Cloeckaert A, Verger J M, Grayon M, Zygmunt M S, Grépinet O
Laboratoire de Pathologie Infectieuse et Immunologie, Institut National de la Recherche Agronomique, France.
Infect Immun. 1996 Jun;64(6):2047-55. doi: 10.1128/iai.64.6.2047-2055.1996.
The nucleotide sequences encoding the major 25-kDa outer membrane protein (OMP) (omp25 genes) of Brucella ovis 63/290, Brucella melitensis 16M, Brucella suis 1330, Brucella canis RM6/66, and Brucella neotomae 5K33 (all reference strains) were determined and compared with that of Brucella abortus 544 (P. de Wergifosse, P. Lintermans, J. N. Limet, and A. Cloeckaert, J. Bacteriol. 177:1911-1914, 1995). The major difference found was between the omp25 gene of B. ovis and those of the other Brucella species; the B. ovis gene had a 36-bp deletion located at the 3' end of the gene. The corresponding regions of other Brucella species contain two 8-bp direct repeats and two 4-bp inverted repeats, which could have been involved in the genesis of the deletion. The mechanism responsible for the genesis of the deletion appears to be related to the "slipped mispairing" mechanism described in the literature. Expression of the 25-kDa outer membrane protein (Omp25) in Brucella spp. or expression from the cloned omp25 gene in Escherichia coli cells was studied with a panel of anti-Omp25 monoclonal antibodies (MAbs). As shown by enzyme-linked immunosorbent assay (ELISA) and immunoelectron microscopy, Omp25 was exported to the outer membrane in E. coli expressing either the truncated omp25 gene of B. ovis or the entire omp25 genes of the other Brucella species. Size and antigenic shifts due to the 36-bp deletion were demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting and by the differences in binding patterns in ELISA of the anti-Omp25 MAbs at the cell surface of E. coli cells harboring the appropriate gene and of cells of B. ovis and other Brucella species. In particular, MAbs directed against discontinuous epitopes of the entire Omp25 showed the absence of, or a significant reduction in, antibody reactivity with the B. ovis truncated Omp25. The results indicated that, as defined by the MAbs, exported Omp25 probably presents similar topologies in the outer membranes of E. coli and Brucella spp. and that the short deletion found in the omp25 gene of B. ovis has important consequences for the expression of surface B-cell epitopes which should be considered for the development of vaccines against B. ovis infection.
测定了绵羊种布鲁氏菌63/290、羊种布鲁氏菌16M、猪种布鲁氏菌1330、犬种布鲁氏菌RM6/66和新墨西哥州布鲁氏菌5K33（均为参考菌株）编码主要25 kDa外膜蛋白（OMP）（omp25基因）的核苷酸序列，并与流产布鲁氏菌544的相应序列进行了比较（P. de Wergifosse、P. Lintermans、J. N. Limet和A. Cloeckaert，《细菌学杂志》177:1911 - 1914，1995年）。发现的主要差异存在于绵羊种布鲁氏菌的omp25基因与其他布鲁氏菌物种的该基因之间；绵羊种布鲁氏菌的基因在基因3'端有一个36 bp的缺失。其他布鲁氏菌物种的相应区域包含两个8 bp的正向重复序列和两个4 bp的反向重复序列，这可能与该缺失的发生有关。导致该缺失发生的机制似乎与文献中描述的“滑动错配”机制有关。利用一组抗Omp25单克隆抗体（MAb）研究了25 kDa外膜蛋白（Omp25）在布鲁氏菌属中的表达情况，以及从克隆的omp25基因在大肠杆菌细胞中的表达情况。如酶联免疫吸附测定（ELISA）和免疫电子显微镜所示，Omp25在表达绵羊种布鲁氏菌截短的omp25基因或其他布鲁氏菌物种完整omp25基因的大肠杆菌中被转运至外膜。通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳和免疫印迹，以及在含有相应基因的大肠杆菌细胞、绵羊种布鲁氏菌细胞和其他布鲁氏菌物种细胞表面，抗Omp25单克隆抗体在ELISA中的结合模式差异，证实了由于36 bp缺失导致的大小和抗原性变化。特别是，针对完整Omp25不连续表位的单克隆抗体显示，与绵羊种布鲁氏菌截短的Omp25的抗体反应性缺失或显著降低。结果表明，根据单克隆抗体的定义，输出的Omp25可能在大肠杆菌和布鲁氏菌属的外膜中呈现相似的拓扑结构，并且在绵羊种布鲁氏菌omp25基因中发现的短缺失对表面B细胞表位的表达具有重要影响，在开发针对绵羊种布鲁氏菌感染的疫苗时应予以考虑。
