| Project/Area Number |
16592067
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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 |
Periodontal dentistry
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| Research Institution | The University of Tokushima |
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Principal Investigator |
SHIBUTANI Takuya The University of Tokushima, University Medical and Dental Hospital, Instructor, 医学部・歯学部附属病院, 助手 (20363153)
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| Co-Investigator(Kenkyū-buntansha) |
NAKAE Hideaki The University of Tokushima, Institute of Health Biosciences of Tokushima Graduate School, Associate Professor, 大学院・ヘルスバイオサイエンス研究部, 助教授 (30227730)
URAYAMA Akihisa The University of Tokushima, Institute of Health Biosciences of Tokushima Graduate School, Instructor, 大学院・ヘルスバイオサイエンス研究部, 助手 (40304539)
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| Project Period (FY) |
2004 – 2005
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| Project Status |
Completed (Fiscal Year 2005)
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| Budget Amount *help |
¥3,100,000 (Direct Cost: ¥3,100,000)
Fiscal Year 2005: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2004: ¥2,100,000 (Direct Cost: ¥2,100,000)
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| Keywords | histon-like protein / Porphyromonas gingivalis / biofilm / GST fused protein / suicide vector / anaerobic system / antibody / histon-like prote / 薬剤耐性遺伝子 |
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| Research Abstract |
To obtain Porphyromonas gingivalis histon-like protein gene we searched the homology between histon-like protein derived from Escherichia coil and P.gingivalis W83 of which chromosomal DNA sequences were determined by TIGER. Based on the high homology sequences between E.coli HLP and P.gingivalis chromosomal DNA sequences, we designed primers. By PCR using the primers we obtained P.gingivalis hlp gene, which had more than 50% identity to HLP which already known in amino acid level. P.gingivalis hlp gene was inserted into the GST fused expression vector in frame and then sub cloned into E.coli. GTS fused HLP proteins were purified with glutathione sepharose column and then removed GST with enzymatic treatment. Recombinant HLP was immunized to rabbits and three times and after the final injection, serum was obtained from the rabbits. By immunoblotting we determined the specificity of obtained anti-HLP serum.
To establish the experimental biofilm mimic to periodontal pockets we cultured P.gingivalis in circulation condition using a peristac pump for HPLC which could control very low flow rate in anaerobic chamber (Thermo Forma, Anaerobic System). Then, we are trying to identify the localization of P.gingivals HLP in experimental biofilm mimic to periodontal pocket using transmission electron microscope.
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