| Project/Area Number |
09660083
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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 |
応用微生物学・応用生物化学
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| Research Institution | KYOTO UNIVERSITY |
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Principal Investigator |
YAMAMOTO Kenji Kyoto Univ., Graduate School Agr., Associate Prof., 農学研究科, 助教授 (70109049)
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| Co-Investigator(Kenkyū-buntansha) |
TAMAKI Hisanori Kyoto Univ., Graduate School Agr., Res.Associate, 農学研究科, 助手 (20212045)
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| Project Period (FY) |
1997 – 1998
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| Project Status |
Completed (Fiscal Year 1998)
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| Budget Amount *help |
¥3,800,000 (Direct Cost: ¥3,800,000)
Fiscal Year 1998: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 1997: ¥2,300,000 (Direct Cost: ¥2,300,000)
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| Keywords | Lactic acid bacteria / Cell adhesion / Glycolipids / Membrane oligosacchariotes / Receptor protein / Lactobacillus casei / Cloning / Lactic acid bacteria therapy / Lactobacillus casel |
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| Research Abstract |
Many viruses, pathogenic bacteria, and bacterial toxins specifically recognize and bind to sugar chains of the eukaryotic cell surface. Their binding to the cells is essential to establish infection or produce a toxic effect. For intestinal bacteria, adherence to cell surfaces for colonizing epithelial tissues is an important initial event. Lactobacillus. a representative useful bacterium, in the intestinal tract was found to bind to some specific glycosphingolipids, like the pathogenic intestinal bacteria. We found that Lactobacillus casei bound to neutral glycosphingolipids such as GAl and trihexosylceramide strongly. We also found and purified the receptor protein of L.casei from surface layer proteins, and attempted to clone the gene of the protein. The results of this investigation are as followed.
We extracted the genomic DNA from L.casei and it was partially digested with Sau3AI.Then, 10-l5kbp DNA fragments was ligated with Charomid cloning vector digested with BamHI using DNA ligase. This recombinant DNA was transduced into Escherichia coli DH5 alpha using phage to construct a genomic library. Colony hybridization was performed with the probe of oligonucleotide mixture corresponding with N-terminal amino acid sequence of the protein. Finally, positive clone was obtained and named CH1, and the 3.0 kbp EcoRI fragment was sequenced and confirmed to contain an open reading frame encoding the protein. It consisted of 465 nucleotides encoding 155 amino acids. When the cell extract of recombinant E.coli was added with GAl and the mixture was subjected to electrophoresis, followed by immunoblotting using anti-GAl-monoclonal antibody, we found a staining band at the position of the protein expressed in recombinant E.coli. The protein was expressed in both the cell surface and cytosol fractions of E.coli.
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