Chong P, Cockle S, Boux H, Klein M
Connaught Centre for Biotechnology Research, Willowdale, Ont., Canada.
Biochem Cell Biol. 1991 May-Jun;69(5-6):336-40. doi: 10.1139/o91-052.
Pertussis toxin (PT), an oligomeric exotoxin of Bordetella pertussis containing five dissimilar subunits, is considered to be an essential immunogen in acellular and component pertussis vaccines against whooping cough. A rapid single-step procedure for isolating PT subunits was developed using reverse-phase high-performance liquid chromatography. Recoveries of individual subunits were 75% (S1), 70% (S2), greater than 90% (S3), greater than 90% (S4), and 50% (S5), as judged by SDS-PAGE and amino acid analysis. Lyophilized subunits were solubilized in urea followed by step-wise dialysis to remove the urea. All subunits were inactive in histamine sensitization, lymphocytosis, and hemagglutination assays. However, purified S1 retained residual NAD-glycohydrolase and ADP-ribosyltransferase activity. A partially active holotoxin could be generated by mixing the five individual subunits. All subunits were immunogenic in rabbits and mice. Monospecific antisera raised in both animal species were able to neutralize the PT-mediated clustering of Chinese hamster ovary cells, but active immunization of mice with single subunits failed to protect them in the intracerebral challenge assay. These subunit preparations therefore retained neutralizing determinants, but did not contain protective epitopes.
百日咳毒素（PT）是百日咳博德特氏菌的一种寡聚外毒素，含有五个不同的亚基，被认为是无细胞百日咳疫苗和百日咳组分疫苗中的一种重要免疫原。采用反相高效液相色谱法开发了一种快速单步分离PT亚基的方法。通过SDS-PAGE和氨基酸分析判断，各个亚基的回收率分别为75%（S1）、70%（S2）、大于90%（S3）、大于90%（S4）和50%（S5）。冻干的亚基溶解在尿素中，然后逐步透析以去除尿素。所有亚基在组胺致敏、淋巴细胞增多和血凝试验中均无活性。然而，纯化的S1保留了残余的NAD-糖苷水解酶和ADP-核糖基转移酶活性。通过混合五个单独的亚基可以产生部分活性的全毒素。所有亚基在兔和小鼠中均具有免疫原性。在这两种动物中产生的单特异性抗血清能够中和PT介导的中国仓鼠卵巢细胞聚集，但用单个亚基对小鼠进行主动免疫在脑内攻毒试验中未能保护它们。因此，这些亚基制剂保留了中和决定簇，但不包含保护性表位。
