基础医学与临床 ›› 2012, Vol. 32 ›› Issue (4): 338-341.

• 错配修复基因与肠癌专题 • 上一篇    下一篇

错配修复基因hMLH1启动子萤光素酶报告基因载体的构建与鉴定

卢俊宇1,盛剑秋1,陆晓娟2,金鹏高巍1,王亚婷1   

  • 收稿日期:2011-11-02 修回日期:2012-01-04 出版日期:2012-04-05 发布日期:2012-03-21
  • 通讯作者: 盛剑秋 E-mail:jianqiu@263.net
  • 基金资助:
    国家自然科学基金

Construction and identification of luciferase reporter gene vector

  • Received:2011-11-02 Revised:2012-01-04 Online:2012-04-05 Published:2012-03-21

摘要: 目的:构建含hMLH1启动子片段的萤光素酶报告基因载体,并检测其在雌激素诱导下的转录活性。方法:以正常人基因组DNA为模板,PCR扩增hMLH1启动子片段(-1953/+53),插入萤光素酶报告基因载体pGL3-Basic,将重组质粒转入HEK293细胞,检测在有和无雌激素诱导下萤光素酶的活性变化。结果:酶切和测序结果证实重组萤光素酶报告基因载体pGL3-Promoter1-luc构建成功。转染pGL3-Promoter1-luc后,雌激素诱导的萤光素酶活性为7.45±0.81,显著高于无水乙醇组的3.28±0.19 (n=3,P<0.001)。结论:hMLH1启动子片段存在与雌激素相关的调控序列。

关键词: 错配修复基因, hMLH1, 启动子, 雌激素, 双萤光素酶报告基因

Abstract: Objective To construct luciferase reporter gene vector containing human MLH1 promoter and to assay the transcriptional activity of hMLH1 promoter induced by E2. Methods hMLH1 promoter(-1953/+53)were amplified from the genomic DNA of human by PCR and cloned into luciferase reporter gene vector, pGL3-Basic. The recombined vector was transfected into HEK293 cells, and the activity of the luciferase was determined after the cells were simulated by E2 or not. Results The results of restriction enzyme digestion and sequencing indicated that the recombinant vector pGL3-Promoter1-luc was successfully constructed. After transcription of pGL3-Promoter1-luc, the activity fold of the luciferase was 7.45±0.81 induced by E2, which is significantly higher than 3.28±0.19 without E2(n=3,P<0.001). Conclusion The hMLH1 promoter contains the regulatory sequence associated with E2.

Key words: 错配修复基因, hMLH1, 启动子, 雌激素, 双萤光素酶报告基因