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首页> 外文期刊>Infection and immunity >Epitope Mapping of Monoclonal Antibodies againstBordetella pertussis Adenylate Cyclase Toxin
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Epitope Mapping of Monoclonal Antibodies againstBordetella pertussis Adenylate Cyclase Toxin

机译：百日咳博德特氏菌腺苷酸环化酶毒素单克隆抗体的抗原表位定位

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摘要

Adenylate cyclase (AC) toxin from Bordetella pertussisis a 177-kDa repeats-in-toxin (RTX) family protein that consists of four principal domains; the catalytic domain, the hydrophobic domain, the glycine/aspartate-rich repeat domain, and the secretion signal domain. Epitope mapping of 12 monoclonal antibodies (MAbs) directed against AC toxin was conducted to identify regions important for the functional activities of this toxin. A previously developed panel of in-frame deletion mutants of AC toxin was used to localize MAb-specific epitopes on the toxin. The epitopes of these 12 MAbs were located throughout the toxin molecule, recognizing all major domains. Two MAbs recognized a single epitope on the distal portion of the catalytic domain, two reacted with the C-terminal 217 amino acids, one bound to the hydrophobic domain, and one bound to either the hydrophobic domain or the functionally unidentified region adjacent to it. The remaining six MAbs recognized the glycine/aspartate-rich repeat region. To localize these six MAbs, different peptides derived from the repeat region were constructed. Two of the six MAbs appeared to react with the repetitive motif and exhibited cross-reactivity with Escherichia coli hemolysin. The remaining four MAbs appeared to interact with unique epitopes within the repeat region. To evaluate the roles of these epitopes on toxin function, each MAb was screened for its effect on intoxication (cyclic AMP accumulation) and hemolytic activity. The two MAbs recognizing the distal portion of the catalytic domain blocked intoxication of Jurkat cells by AC toxin but had no effect on hemolysis. On the other hand, a MAb directed against a portion of the repeat region caused partial inhibition of AC toxin-induced hemolysis without affecting intoxication. In addition, the MAb recognizing either the hydrophobic domain or the unidentified region adjacent to it inhibited both intoxication and hemolytic activity of AC toxin. These findings extend our understanding of the regions necessary for the complex events required for the biological activities of AC toxin and provide a set of reagents for further study of this novel virulence factor.
机译：百日咳博德特氏菌的腺苷酸环化酶（AC）毒素是一种177 kDa毒素重复序列（RTX）家族蛋白，由四个主要域组成;催化结构域，疏水结构域，富含甘氨酸/天冬氨酸的重复结构域和分泌信号结构域。进行了针对AC毒素的12种单克隆抗体（MAb）的表位作图，以鉴定对该毒素的功能活性重要的区域。 AC毒素的框内缺失突变体的先前开发的面板用于在毒素上定位MAb特异性表位。这12个MAb的表位位于整个毒素分子中，可识别所有主要域。两个单克隆抗体识别催化结构域远端的一个表位，两个与C端217个氨基酸反应，一个与疏水域结合，另一个与疏水域或与其相邻的功能未知区域结合。其余六个单克隆抗体识别出富含甘氨酸/天冬氨酸的重复区域。为了定位这六个MAb，构建了源自重复区域的不同肽。六个MAb中有两个与重复基序发生反应，并与大肠杆菌溶血素发生交叉反应。其余四个单克隆抗体似乎与重复区域内的独特表位相互作用。为了评估这些表位在毒素功能上的作用，筛选了每个单克隆抗体对中毒（循环AMP积累）和溶血活性的影响。识别催化结构域远端的两个单克隆抗体阻止了AC毒素对Jurkat细胞的中毒，但对溶血没有影响。另一方面，针对一部分重复区域的MAb引起了AC毒素诱导的溶血的部分抑制，而不影响中毒。此外，识别疏水域或与其相邻的未知区域的单克隆抗体抑制AC毒素的中毒和溶血活性。这些发现扩展了我们对AC毒素生物活性所需的复杂事件所必需区域的理解，并为进一步研究这种新型毒力因子提供了一套试剂。

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