牛乳源性金黄色葡萄球菌nEBPS蛋白表达鉴定及多克隆抗体制备  

Expression and characterization of N-terminal elastin binding protein of Staphylococcus aureus and preparation of its polyclonal antibody

作　　者：布日额[1,2,3] 任晓峰[2] 吴金花[1,3] 王学理[3,4] 刘燕[1,3] 锡林高娃[1,3] 孙立杰[1,3] 刘洋[4] 

机构地区：[1]内蒙古民族大学生命科学学院,内蒙古通辽028043 [2]东北农业大学动物医学院,黑龙江哈尔滨150000 [3]内蒙古民族大学乳源性致病菌研究所,内蒙古通辽028043 [4]内蒙古民族大学动物科技学院,内蒙古通辽028043

出　　处：《中国兽医学报》2013年第11期1674-1678,共5页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(31060352);内蒙古自治区科技厅科技创新引导计划资助项目(20111802[2012])

摘　　要：利用PCR方法扩增乳源致病性金黄色葡萄球菌nEBPS全基因,将其定向克隆至原核表达载体pET30a(+)中,鉴定正确后,转化BL21表达菌,经IPTG诱导获得了以可溶性表达的重组蛋白。通过Ni NTA Purification System纯化重组蛋白,纯化蛋白质量浓度为2.16g/L。纯化蛋白经免疫印迹检测显示重组蛋白能够被牛源金黄色葡萄球菌阳性血清识别,具有良好的反应性。将纯化的重组蛋白免疫新西兰白兔制备多克隆抗体,间接ELISA测定抗体效价为1∶25 600,凝集试验测定抗体效价为1∶128。To expression of N-terminal elastin binding proteins of Staphylococcus aureus （nEBPS） and prepare polyclonal antibody, the nEBPS was amplified by PCR and directionally cloned into the pET30a vector. After characterization, the recombinant plasmid was transformed into E. coli BL21（DE3）,the recombinant nEBPS protein was expressed in soluble body form after induction with IPTG. The concentration of the purified recombinant protein was 2.16 g/L. Western blotting showed that purified protein could react specifically to Staphylococcus aureus positive serum. The polyclonal antibodies to the nEBPS protein was prepared by inoculating rabbits with purified re combinant protein. The polyclonal antibodies was generated with antibody titres of 1 ： 25 600 in ELISA and 1 ： 128 in agglutination test. The recombinant protein and the polyclonal antibody ob- tained in this study could be used for detection Staphylococcus aureus,and could be used to fur ther study the structure,function and epitope mapping of nEBPS.

关 键 词：金黄色葡萄球菌 弹性纤维结合蛋白N端蛋白 原核表达 多克隆抗体 

分 类 号：S852.611[农业科学—基础兽医学]