1994 Fiscal Year Final Research Report Summary
CLONING OF RECEPTOR GENE FOR BOTULINUM TYPE C1 NEUROTOXIN
| Project/Area Number |
05670261
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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 |
Bacteriology (including Mycology)
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| Research Institution | SAPPORO MEDICAL UNIVERSITY |
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Principal Investigator |
FUJII Nobuhiro SAPPORO MEDICAL UNIVERSITY SCHOOL OF MEDICINE,PROF., 医学部, 教授 (90133719)
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| Co-Investigator(Kenkyū-buntansha) |
KIMURA Koichi SAPPORO MEDICAL UNIVERSITY SCHOOL OF MEDICINE,INSTRUCTOR, 医学部, 助手 (90177915)
YOKOSAWA Noriko SAPPORO MEDICAL UNIVERSITY SCHOOL OF MEDICINE,ASSIST.PROF., 医学部, 講師 (50167722)
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| Project Period (FY) |
1993 – 1994
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| Keywords | Botulinum C1 neurotoxin / Binding protein / receptor protein / Gene cloning |
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| Research Abstract |
Messenger RNA was isolated from NG108 cells, and then cDNA was synthesized by ZAP-cDNA synthesis kit to identify the gene encoding receptor for botulinum type Cl neurotoxin. cDNA libraries based on Uni-ZAP XR vector were immunoscreened by botulinum Cl neurotoxin (M.W.150,000) and anti-neurotoxin monoclonal antibodies. We did not get any positive clones because of high level of back ground. On the other hand, ganglyosides, such as GT1b, on cell surface membrane is partly implicated in binding of neurotoxin to cell. Therfore, it is important to consider the posibility of correlation between neurotoxin and ganglyosides.
To decrease the back ground level, the binding domain, which is 650 nucleotide sequences of 3'-terminal region of botulinum C1 neurotoxin gene, is amplificated by PCR (plymerase chain reaction) and cloned into pBleuscript-II.Expression vector pET-3a is used to isolate the protein of binding domain through recloning of the PCR-amplified DNA fragment. The production of the binding protein is confirmed by western-blott analysis using several monoclonal antibodies against botulinum type C1 neurotoxin and by a competitive test.
In addition to this, for immunoscreening of cDAN lybraries, receptor protein is isolated by cross-linker and immunoprecipitation methods to establish antibodies, which make possible to get positive clones.
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