地衣芽孢杆菌2709碱性蛋白酶编码序列的PCR扩增、克隆及序列测定

在地衣芽孢杆菌NCIB 6816菌株碱性蛋白酶基因已知序列的基础上,通过设计合适的引物,利用PCR(Polymerase Chain Reaction)技术从地衣芽孢杆菌2709菌株的柒色体DNA中扩增了2709碱性蛋白酶的编码序列。对两个克隆的PCR片段的全序列分析结果显示,2709碱性蛋白酶的编码序列同相应的NCIB 6816序列相比有3％左右的碱基组成差异。由此推定的2709碱性蛋白酶的氨基酸序列肯定了2709碱性蛋白酶属典型的subtilisin Carlsberg类,同时还表明来源于不同地衣芽孢杆菌菌株的subtilisin Carlsberg存在着若干氨基酸组成上的差异。

PCR Amplifying, Cloning and Sequencing of the Coding Sequence of the Alkaline Protease Gene from Bacillus licheniformis 2709

The coding sequence of the alkaline protease gene from Bacillus licheniformis 2709 has been amplified in vitro using the polymerase chain reaction(PCR) technique, with the B. licheniformis 2709 chromosomal DNA,as template and two PCR primers which have been synthesized according to the published sequence data of the subtilisin Carlsberg gene from B. licheniformis NCIB 6816. The amplified fragment was cloned into the pUC vector and its complete sequence was obtained by sequencing two of these clones. The result of sequencing reveals that approximately 3% of the total nucleotides in the coding region of 2709 alkaline protease gene is different from that of the corresponding NCIB 6816 protease gene. The deduced amino acid sequence implies that 2709 alkaline protease is a typical subtilisin Carlsberg, and that subtilisin Carlsberg proteins from various B. licheniformis strains differ from each other slightly in amino acid composition.