摘要
目的 构建一个鼠碳酸酐酶ⅦcDNA亚克隆 ,用于研究该酶结构与功能的关系。方法 设计两个引物 ,用PCR方法扩增缺失N端 2 3个氨基酸残基的mcA7cDNA片断 ,将扩增的目的基因克隆到 pGEM T载体 ,再用Kunkel方法突变 72 4位的NdeI位点 ,突变后的mCA7b’片断插入表达载体 pET3 1f1(+)。结果与结论 构建了pET3 1f1mcA7b’亚克隆。该克隆顺序正确 ,可高效表达N端缺失 2 3个氨基酸残基的mcA7突变体 ,并适用于mcA7b’基因的点突变研究。
Objective A subolone of Murine Carbonic Anhydrase Ⅶ(mcA7) was constructed to study structure function relationship in CA7. The products of PCR rection were cloned into pGEM T. Two positive clones were then mutated using kunkel. Methods to remove NdeI Site at position 724 in the mCA7 cDNA. The mutated inserts were removed from pGEM T and inserted into pET 31 f 1(+) expression vector. Results and Conclusions pET 31 f 1mCA 7b' contained the correct sequence and allowed efficient high level expression truncation mutant of mCA 7, in which 23 residues from the amine terminal end were deleted and eptimized site directed mutagenisis.
出处
《合肥医学院学报》
2000年第3期198-200,共3页
Journal of Zunyi Medical University
