Analysis of epitope specificity of immune response involved in periodontitis

通过用从牙龈卟啉单胞菌（一种假定的牙周病原体）纯化的天然菌毛免疫BALB/c小鼠产生单克隆抗体（mAb）。这些mAb与菌毛的天然和寡聚形式（二聚体/三聚体）反应，但它们均不与菌毛的单体亚基菌毛蛋白反应。当制备单体并注射到小鼠体内时，可以建立与亚基反应的mAb，但抗菌毛蛋白mAb显示与天然菌毛无反应。这是第一次定性证明牙龈卟啉单胞菌菌毛的免疫显性B细胞表位由寡聚体形式表达，但不存在于亚基中。结果还表明，单体fimbrilin的抗原性与天然蛋白质的抗原性有很大差异。为了确定构象表位的免疫显性特征是否是蛋白质抗原的属特性，使用相同的方法分析了3种不同蛋白质的抗原性。对于2种蛋白质，confor ...更多信息 发现依赖于信息的表位是免疫显性的。因此，高级结构对于许多蛋白质（如果不是全部）表达免疫优势表位可能是至关重要的。选择与二聚体牙龈卟啉单胞菌菌毛上表达的相同或密切相关的表位反应的两个mAb克隆，并进一步阐明表位的性质。利用噬菌体展示的随机肽库来鉴定模拟构象表位的寡肽基序。通过反复的生物淘选，筛选出几个噬菌体克隆，最后确认一个克隆具有特异性。经DNA序列测定，插入肽的氨基酸序列与a.a. 260.还进行了蛋白质化学分析以表征菌毛中的半胱氨酸残基;从DNA序列推断每个菌毛蛋白分子存在3个半胱氨酸。确认了半胱氨酸残基的数量。结果表明，3种化合物中有2种形成了分子内二硫键，另一种形成了游离的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