解淀粉芽孢杆菌中性蛋白酶基因的克隆与表达

摘要

To obtain the efficient neutral protease gene,the neutral protease gene(npr)was amplifier from Bacillus amyloliquefaciens by using PCR technology. After the double enzyme by BamHⅠand SalⅠ,the gene was linked with the express vecter PET-28a and transformed into Escherichia coliBL21. The gene expression product of gene engineering bacteria was analyzed by SDS-PAGE electrophoresis. The length of the amplified specific gene sequences was 1 566 bp,and the gene encoded 522 amino acids and contained the complete ORF. The sequence homology of the gene reached 100%,and the protein molecular weight was 57.4 kDa. By IPTG induction,the neutral protease gene was expressed highly in recombinant strain. By changing the IPTG induction factors such as concentration,temperature and induction time,the best induction conditions were additive IPTG 0.8 mmol·L-1,temperature 30℃and induction 4 h,and the highest enzyme activity was 450 U·mL-1.%为了获得高效的中性蛋白酶基因，试验用PCR方法从解淀粉芽孢杆菌中扩增中性蛋白酶基因（npr），通过BamHⅠ、SalⅠ双酶切后，将该基因与表达载体PET-28a连接，同时转入到大肠杆菌BL21中表达，通过SDS-PAGE电泳技术对构建的工程菌的基因表达产物进行分析。获得特异 npr基因序列含1566 bp，编码522个氨基酸，含有完整的ORF，同源性达到100%。蛋白酶的分子量为57.4 kDa。IPTG能诱导中性蛋白酶基因在重组菌进行表达，通过改变IPTG诱导浓度、诱导温度及诱导时间等因素，得出在添加剂IPTG至终浓度为0.8 mmol·L-1，30℃诱导4 h为最佳诱导条件，获得最高酶活为450 U·mL-1。