人肾组织γ-谷氨酰基羧化酶基因启动子区克隆与活性分析  

Cloning and activity analysis of GGCX promoter region in human kidney

作　　者：杨俊[1] 刘继红[1] 郭小林[1] 王涛[1] 王军凯[1] 陈美元[1] 王少刚[1] 叶章群[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院泌尿外科,武汉430030

出　　处：《中华实验外科杂志》2008年第8期1041-1044,共4页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金资助项目（30400442）

摘　　要：目的克隆人肾组织γ-谷氨酰基羧化酶（GGCX）基因5’侧翼启动子区，并对其转录活性进行分析。方法对GGCX基因5’侧翼区进行生物信息学分析，预测其转录调控区域；从人肾组织中提取基因组DNA，以其为模板扩增GGCX基因启动子区，通过酶切鉴定及测序分析无误后，将其构建至萤光报告基因载体pGL3-basic中，瞬时转染至Hela细胞，检测萤光素酶水平，从而分析扩增启动子区的转录活性。结果成功克隆长度为804bp（-891～-88）的GGCX基因启动子片段，并构建相应的萤光素酶重组子pGL3-GGCX Promoter，转染Hela细胞48h后检测萤光素酶活性显示，pGL3-GGCX Promoter与空质粒对照pGL3-basic相对萤光素酶值分别为16．924-4、01与0．02±0．01，pGL3-GGCX Promoter萤光素酶活性显著增强（P〈0．05）。Matlnspector等分析显示该片段具有多个转录因子结合位点。结论成功构建含有GGCX基因启动子序列的萤光素酶报告系统，为进一步研究GGCX基因功能及其转录调控奠定了基础。Objective To clone 5＇ flank promoter region of GGCX in human kidney and analyze its transcriptional activity. Methods Approximate 3800-bp fragment of 5＇ flanking region of GGCX was bioinformatically analyzed with computer to predict the promoter region. The promoter segment was obtained from human kidney genomic DNA by nested PCR method and identified by restriction enzyme digestion and sequencing. Its promoter activity was examined by the dual luciferase reporter assay after the promoter was cloned into pGL3-basic vector generating pGL3-GGCX and transfected into Hela cells, pGL3- basic and pGL3-control were respectively used as the negative and positive controls, pRL-TK, a Renilla luciferase reporter vector,was co-transfected into Hela cells as an internal control. Results We successfully cloned and indentified a 804-bp （ - 891 - - 88） promoter region of GGCX gene and constructed its luciferase report plasmid. Dual luciferase reporter assay revealed that transient transfection of pGL3-GGCX resulted in a significant increase in luciferase activity compared to the empty vector control, being 16.92±4. 01 and 0.02±0.01 respectively. Sequence analysis with MatInspector database showed that a number of putative transcription factors may locate on this 804-bp promoter region. Conclusion The luciferase report system of GGCX gene promoter we have constructed can be applied for further study on the function and transcriptional regulation of GGCX.

关 键 词：γ-谷氨酰基羧化酶 启动子 生物信息学 萤光素酶 

分 类 号：R692[医药卫生—泌尿科学]