL3-Basic to construct luciferase reporter vectors. The mutation or deletion of NF-κB binding site within FLK-1 promoter was prepared. The recombinant reporter vectors were transiently transfected into mouse vascular endothelial cell line SVEC4-10 by Lipofectamine 2000. Results showed that all these FLK-1 promoters presented activities in mouse vascular endothelial cells. The region of - 71 - - 36 bp contained the core element of FLK-1 promoter. Mutation or deletion of NF-κB binding site within this region resulted in significant decrease of promoter activity. EMSA demonstrated that this region could bind NF-κB. These results suggest that the upstream promoter sequences of FLK-1 with activities in vascular endothelial ceils, were obtained successfully, in which NF-κB was an important transcription factor determining its basic promoter activity. These results established a basis for further studying transcriptional regulation mechanisms of FLK-1 gene.

关 键 词：胎肝激酶-1 血管内皮细胞 启动子 报告基因 

分 类 号：R57[医药卫生—消化系统]