作　　者： ; ; ; ; ;

机构地区： 南方医科大学基础医学院病理学系

出　　处： 《南方医科大学学报》 2010年第9期2025-2029,共5页

摘　　要： 目的鉴定人肺特异性X蛋白(lung specific X protein,LUNX)基因的增强子及其调控活性。方法以人基因组DNA为模板,PCR扩增生物信息学预测的3个增强子片段(E1:+3770～+3959bp;E2:+6454～+6555bp;E3:+14553～+14652bp),通过荧光素酶报告基因表达体系检测转录活性。结果 PCR产物经测序证实后,分别定向连接至pGL3-Promoter载体中报告基因启动子上游的Kpn I和Xho I酶切位点和报告基因下游的BamHI和Sal I酶切位点上,经酶切鉴定后,构建了6种荧光素酶报告基因表达体系。以pSV-β-Galactosidase质粒为内对照,瞬时转染HEK293细胞,培养48h后检测细胞裂解液中荧光素酶活性。当3个DNA片段位于报告基因启动子上游时,均不具有增强转录的能力。将它们分别连接于报告基因下游时,E1和E3所调控的荧光素酶活性分别是对照pGL3-Promoter的2.83倍(P<0.05)和1.59倍(P<0.05)。结论 LUNX基因的+3770～+3959bp和+14553～+14652bp序列具有增强转录的能力,为LUNX表达调控机制的深入研究奠定了基础。 Objective To identify the enhancers of human lung specific X protein （LUNX） and their regulation at the transcription level in vitro. Methods Three enhancer fragments （E1：＋3770-＋3959bp; E2： ＋6454-＋6555bp; E3： ＋14553-＋14652 bp） predicted by bioinformatics software were isolated from the human genomic DNA by PCR amplification. Luciferase assay was performed to detect the activities of the enhancers in transcriptional regulation. Results PCR products were confirmed by DNA sequencing. The amplified enhancers digested by Kpn I/Xho I and BamH I/Sal I, to generate the sticky-end fragments were inserted into PGL3-promoter in a reporter vector, and 6 luciferase expression vectors were obtained. All the reporter plasmids and pGL3-promoter were transiently transfected into HEK293 ceils with an internal control of p SV-β-Galactosidase reporter vector. The enhancer activity of each construct was evaluated by luciferase assay of the cell extracts after transfection for 48 h. The results showed that the 3 fragments, when located upstream, did not increase transcription of reporter gene, but when at the downstream, E1 and E3 increased the transcription by 2.83 and 1.59 folds of that ofpGL3-promoter, respectively. Conelusion LUNX gene sequences from ＋3770 to ＋3959 bp and ＋14553 to ＋14652 bp possess the capacity to enhance gene transcription.

关 键 词： 基因 增强子 转录调控 荧光素酶活性

领　　域： [生物学]