人细胞色素P450 1A2的异源表达及代谢活性测定

Expression and Characterization of Human Cytochrome P450 1A2

关键词:  细胞色素P450 1A2  杆状病毒  草地夜蛾细胞  非那西丁  活性测定

Key words:  cytochrome P450 1A2  baculovirus  Spodoptera frugiperda  phenacetin  activity assay

摘要:

目的获得单一的具有活性的人细胞色素P450 1A2(CYP1A2)重组酶，以进一步进行该酶参与的药物代谢研究。方法以人肝组织提取的总RNA为模板，RT- PCR扩增全长的人类CYP1A2基因片段，插入转座载体pFastBac构建重组转座质粒pFastBac-CYP1A2，转化入DH10Bac 大肠杆菌，获得重组杆状病毒穿梭质粒Bacmid-CYP1A2，以之转染草地夜蛾细胞(Sf9)，与细胞色素氧化还原酶(CYPOR)进行共表达。利用蛋白质印迹分析鉴定其表达，并以非那西丁为底物，利用HPLC对其进行代谢活性测定。 结果蛋白质印迹分析表明人CYP1A2蛋白在Sf9细胞中成功表达，且该重组酶对非那西丁的Km值为(82.04±11.39)μmol·L-1(n=3)，Vmax值为(1.78±0.26)nmol·min-1·mg-1蛋白(n=3)，表观清除率Clint值为0.02 mL·min-1·mg-1蛋白。结论利用杆状病毒/昆虫细胞系统，可获得具代谢活性的人CYP1A2重组酶，该酶可进一步用于其他底物的I相代谢研究。

Abstract:

OBJECTIVE To obtain a single active recombinant human CYP1A2, in order to take further study of drug metabolism. METHODS The full human CYP1A2 gene was got by RT-PCR from the total RNA being isolated from the human liver. This gene was subcloned into the expression vector pFastBac. Through transposition, recombinant bacmid-CYP1A2 DNA was formed and was then transfected into Spodoptera frugiperda 9 (Sf9) insect cells to generate the target protein with cytochrome P450 reductase (CYPOR). Western Blot analysis was used to identify the expression of CYP1A2, and the enzyme activity was assayed by HPLC with phenacetin as substrate. RESULTS The expression of recombinant human CYP1A2 was detected by Western Blot analysis, and the Km, Vmax and Clint with phenacetin as substrate were (82.04±11.39)μmol·L-1 (n=3), (1.78±0.26)nmol·min-1·mg-1 protein (n=3) and 0.02 mL·min-1·mg-1 protein respectively. CONCLUSION Active human recombinant CYP1A2 was obtained with baculovirus/insect cells expression system and it can be used to study phase I metabolism of other substrates.