布鲁菌核糖体蛋白L7/L12的表达纯化及生物活性鉴定  

Expression and Purification of Ribosomal L7/L12 Protein of Brucella melitensis and its Characterization of Bioactivity

作　　者：司瑞[1] 白文涛[1] 张芳琳[1] 刘艳丽[1] 胡刚[1] 吴兴安[1] 阎岩[1] 徐志凯[1] 

机构地区：[1]第四军医大学微生物学教研室,陕西西安710032

出　　处：《生物技术通讯》2006年第6期872-874,共3页Letters in Biotechnology

基　　金：军队科技攻关项目(06G091);第四军医大学科研重点项目(05XJZ006)

摘　　要：目的:原核表达系统表达布鲁菌核糖体蛋白L7/L12与GST的融合蛋白GST-L7/L12,并纯化蛋白L7/L12,建立检测特异性抗体的间接ELISA方法。方法:对含有L7/L12的原核表达载体pGEX-4T-1-L7/L12进行了原核表达。利用亲和层析柱分别纯化融合蛋白GST-L7/L12和蛋白L7/L12,并用SDS-PAGE及Western印迹分析鉴定。以L7/L12为抗原包被微量板,优化抗原包被浓度和羊抗鼠IgG-HRP稀释度,建立间接ELISA方法,并检测其特异性。结果:SDS-PAGE结果显示在相对分子质量为38000和12000处可见纯化蛋白的条带,Western印迹分析表明这2条带均能被免疫兔血清识别,表明获得了纯化的有生物活性的融合蛋白GST-L7/L12和蛋白L7/L12。间接ELISA方法的L7/L12抗原包被浓度为5μg/mL,羊抗鼠酶标二抗稀释度为1∶1000。小鼠免疫血清与L7/L12抗原出现阳性反应,而与布鲁菌融合蛋白OMP31、结核分枝杆菌抗原85b及牛血清白蛋白则呈阴性。结论:成功地对布鲁菌核糖体蛋白L7/L12进行了原核表达和纯化,以其为基础建立的间接ELISA方法稳定且特异。Objective： To obtain fusion protein GST-L7/L12 and purified ribosomal L7/L12 protein of BruceUa melitensis, and to establish indirect ELISA method for detecting antibodies against L7/L12 agent. Methods： To express the GST- L7/L12 protein in Escherichia coli. The protein was purified with affinity chromatography and analyzed by SDS-PAGE and Western-blot. Optimize suitable concentration of coating antigen and dilution of the goat-anti-mouse IgG-HRP antibody required in the indirect ELISA and detect the specificity of the method. Results： SDS-PAGE analysis showed that proteins could be visualized on gel at molecular mass about 38 kD and 12 kD and could be detected by the positive serum from immuned rabbit via Western-blot. The most suitable concentration of coating antigen was 5μg/mL and the dilution of goat-anti-mouse IgG-HRP antibody was 1：1 000. This method showed a positive reaction with positive immuned mouse serum and did not react when coated with Brucella OMP31, Mycobacterium tuberculosis 85b and BSA. Conclusion： The fusion protein GST-L7/L12 and ribosomal L7/L12 protein has been expressed and purified successfully. The iELISA method established here is stable and specific.

关 键 词：布鲁菌 核糖体蛋白L7/L12 ELISA 

分 类 号：Q786[生物学—分子生物学] Q503