副结核分枝杆菌MAP1068胞外蛋白酶的特性研究  

The activity of extracellular MAP1068 protease from Mycobacterium avium subsp. Paratuberculosis

作　　者：王忠星[1,2] 党光辉 刘虹秀[2] 臧鑫鑫 崔莹莹 崔子寅[2] 宋宁宁 陈利苹[2] 刘思国[2] WANG Zhong-xing;DANG Guang-hui;LIU Hong-xiu;ZANG Xin-xin;CUI Ying-ying;CUI Zi-yin;SONG Ning-ning;CHEN Li-ping;LIU Si-guo(College of Animal Science and Technology,Jilin Agricultural University,Changchun 130118,China;State Key Laboratory of Veterinary Biotechnology,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,China)

机构地区：[1]吉林农业大学动物科学技术学院,吉林长春130118 [2]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室,黑龙江哈尔滨150069

出　　处：《中国预防兽医学报》2019年第5期505-508,共4页Chinese Journal of Preventive Veterinary Medicine

基　　金：牛羊重要疫病免疫防控新技术研究/牛羊疫苗新型佐剂及工艺研究(2017YFD0500906);黑龙江省青年科学基金项目(QC201834)

摘　　要：为研究副结核分枝杆菌(MAP)1068蛋白的特性,本研究以MAP参考株K10基因组为模板,PCR扩增其map1068基因,构建p ET28a-map1068重组载体,将其转化BL21后诱导表达并纯化MAP1068蛋白。利用纯化的重组蛋白免疫(100μg/只/0.1 m L)小鼠制备多克隆抗体,应用该抗体对MAP1068蛋白进行亚细胞定位。以酪蛋白为底物测定MAP1068蛋白的酶活性。结果显示,MAP1068蛋白以包涵体形式表达;经变性、复性、亲和层析纯化后获得目的蛋白;将该蛋白免疫小鼠后其血清抗体效价可达1∶204 800;该蛋白主要定位于MAP细胞壁,可降解酪蛋白,为胞外蛋白酶。本实验为进一步研究MAP的致病机制奠定了基础。In order to explore the characteristics of MAP1068 protein from Mycobacterium avium subsp.Paratuberculosis(MAP),this experiment used the MAP reference strain K10 genome as a template to specifically amplify the map1068 gene,which was subcloned into to the pET28 a vector after digestion.The resulting recombinant vector pET28 a-map1068 was then transformed to E.coli BL21 to induce the expression of MAP1068 protein.After purification the recombinant protein was used to immunize(100μg/0.1 mL)mice to prepare polyclonal antibody against MAP1068 protein,and applied the antibody for subcellular localization.The enzymatic activity of the MAP1068 protein was assayed using casein as the substrate.The results showed that MAP1068 protein was expressed in the form of inclusion bodies,and target protein was obtained by denaturing renaturation and affinity chromatography.After immunized mice,1∶204,800 high titer antibody was obtained.The protein is mainly localized on the cell wall and it is an extracellular protein possessing the enzymatic activity of casein degradation.The results of this experiment lay the foundation for further study of the pathogenic mechanism of MAP.

关 键 词：副结核分枝杆菌 MAP1068 特性 酶活性 

分 类 号：S852.61[农业科学—基础兽医学]