金黄色葡萄球菌肠毒素A在枯草芽孢杆菌中的表达及其活性分析  

Expression and activity of Staphylococcus aureus enterotoxin A in Bacillus subtilis

作　　者：韩佳丽[1,2] 李昌[2] 沈国顺[1] 胡博[2] 李霄[2] 田明尧[2] 孙珊珊[1] 叶明[3] 金宁一[2] 

机构地区：[1]沈阳农业大学畜牧兽医学院,辽宁沈阳110161 [2]军事医学科学院军事兽医研究所,吉林长春130062 [3]延边大学农学院,吉林龙井133400

出　　处：《中国兽医学报》2010年第3期327-331,共5页Chinese Journal of Veterinary Science

基　　金：科技部863计划(2006AA02Z447);军内十一五科技攻关项目(06G127);长春市科技局重点项目(2007KZ19)

摘　　要：利用优化后的SEA基因片段与带有枯草芽孢杆菌启动子的表达载体pBC38c,成功构建了含有SEA基因的重组表达质粒pBC38c-SEA。通过电转化的方法,将重组质粒pBC38c-SEA转入枯草芽孢杆菌1A751中。对目的蛋白进行表达,采用硫酸铵沉淀法浓缩上清中的蛋白,超声波裂解法裂解菌体沉淀,SDS-PAGE凝胶电泳法检测蛋白表达情况,Western-blot对表达的目的蛋白进行活性分析,并对表达条件与硫酸铵浓度进行优化。结果表明,成功构建了枯草芽孢杆菌表达质粒pBC38c-SEA,并在枯草芽孢杆菌中获得了表达,目的蛋白存在于培养基上清中,且具有抗原性。随着培养时间的延长,目的蛋白表达产量增多,在35h时达到最多,同时硫酸铵终浓度为50%,目的蛋白得到最好沉淀。本试验结果表明,SEA基因得以分泌表达,为进一步研究其生物活性并作为佐剂在临床中应用奠定了基础。The recombinant plasmid pBC38c-SEA containing gene fragment of Staphylococcal enterotoxin A（SEA）was constructed by the expression vector pBC38c with the promoter of Bacillus subtilis.Then the recombinant plasmid pBC38c-SEA was transformed into Bacillus subtilis 1A751 by electrotransformation.For the purpose of protein expression,supernatant protein was concentrated by ammonium sulfate precipitation and bacterial precipitation was lysised by sonic disruption.SDS-PAGE gel electrophoresis was used to detect protein expression,and then the activity of the interest protein was analyed by Western-blot.Finally,expression conditions and concentration of ammonium sulfate were optimized.The results showed that the expression plasmid pBC38c-SEA was successfully constructed and expressed in Bacillus subtilis 1A751,and target protein was secreted in the culture medium supernatant and had antigenicity.As the cultivation time extended,the target protein secreted in the culture was increased,and the highest expression product was detected at 35 h.And the optimization of final concentration of ammonium sulfate was 50%.Result indicated that SEA gene was a secretory expression,suggesting that SEA should be of great value for further investigation of immunogenic adjuvant in the clinical application.

关 键 词：金黄色葡萄球菌肠毒素A 枯草芽孢杆菌 分泌表达 

分 类 号：S852.61[农业科学—基础兽医学]