含hITF启动子的荧光素酶报告基因质粒的构建及.doc

PAGE PAGE 1 含hITF启动子的荧光素酶报告质粒的构建及活性检测* 王臻，王林，万千雪，金星，梁光萍，彭曦 (第三军医大学西南医院全军烧伤研究所,创伤、烧伤与复合伤国家重点实验室,重庆400038) 摘 要：目的 构建含有人肠三叶因子 (hITF)启动子的荧光素酶表达载体,并检测启动子活性。方法 采用PCR 技术从LS-174T细胞基因组DNA中扩增出长约2.5kb的含hITF基因启动子片段（-2331/+53）,而后在此基础上构建了10个不同长度的启动子序列至pGL-3 basic荧光素酶表达载体，然后将这些重组质粒转染HEK-293细胞并在48小时后检测其荧光素酶活性。结果 插入的hITF启动子片段测序结果显示正确，确定了最小活性功能域位于-300/+53区域，且在-300/-200区域有潜在的具有较强活性的元件存在。结论 成功构建了含hITF启动子的萤火虫荧光素酶报告基因质粒, 鉴定出人肠三叶因子(hITF)启动子活性位于-300/-200区域。 关键词: 肠三叶因子；启动子；pGL-3 basic；HEK-293细胞 The construction of a luciferase reporter vector directed by hITF promoter WANG Zhen，WANG Lin，WAN Qian-xue，JIN Xing，LIANG Guang-ping，PENG Xi (State Key Laboratory of Trauma,Burns and Combined Injury, Institute of Burns, Southwest Hospital,Third Military Medical University, Chongqing 400038, China) Abstract： Objective To construct a hITF-promoter-directed luciferase reporter vector and to evaluate its activity in LS-174T and HEK-293 cells. Methods A 2.5 kb fragment spanning the region -2331 to +53 of hITF gene was amplified from genomic DNA extracted from LS-174T by PCR, and substantially constructed 10 fragments containing different region of the promoter to promoter-less pGL-3 basic luciferase reproter vector and transfected into HEK-293 cells. 48 hours after the transfection, cells were lysed and the luciferase activity was measured. Resluts The inserted hITF promoter was verified by DNA sequencing and the minimal functional promoter was located in the -300/+53,and there were some potential active elements. Conclusion The recombinant plasmid containing luciferase reporter gene driven by hITF promoter was constructed successfully and the region spanning -300/-200 play a important role in facilitating the initiation of transcription. Key words: ITF; promoter; pGL-3 basic; HEK-293细胞 基金项目： 国家自然科学基金重点项目(批准号 supported by National Natural Science Foundation 作者简介：王臻，男，陕西省周至县人，硕士研究生，主要从事烧伤代谢营养方面的研究工作，E-mail：rico_azerni@126.com 通讯作者：彭曦，电话：023－6875414