豆豉纤溶酶基因的克隆及其在枯草杆菌中的表达

Cloning of Douchi Fibrinolytic Enzyme Gene and Its Expression in Bacillus subtilis

以实验室筛选的UD8豆豉芽孢杆菌的总DNA为模板,应用PCR技术根据豆豉纤溶酶基因DNA序列(GeneBank AY720895.2)设计并合成1对引物,扩增出了豆豉纤溶酶基因,将该基因克隆到大肠杆菌-枯草芽孢杆菌穿梭质粒EC-BS8上,构建成表达质粒EC-BS8,再将其转化到枯草芽孢杆菌Bs6中。筛选表达菌株,实现了豆豉纤溶酶基因在枯草杆菌中高效表达。获得重组菌纤溶酶活力高达9 830 IU.mL-1,是出发菌株活力的2.9倍。经纯化SDS-PAGE鉴定重组豆豉纤溶酶相对分子质量为31.0 kDa。

With the total DNA extracted from Bacillaceae UD8 as template,one pair of primers were designed according to DNA sequence of Douchi fibrinolytic enzyme（DFE）gene（AY720895.2）on Genbank website,to amplify DFE gene by PCR.The PCR prodcuct was inserted into expression plasmid EC-BS8,and the plasmid was transformed into the host strain Bacillus subtilis.DFE gene was successfully expressed in Bacillus subtilis and the maximum Douchi fibrinolytic enzyme level reached 9 830 IU·mL-1,which was 2.9 fold higher of the initial strain.The cloned Douchi fibrinolytic enzyme purified molecular weight was 31.0 kDa with a single band from SDS-PAGE.