| Budget Amount *help |
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1987: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1986: ¥1,600,000 (Direct Cost: ¥1,600,000)
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| Research Abstract |
Streptococcus mutans is known to be cariogenic in animal as well as human. Some strains of Streptococcus mutans have been known to produce some bacteriocin-like substances, which are termed "mutacin" by Hamada and Ooshima. We attempted to purity a mutacin produced by S. mutans LM7(sero-type e) and clone its gene. When cells were cultured to stationaly phase in Berman's broth containing 5% yeast extract(BBL) sterilized by filtration, the total amount of mutacin became maximum. The activity was recovered in the precipitate at 60% saturation of ammonium sulfate. The precipitate was dissolved in 10 mM Na-phosphate buffer and dialyzed against the phosphate buffer. Then, the resultant solution was ultra-centrifuged at 200,000xg for 15 h. The activity was recovered in the precipitate. Since the molecular weight was estimated to be more than 1000,000 dal., the mutacin may be dissolved as an aggregate form. So far, further purification has not been succeeded yet.With the crude mutacin containing 4-5 proteins with different molecular weight, antiserum against mutacin was obtained with a rabbit. We found that a plasmid pAM7 of S. mutans LM7 codes no bacteriocin-like activity. Thus, the genomic DNA library for S. mutans LM7 was screened immunologically with the antiserum against the crude mutacin. We examined 10 positive clones for bacteriocin activity. All clone was found to produce a surface protein antigen(spa) of molecular weight 180,000-190,000 , but not mutacin from the results obtained by immunoblot analysis with antiserum against spa. We supposed that since the crude mutacin should contains spa, the serum against mutacin also contains antibody against spa. Thus, we intend now to absorb the spa antibodies fro the serum and then screen the DNA library again with the absorbed serum. The results will publish elsewhere.
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