1992 Fiscal Year Final Research Report Summary
Basic studies on development of DNA diagnosis for periodontal disease associated bacteria
| Project/Area Number |
02454430
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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 |
Functional basic dentistry
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| Research Institution | Nihon university |
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Principal Investigator |
SAITO Shigeno Nihon university, Sch. Dent. at Matsudo Lecturer, 松戸歯学部・講師(専任攻) (60072394)
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| Co-Investigator(Kenkyū-buntansha) |
HAYAKAWA Mitsuo Nihon university, Sch. Dent. at Matsudo Lecturer, 松戸歯学部, 専任講師 (10112955)
TAKIGUCHI Hisashi Nihon university, Sch. Dent. at Matsudo Part-time Lecturer, 松戸歯学部, 非常勤講師 (00050013)
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| Project Period (FY) |
1990 – 1992
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| Keywords | Periodontal disease / Recombinant technology / DNA diagnosis / DNA probes |
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| Research Abstract |
We have done cDNA cloning of ribosomal RNA of Porphylomonas gingivalis. The clone DNA hybridized with DNA from several periodontal disease associated bacteria. As rDNA of P. gingivalis had homology with other periodontal disease associated bacteria, it is difficult to identify the gene specific regions. We therefore attempted to isolate a gene clone encoding the fusion protein of the major antigenic region of 200 kDa specific protein of P. gingivalis and E. coli maltose binding protein. A recombinant clone produced 65 kDa fusion protein. The fusion protein could be purified amylose regin affinity column chromatography. After clevage of the fusion protein to the major antigenic region of 65 kDa protein and the maltose binding protein using Factor Xa, 65 kDa fusion protein could be eluted and be recovered by the same affinity column. These data suggest that the major antigenic region of P. gingivalis 200 kDa specific protein was successfully cloned in pMal vector plasmid and the gene clone is useful for large quantity production of antigen for serodiagnosis of P. gingivalis infection.
We also have attempted gene cloning of specific antigens of other periodontal disease associated bacteria such as Prevotella loescheii, P. endodontalis, Treponema denticola, Camphylobacter rectus. Since human sera from adult periodontitis patients recognized each recombinant protein, these proteins will be useful antigens for the development of immunological diagnosis of periodontal diseases.
In another approach we have attempted to make DNA probe to use the DNA diagnosis of periodontal diseases. We have isolated DNA probe hybridized with only P. gingivalis chromosomal DNA. The DNA probe was labeled with digoxigenin instead of labeling with isotope which has been using for labeling DNA. The non-isotope labeled DNA probe must be useful tool for routinely use in diagnosis of periodontal diseases.
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