DEMONSTRATION OF PEPTIDOGLYCAN-ASSOCIATED BRUCELLA OUTER-MEMBRANE PROTEINS BY USE OF MONOCLONAL-ANTIBODIES
DEMONSTRATION OF PEPTIDOGLYCAN-ASSOCIATED BRUCELLA OUTER-MEMBRANE PROTEINS BY USE OF MONOCLONAL-ANTIBODIES
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DOI：
10.1099/00221287-138-7-1543
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发表时间：
1992-07-01
期刊：
影响因子：
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通讯作者：
LIMET, JN
中科院分区：
文献类型：
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作者：
CLOECKAERT, A;ZYGMUNT, MS;LIMET, JN
产生的单克隆抗体 (3D6) 仅与超声处理后经过溶菌酶处理的布鲁氏菌超声处理细胞提取物发生反应。在蛋白质印迹中，单克隆抗体 (mAb) 与 B. melitensis B115 的三种主要外膜蛋白 (OMP) 发生反应。经溶菌酶处理的大肠杆菌和小肠结肠炎耶尔森氏菌超声处理的细胞提取物中存在大量 12 至 43 kDa 的反应带。在乳胶凝集抑制免疫测定中，mAb 3D6 与 B. melitensis B115 的纯化肽聚糖 (PG) 表现出比与大肠杆菌更好的反应性。该 mAb 还用于免疫金电子显微镜观察整个布鲁氏菌细胞和切片。在整个细胞上没有观察到结合，并且在靠近外膜、周质空间和细胞质的切片中观察到免疫金标记。这些发现表明 mAb 3D6 对 PG 亚基具有特异性。使用分子量为 10、16.5、19、25-27、31-34、36-38 和 89 kDa 的布鲁氏菌 OMP 特异性单克隆抗体，对 SDS-PAGE 后的羊种布鲁氏菌 B115 粗超声处理细胞提取物进行免疫印迹分析，针对 PG 和粗脂多糖 (R-LPS) 以及平滑脂多糖 (R-LPS)脂多糖（S-LPS）。 25-27、31-34 和 36-38 kDa OMP 特异性的 mAb 与 3 至 6 个条带发生反应。除最低分子量带外，所有这些都与 PG 特异性 mAb 反应，而不与 R-LPS 和 S-LPS 特异性 mAb 反应。因此，我们认为主要 OMP（25-27,31-34 和 36-38 kDa）的表观分子量的变化是由于 PG 亚基残基数量的变化而不是附着的 R-LPS 或 S-LPS 分子造成的。我们的结果表明主要 OMP（25-27、31-34 和 36-38 kDa）与 PG 存在非常强的、可能是共价的相互作用。没有证据表明次要 OMP（10、16.5、19 和 89 kDa）与 PG 相关，因为这些蛋白质特异性的 mAb 与单条带发生反应，而与抗 PG mAb 反应时这些条带并不明显。此外，溶菌酶处理不影响这些蛋白质的电泳迁移率。
A monoclonal antibody (3D6) was produced which reacted only with Brucella sonicated cell extracts that had been lysozyme-treated after sonication. The monoclonal antibody (mAb) reacted with the three major outer-membrane proteins (OMPs) of B. melitensis B115 in Western blots. A large number of reactive bands ranging from 12 to 43 kDa were present in lysozyme-treated Escherichia coli and Yersinia enterocolitica sonicated cell extracts. In a latex agglutination inhibition immunoassay, mAb 3D6 showed better reactivity with purified peptidoglycan (PG) of B. melitensis B115 than with that of Escherichia coli. This mAb was also used in immunogold electron microscopy with whole Brucella cells and sections. No binding was observed on whole cells and immunogold labelling in sections was observed close to the outer membrane, in the periplasmic space and in the cytoplasm. These findings indicate that mAb 3D6 is specific for PG subunits. Immunoblot analysis of B. melitensis B115 rough sonicated cell extracts after SDS-PAGE, with or without lysozyme treatment, was performed using mAbs specific for Brucella OMPs of molecular masses of 10, 16.5, 19, 25-27, 31-34, 36-38 and 89 kDa, for PG and for rough lipopolysaccharide (R-LPS) and smooth lipopolysaccharide (S-LPS). mAbs specific for the 25-27, 31-34 and 36-38 kDa OMPs reacted with three to six bands. All of them except the band of lowest molecular mass reacted with the PG-specific mAb and not with R-LPS- and S-LPS-specific mAbs. Therefore we propose that variation in the apparent molecular mass of the major OMPs (25-27,31-34, and 36-38 kDa) is due to varying numbers of PG subunit residues rather than attached R-LPS or S-LPS molecules. Our results suggest a very strong, possibly covalent, interaction of the major OMPs (25-27, 31-34 and 36-38 kDa) with PG. There is no evidence of association of the minor OMPs (10, 16.5, 19 and 89 kDa) with PG, since mAbs specific for these proteins reacted with single bands that were not apparent when reacted with the anti-PG mAb. Furthermore, lysozyme treatment did not affect the electrophoretic mobility of these proteins.