1991 Fiscal Year Final Research Report Summary
Molecular biological Analysis of Periodontopathic Bacteria
| Project/Area Number |
01480425
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
Morphological basic dentistry
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| Research Institution | Tokyo Dental College |
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Principal Investigator |
ICHIRO Takazoe 東京歯科大学, 微生物学講座, 教授 (80085696)
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| Co-Investigator(Kenkyū-buntansha) |
YAMANAKA Ayumi 東京歯科大学, 微生物学講座, 副手
ISHIHARA Kazuyuki 東京歯科大学, 微生物学講座, 助手 (00212910)
KATO Tetsuo 東京歯科大学, 微生物学講座, 助手 (00159253)
NAITO Yuko 東京歯科大学, 微生物学講座, 講師 (00147258)
OKUDA Katsuji 東京歯科大学, 微生物学講座, 教授 (40085741)
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| Project Period (FY) |
1989 – 1991
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| Keywords | Periodontal Disease / Spirochte / Trponeme denticola / Gene cloning / ATCase / 57.5 KDa protein / Diagnosis / DNA probe |
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| Research Abstract |
The mechanisms responsible for periodontal disease have not been clarified, but subgingival bacterial infection by organisms such as gram-negative rods and spirochetes is a major etiological factor in the disease process. Treponema denticola has been demonstrated to be predominant in subgingival plaque of patients with periodontitis. We have been attempting to clone and sequence several antigenic proteins of T. denticola. T. denticola ATCC 33520 was grown in TYGVS medium. Chromosomal DNA was prepared and digested with Hind III. and ligated to the phage vector L47.1. The phage particles were infected with Escherichia coli Q358. Clone of the surface antigen was pick up using rabbit antiserum against whole cells of T. denticola. The obtained clone was subcloned into plasmid vector PACYC 184 and transformed into E. coli HB101. Sonicated extracts of the cloned TD12 was approximately 57.5 KDa protein. Southern blot analysis showed that the gene exists in common in T. denticola ATCC 33521, ATCC 45404, and ATCC 35405. but does not exist in other oral treponema species. The clone protein possessed aspartate carbamoyltransferase activity and strong antigenicity. The DNA sequencing was carried out by the dideoxy chain termination method using an Applied Biosystems Model 373 automated DNA sequencer. The cloned protein contains 486 amino acids and has a calculated molecular weight of 54, 919. The cloned gene encoding for aspartate carbomyltransferase may be an useful DNA probe for identification of T. denticola and applicable for clinical examinations.
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