PURIFICATION OF THE 69KDA PROTEIN OF BORDETELLA PERTUSSIS

百日咳博德特氏菌 69KDA 蛋白的纯化

• 批准号: 3811012
• 负责人: D L BURNS
• 金额: --
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3811012
• 关键词: Bordetella pertussis; affinity chromatography; bacterial antigens; gel electrophoresis; molecular weight; pertussis; pertussis vaccine; protein purification

Concern about the safety of pertussis vaccines composed of killed whole cells of Bordetella pertussis has led to decreased use of these vaccines in several countries. Thus an urgent need exists for a safer pertussis vaccine. For this reason, efforts are underway to develop pertussis vaccines which consist of purified proteins from B. pertussis. One of the antigens which is being considered for inclusion in these vaccines is a 69 kDa protein associated with the outer membrane of virulent strains of B. pertussis. Relatively large quantities of this protein will be needed to evaluate its role in the disease process. We have therefore devised a purification scheme which is easily reproduced and which can be scaled-up in order to make sufficient quantities of the 69 kDa protein for possible vaccine use. B. pertussis cells were heated at 60 degrees C for 1 h in order to extract the 69 kDa protein. This extract was centrifuged, the supernatant was applied to a column containing DEAE-Sepharose and was eluted from the column by a linear gradient of NaCl. The fractions containing the 69 kDa protein were then pooled and applied to a column containing Affi-Gel Blue. Contaminating protein was eluted with 0.5 M potassium phosphate followed by elution of the 69 kDa protein with buffer containing 4 M urea. This preparation which was homogenous as determined by SDS-polyacrylamide gel electrophoresis was determined to be free of lipooligosaccharide and pertussis toxin. This purification scheme is simple and the protein produced by this method appears to be suitable for the study of the biochemical and immunological properties of this protein as well as for the evaluation of this protein as a vaccine candidate.

对由灭活全百日咳疫苗组成的百日咳疫苗安全性的关注 百日咳博德特氏菌的细胞导致这些疫苗的使用减少 在几个国家。 因此迫切需要一种更安全的百日咳疫苗 疫苗 出于这个原因，正在努力开发百日咳 由来自B的纯化蛋白组成的疫苗。百日咳。 之一 考虑纳入这些疫苗的抗原 是一种69 kDa的蛋白质，与毒力 B.百日咳。 相对大量的这种蛋白质 将需要评估其在疾病过程中的作用。 我们有 因此，设计了一种易于重现的纯化方案， 其可以按比例放大以制备足够量的69 kDa的蛋白质用于可能的疫苗用途。 B。百日咳细胞在60 ℃下加热1小时， 提取69 kDa蛋白质。 将该提取物离心， 将上清液加到含有DEAE-Sepharose的柱上， 通过NaCl的线性梯度从柱中洗脱。 的级分 然后将含有69 kDa蛋白的混合物合并并施加到柱上 含有Affi-Gel Blue。 用0.5 M洗脱剂洗脱污染的蛋白质。 磷酸钾，然后用洗脱剂洗脱69 kDa蛋白质。 缓冲液含有4 M尿素。 该制剂是均匀的， 通过SDS-聚丙烯酰胺凝胶电泳测定， 不含脂寡糖和百日咳毒素。 该纯化方案简单，并且由此产生的蛋白质可用于纯化。 该方法似乎适合于研究生物化学和 该蛋白的免疫学特性以及用于评价 这种蛋白质作为候选疫苗。