天津医药

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小鼠核结合因子a1基因启动子报告载体的构建及鉴定

郑纺1,崔壮2,王宝利3, 王洪武4   

  1. 1. 天津中医药大学
    2. 天津医科大学公共卫生学院
    3. 天津医科大学代谢病医院内分泌研究所、卫生部激素与发育重点实验室
    4. 天津中医药大学中医学院
  • 收稿日期:2011-08-03 修回日期:2011-09-24 出版日期:2011-12-15 发布日期:2011-12-15
  • 通讯作者: 郑纺

Reporter Vector Construction and Identification of Mice Cbfa1 Gene Promoter

  • Received:2011-08-03 Revised:2011-09-24 Published:2011-12-15 Online:2011-12-15
  • Contact: ZHENG Fang

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摘要： 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（-1 000～+200 bp）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奠定了实验基础。

关键词: 基因, 报告, 萤光素酶类, 核心结合因子类, 转录启动子

Abstract: Abstract Objective: To construct a specific reporter vector 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 (-1000bp-+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 (-1000bp-+200bp). Results: pGL3-Cbfa1 was the same as the design confirmed by restriction digestion and sequence analysis. High luciferase activity (35 fold greater than that of contro vector pGL3-Basic) was found in the pGL3-Cbfa1 promoter constructs. Conclusion: Mouse cbfa1 gene promoter luciferase reporter vector was successfully constructed and the reporter vetor laid the experiment foundation for screening of anti-osteoporosis medicine.