多序列比对在克隆表达一株未知芽孢杆菌角蛋白酶基因中的应用

为克隆表达一株未知芽孢杆菌的角蛋白酶基因,本文对芽孢杆菌属来源的13个角蛋白酶基因进行了分类比对,并设计简并引物,成功筛选出一株未知芽孢杆菌的角蛋白酶基因,在大肠杆菌中克隆表达,最后通过SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis)分析和酶活测定验证表达成功,同时还确立重组大肠杆菌BL21(pET-28a-Ker)的最佳诱导温度为20℃,经过0.5 mmol/L IPTG (isopropylthio-galactoside)诱导16 h后,粗酶液中的角蛋白酶酶活达到389.7 U/mL。本文证明了通过多序列比对而设计的角蛋白酶简并引物的有效性,且该方法能够简化角蛋白酶的研究和开发。

Application of Multi-sequence Alignment in Cloning and Expression of Keratinase Gene from an Unknown Bacillus Strain

In order to clone and express a keratinase gene from a Bacillus strain,13 keratinase genes form Bacillus was classified to do multiple sequence alignments. The keratinase gene was cloned and expressed successfully in Escherichia coli by using degenerate primers. Finally,the expression of recombinant enzyme was confirmed by SDS-PAGE(sodium dodecyl sulfate-polyacrylamide gel electrophoresis)and enzyme activity assay. Simultaneously,the results of temperature optimization experiment showed that the best induction temperature of the recombinant strain E. coli BL21(pET-28a-ker)was 20 ℃,and the activity of crude enzyme solution reached 389.7 U/mL after induction of 0.5 mmol/L IPTG for 16 h. This paper demonstrates the effectiveness of degenerate primers for keratinase designed by multiple sequence alignments,and this method can simplify the research and development of keratinase.