抑癌基因BRCA1启动子荧光素酶报告基因的构建及活性测定

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

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.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-Li^1,2, WANG Da-peng^1,2, FAN Fei-YUE², ZHAN Qi-Min¹

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/Abstract

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:

贾立立, 王大鹏, 樊飞跃, 詹启敏. 抑癌基因BRCA1启动子荧光素酶报告基因的构建及活性测定[J]. J4, 2010, 36(3): 465-468.

JIA Li-Li, WANG Da-peng, FAN Fei-YUE, ZHAN Qi-Min. Construction and identification of human tumor suppressor gene BRCA1 promotor luciferase report gene vector[J]. J4, 2010, 36(3): 465-468.

[1]	孔宁\|高海成\|刘国民\|陈月\|周余来\|颜炜群. 巴斯德毕赤酵母表达的人抑瘤素M发酵条件及发酵产物分析[J]. J4, 2012, 38(5): 943-947.
[2]	薛萍\|朱小静\|刘孝菊\|李一兰\|南佳\|姜潮\|李校堃. 重组截短型人角质细胞生长因子1在昆虫细胞中的表达[J]. J4, 2012, 38(4): 633-639.
[3]	徐晓芳\|宋晗星\|邢沈阳\|吕强\|高江明\|赵娜\|常乐\|高峰\|宫鹏涛\|李建华\|张国才\|张西臣. RNA干扰IDH2基因对HCT-8细胞增殖能力的影响[J]. J4, 2012, 38(4): 649-652.
[4]	段秀梅\|邹亚斌\|马洪喜\|李树蕾\|王银萍\|王超\|李玉琴. 结核杆菌热休克蛋白70刺激表位在融合基因疫苗中的佐剂作用[J]. J4, 2012, 38(3): 506-511.
[5]	张耀方, 万晓珊, 赵宏鑫, 邵明龙, 杨苹, 孔祥鑫, 刘敏, 李校堃, 王会岩. FGF21［Tyr²⁰］突变体的克隆、表达及纯化[J]. J4, 2010, 36(6): 1088-1093.
[6]	梁作文, 赵丽晶, 贾慧婕, 李馨, 高丽芳, 张灵, 赵雪俭. 共表达siRNA-Stat3与GRIM-19质粒的构建及其功能研究[J]. J4, 2010, 36(5): 858-861.
[7]	邓林, 刘孝菊, 龚守良, 王会岩, 田海山, 王晓杰, 马吉胜, 李校堃. 重组截短型人角质细胞生长因子-1的表达及纯化[J]. J4, 2010, 36(4): 629-633.
[8]	邵明龙, 赵宏鑫, 杨苹, 万晓珊, 孔祥鑫, 王会岩, 李校堃. 人成纤维细胞生长因子21基因的克隆及在毕赤酵母中的表达[J]. J4, 2010, 36(4): 644-650.
[9]	张璐, 尹战海, 史学涛. pLenti6/V5-DEST-TAZ载体的构建及其对成骨前体细胞MC3T3-E1分化的影响[J]. J4, 2010, 36(3): 429-433.
[10]	李玉香, 张凯宇, 张一宁, 王峰, 姜艳芳, 牛俊奇. 人肝细胞生长因子基因表达对CCl4损伤的人肝细胞及大鼠肝星状细胞株的影响及其机制[J]. J4, 2010, 36(3): 505-509.
[11]	王会岩, 尹海燕, 尹翌秋, 刘孝菊, 赵宏鑫, 秦玉侠, 刘敏, 李校堃. 抗菌肽CM与血管内皮生长因子VEGF121在大肠杆菌中表达及鉴定[J]. J4, 2010, 36(2): 285-290.
[12]	田海山, 唐禄, 王晓杰, 王会岩, 刘笑菊, 冯秀萍, 王一, 李校堃. 乳糖诱导角质细胞生长因子-2在大肠杆菌中的表达[J]. J4, 2010, 36(2): 262-266.
[13]	盖晋宏, 牟特, 邢沈阳, 倪劲松, 李建华, 张西臣. RNA干扰乳腺癌MCF-7细胞HSPA8基因表达[J]. J4, 2010, 36(2): 271-275.
[14]	张勇, 刘宁, 周蔷, 李洪岩, 刘全. 衔接蛋白2基因siRNA表达载体的构建及鉴定[J]. J4, 2010, 36(2): 331-335.
[15]	吴扬, 李玉琴, 王智昊, 臧秀贤. 人纤溶酶真核表达重组质粒的构建[J]. J4, 2009, 35(5): 898-901.