藤黄微球菌纤溶酶基因在枯草芽孢杆菌WB600中的表达

Gene Expression of the Micrococcus luteus Fibrinolytic Enzyme (MLFE) in Bacillus subtilis WB600

MLFE(Micrococcus luteus fibrinolytic enzyme)是由一株新的纤溶酶产生菌藤黄微球菌ML909分泌的胞外纤溶酶。从一株解淀粉芽孢杆菌(Bacillus amyloliquefaciens)DC-4中克隆α-淀粉酶基因的启动子-信号肽序列,将其与mlfe基因(GenBank,登录号为EU232121)的成熟肽编码序列相融合,构建成融和基因amymlfe。将该基因克隆到大肠杆菌-枯草芽孢杆菌穿梭质粒pSUGV4上,构建成表达质粒pSU-AmyMLFE,再将其转化到枯草芽孢杆菌WB600中。转化子在纤维蛋白平板上可产生清晰的水解圈,24h发酵液中纤溶酶活性达到238UKU/mL;SDS-PAGE分析显示,在发酵上清液可见表达蛋白;Western印迹分析表明,表达产物的分子量大小与预期完全一致。所有结果表明,融合基因amymlfe在枯草芽孢杆菌WB600中获得了表达。

MLFE （Micrococcus luteus fibrinolytic enzyme） is a fibrinolytic enzyme produced by Micrococcus luteus ML909 strain. The promoter and signal peptide-coding sequence of α-amylase gene from Bacillus amyloliquefaciens DC-4 was cloned and fused to the sequence coding for mature peptide of MLFE （GenBank： EU232121）, forming the fusion gene called amymlfe. This hybrid gene was inserted into the Escherichia coil-Bacillus subtilis shuttle plasmid vector pSUGV4 and expression plasmid pSU-AmyMLFE was constructed, After transformation with B. subtilis WB600, transformant WB600/pSU-AmyMLFE was obtained and produced clear hydrolyzed zones on fibrin plates. The fibrinolytic activity in supernatants of WB600/pSU-AmyMLFE fermented for 24 hours was tested and found to be 238 UKU/mL. The results of SDS-PAGE analysis showed that there was indeed recombinant protein in supernatants. The Western blotting showed that the molecular weight of the expressed protein was the same as expected. These results indicate that the gene, amymlfe, is successfully expressed in B. subtilis WB600.