过氧化氢酶基因定向进化文库的构建及其评价

Construction and Evaluation of a Directed Evolution Library of Catalase

采用易错聚合酶链反应和DNA改组技术构建野生型梅花鹿过氧化氢酶(CAT)基因的突变体文库,并随机对两种方法所得产物各5个样品做序列测定。序列分析结果表明突变率分别为0.329%和27.58%,易错聚合酶链反应体系的错配率可以比普通PCR体系提高约10倍,DNA改组的突变率则更高,但是难以避免由于突变率太高造成的目的基因无法正确翻译这一情况。另外,应用邻接法(neighbor-joining,NJ)对随机选择的过氧化氢酶基因突变体序列和野生型序列做核酸和蛋白质序列的NJ进化树,进化关系与突变率分析基本一致。

The directed evolution of wild-type catalase from Cervus nippon was studied,which mimicked evolution on a laboratory timescale.The mutant library of catalase gene was constructed using error-prone PCR and DNA shuffling,and its sequencing result showed mutant rate to be 0.329% and 27.58%.The mutant rate using error-prone PCR was 10 fold higher than normal PCR,while the mutant rate using DNA shuffling was much more higher.However,tremendous mutant rate may affect or even disable gene transcription and translation.A suitable mutant rate is needed to guarantee fluent gene expression.Furthermore,both wild-type and mutant catalase sequences selected randomly were analysed to obtain their Neighbor-Joining phylogenetic trees.