人血小板源性生长因子受体β启动子的结构与功能分析

Structural and Functional Analysis of Human PDGFR-β Promoter

通过PCR扩增人血小板源性生长因子受体β(PDGFR-β)基因启动子片段,经双酶切后克隆到pGL3-basic载体构建了5个PDGFR-β基因启动子5′系列缺失重组载体,与作为内参的pRL-TK共转染人脐静脉内皮细胞(ECV304,HUVEC)后,通过荧光素酶活性检测鉴定出人PDGFR-β启动子中具有转录调控作用的2个活性区(-983～+53)和(+540～+1457),前者执行正调控,后者则起负调控作用.这2个转录活性区分别含有在人、大鼠、小鼠PDGFR-β基因启动子区都高度保守的区域A-box、B-box和C-box,凝胶迁移或电泳迁移率检测(EMSA)证实,核因子能与B-box*、C-box*特异结合.

The promoter of human platelet-derived growth factor-β（PDGFR-β） gene tightly regulated during embryonic development and in several physiological and pathological situations,was identified and characterized.Several deletions of the human PDGFR-β promoter were obtained by PCR from human genomic DNA and then subcloned into pGL3-basic vector containing a luciferase reporter.The recombinant reporters were transiently transfected into the human umbilical vein endothelial cells（ECV304,HUVEC） for the studies of the expression regulatory of this receptor.Two regions（-983 ～ ＋ 53） and（＋ 540 ～ ＋ 1457） were identified to regulate human PDGFR-β promoter transcription by luciferase activity assays.The former showed a positive regulatory role,while the latter was negative.The highly conserved regions of human,mouse and rat PDGFR-β core promoters were marked as A-box,B-box and C-box and were found within these two regions.The B-box＊ and C-box＊ were observed to specifically bind nuclear factors by electrophoretic mobility shift assays.