Cloning of cytochrome P-450 2C9 cDNA from human liver and its expression in CHL cells

查看全文 作　　者：Ge-Jian Zhu Ying-Nian Yu,Department of Pathophysiology and Laboratory of Medical Molecular Biology,Zhejiang University School of Medicine,Hangzhou 310031,Zhejiang Province,China Xin Li,Department of pharmaceutical analysis & drug metabolism,College of Pharmacology Science,Zhejiang University,Hangzhou 310031,Zhejiang Province,China Yu-Li Qian, Present address:Center of laboratory,Women’s hospital,School of Medicine,Zhejiang University,Hangzhou 310031,Zhejiang Province,China

出　　处：《World Journal of Gastroenterology》索引2002年第8卷第2期,共5页

基　　金：National Natural Science Foundation of China,No.39770868;Natural Science Foundation of Zhejiang Province,No.397490 摘　　要：AIM: Using bacterial, yeast, or mammalian cell expressing a human drug metabolism enzyme would seem good way to study drug metabolism-related problems. Human cytochrome P-450 2C9 ( CYP2 C9) 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 humans. In order to provide a sufficient amount of the enzyme for drug metabolic research, the CYP2 C9 eDNA was cloned and expressed stably in CHL cellsMETHODS: After extraction of total RNA from human livertissue, the human CYP2C9 eDNA was amplified withreverse transcription-polymerase chain reaction (RT-PCR),and cloned into cloning vector pGEM-T. The cDNA fragmentwas identified by DNA sequencing and subcloned into amammalian expression vector pREP9. A transgenic cell linewas established by transfecting the recombinant vector ofpREP9-CYP2C9 into CHL cells. The enzyme activity ofCYP2C9 catalyzing oxidation of tolbutamide to hydroxytolbutamide in S9 fraction of the cell was determined by highperformance liquid chromatography(HPLC).RESULTS: The amino acid sequence predicted from theeDNA segment was identical to that of CYP2 C9 * 1, the wildtype CYP2 C9. However, there were two base differences, i.e. 21T > C, 1146C > T, but the encoding amino acidsequence was the same, L7, P382. The S9 fraction of theestablished cell line metabolizes tolbutamide to hydroxytolbutamide; tolbutamide hydroxylass activity was found to be0.465 ± 0.109 μmol@ min-1 . g1 S9 protein or 8.62 ± 2.02 mol@ min 1 ~mol-1 CYP, but was undetectable in parental CHL cell.CONCLUSION: The cDNA of human CYP2C9 was successfullycloned and a cell line of CHL- CYP2C9, efficiently expressingthe protein of CYP2C9, was established. 关键词：人肝细胞色素 CDNA 克隆 CHL细胞

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Cloning of cytochrome P-450 2C9 cDNA from human liver and its expression in CHL cells

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