Frisk A, Roggen E L, Lagergård T
Department of Medical Microbiology and Immunology, University of Gothenburg, Sweden.
J Med Microbiol. 1995 Sep;43(3):192-200. doi: 10.1099/00222615-43-3-192.
The cell wall and outer structures of Haemophilus ducreyi bacteria were investigated. The 24-kDa outer protein from two strains was purified with an SDS-PAGE preparative continuous-elution electrophoresis cell. The protein was further characterised by SDS-PAGE and immunoblotting, and the immunological properties were investigated by ELISA. Localisation on the bacterial surface was investigated by immuno-electron-microscopy with a polyclonal antiserum raised against the purified protein. A triple-laminar cell wall typical of gram-negative bacteria, close cellular contact between bacterial cells and outer blebs were seen on thin sections. An additional high mol. wt band of c. 165 kDa was seen when not treated by heating to 100 degrees C. A high density fibrilla-like material was detected on the bacterial cell and in the environment by negative staining and immuno-electron-microscopy with antisera specific for the 24-kDa protein. The surface localisation of the 24-kDa protein was confirmed by an ELISA technique with the specific antiserum and whole bacterial cells as antigen. The presence of antibodies to the 24-kDa protein was demonstrated in antisera to 13 strains of H. ducreyi, indicating antigenic identity or within-species cross-reactivity. Low titres of antibodies to this protein were also detected in 19 antisera raised against different strains of gram-negative bacteria, indicating cross-reactivity with other species. Antibody response to the 24-kDa protein in rabbits immunised subcutaneously with live bacteria resulted in a secondary IgG response. Of 28 sera from patients with culture-verified chancroid, 26 manifested high titres of IgG antibodies to the 24-kDa protein, thus indicating the involvement of this antigen in the disease process in man.
对杜克雷嗜血杆菌的细胞壁和外部结构进行了研究。用SDS-PAGE制备型连续洗脱电泳槽从两株菌株中纯化出24 kDa的外膜蛋白。通过SDS-PAGE和免疫印迹对该蛋白进行进一步表征，并通过ELISA研究其免疫学特性。用针对纯化蛋白产生的多克隆抗血清通过免疫电子显微镜研究其在细菌表面的定位。在薄切片上可见革兰氏阴性菌典型的三层细胞壁，细菌细胞与外部泡之间紧密的细胞接触。未加热至100℃处理时可见一条约165 kDa的额外高分子量条带。通过负染色以及用针对24 kDa蛋白的抗血清进行免疫电子显微镜观察，在细菌细胞及其周围环境中检测到高密度的纤维状物质。用特异性抗血清和完整细菌细胞作为抗原的ELISA技术证实了24 kDa蛋白的表面定位。在针对13株杜克雷嗜血杆菌的抗血清中证实存在针对24 kDa蛋白的抗体，表明抗原一致性或种内交叉反应性。在针对不同革兰氏阴性菌菌株产生的19份抗血清中也检测到低滴度的针对该蛋白的抗体，表明与其他菌种存在交叉反应性。用活细菌皮下免疫的兔子对24 kDa蛋白的抗体反应导致了二次IgG反应。在28份经培养证实为软下疳的患者血清中，26份表现出针对24 kDa蛋白的高滴度IgG抗体，因此表明该抗原参与了人类疾病过程。
