Purification and identification of Haemophilus ducreyi cytotoxin by use of a neutralizing monoclonal antibody
Purification and identification of Haemophilus ducreyi cytotoxin by use of a neutralizing monoclonal antibody
复制标题
使用中和单克隆抗体纯化和鉴定杜克雷嗜血杆菌细胞毒素
作者：
M. Purven;A. Frisk;I. Lönnroth;T. Lagergård
杜克雷嗜血杆菌产生一种细胞毒素，负责杀死培养的人类上皮细胞。在经培养证实的软下疳患者的大多数血清中检测到了细胞毒素中和抗体，并且在疾病流行地区和非流行地区都发现，患者中此类抗体的水平显着高于献血者。我们用细胞毒素的粗渗透制剂在小鼠体内生产了中和单克隆抗体 (MAb)。这些抗体具有高中和细胞毒性的能力，可用于细胞毒素的纯化和鉴定。通过两步程序进行纯化，包括Sephacryl S-200过滤，然后进行免疫亲和层析。纯化导致细胞毒素蛋白回收率较差，并被亲和柱中的单克隆抗体污染。凝胶过滤实验和免疫印迹的结果表明，活性细胞毒素由分子量约为 20 kDa 的单一小蛋白质组成。来自不同菌株的细胞毒素似乎具有相同或相似的表位。在无毒菌株的制剂中未检测到细胞毒素蛋白。 20kDa条带的N端氨基酸序列为E-S-N-P-D-P-T-T-Y-P-D-V-E-L-S-P-P-P。该序列与任何目前已知的细菌蛋白的序列都不相似。
Haemophilus ducreyi produces a cytotoxin responsible for the killing of cultured human epithelial cells. Cytotoxin-neutralizing antibodies were detected in the majority of sera from patients with culture-proven chancroid, and a significantly higher level of such antibodies in patients than in blood donors was noted both in areas where the disease is endemic and those where it is not. We produced neutralizing monoclonal antibodies (MAbs) in mice with a crude osmotic preparation of the cytotoxin. These antibodies, with high capacity to neutralize cytotoxicity, were used for purification and identification of the cytotoxin. Purification was performed by a two-step procedure which included Sephacryl S-200 filtration followed by immunoaffinity chromatography. The purification resulted in poor cytotoxin protein recovery and contamination with MAbs from the affinity column. The results of the gel filtration experiments and immunoblotting indicate that the active cytotoxin consists of a single, small protein with an approximate molecular mass of 20 kDa. Cytotoxins from different strains seem to have the same or similar epitopes. The cytotoxin protein was not detected in preparations from nontoxic strains. The N-terminal amino acid sequence of the 20-kDa band was E-S-N-P-D-P-T-T-Y-P-D-V-E-L-S-P-P-P. This sequence does not resemble that of any currently known bacterial protein.