| Project/Area Number |
14370601
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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 |
病態科学系歯学(含放射線系歯学)
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| Research Institution | Tokyo Medical and Dental University |
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Principal Investigator |
NAKAJIMA Takuma Graduate School, Tokyo Medical and Dental University, Dept. of Molecular Cellular Oncology and Microbiology, Assistant Professor, 大学院・医歯学総合研究科, 助教授 (90256678)
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| Co-Investigator(Kenkyū-buntansha) |
TSUCHIDA Nobuo Graduate School, Tokyo Medical and Dental University, Dept. of Molecular Cellular Oncology and Micirobiology, Professor, 大学院・医歯学総合研究科, 教授 (60089951)
ARAKAWA Shinichi Faculty of Dentistry, Tokyo Medical and Dental University, Dental Hospital, Periodontology. Assistant, 歯学部附属病院, 助手 (20302888)
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| Project Period (FY) |
2002 – 2003
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| Project Status |
Completed (Fiscal Year 2003)
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| Budget Amount *help |
¥14,000,000 (Direct Cost: ¥14,000,000)
Fiscal Year 2003: ¥5,900,000 (Direct Cost: ¥5,900,000)
Fiscal Year 2002: ¥8,100,000 (Direct Cost: ¥8,100,000)
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| Keywords | Tannerella forstyensis / Actinobacillus actinomyctemcomitans / cytocidal toxin(CCT) / cytolethal istending loxin(CDT) / Cell death / cytopathy / Bacterial pathogen / monoclonad antibody / Tannerella forsythensis / cellular toxicity / Cytolethal distending toxin(Cdt) / Cytocidal toxin / p53 / monoclonal antibody / ELISA / 歯周病 / 病原因子 / 遺伝子 / ORF / 組換え蛋白質 / 精製 |
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| Research Abstract |
[I] We had detected a novel activity of Tannerella forsyhtensis cytocidal toxin (Tf-CCT) from an extract of the periodontopathic bacteria that exhibited Cytolethal distending toxin (CDT)-like activity. To identify what is Tf-CCT, and to investigate the mechanism how Tf-CCT exhibits a cytopathy, we cloned its gene on the basis of amino acid sequence of purified protein, and subsequently, we confirmed the cytopathic activity of the cloned gene product. Moreover, we estimated a domain which contributes to the cytopathy of Tf-CCT. Details of the report are following;
(a) Identification of Tf-CCT protein purified from T. forsytliensis extract. Tf-CCT protein purified by anion-exchange, size-exclusion and heparin-affinity was identified by the monoclonal antibody Be-24 that was established for neutralization against the cytopathy of T. forsydiensis extract. An 28.5 kDa fragment of Tf-CCT was chosen for the N-terminal amino acid sequence analysis, and a degenerative primer was prepared for the
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resulted sequence APQNMDVLL. The sequence analysis for both PCR product and cloned gene obtained from T.forsythensis genomic DNA library revealed that Tf-CCT was identical with prtH protease (prtH). However, the translation initiation site resides in upstream of the reported site hence the molecular size of natural Tf-CCT detected in the purified fraction was larger than estimated size of pt-Il-I.
(b) To identify the translation initiation site, tirst (without ribosome binding motif; RBS), fifth (reported site with RBS) and eighth (with more typical RBS) were chosen as the first ATG to construct the recombinant gene, respectively. Albeit the extensive trial with different types of expression vectors for Tf-CCT was examined, no recombinant Tf-CCT was expressed in Esclmerichia coli whereas the significant expression was detected by in vitro expression system such as RTS and PURESYSTEM.
(c) The recombinant Tf-CCT (rTf-CCT) expressed from fifth ATG (identical with reported prtH) exhibited the greatest cytopathy.
(d) The protein BLAST revealed that Tf-CCT shares no similarity with any reported CDT besides a distinct homology with third domain of human vesicle transport-related protein slylp in the C-terminal side. Thus, the cytopathy of Tf-CCT is likely to exhibit through interaction with proteins that contribute regulation of intracellular membrane fusion.
(e) To get the expression of rTf-CCT in vivo, we tried to fuse recombinant with several kinds of protein-tags, and eventually we found that C-terminal strep-tag fusion achieved sufficient expression of rTf-CCT in E. coli.
(f) An ELISA system was examined for detection of anti-Tf-CCT antibody contained by humor in gingival sulcus from the patients of periodontitis.
[II] We cloned and identified and cdtB gene of Actinobacillus actinomycetemcomilans serotype
(a) (Aaa-cdtB gene). The amino acid sequence of Aaa-CdtB vas revealed to be identical with that of A. actinomycetemcotnitans serotype (b), while the DNA sequence contains two substitutions. We also examined an ELISA system for detection of anti-Aa-CdtB protein antibody contained by humor in gingival sulcus from the patients of periodontitis. Less
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