Cloning of cytochrome P-450 2C9 cDNA from XXXX liver and its expression in @entity978 cells.

@entity1 @entity70700 @entity11243 @entity5060 @entity70701 @entity13323 @entity70702 @entity375 @entity3689 @entity978
AIM: Using bacterial, @entity375 , or @entity1 cell expressing a @entity1 drug metabolism enzyme would seem good way to study drug metabolism-related problems. @entity1 cytochrome P-450 2C9( @entity5060 ) is a polymorphic enzyme responsible for the metabolism of a large number of clinically important drugs. It ranks among the most important drug metabolizing enzymes in @entity1 . In order to provide a sufficient amount of the enzyme for drug metabolic research, the @entity5060 cDNA was cloned and expressed stably in @entity978 cells. METHODS: After extraction of total RNA from @entity1 liver tissue, the @entity1 @entity5060 cDNA was amplified with reverse transcription-polymerase chain reaction (RT-PCR), and cloned into cloning vector pGEM-T. The cDNA fragment was identified by DNA sequencing and subcloned into a @entity1 expression vector pREP9. A transgenic cell line was established by transfecting the recombinant vector of pREP9- @entity5060 into @entity978 cells. The enzyme activity of @entity5060 catalyzing oxidation of @entity11243 to @entity70700 in S9 fraction of the cell was determined by high performance liquid chromatography(HPLC). RESULTS: The @entity3689 sequence predicted from the cDNA segment was identical to that of @entity5060 *1, the wild type @entity5060 . However, there were two base differences, i.e. @entity70701 , @entity70702 , but the encoding @entity3689 sequence was the same, L7, P382. The S9 fraction of the established cell line metabolizes @entity11243 to @entity70700 ; @entity11243 hydroxylase activity was found to be 0.465 +/- 0.109 micromol.min(-1).g(-1) S9 protein or 8.62 +/- 2.02mol.min(-1).mol(-1) @entity13323 , but was undetectable in parental @entity978 cell. CONCLUSION: The cDNA of @entity1 @entity5060 was successfully cloned and a cell line of @entity978 - @entity5060 , efficiently expressing the protein of @entity5060 , was established.
