MAPPING OF A PROTECTIVE EPITOPE OF PERTUSSIS TOXIN BY INVITRO REFOLDING OF RECOMBINANT FRAGMENTS
MAPPING OF A PROTECTIVE EPITOPE OF PERTUSSIS TOXIN BY INVITRO REFOLDING OF RECOMBINANT FRAGMENTS
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DOI：
10.1038/nbt0688-709
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发表时间：
1988-06-01
期刊：
影响因子：
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通讯作者：
RAPPUOLI, R
中科院分区：
文献类型：
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作者：
BARTOLONI, A;PIZZA, M;RAPPUOLI, R
抗原决定簇通常由蛋白质表面相邻的不连续的多肽片段组成，很难通过常规技术进行定位。在这里，我们表明，尽管单克隆抗体不能识别仅含有蛋白质的氨基末端或仅含有羧基末端部分的重组分子，但在体外重折叠后，它们能够识别这两种分子的混合物。这表明两个分子，每个分子都含有不连续表位的一部分，能够在体外重建单克隆抗体识别的构象。因此，可以通过重组分子的体外重折叠来绘制抗体结合所需区域的图谱。然后使用定点诱变来进一步定义参与抗体识别的序列。这种新方法用于绘制百日咳毒素 S1 亚基上不连续的构象表位。分析显示，百日咳毒素可能只含有一种免疫显性表位，我们可用的所有七种保护性单克隆抗体都可以识别该表位。理论上，所描述的方法可用于绘制任何蛋白质中的构象表位。
Antigenic determinants are often composed of discontiguous polypeptide segments, adjacent on protein surfaces, which are difficult to map by conventional techniques. Here we show that although monoclonal antibodies did not recognize recombinant molecules containing only the aminoterminal or only the carboxyterminal part of a protein, after in vitro refolding they are able to recognize a mixture of the two molecules. This suggests that two molecules, each containing part of a discontiguous epitope, are able to reconstitute in vitro the conformation recognized by a monoclonal antibody. Mapping of the regions required for antibody binding can therefore be achieved by in vitro refolding of recombinant molecules. Site-directed mutagenesis is then used to further define the sequencs involved in antibody recognition. This new approach was used to map a discontiguous, conformational epitope on the S1 subunit of pertussis toxin. The analysis revealed that pertussis toxin is likely to contain only one immunodominant epitope, which is recognized by all seven protective monoclonal antibodies available to us. In theory, the method described can be used to map conformational epitopes in any protein.