Novotny P, Chubb A P, Cownley K, Montaraz J A
Infect Immun. 1985 Oct;50(1):199-206. doi: 10.1128/iai.50.1.199-206.1985.
A method was developed which is suitable for the isolation of substantial quantities of outer membrane proteins of Bordetella species in a water-soluble form. The extracted material may then be further fractionated in the absence of detergents by ion-exchange chromatography and preparative flat-bed isoelectrofocusing. These procedures facilitated the isolation of one of the proteins, of molecular weight 68,000, for which the antibody titer correlated with the degree of protection against nasal changes induced in specific-pathogen-free piglets by Bordetella bronchiseptica infection (P. Novotny, M. Kobisch, K. Cownley, A. P. Chubb, and J. A. Montaraz, Infect. Immun. 50:190-198). This protein, which banded between 7.0 and 7.6 pH in preparative isoelectrofocusing, may be further purified with a monoclonal immunosorbent. Immunopurified protein showed adenylate cyclase activity. The enzymatic activity was found to be unstable during processing; i.e., although the crude extract showed up to 150 nmol of cyclic AMP per mg/min, the immunopurified protein showed a maximum of only 200 nmol of cyclic AMP per mg/min. Two strains of B. bronchiseptica, isolated from herds of healthy pigs showing no signs of atrophic rhinitis, did not produce the 68,000-molecular-weight protein and were negative for adenylate cyclase. However, it is not known whether the 68,000-molecular-weight protein is a component of adenylate cyclase or whether it is an unrelated protein associated with this enzyme in some unknown way. Adenylate cyclase activity from culture supernatants of B. bronchiseptica, B. pertussis, and B. parapertussis can be absorbed equally to the same monoclonal immunosorbent.
已开发出一种方法，该方法适用于以水溶性形式分离大量博德特氏菌属的外膜蛋白。然后，提取的物质可在不存在去污剂的情况下通过离子交换色谱和制备型平板等电聚焦进一步分级分离。这些程序有助于分离出一种分子量为68,000的蛋白质，其抗体滴度与无特定病原体仔猪抵抗支气管败血博德特氏菌感染引起的鼻腔变化的保护程度相关（P.诺沃特尼、M.科比施、K.考恩利、A.P.查布和J.A.蒙塔拉兹，《感染与免疫》50:190 - 198）。这种在制备型等电聚焦中在pH 7.0至7.6之间出现条带的蛋白质，可用单克隆免疫吸附剂进一步纯化。免疫纯化的蛋白质显示出腺苷酸环化酶活性。发现该酶活性在处理过程中不稳定；即，尽管粗提取物显示每毫克/分钟高达150纳摩尔的环磷酸腺苷，但免疫纯化的蛋白质每毫克/分钟最多仅显示200纳摩尔的环磷酸腺苷。从无萎缩性鼻炎迹象的健康猪群中分离出的两株支气管败血博德特氏菌不产生分子量为68,000的蛋白质，且腺苷酸环化酶呈阴性。然而，尚不清楚分子量为68,000的蛋白质是腺苷酸环化酶的一个组分，还是以某种未知方式与该酶相关的无关蛋白质。支气管败血博德特氏菌、百日咳博德特氏菌和副百日咳博德特氏菌培养上清液中的腺苷酸环化酶活性可被同等地吸附到同一单克隆免疫吸附剂上。
