1987 Fiscal Year Final Research Report Summary
Studies on P. cepacia nemolysin by molecular cloning
| Project/Area Number |
61570211
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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 | Yamaguchi University |
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Principal Investigator |
NAKAZAWA Teruko Department of Microbiology, Yamaguchi University School of Medicine, Professor, 医学部, 教授 (40053053)
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| Co-Investigator(Kenkyū-buntansha) |
ISHIBASHI Masahiro School of Allied Health Sciences, Yamaguchi University, Assistant, 医療技術短期大学部, 助手 (90176222)
ABE Mitsuko Department of Microbiology, Yamaguchi University School of Medicine, Assistant, 医学部, 助手 (60107737)
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| Project Period (FY) |
1986 – 1987
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| Keywords | Pseudomonas cepacia / hemolysin / molecular cloning / cloning vector / 白血球毒 |
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| Research Abstract |
1. A gene library of a hemolytic strain of P. cepacia, JN106, was made in E. coli, and hemolytic clones were obtained. The plasmid in one of the clones was isolated, and the restriction and genetic maps were determined. The partial DNA sequence determined revealed an open reading frame corresponding to the 447 amino acid sequence. From the molecular weight of P. cepacia hemolysin, the deduced amino acid sequence was assumed to correspond to 90% of the whole sequence lacking the N terminus.
2. To analyze the hemolysin gene in P. cepacia, a host-vectorsystem was developed. A shuttle vector, pTS1209 was constructed which could be transferred from E. coli to P. cepacia by the aid of a helper plasmid, pRK2013.
3. An anti-hemolysin antibody was made in rabbits. The antibody specifically inhibited the hemolysin activity . Hemolysins produced by two other strains were shown to have common antigenicity to JN106 hemolysin.
4. Binding of hemolysin to red blood cells ocured at 2C followed by hemolytic reaction which occured at 37C.
5. Toxicity of hemolysin to leukocytes were investigated. Mixed leukocytes, particularly granulocytes were susceptible to hemolysin. The cytotoxicity was inhibited by preincubation of hemolysin with cholesterol which inhibited hemolysis. The hemolysin activity decreased by heat treatment which accompanied by the decrease of leukocyte toxicity.
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