1992 Fiscal Year Final Research Report Summary
PREPARATION OF ANTIBODY PEPTIDE INHIBITING POLYPHENOL OXIDASE
| Project/Area Number |
03660143
|
|---|
| Research Category |
Grant-in-Aid for General Scientific Research (C)
|
| Allocation Type | Single-year Grants |
|---|
| Research Field |
製造化学・食品
|
|---|
| Research Institution | KYUSHU UNIVERSITY |
|---|
Principal Investigator |
YOSHIMOTO Makoto KYUSHU UNIVERSITY,FACULTY OF AGRICULTURE,ASSOCIATE PROFESSOR, 農学部, 助教授 (90182831)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
MIYAMOTO Takahisa KYUSHU UNIVERSITY,FACULTY OF AGRICULTURE,ASSISTANT PROFESSOR, 農学部, 助手 (70190816)
HATANO Shoji KYUSHU UNIVERSITY,FACULTY OF AGRICULTURE,PROFESSOR, 農学部, 教授 (30038260)
|
|---|
| Project Period (FY) |
1991 – 1992
|
| Keywords | Polyphenol oxidase / Monoclonal antibody / Inhibitory antibody / Antibody peptide / PCR / Fv fragment |
|---|
| Research Abstract |
We planned to make use of an inhibitory peptide prepared from a monoclonal antibody as an inhibitor of food-deteriorating enzyme. Polyphenol oxidases (EC 1.14.18.1),which catalyze browning reaction in foods and are widely distributed throughout plants and mammals, were employed as food-deteriorating enzyme. 1) Since the enzyme preparation (mushroom) purchased from Sigma was brown-colored and contained the contaminating protein, polyphenol oxidase was purified to electrophoretic homogeneity by DEAE-Sepharose and TSKgel-DEAE 5PW ion exchange chromatography and gel filtration on ZORBAX GF-1250.2) Eight stable hybridoma cell lines which secrete monoclonal antibodies against purified polyphenol oxidase were obtained in ten separate fusions. These antibodies were inhibitory or stimulatory or had no effect on the enzyme activity. However, the inhibitory activity was very weak. Therefore, we investigated the term and the number of immunization and the kind of adjuvant, but we could not successfully obtain the antibody which strongly inhibits the enzyme. 3) We attempted to directly prepare Fv fragment, a minimum antigen-binding unit, from E. coli. Poly (A)^+RNA was purified from the spleen of the mouse immunized with the enzyme and were reversed to cDNA. Fv genes were successfully amplified using appropriate primers and polymerase chain reaction (PCR). Expression of the Fv fragment from E. coli is in progress.
|
