XJT-9503高温中性蛋白酶基因Gp1的克隆、表达及序列分析

Coloning,Expressing and Sequence Analysis of the Gene Gp1 of 27K Thermophilic Neutral Protease from Bacilius Licheniformis XJT-9503

试验根据地衣芽孢杆菌XJT9503的高温中性蛋白酶酶蛋白所测定的N-端及部分肽段氨基酸序列及质谱分析结果设计了PCR引物,用PCR方法从地衣芽孢杆菌XJT9503中扩增了高温中性蛋白酶基因Gp1,对所获得的基因进行序列分析测定,并与蛋白酶的氨基酸序列分析对比。GP1基因全序列共1129bp,包括整个阅读框,共编码376个氨基酸。将扩增的DNA片段插入到大肠杆菌载体pET-28a中,构建成重组分泌型表达载体pGp1。并在大肠杆菌宿主BL21中得到表达。SDS-PAGE分析显示产物的分子量为27.0×103,同核酸序列测定所推导的值相符。同时,对地衣芽孢杆菌XJT9503中性蛋白酶基因序列进行了测定和比较分析,发现与地衣芽孢杆菌6816丝氨酸蛋白酶和地衣芽孢杆菌1411T角蛋白水解酶基因的同源性为97%。

Thermophilic neutral protease genes Gp1 was amplified from Bacillus licheniformis XJT9503 by using PCR technique. The PCR primers used is designed by N-terminal amino acid sequence and mass spectrum analysis of the 27K protease from the Bacillus licheniformis XJT9503. The gene was contained 1129 nucleotide with 376 amino acids. The amplified DNA fragments were inserted into an E. coli vector pET-28a to yield the recombinant secrete plasmid pGp1 . The Gp1 in pET-28 was expressed in E. coli BL21. SDS-PAGE analysis showed that the molecular weight of the expressed recombinant products was about 27.0 10^3 kda, which was corresponded with prediction by gene sequenc. Neutral protease gene from B. licheniformis XJT9503 was compared with that of B. licheniformis and the result showed that there was 96% identity for the amino acid sequence of the gene with B. licheniformis 6816 SerA protease and B. licheniformis 1411T KerA orotease.