1989 Fiscal Year Final Research Report Summary
Characterization of regular array protein in the cell wall of Lactobacillus
| Project/Area Number |
63570195
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
細菌学
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| Research Institution | TOKUSHIMA UNIVERSITY |
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Principal Investigator |
KOGA Tetsuro TOKUSHIMA UNIV., MEDICINE RESEARCH ASSISTANT, 医学部, 助手 (20093859)
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| Co-Investigator(Kenkyū-buntansha) |
YAMATO Masayuki TOKUSHIMA UNIV. MEDICINE RESEARCH ASSISTANT, 医学部, 助手 (90210492)
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| Project Period (FY) |
1988 – 1989
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| Keywords | Lactobacillus / Regular array / Gene library / Immunoscreening |
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| Research Abstract |
1. Lactobacillus acidophilus was composed of many bacterial strains with very heterogeneous biological and biochemical characteristics. We showed possibility that in addition to classification by DNA-DNA homology,character of regular array protein of the organism could be used to classify L. acidophilus strains. Only the strains of DNA homology groupA produced RA proteins and no other groups produced. All A-1 subgroup strains produced RA proteins with same molecular weight and peptidemapping. Furthermore, they showed high homology between their immunological properties. In contrast, RA proteins of subgroup A-2, A-3 and A-4 were differed from each other in their molecular weight and peptide mapping. Although, immunological properties of subgroup A-2 RA proteins showed considerably homogeneous, those of subgroup A-3 and A-4 strains appeared heterogeneous. We conclude that there are necessity to classify subgroup A-3 and A-4 by means of properties of RA proteins.
2. We tried to clone the regular array protein gene of L. brevis ATCC8287. The chromosomal DNA fragments(3-9 kb) produced bypartial digestion with EcoRI was ligated with gt 11 EcoRI digested DNA and then packaged in vitro. Sixteen positive clones were screened from 2638 plaques by immunoscreening with anti-RA protein antibody. When we examined the lysate of ten positive clones by Western blotting, seven clones produced immunopositive protein with molecular weight more than 130 kDa, and three clones made positive protein with a molecular weight lower than 130 kDa. The former phage DNA contained 2.4 kb EcoRI fragment and the latter contained 3.2 kb EcoRI fragment. The 2.4 kb EcoRI fragment was cloned in the plasmid pU19 to construct pLB1, which was expressed in E.coli JM109 and produced the RA protein identical in molecular weight to the purified RS protein. The delated mutant of pLB1 was produced and work is in progress to sequence the 2.4 kb fragment of the cloned DNA.
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