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细胞分裂周期蛋白2启动子报告基因载体的构建与鉴定

周辉1,吴炜2,赵明1   

  1. 1. 南方医科大学 病理生理学教研室
    2. 中国科学院广州生物医药与健康研究院
  • 收稿日期:2013-05-09 修回日期:2013-09-29 出版日期:2014-02-15 发布日期:2014-02-15
  • 通讯作者: 周辉

Construction and ldentification of Cell Division Cycle 2 Promoter Reporter Gene Vector

  • Received:2013-05-09 Revised:2013-09-29 Published:2014-02-15 Online:2014-02-15
  • Contact: Hui ZHOU

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摘要： 摘要 目的：构建细胞分裂周期蛋白2Cdc2 基因启动子荧光素酶报告基因载体 pGL3-Cdc2-promoter，并检测其转录活性。方法：根据 UCSC 软件查找的人基因组 DNA 的 Cdc2 启动子序列并设计两端引物，扩增人基因组 DNA 中的 Cdc2 启动子。用限制性内切酶 ScaⅠ和 XhoⅠ双酶切质粒 pGL3-Basic 和 Cdc2 启动子后，将 Cdc2 基因启动子插入到 pGL3-Basic 报告基因载体上。重组质粒命名为 pGL3-Cdc2-promoter。将其与内参质粒 pRL-SV40 瞬时共转染骨肉瘤细胞 U2OS，检测双荧光素酶活性。结果：成功构建 Cdc2 基因启动子荧光素酶报告基因载体 pGL3-Cdc2-promoter，质粒酶切及测序结果完全正确。瞬时转染实验显示，Cdc2 启动子具有转录活性，pGL3-Cdc2-promoter 的荧光素酶水平约是 pGL3-Basic 的 32 倍。结论：pGL3-Cdc2-promoter 在骨髓瘤细胞 U2OS 细胞中能被转录激活，为进一步将其用于抗肿瘤药物的筛选与评价奠定了基础。

关键词: 细胞分裂周期蛋白2, Cdc2, 细胞周期素依赖性激酶1, CDK1, 细胞周期, 启动子, 双荧光素酶

Abstract: Abstract Objective: To construct the luciferase reporter gene vector of cell division cycle 2 gene promoter and determine its transcriptional activity. Methods: Primers were designed based on human Cdc2 promoter sequence from UCSC software. Then Cdc2 promoter from human genome DNA was replicated. After pGL3-Basic Vector and Cdc2 promoter were digested with restriction enzymes ScaⅠand XhoⅠseparately, Cdc2 promoter was inserted into pGL3-Basic vector. The recombinant plasmid named pGL3-Cdc2-promoter was transiently co-transfected into U2OS cells with control vector pRL-SV40, then the activity of dual luciferase was detected. Results: pGL3-Cdc2- promoter was constructed successfully. Restriction analysis and sequencing proved the same as the design . Promoter activity of pGL3-Cdc2-promoter in U2OS was 32 times than that in negative control group. Conclusion: pGL3-Cdc2-promoter can be transcribed and activated in U2OS cells. This research provided an important basis for antitumor drug screening and evaluation.

Key words: cell division cycle 2, Cdc2, cyclin-dependent kinase 1, CDK1, cell cycle, promoter, dual-Luciferase