BORDETELLA CYCLASE: STRUCTURE AND BIOLOGICAL ACTIVITY

百日咳（百日咳）是一种主要的儿童疾病， 由于对百日咳的关注， 疫苗反应原性，在许多地区的发病率增加 发达国家的。 在非完整细胞的发育过程中 百日咳疫苗，几种重要毒素和毒力因子 被认为是潜在的疫苗抗原。 一个主要 其中的候选者是腺苷酸环化酶（AC）毒素， 是这个研究项目的重点。 百日咳杆菌AC毒素是一种钙调素激活酶 其在细胞质外部的位置是新颖的， 细菌的膜及其结合和进入的能力 靶向真核细胞，催化cAMP的产生 来自内源性ATP。 在这个实验室里， 已部分纯化至极高的酶特异性 活性，同时保留其毒素活性， 已经制备了抗体。 在本文提出的工作中， 单克隆抗体将被进一步表征并用于 免疫纯化AC毒素。 毒素亚基结构将是 使用单克隆抗体和其他 技术. AC毒素基因将被鉴定和测序 以及用于获得体外表达的分离的基因， 蛋白质。 工作的一个主要重点是调查 毒素的传递、结合和进入细胞的机制。 免疫电子显微镜将使这些过程的研究 以及确定毒素的细胞内命运。 为配合新百日咳疫苗的研制， 纯化的AC毒素（或其类毒素）和针对 对抗毒素的能力将被测试， 感染B。小鼠脑内激发百日咳 和小鼠气溶胶感染模型。 患者血清 百日咳后恢复期将检查抗毒素 活动 最后是B。将比较百日咳AC毒素， 与唯一已知的来自芽孢杆菌的AC毒素相比， 炭疽病 DNA序列同源性、免疫交叉 反应性，受体和进入机制之间的关系 将被审查，使审议的演变 两者的发展和关联。 本申请中提出的工作和目前正在进行的工作将 提供疫苗开发中使用的信息， 了解细菌毒素及其 在细菌性疾病发病机制中的作用。

Pertussis (whooping cough) is a major childhood illnesses in the developing world and, as a result of concerns about pertussis vaccine reactogenicity, has increased in incidence in many areas of the developed world. In the development of a non-whole cell pertussis vaccine, several important toxins and virulence factors are being considered as potential vaccine antigens. A major candidate among these is the adenylate cyclase (AC) toxin, which is the focus of this research project. Bordetella pertussis AC toxin is a calmodulin-activated enzyme which is novel in its location external to the cytoplasmic membrane of the bacterium and its ability to bind to and enter target eukaryotic cells where it catalyzes the production of cAMP from endogenous ATP. In this laboratory the enzyme/toxin has been partially purified to extremely high enzymatic specific activity while retaining its toxin activity, and monoclonal antibodies have been prepared. In the work proposed herein, the monoclonal antibodies will be characterized further and used to immunopurify the AC toxin. The toxin subunit structure will be evaluated with the use of the monoclonal antibodies and other techniques. The AC toxin gene will be identified and sequenced and the isolated gene employed to obtain in vitro expression of the protein. A major focus of the work will be investigation of the delivery, binding and cell entry mechanisms for the toxin. Immuno-electron microscopy will enable study of these processes and determination of the intracellular fate of the toxin. In line with the development of a new pertussis vaccine, the purified AC toxin (or toxoid thereof) and antibodies directed against the toxin will be tested for their ability to protect against infection with B. pertussis in the mouse intracerebral challenge and the mouse aerosol infection model. Sera from patients convalescent after pertussis will be examined for antitoxin activity. Finally, B. pertussis AC toxin will be compared and contrasted with the only other known AC toxin, from Bacillus anthracis. The homology of DNA sequence, immunologic cross reactivity, relationships between receptors and entry mechanisms will be examined, enabling consideration of the evolutionary development and relatedness of the two. The work proposed in this application and ongoing at present will provide information of use in vaccine development and of importance to the understanding of bacterial toxins and their roles in pathogenesis of bacterial disease.