1994 Fiscal Year Final Research Report Summary
STUDIES ON EXPRESSION AND FUNCTION OF ENDOZEPINE IN ADRENOCORTICAL FASCICULATA CELLS.
| Project/Area Number |
05670104
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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 |
General pharmacology
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| Research Institution | THE JIKEI UNIVERSITY SCHOOL OF MEDICINE |
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Principal Investigator |
OHNO Yuji THE JIKEI UNIVERSITY SCHOOL OF MEDICINE,LECTURER, 医学部, 講師 (60142478)
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| Project Period (FY) |
1993 – 1994
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| Keywords | adrenal cortex / endozepine / polymerase chain reaction / fusion protein / monoclonal antibody / steroidogenic protein |
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| Research Abstract |
By means of polymerase chain reaction (PCR), we have clarified endozepine was expressed in bovine adrenocortical fasciculata cells. Then two primers for sticky end cloning of endozepine were synthesized. Between BamHI site and coding region of endozepine, the upper primer had nucleotide sequences encoding amino acids that recognized and cleaved by Factor X.The PCR product of endozepine cDNA was digested with BamHI and EcoRI and inserted into a plasmid vector of pGEX-2T.the cholesterol transport and transform into pregnenolone or not. The protein could dosedThe vector (pGEX/EDZ) was transfected to E.coli. The transfected E.coli were induced the production of GST-endozepine fusion protein. The fusion protein could purified by the glutathione-Sepharose 4B affinity column. The fusion protein could cleaved into GST and endozepine by Factor X.
Then we have tried to make monoclonal antibodies against the fusion protein. Intraperitoneally, we injected the protein to five mice. All mice made antibody against the protein. We prepared immune cells from spleen and made hybridoma cells by polyethylene glycol (PEG) 1500. We exploited single cell cloning technique by limiting dilution.
We also checked the steroidogenic activity of cloned endozepine in rat adrenocortical mitochondria. The mitochondria prepared from rats treated with ACTH (0.5IU/rat) and cycloheximide (10mg/rat) accumulated cholesterol in outer mitochondrial membrane. A protein was known that could accelerate the cholesterol transport from outer to inner mitochondrial membrane. We examined whether the cloning endozepine could facilitateependently stimulated pregnenolone production.
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