Analysis of epitope specificity of immune response involved in periodontitis

单克隆抗体（mAb）是用从假定的牙周病原体牙龈卟啉单胞菌（Porphyromonas gingivalis）中纯化的天然菌毛免疫BALB/c小鼠产生的。这些单抗与原生和低聚形式的菌毛（二聚体/三聚体）反应，但没有一个与菌毛的单体亚基——纤毛蛋白反应。制备该单体并注射到小鼠体内可建立与亚基反应的单抗，但抗纤原蛋白单抗对天然纤原膜无反应。这是首次定性地证明了牙龈假单胞菌菌毛的免疫显性B细胞表位是以寡聚体形式表达的，而不是驻留在亚基中。结果还表明，单体纤维蛋白的抗原性与天然蛋白的抗原性有很大差异。为了确定构象表位的免疫优势性是否为蛋白质抗原的属性，采用相同的方法分析了3种不同蛋白质的抗原性。对2种蛋白，发现更多的结构依赖表位具有免疫优势。因此，高阶结构可能对许多蛋白质（如果不是全部的话）表达免疫显性表位至关重要。选择了两个与二聚体牙龈卟啉菌毛上表达的相同或密切相关的表位反应的单抗克隆，并进一步阐明了表位的性质。利用噬菌体展示的随机肽文库来鉴定模仿构象表位的寡肽基序。通过反复筛选筛选出多个噬菌体克隆，最终确定一个克隆具有特异性。通过噬菌体基因的DNA测序，推断出插入肽的氨基酸序列与a.a.260的纤原蛋白氨基酸序列部分同源。还进行了蛋白质化学分析，以表征半胱氨酸残基在菌毛；从DNA序列推断，每一个纤原蛋白分子中存在3个半胱氨酸。确定了半胱氨酸残基的数量。进一步表明，3个分子中有2个形成了分子内二硫键，另一个给出了游离SH-，没有形成分子间二硫键。少

Monoclonal antibodies (mAb) were generated by immunizing BALB/c mice with native fimbriae purified from Porphyromonas gingivalis, a putative periodontal pathogen. These mAb reacted with native and oligomeric forms of fimbriae (dimer/trimer), but none of them reacted with fimbrilin, the monomeric subunit of fimbriae. mAb reacting to the subunit could be established when the monomer was prepared and injected into mice, but the anti-fimbrilin mAb showed no reaction to the native fimbriae. This is the first qualitative demonstration that immunodominant B cell epitopes of P.gingivalis fimbriae are expressed by the oligomeric form, but do not reside in the subunit. The results also suggest that antigenicity of monomeric fimbrilin greatly differs from that of native protein. To determine whether the immunodominant character of conformational epitopes is a genera] property of protein antigens, antigenicity of 3 different proteins were analyzed using the same methodology. For 2 proteins, confor … More mation dependent epitopes were found to be immunodominant. Thus, higher order structures are likely to be crucial for many proteins, if not all, to express the immunologically dominant epitopes. Two mAb clones which reacted with the same or closely related epitopes expressed on the dimeric P.gingivalis fimbriae were selected and nature of the epitope was further elucidated. A phage-displayed random peptide library was utilized to identify a oligopeptidic motif that mimics the conformational epitope. Several phage clones were selected by repeated biopanning, and finally one clone was confirmed for the specificity. The deduced amino , acid sequence of inserted peptide obtained by DNA sequencing of the phage gene showed a partial homology with the amino acid sequence of fimbrilin from the a.a. 260. Protein chemical analyses were also performed to characterized the cysteine residues in the fimbriae ; presence of 3 cysteines per one fimbrilin molecule is deduced from the DNA sequence. The number of cysteine residues was confirmed. It was further suggested that 2 of the 3 made a intramolecular disulfide bond, another gave a free SH-, and no intermolecular disulfide bond was formed. Less