Analysis of epitope specificity of immune response involved in periodontitis
| Project/Area Number |
09671924
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
病態科学系歯学(含放射線系歯学)
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| Research Institution | Kagoshima University (1998)
Kyushu University (1997) |
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Principal Investigator |
ITO Hiro-O Kagoshima Univ.Dental Sch., Associate Professor, 歯学部, 助教授 (40213079)
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| Co-Investigator(Kenkyū-buntansha) |
HIRATA Masato Kyushu Univ.Fac.Dent., Professor, 歯学部, 教授 (60136471)
UEDA Tadashi Grad.Sch.Pharm.Kyushu Univ., Associate Professor, 大学院・薬学研究科, 助教授 (90184928)
INOUE Masakazu Kagoshima Univ.Dental Sch., Professor, 歯学部, 教授 (30028740)
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| Project Period (FY) |
1997 – 1998
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| Project Status |
Completed (Fiscal Year 1998)
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| Budget Amount *help |
¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 1998: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1997: ¥2,000,000 (Direct Cost: ¥2,000,000)
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| Keywords | 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 |
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| Research Abstract |
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
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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
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[Publications] Yanagita, M., Hiroi, T., Kitagaki, N., Hamada, S., Ito, H., Shimauchi, H., Murakami, S., Okada, H., Kiyono, H.: "Nasopharyngeal-associated lymphoreticular tissue (NALT) immunity : fimbriae-specific Th1 and Th2 cell-regulated IgA responses for the inhibition of bacterial attachment to epithelial cells and subsequent inflammatory cytokine production" J.Immunol.162-6. 3559-3565 (1999)
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