摘要
目的构建人牙釉质蛋白(Amelotin,AMTN)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及Hela细胞中的活性,为进一步判定上游启动子的转录调控区奠定基础。方法以PCR方法荻取基因上游启动子片段,将其构建至报告基因载体pGL3-Basic中,瞬时转染成釉细胞及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Amelotin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,-190～-358与-35～-93区域为特异的转录调控作用区。结论成功构建了不同长度的Amelotin基因启动子的pGL3-Basic荧光素酶报告基因载体,初步确定Amelotin基因启动子的转录活性区,为进一步研究Amelotin基因转录调控特点奠定基础。
Objective Constructing luciferase report gene vectors with different promoter segments of human Amelotin,and comparing the luciferase activitiy of different length segments of human Amelotin in Ameloblast and Hela cells, so as to provide a foundation for confirming transcription regulation district of promoter sequence in further study. Methods The different-length desired promoter segments were obtained by PCR method. They were cloned into luciferase report gene vectors pGL3-Basic, and transiently transfected into ameloblast and Hela cells. The transcriptional regulatory capacity of the promoter segments was analyzed by detecting luciferase activity. Results Different-length promoter segments of Amelotin gene were obtained. The different-length promoter recombinant luciferase reporter vectors were con- structed successfully by cutting them with two different restrict enzymes. Different-length promoters had different activity in different-type cells, -424 -267 and - 128 -37 regions are specific transcriptional regulatory district. Conclusion Different-length recombinant pGL3- Basic luciferase reporter gene vector were successfully constructed. The transcriptional activitive regions of Amelotin gene promoter were initially determined ,which will provide a foundation for further studying the transcriptional regulatory characteristics of Amelotin gene.
出处
《潍坊医学院学报》
2009年第6期401-404,共4页
Acta Academiae Medicinae Weifang
基金
国家自然科学基金资助课题(课题编号:30672316)
山东省自然科学基金资助课题(课题编号:Y2006C106)
