Kittelberger R, Diack D S, Vizcaíno N, Zygmunt M S, Cloeckaert A
Central Animal Health Laboratory, Wallaceville Animal Research Centre, Upper Hutt, New Zealand.
Vet Microbiol. 1998 Jan 16;59(2-3):213-27. doi: 10.1016/s0378-1135(97)00196-x.
A panel of 45 Brucella ovis serologically positive sera were tested in immunoblots against B. ovis outer membrane proteins Omp31 and Omp25, purified by preparative SDS-gel electrophoresis. Forty-three sera reacted with Omp31, while only 11 reacted with Omp25, suggesting that Omp31 is identical to the previously reported immuno-dominant 29-kDa protein. Attempts to purify Omp31 on a larger scale by using procedures such as ion exchange-, reversed phase-, affinity- and gel filtration chromatography suggested that the outer membrane proteins were aggregated with rough lipopolysaccharide. Only denaturing SDS-gel filtration chromatography was able to separate proteins of about 29 kDa from rough lipopolysaccharide but did not separate Omp31 from Omp25 in B. ovis preparations. When used in an enzyme-linked immunosorbent assay, this 29-kDa protein preparation was less sensitive and less specific than the routinely used heat-extracted B. ovis antigen. A readily available recombinant E. coli, expressing the gene for Omp31 from Brucella melitensis 16 M, was used to extract and enrich recombinant Omp31 by a temperature-dependent Triton X-114-based technique. When this material was used in immunoblots with the 45 sera from B. ovis-infected sheep and with 10 monoclonal antibodies, raised against B. ovis Omp31, major differences in the antibody reactivity between the recombinant B. melitensis Omp31 and the B. ovis Omp31 were found. Such differences were unexpected because of the known structural and immunological relatedness of outer membrane proteins from various Brucella species. These results indicated that the antibody-response in B. ovis naturally-infected sheep against the immuno-dominant Omp31 was directed against epitopes which were only accessible when the protein was aggregated with rough lipopolysaccharides, or which were formed after aggregation but were not present in the recombinant protein.
用通过制备性SDS凝胶电泳纯化的布鲁氏菌羊种外膜蛋白Omp31和Omp25，对一组45份布鲁氏菌羊种血清学阳性血清进行免疫印迹检测。43份血清与Omp31反应，而只有11份与Omp25反应，这表明Omp31与先前报道的免疫显性29 kDa蛋白相同。尝试通过离子交换色谱、反相色谱、亲和色谱和凝胶过滤色谱等方法大规模纯化Omp31，结果表明外膜蛋白与粗糙脂多糖聚集在一起。只有变性SDS凝胶过滤色谱能够将约29 kDa的蛋白质与粗糙脂多糖分离，但在布鲁氏菌羊种制剂中不能将Omp31与Omp25分离。当用于酶联免疫吸附测定时，这种29 kDa蛋白制剂的敏感性和特异性低于常规使用的热提取布鲁氏菌羊种抗原。利用一种基于温度依赖性Triton X-114的技术，使用一种易于获得的表达来自布鲁氏菌 melitensis 16 M的Omp31基因的重组大肠杆菌来提取和富集重组Omp31。当将该材料用于与来自感染布鲁氏菌羊种的绵羊的45份血清以及与针对布鲁氏菌羊种Omp31产生的10种单克隆抗体进行免疫印迹时，发现重组布鲁氏菌 melitensis Omp31和布鲁氏菌羊种Omp31之间的抗体反应性存在主要差异。由于已知各种布鲁氏菌属物种的外膜蛋白在结构和免疫方面具有相关性，这些差异是出乎意料的。这些结果表明，在自然感染布鲁氏菌羊种的绵羊中，针对免疫显性Omp31的抗体反应针对的表位只有在该蛋白与粗糙脂多糖聚集时才可及，或者是在聚集后形成但不存在于重组蛋白中的表位。
