| Project/Area Number |
08457484
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Morphological basic dentistry
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| Research Institution | TOKYO DENTAL COLLEGE |
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Principal Investigator |
OKUDA Katuji TOKYO DENTAL COLLEGE,DEPARTMENT OF DENTISTRY,PROFESSOR, 歯学部, 教授 (40085741)
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| Co-Investigator(Kenkyū-buntansha) |
YAMANAKA Ayumi TOKYO DENTAL COLLEGE,DEPARTMENT OF DENTISTRY,ASSISTANT, 歯学部, 助手 (40231667)
MIURA Tadashi TOKYO DENTAL COLLEGE,DEPARTMENT OF DENTISTRY,ASSISTANT, 歯学部, 助手 (10266570)
ISHIHARA Kazuyuki TOKYO DENTAL COLLEGE,DEPARTMENT OF DENTISTRY,LECTURER, 歯学部, 講師 (00212910)
KATO Tetsuo TOKYO DENTAL COLLEGE,DEPARTMENT OF DENTISTRY,ASSISTANT PROFESSOR, 歯学部, 助教授 (00159253)
君塚 隆太 東京歯科大学, 歯学部, 助手 (90287178)
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| Project Period (FY) |
1996 – 1997
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| Project Status |
Completed (Fiscal Year 1997)
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| Budget Amount *help |
¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 1997: ¥1,700,000 (Direct Cost: ¥1,700,000)
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| Keywords | Periodontal disease / Antibody / Attachment factor / fop gene / cloning / A.actinomycetemcomitans / fimbrial / vaccination / 感染防御抗体 / 遺伝子 / fap / Actinobacillus actinomycetemcomitans / 線毛抗原 / 合成ペプチド / 感染防御 / 分泌型IgA / 遺伝学的制御 |
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| Research Abstract |
Fundamental advances to maintain oral health such as preventing periodontal disease are required in the elderly society. Actinobacillus actinomycetemcomitans is considered to be one of the most important periodontopathic bacteria. We found that sera from A.actinomycetemcomitans culture-negative periodontitis patients with elevated titers of IgG against fimbriae had significantly higher avidity for the fimbrial antigen than did sera from culture-positive patients with elevated titers. We extracted A.actinomycetemcomitans fimbrial antigen and determined the N-terminal amino acid sequence. We prepared primers according to the N-terminal aminoacid sequence and fimbrial-associated protein was cloned. The plasmid obtained was found to harbor a 1.7kb fragment which contained a 228bp open reading frame encoding 75 amino acids. The fimbriae associated protein gene (fap) was strongly expressed in fimbriated A.actinomycetemcomitans 310a but not in non-fimbriated strains. We sunthesized an oligope
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ptide, according to the amino acid sequence and the synthetic peptide was conjugated by the branched lysine polymer resin beads method, which has the advantage of Increasing molecular size and immunogenicity. An synthetic peptide with the sequence of V-A-V-F-Y-S-N-N-G-F-I-A-N-L-Q-S-K-F-F-S-L produced highest anti-fimbrial antibody levels in the ELISA test. We found that rabbit antiserum against a synthetic peptide inhibited the attachment of A.actinomycetemcomitans strains to saliva-coated hydroxyapatite beads, human buccal epithelial cells, and cultured gingival fibroblasts in vitro. These findings suggest that the synthetic fimbrial antigen acts as a vaccine for inducing an antibody response that inhibits A.actinomycetemcomitans colonization.
We demonstrated the genetic regulation of immune responses to the synthetic A.actinomycetemcomitans fimbrial antigen using various inbred strains of mice. We examined the effective adjuvant activity and immunization route to produce s-IgA against the synthetic A.actinomycetemcomitansfimbrial antigen in saliva using BALB/c mice. The synthetic peptide antigen suspended in PBS was injected with Freund adjuvant Ribi adjuvant, liposome, plasmid DNA coding mouse IL-4, or/and cholera toxin. BALB/c mice were immunized through subcutaneous, nasal mucosa or oral route. We found that a group of mice immunized through nasal mucosa with a mixture of liposomes, the plasmid DNA and cholera toxin. Mice immunized through nasal mucosal route resulted in higher s-IgA production against the synthetic antigen, and delayd type hypersensitivity was also significantly enhanced in the group. We are now studying the role of s-IgA antibody against the fimbrial antigen in colonization inhibition by A.actinomycetemcomitans. Less
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