PURIFICATION OF THE 69KDA PROTEIN OF BORDETELLA PERTUSSIS

百日咳全灭活疫苗安全性令人担忧 百日咳杆菌的细胞已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。出于这个原因，正在努力发展百日咳。 由百日咳杆菌纯化蛋白组成的疫苗。其中之一 考虑包括在这些疫苗中的抗原 是一种69 kDa的蛋白，与强毒的外膜有关 百日咳杆菌菌株。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和 可以放大以生产足够数量的69 可能用于疫苗用途的KDA蛋白。 B.百日咳细胞在60摄氏度下加热1小时，以便 提取69 kDa蛋白。这个提取物是经过离心的， 将上清液加到含有DEAE-Sepharose的柱子上， 用氯化钠的线性梯度从柱子上洗脱。分数 然后将含有69 kDa蛋白的蛋白质混合在一起并应用到柱上 含有阿菲-凝胶蓝。污染蛋白质用0.5M洗脱 磷酸二氢钾洗脱69 kDa蛋白 含有4M尿素的缓冲液。该制剂是均一的AS 采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法测定 不含脂低聚糖和百日咳毒素。 这种纯化方案很简单，由这种方法生产的蛋白质 该方法适合于生化和生物化学的研究。 该蛋白的免疫学性质及其评价 将这种蛋白质作为疫苗候选。

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.