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  

Co-expression model and inhibitor screening of the human P450 CYP2C19 with the CYPOR and CYPb5

作　　者：孔丽敏[1] 胡海红[1] 曾苏[1] 

机构地区：[1]浙江大学药学院药物分析与药物代谢实验室,杭州310058

出　　处：《药物分析杂志》2012年第12期2099-2107,共9页Chinese Journal of Pharmaceutical Analysis

基　　金：国家重大专项(2012ZX09506001-004;2009ZX09304-003);国家自然科学基金(81173120)资助项目

摘　　要：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Km为(4.99±0.22)μmol.L-1,最大反应速率Vmax为(0.2539±0.0024)μmol.min-1.mg-1蛋白,表观清除率CLint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 suhstrate, and to employ it for in vitro inhibitor screening for the natural product. Method： The Bac - to - Bac baculovirns expression system was used to co - express recombinant CYP2C19 with CYPOR and CYPbS. The His - tagged recombinant CYP2C19 was detected by Western Blot using anti - His antibody. The enzyme activity was de- termined by HPLC using omeprazole as a probe substrate. Then the recombinant enzyme was used to screen the inhi- bition activities to CYP2C19 of 70 phytochemicals. Result：The expression of CYP2C19 was detected by Western blot analysis. The Kin, Vmax and CLant were （4.99 ± 0.22 ）μ mol · L^- 1, （0. 2539 ± 0. 0024 ） μmol · min ^- 1 . mg ^- 1 protein, and 0. 0509 L·main ^-1·mg^- 1 protein, respectively. The inhibitor screening analyses revealed that resvera- trol, 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 screen- ing in vitro.

关 键 词：人细胞色素 蛋白重组酶 昆虫细胞-杆状病毒表达系统 蛋白印迹分析 HPLC法酶活性鉴定 奥美拉唑底物 天然产物抑制剂筛选 酶动力学参数 

分 类 号：R917[医药卫生—药物分析学]