流产布鲁菌免疫原性蛋白SSB的鉴定及其反应原性分析

Identification of Immunogenic Protein SSB of Brucella abortus and Analysis of Its Reactivity with Serum Samples

本研究通过免疫印迹试验分析流产布鲁菌感染小鼠不同阶段的血清中以及临床牛羊布病阳性血清中抗体生成规律和特点,确定布鲁菌主要免疫原性蛋白的相对区域。结果显示:布鲁菌感染小鼠2周后抗体逐渐产生,在6~8周达到顶峰,之后维持稳定状态;结合临床牛羊血清分析发现布鲁菌免疫原性蛋白主要集中在相对分子量40~70 kDa和7~20 kDa的两个区域;通过SDS-PAGE分离布鲁菌总蛋白,对照免疫印迹试验结果,切取免疫原性蛋白条带进行质谱分析,共鉴定到10个布鲁菌免疫原性蛋白;在大肠杆菌中成功表达和纯化了免疫原性蛋白SSB;免疫印迹试验显示,纯化的SSB蛋白可与临床牛布病阳性血清产生较好的反应,但检测阳性样本时也会产生阴性反应,检测效率与虎红平板凝集试验相比略差,在检测阳性样本时,与虎红平板凝集试验的符合率约为82%。该研究为布病诊断试剂研发奠定基础。

In the present study,antibody production roles were assessed in mice infected with Brucella abortus at different times.Immunogenic protein regions in Brucella were confirmed using Western blot by reacting with clinical bovine and caprine positive sera.The results showed that antibodies against Brucella were gradually produced after 2 weeks post-infection(p.i.)and remained at stable level until 22 weeks p.i.with the peak at 6-8 weeks p.i..Analysis of bovine and caprine sera showed that immunogenic proteins were mainly located in two molecular weight regions of Brucella proteins,40-70 kDa and 7-20 kDa as demonstrated in Western blot.Total Brucella proteins were separated by SDS-PAGE and the immunogenic protein bands were incised from the gels according to the results of Western blot.Total 10 immunogenic proteins were identified with mass spectrometry analysis and the SSB protein was further expressed in E.coli and purified.The purified SSB protein reacted with the majority of clinical bovine Brucella positive sera as visualized in Western blot,which indicating a slightly lower detection than Rose-Bengal plate test(RBPT)as the agreement with RBPT was about 82%for the tested Brucella-positive samples.In conclusion,the present study provided a novel research strategy for diagnosis of brucellosis.