小鼠核结合因子a1基因启动子报告载体的构建及鉴定  

Construction and Identification of Mouse Cbfa1 Gene Promoter Reporter Vector

作　　者：郑纺[1] 崔壮[2] 王宝利[2] 王洪武[1] 

机构地区：[1]天津中医药大学中医学院,300193 [2]天津医科大学

出　　处：《天津医药》2011年第12期1133-1135,共3页Tianjin Medical Journal

基　　金：国家自然科学基金项目(项目编号:81102543);教育部高等学校博士学科点专项科研基金项目(项目编号:20091210120003);天津市高等学校科技发展基金计划项目(项目编号:20080219);天津市应用基础及前沿技术研究计划项目(项目编号:11JCYBJC13400)

摘　　要：目的:构建小鼠核结合因子a1(Cbfa1)基因启动子萤光素酶报告载体,并检测其驱动报告基因的转录活性。方法:以小鼠基因组DNA为模板,巢式PCR扩增含有Cbfa1基因启动子(-1000～+200bp)的序列。限制性内切酶MluI和XhoI酶切这段序列后定向克隆到不含启动子的PGL3-Basic报告基因载体上,重组质粒命名为pGL3-Cbfa1。pGL3-Cbfa1瞬时转染成骨细胞系MC3T3-E1,检测其能否在Cbfa1基因启动子的调控下表达报告基因并提高萤光素酶活性。结果:pGL3-Cbfa1经酶切鉴定和序列分析证实与设计完全一致。细胞瞬时转染结果表明Cbfa1启动子具有转录活性,pGL3-Cbfa1的萤光素酶水平约是pGL3-Basic的35倍。结论:小鼠Cbfa1启动子报告基因载体的成功构建,为进一步将其用于抗骨质疏松药物的筛选与评价奠定了实验基础。Objective： To construct a luciferase reporter vector used to monitor the activity of mouse core binding factor a1 （Cbfa1） gene promoter and to analyze its transcriptional activity. Methods： A DNA segment of Cbfa1 gene promoter （-1 000bp-＋200bp） was amplified by nested-PCR from mouse genome DNA, and correctly connected to promoterless vector PGL3-Basic by restriction enzyme MluI and XhoI. Recombinant plasmid pGL3-Cbfa1 was transiently transfected into osteoblastic cell line MC3T3-E1. The expression of luciferase was detected under the monitor of mouse Cbfa1 gene promoter. Results： pGL3-Cbfa1 was confirmed the same as the design by restriction digestion and sequence analysis. High luciferase activity （35 fold greater than that of control vector pGL3-Basic） was found in the pGL3-Cbfa1 promoter constructs. Conclusion： The mouse cbfa1 gene promoter luciferase reporter vector was successfully constructed, and the reporter vector laid the experimental foundation for anti-osteoporosis drug screening and evaluation.

关 键 词：基因 报告 萤光素酶类 核心结合因子类 转录启动子 

分 类 号：R737.250.2[医药卫生—肿瘤]