重组枯草芽孢杆菌猪尿酸氧化酶基因工程菌的构建和分泌表达  

Construction of recombinant Bacillus subtilis for secreted expression of the porcine urate oxidase

作　　者：李辉[1] 陈思[1] 马娇颖[1] 戴君[1] 谢光蓉[1] 陈建华[1] 

机构地区：[1]中国药科大学生命科学与技术学院,南京210009

出　　处：《中国医药生物技术》2012年第3期187-190,共4页Chinese Medicinal Biotechnology

基　　金：国家自然科学基金(81072560)

摘　　要：目的构建重组枯草芽孢杆菌猪尿酸氧化酶基因工程菌及其目的蛋白的分泌表达,并对其酶活性进行测定。方法从pET-22b/pUOX重组质粒上PCR获得pUOX基因,然后通过重叠延伸PCR技术将pUOX与一段nprB信号肽基因相连,导入穿梭载体pP43X,构建重组载体pP43X/pUOX,运用化学转化法转化枯草芽孢杆菌WB800中。结果发酵表达后胞外分泌液经SDS-PAGE检测,在约34kD处有一清晰蛋白条带,这与预期分子量一致。发酵液中该重组蛋白的酶活力达2.322U/ml。结论成功构建重组枯草芽孢杆菌猪尿酸氧化酶基因工程菌,目的蛋白分泌表达并具有较高活性。Objective Construction of recombinant Bacillus subtilis for secreted expression of the porcine urate oxidase （pUOX）. Methods pUOX-encoding gene pUOX was placed under the control of the P43 promoter and Bacillus subtilis signal peptide-encoding sequence. The pUOX was connected with a nprB signal peptide by SOE-PCR, then imported the shuttle vector pP43X. High-level expression and secretion of mature, authentic, and stable pUOX were achieved using the protease-deficient strain B. subtilis WB800 as the host. Results SDS-PAGE detected shows a clear protein band of about 34 kD, which is consistent with the expected molecular weight. Determination of enzyme activity of the recombinant protein is 2.322 U/ml. Conclusions Construction of recombinant Bacillus subtilis for secreted expression of the porcine urate oxidase is successful. Porcine urate oxisase can be expressed with a high activity.

关 键 词：芽孢杆菌 枯草 尿酸氧化酶 高尿酸血症 分泌表达 

分 类 号：R378[医药卫生—病原生物学]