人细胞色素P450_CYP2C19与CYPOR、CYPb5共表达模型的构建及其抑制剂筛选

目的: 获得人细胞色素P450 2C19(CYP2C19)蛋白重组酶,用特征性底物表征重组酶活性,并将该重组酶用于天然产物CYP2C19抑制剂的筛选。 方法: 构建含人CYP2C19基因的重组杆状病毒穿梭质粒,使用昆虫细胞-杆状病毒表达系统共表达人CYP2C19、细胞色素氧化还原酶(CYPOR)和细胞色素b5(CYPb5);用蛋白印迹分析(Western Blot)鉴定重组酶的表达;以奥美拉唑为底物进行代谢孵育,HPLC分析鉴定酶的活性;并使用该重组蛋白对70种天然产物进行抑制能力的筛选。 结果: Western Blot结果显示重组人CYP2C19蛋白在Sf 9细胞中成功表达,该重组酶代谢经典底物奥美拉唑的酶动力学参数分别为:米氏常数K_m为(4.99±0.22) μmol·L^-1,最大反应速率V_max为(0.2539±0.0024) μmol·min^-1·mg^-1蛋白,表观清除率CL_int值为0.0509 L·min^-1·mg^-1蛋白。对70种天然产物的抑制筛选实验显示白藜芦醇、大黄素、异土木香内酯、莪术醇、薄荷脑、鬼臼毒素、补骨脂素、五味子甲素、五味子乙素、新狼毒素B等对人CYP2C19有比较明显的抑制能力。 结论: 利用昆虫细胞-杆状病毒表达系统,可获得代谢活性良好的重组人CYP2C19蛋白,该重组酶可进一步用于其他底物的Ⅰ相代谢研究以及各种抑制剂的筛选。

Objective: To obtain recombinant human CYP2C19,in order to determine its activity by using a specific probe substrate,and to employ it for in vitro inhibitor screening for the natural product. Method: The Bac-to-Bac baculovirus expression system was used to co-express recombinant CYP2C19 with CYPOR and CYPb5.The His-tagged recombinant CYP2C19 was detected by Western Blot using anti-His antibody.The enzyme activity was determined by HPLC using omeprazole as a probe substrate.Then the recombinant enzyme was used to screen the inhibition activities to CYP2C19 of 70 phytochemicals. Result: The expression of CYP2C19 was detected by Western blot analysis.The K_m,V_max and CL_int were (4.99±0.22) μmol·L^-1,(0.2539±0.0024) μmol·min^-1·mg^-1 protein,and 0.0509 L·min^-1·mg^-1 protein,respectively.The inhibitor screening analyses revealed that resveratrol,emodin,isoalantolactone,curcumol,DL-menthol,podophyllotoxin,psoralen,schisandrin A,schisandrin B and chamaejasmine B showed inhibition to CYP2C19. Conclusion: Active recombinant CYP2C19 was obtained by Bac-to-Bac expression system,and it would facilitate much in studies of phase Ⅰ metabolism and inhibitors screening in vitro.