大肠杆菌多重耐药调控基因rob的原核表达及其表达蛋白多克隆抗体的制备  

Prokaryotic expression of multidrug resistant regulator gene rob of Escherichia coli and preparation of the polyclonal antibody against the expressed protein

作　　者：陈立芳[1] 马红霞[1] 刘玉堂[1] 刘树明[1] 

机构地区：[1]吉林农业大学动物科学技术学院,吉林长春130118

出　　处：《中国兽医科学》2010年第5期486-489,共4页Chinese Veterinary Science

基　　金：国家自然科学基金资助项目(30400326);吉林省科技厅项目(20010538;20030425;20050124;20090239);吉林省教育厅项目(2005042)

摘　　要：提取阳性克隆质粒pMD18-T-rob,经SacⅠ+XhoⅠ双酶切,回收目的片段,定向克隆到pET-28a(+)原核表达载体中,提取阳性质粒,转化至大肠杆菌BL21(DE3)中,获得阳性克隆。经IPTG诱导阳性菌,收集表达产物,SDS-PAGE分析证实pET-28a-rob可成功地在大肠杆菌中表达出Rob蛋白。Western-blot分析证明,Rob蛋白具有良好的反应原性。表达出的Rob蛋白经侧带法纯化后免疫獭兔3次,制备抗Rob蛋白的多克隆抗体。通过间接ELISA方法检测抗体效价,结果表明抗体血清1∶40稀释时抗体效价较高。The positive clone pMD18-T-rob was digested with SacⅠ and XhoⅠ and the target gene rob was ligated into pET-28a（＋） vector.Then the plasmid pET-28a-rob was transformed into the competent cell of BL21（DE3）.The positive clone was induced with IPTG and SDS-PAGE analysis showed that the target gene was expressed successfully.Western-blot analysis indicated that the expressed protein had good reactigenicity.The purified Rob protein was injected into Rex rabbits three times and the polyclonal antibody was prepared.The D490nm value of the rabbit serum was determined by ELISA.Results showed that antibody titer of the rabbit serum diluted at 40-fold was higher.

关 键 词：大肠杆菌 rob基因 原核表达 多克隆抗体 

分 类 号：S852.612[农业科学—基础兽医学]