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细胞色素P450 2D6(cytochrome P450 2D6,CYP2D6)是一种重要的氧化代谢酶,呈基因多态性,对药物的代谢呈现明显的个体差异和种族差异,CYP2D6*10在中国人群中的发生率高达51.3%。采用杆状病毒昆虫表达系统,采用与细胞色素氧化还原酶(cytochrome oxidoreductase,CYPOR)共表达的方式获得具有代谢活性的CYP2D6*1/CYP2D6*10,并初步研究其催化右美沙芬的代谢活性差异。构建重组质粒pFastBac-CYP2D6*1,pFastBac-CYP2D6*10和pFastBac-CYPOR,并分别转化DH10Bac菌株构建重组Bacmid-CYPOR,Bacmid-CYP2D6*1和Bacmid-CYP2D6*10,转染Sf9昆虫细胞,获得重组杆状病毒用于病毒扩增和感染Sf9昆虫细胞。通过测定混悬蛋白催化右美沙芬的速率来优化重组CYPOR和CYP2D6病毒感染Sf9昆虫细胞的感染复数(multiplicity of infection,MOI)值和其比例,获得活力较高的重组CYP2D6*1和CYP2D6*10。重组CYP2D6*1催化右美沙芬的Km为(26.67±2.71)μmol·L-1(n=3),Vmax为(666.7±56.78)pmol·nmol-1(CYP2D6)·min-1(n=3),清除率为25.0;CYP2D6*10催化右美沙芬的Km为(111.36±10.89)μmol·L-1(n=3),Vmax为(222.2±20.12)pmol·nmol-1(CYP2D6)·min-1(n=3),清除率为2.0。利用杆状病毒昆虫细胞系统表达的重组CYP2D6*1和CYP2D6*10代谢药物具有活性差异,可用于体外研究其催化某些药物的代谢差异,这有助于理解药物代谢的个体差异、预测药物之间的相互作用。
Cytochrome P450 2D6 (CYP2D6) is an important oxidative metabolic enzyme with genetic polymorphism. It shows significant individual differences and racial differences in the metabolism of CYP2D6 * 10. The incidence of CYP2D6 * 10 in Chinese population is as high as 51.3%. The baculovirus expression system was used to obtain the metabolic activity of CYP2D6 * 1 / CYP2D6 * 10 by co-expression with cytochrome oxidoreductase (CYPOR), and preliminary study of its catalytic metabolic dextromethorphan difference . The recombinant plasmids pFastBac-CYP2D6 * 1, pFastBac-CYP2D6 * 10 and pFastBac-CYPOR were constructed and transformed into DH10Bac strain to construct recombinant Bacmid-CYPOR, Bacmid-CYP2D6 * 1 and Bacmid-CYP2D6 * 10, Baculovirus is used for virus amplification and infection of Sf9 insect cells. The multiplicity of infection (MOI) value and its proportion of Sf9 insect cells infected by recombinant CYPOR and CYP2D6 virus were optimized by measuring the rate of the enzyme-catalyzed dextromethorphan by the canned protein, and the higher viable recombinant CYP2D6 * 1 and CYP2D6 * 10 . The Km of recombinant CYP2D6 * 1 was (26.67 ± 2.71) μmol·L-1 (n = 3) and the Vmax was (666.7 ± 56.78) pmol · nmol-1 (CYP2D6) · min- ), And the clearance rate was 25.0. The Km of dextromethorphan was (111.36 ± 10.89) μmol·L-1 (n = 3) and the Vmax was (222.2 ± 20.12) pmol · nmol-1 (CYP2D6) · min -1 (n = 3) with a clearance of 2.0. The differences in activity of recombinant CYP2D6 * 1 and CYP2D6 * 10 metabolites expressed in the baculovirus insect cell system can be used in vitro to study their catalytic differences in the metabolism of certain drugs, which helps to understand individual differences in drug metabolism, Interaction between.