上海交通大学学报（医学版）

›› 2013, Vol. 33 ›› Issue (4): 404-.doi: 10.3969/j.issn.1674-8115.2013.04.005

• 论著（基础研究） • 上一篇    下一篇

GAL4/VP16融合人工转录因子的活性研究

张 萍1, 应 磊1, 钱关祥1, 徐 让2   

  1. 1.上海交通大学 基础医学院生物化学与分子细胞生物学系, 上海 200025； 2.上海交通大学 医学院附属新华医院科研中心, 上海 200092
  • 出版日期:2013-04-28 发布日期:2013-05-03
  • 通讯作者: 徐 让, 电子信箱: rang_xu@yahoo.com.cn。
  • 作者简介:张 萍（1976—）, 女, 博士生, 副教授； 电子信箱: ssmuzp@yahoo.com.cn。
  • 基金资助:

    国家自然科学基金项目（81070135）

Study on activity of GAL4/VP16 fusion artificial transcription factor

ZHANG Ping1, YING Lei1, QIAN Guan-xiang1, XU Rang2   

  1. 1.Department of Biochemistry and Molecular Cellular Biology, Basic Medical College, Shanghai Jiaotong University, Shanghai 200025, China； 2.Scientific Research Center, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200092, China
  • Online:2013-04-28 Published:2013-05-03
  • Supported by:

    National Natural Science Foundation of China, 81070135

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摘要：

目的 构建含有融合转录因子GAL4/VP16的表达载体和含有上游激活序列（UAS）启动子驱动报告基因增强型绿色荧光蛋白（EGFP）的表达载体组成的GAL4/VP16-UAS系统，观察融合转录因子GAL4/VP16的活性。方法 构建含有融合转录因子GAL4/VP16的表达载体pcDNA3-GAL4/VP16，含有5个拷贝UAS串联排列序列和腺病毒E1b基因核心启动子的人工杂合启动子驱动报告基因EGFP的表达载体pGL3-G5E1b-TATA-EGFP，利用脂质体将两种载体瞬时转染Hep3B和HEK293细胞，48 h后采用RT-PCR和流式细胞术检测EGFP的表达，观察GAL4/VP16融合转录因子对G5E1B-TATA的转录调节作用。结果 在GAL4/VP16融合转录因子存在的情况下，G5E1b-TATA启动子活性明显上调，甚至可以达到巨细胞病毒（CMV）启动子的活性程度。结论 GAL4/VP16融合转录因子具有上调G5E1B-TATA启动子转录活性的作用，并且该转录活性足以达到驱动下游目的基因高效表达的水平，在基因治疗和基因功能的研究中具有一定的应用潜力。

关键词: GAL4/VP16, 人工转录因子, 增强型绿色荧光蛋白

Abstract:

Objective To construct GAL4/VP16-up-stream activating sequence (UAS) system, which is composed of expression vector containing GAL4/VP16 fusion transcriptional factor and expression vector containing UAS promoter driving reporter gene enhanced green fluorescent protein (EGFP), and evaluate the activity of GAL4/VP16 fusion transcriptional factor. Methods The expression vector pcDNA3-GAL4/VP16 containing GAL4/VP16 fusion transcriptional factor was constructed, and the expression vector pGL3-G5E1b-TATA-EGFP was also constructed, in which the reporter gene EGFP was driven by G5E1b-TATA promoter consisted of five tandem-repeat copies of UAS and E1b core promoter. Hep3B and HEK 293 cells were transiently transfected by these two vectors. After 48 h, the expression of EGFP was determined by RT-PCR and flow cytometry, and the effect of GAL4/VP16 fusion transcriptional factor on the G5E1b-TATA promoter activity was observed. Results The activity of G5E1b-TATA promoter was significantly up-regulated by GAL4/VP16 fusion transcriptional factor, and might even reach the level of cytomegalovirus (CMV) promoter. Conclusion In transcriptional level, the GAL4/VP16 fusion transcriptional factor can significantly up-regulate the activity of G5E1b-TATA promoter, and the G5E1b-TATA promoter can drive the efficient expression of downstream target genes in the presence of GAL4/VP16 protein. It is expected to make a valuable contribution in the study of gene function and gene therapy.

Key words: GAL4/VP16, artificial transcription factor, enhanced green fluorescent protein