人成釉蛋白基因的启动子活性区域分析

摘要

目的:构建人成釉蛋白(ameloblastin,AMBN)基因不同长度的上游启动子荧光素酶报告基因载体,比较不同的启动子片段在成釉细胞、Hela细胞中的活性.方法:以PCR方法获取基因上游启动子片段,将其构建至报告基因载体pGL3-Basic中,瞬时转染成釉细胞、Hela细胞,通过检测荧光素酶活性来分析启动子区域的转录调控能力.结果:成功地获得了不同长度的ameloblastin基因启动子目的片段,酶切鉴定表明不同长度启动子荧光素酶报告基因载体构建成功,不同长度的启动子在不同的细胞中活性不同,-424-267与-128-37区域为特异性转录调控作用区.结论:成功构建了不同长度的ameloblastin基因启动子的pGL3-Basic荧光素酶报告基因载体,初步确定ameloblastin基因启动子的转录活性区,为进一步研究ameloblastin基因转录调控特点奠定基础.%AIM:To construct luciferase report gene vectors with different promoter segments of human amelo blastin, and to compare the luciferase activities of different length segments of human ameloblastin in ameloblast and Hela cells. METHODS: The different -length desired promoter segments were obtained by PCR method,cloned into lucifer ase report gene vectors pGL3 - Basic, and transiently transfected into ameloblast and Hela cells. We analyzed the tran scriptional regulatory capacity of the promoter segments by detecting luciferase activity. RESULTS: We have obtained different - length promoter segments of ameloblastin gene, and the different - length promoter recombinant luciferase re porter vectors were constructed successfully by cutting with two different restrict enzymes. Different - length promoters had different activities in different type cells, and -424 -267 and -128-37 regions were the specific transcriptional regulatory district. CONCLUSION: We successfully constructed the different - length recombinant pGL3 - Basic lucif erase reporter gene vector. The transcriptional activity regions of ameloblastin gene promoter were determined initially , which will provide a foundation for further studying the transcriptional regulatory characteristics of ameloblastin gene.