Bogdan John A, Yuan Wei, Long-Rowe Karen O, Sarwar Jawad, Brucker Eric Allen, Blake M S
Baxter Healthcare Corporation, Columbia, Maryland 21046, USA.
Appl Environ Microbiol. 2003 Oct;69(10):6272-9. doi: 10.1128/AEM.69.10.6272-6279.2003.
The introduction of acellular pertussis vaccines has greatly enhanced the safety profile of vaccines to prevent whooping cough. Pertussis toxin (Ptx) is one component produced by Bordetella pertussis that is contained in all of these vaccines, either in combination with other known pertussis virulence factors or as the sole pertussis component, combined with tetanus and diphtheria toxoids. A hydrogen peroxide toxoid of Ptx has been shown to be efficacious in preventing pertussis infections in a mass vaccination trial and is presently licensed in the United States and Europe (B. Trollfors, J. Taranger, T. Lagergard, L. Lind, V. Sundh, G. Zackrisson, C. U. Lowe, W. Blackwelder, and J. B. Robbins, N. Engl. J. Med. 333:1045-1050, 1995). The industrial production of Ptx can be performed through the cultivation of B. pertussis in well-defined growth media, in which the components can be well characterized and their origins can be documented. Once the bacteria are removed from the culture, Ptx can be isolated from the supernatant and purified by using the technique described by Sekura et al. (R. D. Sekura, F. Fish, C. R. Manclark, B. Meade, and Y. L. Zhang, J. Biol. Chem. 258:14647-14651, 1983). The only drawback of this procedure, which combines two affinity chromatography steps, one with Blue Sepharose and a second with matrix-bound bovine fetuin (BF), is the source and purity of the BF. Concern about vaccine preparations that may possibly risk contamination by material associated with bovine spongioform encephalopathy has continued to increase. We thus sought a replacement for the BF affinity chromatography and, more specifically, for the glycosidic moiety on BF. We describe here the identification of a seven-amino-acid peptide that mimics the glycosidic moiety on BF to which Ptx binds. Furthermore, we have constructed an affinity column containing this peptide that can be used to replace BF in Ptx purification. Finally, we used the X-ray crystallographic structure of Ptx bound to the oligosaccharide moiety of BF as a scaffold and replaced the oligosaccharide with the peptide.
无细胞百日咳疫苗的引入极大地提高了预防百日咳疫苗的安全性。百日咳毒素（Ptx）是百日咳博德特氏菌产生的一种成分，包含在所有这些疫苗中，要么与其他已知的百日咳毒力因子结合，要么作为唯一的百日咳成分，与破伤风类毒素和白喉类毒素结合。在一项大规模疫苗接种试验中，Ptx的过氧化氢类毒素已被证明对预防百日咳感染有效，目前在美国和欧洲已获许可（B. Trollfors、J. Taranger、T. Lagergard、L. Lind、V. Sundh、G. Zackrisson、C. U. Lowe、W. Blackwelder和J. B. Robbins，《新英格兰医学杂志》333:1045 - 1050，1995年）。Ptx的工业生产可通过在成分明确的生长培养基中培养百日咳博德特氏菌来进行，其中的成分可得到很好的表征，其来源也可记录在案。一旦从培养物中去除细菌，Ptx可从上清液中分离出来，并使用Sekura等人描述的技术进行纯化（R. D. Sekura、F. Fish、C. R. Manclark、B. Meade和Y. L. Zhang，《生物化学杂志》258:14647 - 14651，1983年）。该方法结合了两个亲和色谱步骤，一个使用蓝色琼脂糖凝胶，另一个使用与基质结合的牛胎球蛋白（BF），其唯一的缺点是BF的来源和纯度问题。对可能因与牛海绵状脑病相关的物质而有污染风险的疫苗制剂的担忧持续增加。因此，我们寻求一种替代BF亲和色谱的方法，更具体地说，是替代BF上的糖苷部分。我们在此描述了一种七氨基酸肽的鉴定，该肽模拟了Ptx与之结合的BF上的糖苷部分。此外，我们构建了一个含有该肽的亲和柱，可用于在Ptx纯化过程中替代BF。最后，我们以Ptx与BF的寡糖部分结合的X射线晶体结构为支架，用该肽取代了寡糖。
