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Analysis of epitope specificity of immune response involved in periodontitis

牙周炎免疫反应表位特异性分析

基本信息

批准号：
09671924
负责人：
ITO Hiro-O
金额：
$ 1.92万
依托单位：
Kagoshima University (1998)Kyushu University (1997)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09671924/
关键词：
Porphyromonas gingivalis fimbriae monoclonal antibodies B cell epitopes phage-displayed peptide library higher order structure of proteins periodontal diseases Immunodominance タンパク質立体構造変性未変性

项目摘要

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

使用从牙龈卟啉单胞菌（一种推定的牙周病原体）纯化的天然菌毛对 BALB/c 小鼠进行免疫接种来产生单克隆抗体 (mAb)。这些单克隆抗体与天然和寡聚形式的菌毛（二聚体/三聚体）反应，但它们都不与菌毛单体亚基菌毛蛋白反应。当制备单体并将其注射到小鼠体内时，可以建立与亚基反应的单克隆抗体，但抗菌毛蛋白单克隆抗体对天然菌毛没有反应。这是首次定性证明牙龈卟啉单胞菌菌毛的免疫显性 B 细胞表位以寡聚形式表达，但不存在于亚基中。结果还表明单体纤毛蛋白的抗原性与天然蛋白的抗原性有很大不同。为了确定构象表位的免疫显性特征是否是蛋白质抗原的一般特性，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，发现一致性依赖表位具有免疫优势。因此，高阶结构可能对于许多蛋白质（如果不是全部）表达免疫显性表位至关重要。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。通过重复淘选筛选出数个噬菌体克隆，最后确认1个克隆的特异性。通过噬菌体基因的DNA测序获得的推导的插入肽的氨基酸序列显示出与来自a.a.的菌毛蛋白的氨基酸序列部分同源。 260.还进行了蛋白质化学分析来表征菌毛中的半胱氨酸残基；从 DNA 序列推断出每个纤毛蛋白分子存在 3 个半胱氨酸。确认半胱氨酸残基的数量。进一步表明，3个中的2个形成分子内二硫键，另一个形成游离的SH-，并且没有形成分子间二硫键。较少的