Id1启动子的荧光素酶报告基因载体的构建及其在肝癌HepG2细胞中的活性检测  

Construction of luciferase reporter gene vectors containing Id1 promoter region and Id1 promoter activity in HepG2 cells

作　　者：丁睿[1] 韩霜[2] 雷婷[2] 刘杰[2] 窦科峰[1] 

机构地区：[1]第四军医大学西京医院肝胆外科,陕西西安710033 [2]第四军医大学西京医院全军消化病研究所,陕西西安710033

出　　处：《第四军医大学学报》2007年第9期798-801,共4页Journal of the Fourth Military Medical University

摘　　要：目的:扩增Id1启动子基因,构建Id1启动子的报告基因载体.方法:通过PCR及双酶切方法,从HepG2细胞基因组DNA中获得Id1基因编码序列上游包含不同长度碱基的两段Id1启动子序列;分别克隆到报告基因pGL3-enhancer载体上,构建包含两段不同长度Id1启动子的报告基因载体;用脂质体转染法将两报告基因载体分别转染至肝癌HepG2细胞系;采用双荧光素酶报告基因系统评估Id1启动子活性.结果:经PCR方法扩增出大小分别为1096bp和266bp的两段不同长度的Id1启动子片段,序列测定其编码序列及读框正确,酶切鉴定亚克隆序列正确.瞬时转染HepG2后经报告基因检测,两段启动子均具有转录活性.结论:成功构建了Id1启动子的报告基因载体,为进一步研究Id1启动子和肝癌的关系奠定了实验基础.AIM： To amplify the Id1 promoter gene region and to construct two luciferase reporter gene vectors containing Id1 promoter regions. METHODS： Two DNA fragments of the 5＇ flanking region of human Id1 gene were isolated from genomic DNA of HepG2 cells by polymerase chain reaction （PCR）. Pro- duce two recombinant constructs containing the fragments of the Id1 promoter. The two DNA upstream fragments of the transcrip- tion start site of Id1 gene were subcloned into the luciferase （Luc） reporter vector PG123-enhancer. Then the recombinant constructs were transiently transfected into HepG2 cells. After 24 h of transfection, cells were lysed and the Luc activity in lysates was assayed. RESULTS： High promoter activities （6 ～10 fold greater than those of empty vector control） were found in the both promoter constructs, with the higher activity in the Idlpl con- struct. CONCLUSION： The DNA region of -189～ ＋76 con- tains the core fragments of Id1 promoter and there may exist posi- tive regulator in the region of - 1019 -～- 189.

关 键 词：分化抑制因子(Id) 启动子 双荧光素酶报告基因 癌 肝细胞 

分 类 号：Q782[生物学—分子生物学]