体外重组人细胞色素P450 2C9酿酒酵母表达体系的构建及其基因多态性对药物相互作用影响的初探

Establishment of the recombinant human cytochrome P450 2C9 Saccharomyces cerevisiae expression systems in vitro and the impact of gene polymorphisms on drug-drug interactions

目的体外构建重组人细胞色素P450 2C9(CYP2C9)酿酒酵母(Saccharomyces cerevisiae)表达体系,且利用该体系研究药物对CYP2C9基因多态性酶抑制程度的差异性。方法将CYP2C9*1(野生型)和通过定点突变PCR法获得的等位基因突变克隆CYP2C9*2(R144C突变体)和CYP2C9*3(I359L突变体)电穿孔转化酿酒酵母后,差速离心法制备酿酒酵母微粒体,蛋白免疫印迹法检测3种CYP2C9微粒体蛋白的表达;HPLC法检测CYP2C9*1与特异性底物双氯芬酸的反应,记录产物峰面积值,利用Prism Demo软件计算米氏常数(Km)值;利用荧光底物BOMCC对3个等位基因进行酶活性分析,分别计算Km、最大酶促反应速度(Vmax)和内在清除率。采用荧光高通量方法测定5种药物(甲苯磺丁脲、磺胺苯比唑、酮康唑、氟西汀和泰洛平)对酶的抑制程度。结果蛋白免疫印迹结果表明,3个等位基因均表达目的蛋白。CYP2C9*1代谢双氯芬酸的Km值为5.34±0.96μmol/L;以BOMCC为底物时,CYP2C9*1和CYP2C9*2的Km值分别为16.94±4.78、34.73±5.51μmol/L,Vmax分别为0.21±0.10、0.12±0.01pmol/(min·pmolP450),内在清除率分别为0.012±0.003、0.003±0.0001μl/(min·pmolP450);CYP2C9*3无催化活性。5种药物对CYP2C9*1的抑制程度:磺胺苯比唑>酮康唑>氟西汀>甲苯磺丁脲>泰洛平;对CYP2C9*2的抑制程度:磺胺苯比唑>甲苯磺丁脲>酮康唑>氟西汀>泰洛平。结论成功构建了重组人细胞色素CYP2C9及其多态性等位基因(CYP2C9*2、CYP2C9*3)酿酒酵母表达系统;初步建立了药物对CYP2C9基因多态性酶的抑制作用体外检测体系,为指导临床联合用药奠定了基础。

Objective Recombinant human cytochrome P450 2C9 （CYP2C9） Saccharomyces cerevisiae expression systems are constructed in vitro, and drug inhibition degrees on the different CYP2C9 gene enzymes were measured respectively. Methods Recombinant human CYP2C9^＊1 （wild type） and two mutant （CYP2C9^＊2, CYP2C9^＊3） clones were expressed in Saccharomyces cerevisiae. The microsomals were prepared by differential centrifugation. The proteins were identified by Western blotting. The reaction of CYP2C9^＊ 1 and the specificity substrate Diclofenac was detected by HPLC, and recorded the value of the product peak area, calculated Km value using PrismDemo software. The activity of three alleles were analyzed using fluorescent substrate BOMCC. Km, the maximum enzymatic reaction rates （Vmax） and the intrinsic clearance rates were calculated using software PrismDemo. Five kinds of drugs （Tolbutamide, Sulfaphenazole, Ketoconazole, Fluoxetine, and Pioglitazone） on the enzyme inhibition were measured by fluorescent high-throughput. Results The expression of enzymes were confirmed by Western blotting. Diclofenac as a substrate ,the Km of CYP2C9^＊ 1 was 5.34 ± 0.96 μmol/L, which was in the range of FDA recommended, BOMCC as a fluorescent substrate, the Km Vmax, and the intrinsic clearance rates of CYP2C9^＊1 and CYP2C9^＊2 were 16.94 ± 4.78, 34.73 ±5.51 μmol/L; and 0.21±0.10, 0.12 ±0.01 pmol/（min·pmol P450）; 0.012 ± 0.003, 0.003 ± 0.0001 μl/（min·pmol P450）, respectively. The inhibition degree of five drugs to CYP2C9^＊1 were Sulfaphenazole 〉 Ketoconazole 〉 Fluoxetine 〉 tolbutamide 〉 pioglitazone.The inhibition degree of five drugs on CYP2C9^＊2 were Sulfaphenazole 〉 tolbutamide 〉 Ketoconazole 〉 Fluoxetine 〉 pioglitazone. Conclusions The Saccharomyces cerevisiae expression system of CYP2C9 and its genetic polymorphism were constructed successfully. The detection system of inhibition on drugs to CYP2C9 enzymes were established in vitro, which will give the basis of clinical combination.