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

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

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 pGL3 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。

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.[