J4 ›› 2010, Vol. 36 ›› Issue (3): 465-468.

• 基础研究 • 上一篇    下一篇

抑癌基因BRCA1启动子荧光素酶报告基因的构建及活性测定

贾立立1,2|王大鹏1,2|樊飞跃2|詹启敏1   

  1. 1.北京协和医学院肿瘤研究所 分子肿瘤学国家重点实验室|北京 100021;2.北京协和医学院放射医学研究所生物学研究室|天津 300192
  • 收稿日期:2010-01-05 出版日期:2010-05-28 发布日期:2010-05-28
  • 通讯作者: 樊飞跃(Tel:022-85683036,E-mail: faithyfan@yahoo.com);詹启敏(Tel:010-67715058,E-mail: Zhanqimin@pumc.edu.cn) E-mail:faithyfan@yahoo.com Zhanqimin@pumc.edu.cn
  • 作者简介:贾立立(1981-)|女|吉林省松原市人|医学博士|主要从事分子肿瘤学研究。
  • 基金资助:

    国家自然科学基金资助课题(30730046,30721001);973国家重点基础研究项目资助课题(2002 CB513101)

Construction and identification of human tumor suppressor gene BRCA1 promotor luciferase report gene vector

 JIA Li-Li1,2, WANG Da-peng1,2, FAN Fei-YUE2, ZHAN Qi-Min1   

  1. 1.State Key Laboratory of Molecular Oncology, Cancer Institute, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100021|China|2. Department of Biology Laboratory,Institute of Radiation Medicine, Chinese Academy of Medical Sciences and Peking Union Medical College,Tianjin 300192|China
  • Received:2010-01-05 Online:2010-05-28 Published:2010-05-28

摘要:

目的:克隆人BRCA1基因的启动子,构建荧光素酶报告基因载体,并在细胞内检其活性,为其后续基因凋控研究提供依据。方法:采用PCR技术,从人正常宫颈组织细胞中扩增出BRCA1启动子,插入荧光素酶报告基因载体pGL3-basic中,测序所扩增的DNA序列,将其转染入HCT 116细胞中并检测其活性。结果:酶切及基因测序方法证实所构建质粒含有pGL3-basic全序列及BRCA1启动子上游调控序列,扩增的BRCA1启动子序列正确;双报告基因实验检测荧光素酶活力表明,p53缺失的HCT116细胞中BRCA1启动子明显增加(P<0.05),构建的报告基因具有启动子活性。结论:克隆BRCA1启动子及成功构建人BRCA1启动子报告基因,可实现快速、经济和准确地克隆已知基因启动子分子和构建启动子载体的目的。

关键词: BRCA1;启动子;荧光素酶;报告基因

Abstract:

Abstract:Objective To construct the human BRCA1 promotor luciferase report gene vector and detect its activity in cells. Methods The BRCA1 promoter from human normal cervix tissues                 
  was amplified by PCR, and was inserted into the luciferase report gene pGL3-basic vector. The amplified DNA sequence was confirmed by sequencing and then the constructed vector was transfected into  HCT116 cells to detect its activity by Premaga Dual-luciferase report gene detection system. Results The recombinant plasmid was tested by gel electrophoresis and sequencing analysis, it was proved that the plasmid included pGL3-basic DNA sequence and PRL regulating sequence.The sequencing results indicated that the amplified sequence was correct, in p53 minus HCT116 cells the number of BRCA1 promoter was increased (P<0.05), and the luciferase activity detection result demonstrated that the constructed vector had the promotor activity.Conclusion The human BRCA1 promotor luciferase report gene vector has been constructed successfully, and it will become essential material for further study on the function of BRCA1 regulation.

Key words: BRCA1, promotor, luciferase, report gene

中图分类号: 

  • Q78