癌变·畸变·突变 ›› 2003, Vol. 15 ›› Issue (1): 10-12.doi: 10.3969/j.issn.1004-616x.2003.01.003

• 论著 • 上一篇    下一篇

通过定点诱变构建带有突变的载脂蛋白CⅡ启动子的表达载体

陈春华1, 曹英林1, 胡维诚2, 赵　丽1   

  1. 1. 山东大学医学院免疫学研究所， 山东　济南　250012；　2. 山东大学医学院病理与病理生理学研究所， 山东　济南　250012
  • 收稿日期:2002-07-23 修回日期:2002-09-28 出版日期:2003-01-30 发布日期:2003-01-30
  • 通讯作者: 曹英林

CONSTRUCTION OF THE EXPRESSION VECTOR WITH THE MUTANT APOCⅡ PROMOTER USING SITE- DIRECTED MUTAGENESIS

CHEN Chun- hua 1, CAO Ying- lin 1, HU Wei- cheng 2, ZHAO li 1   

  1. 1. Institute of Immunology; 2. Institute of Pathology and Pathophysiology, School of Medicine, Shandong University, Jinan 250012 , China
  • Received:2002-07-23 Revised:2002-09-28 Online:2003-01-30 Published:2003-01-30
  • Contact: CAO Ying- lin

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摘要： 目的： 为研究载脂蛋白CⅡ启动子- 190处的碱基突变对其转录活性的影响，构建带有突变的载脂蛋白CⅡ启动子的表达载体。 方法： 以插入有正常ApoCⅡ启动子的质粒pGL 3为模板，设计一对带有预期突变的完全互补的引物，利用Stratagene的定点诱变试剂盒对ApoCⅡ启动子- 190处的碱基进行突变，扩增出带有缺刻的突变质粒，转化入XL- Blue超感受态细胞中修复缺刻。 结果： 成功地把ApoCⅡ启动子- 190处的碱基由T突变为A，构建了带有突变的ApoCⅡ启动子的表达载体。 结论： 用一种快速、高效的定点诱变方法，获得了带有预期突变的环状质粒，为进一步检测突变的载脂蛋白CⅡ启动子的活性奠定了基础。

关键词: 载脂蛋白CⅡ, 定点诱变, 启动子

Abstract: Purpose: To construct the expression vector with the mutant apolipoprotein CⅡ(apoCⅡ) promoter for studying the transcript activity of the mutant apoCⅡ promoter on - 190 base pair. Methods: The plasmid pGL 3 with normal apoCⅡ promoter was used as the template, and a pair of completely complementary primers with the desired mutation was designed, which make the plasmid amplified and mutated on - 190 base pair of the apoCⅡ promoter using Stratagene's site- directed mutagenesis kit. After being transformed into XL- Blue supercompetent cells, the nicked mutant plasmid was repaired in these cells. Results: The normal apoCⅡ promoter was successfully changed from T to A on the - 190 base pair, the expression vector with the mutant apoCⅡ promoter was constructed. Conclusion: The whole circled plasmid with the desired mutation was obtained by a rapid and efficient method for site- directed mutagenesis, which has an implication for detecting the activity of the mutant apoCⅡ promoter.

Key words: Apolipoprotein CⅡ, Site- directed mutation, Promoter

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