Brooks-Worrell B M, Splitter G A
Department of Animal Health and Biomedical Sciences, University of Wisconsin, Madison 53706.
Infect Immun. 1992 Jun;60(6):2459-64. doi: 10.1128/iai.60.6.2459-2464.1992.
Cellular immune responses are influential for protection against intracellular bacteria such as brucellae. Therefore, identification of Brucella abortus antigens that activate primed bovine lymphocytes is fundamental for discerning the breadth of cellular response in bovine brucellosis. Potentially antigenic components of B. abortus S19 were isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by nitrocellulose blotting. Specific one-dimensional blot segments induced proliferation of peripheral blood lymphocytes from all 25 of the vaccinated cattle tested and were defined as immunodominant. Individual proteins that stimulated lymphocyte proliferation were further characterized by two-dimensional cellular immunoblotting by two different approaches. Individual one-dimensional stimulatory blot segments were eluted, concentrated, and then subjected to two-dimensional cellular immunoblotting. Alternatively, entire two-dimensional gels containing all of the B. abortus components were blotted and nitrocellulose sections containing individual proteins were assayed for lymphocyte activation. Thirty-eight Brucella proteins that induced lymphocyte proliferation were resolved by both procedures. Phenotypic analysis of the proliferating cell population demonstrated the presence of CD4+, CD8+, and immunoglobulin M+ lymphocytes. Two immunogenic proteins, 12 and 31 kDa, identified by two-dimensional cellular immunoblotting, were subjected to partial N-terminal amino acid analysis. The 12-kDa protein was within the area of greatest lymphocyte proliferation, while the 31-kDa protein was chosen for comparison with a 31-kDa protein previously reported by others. A search of the National Biomedical Research Foundation protein data bank showed that the sequences were not homologous with other known proteins. Identification of Brucella proteins immunogenic for bovine lymphocytes provides an important step in distinguishing the various proteins involved in pathogenicity and/or disease resistance.
细胞免疫反应对于抵抗细胞内细菌（如布鲁氏菌）具有重要作用。因此，鉴定能激活经致敏的牛淋巴细胞的流产布鲁氏菌抗原，对于了解牛布鲁氏菌病细胞反应的广度至关重要。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳，随后进行硝酸纤维素膜印迹，分离出了流产布鲁氏菌S19潜在的抗原成分。特定的一维印迹片段能诱导所有25头受试接种牛的外周血淋巴细胞增殖，这些片段被定义为免疫显性片段。通过两种不同方法的二维细胞免疫印迹，进一步对刺激淋巴细胞增殖的单个蛋白质进行了表征。将单个一维刺激印迹片段洗脱、浓缩，然后进行二维细胞免疫印迹。或者，对包含所有流产布鲁氏菌成分的完整二维凝胶进行印迹，对含有单个蛋白质的硝酸纤维素膜切片进行淋巴细胞激活检测。两种方法都鉴定出了38种能诱导淋巴细胞增殖的布鲁氏菌蛋白质。对增殖细胞群体的表型分析表明存在CD4 +、CD8 +和免疫球蛋白M +淋巴细胞。通过二维细胞免疫印迹鉴定出的两种免疫原性蛋白质，12 kDa和31 kDa，进行了部分N端氨基酸分析。12 kDa蛋白质位于淋巴细胞增殖最显著的区域，而选择31 kDa蛋白质与其他人先前报道的一种31 kDa蛋白质进行比较。检索国家生物医学研究基金会蛋白质数据库发现，这些序列与其他已知蛋白质无同源性。鉴定对牛淋巴细胞具有免疫原性的布鲁氏菌蛋白质，是区分参与致病性和/或抗病性的各种蛋白质的重要一步。
