人细胞色素P450 3A4突变体CYP3A4.3,CYP3A4.4,CYP3A4.5和CYP3A4.18重组酶的异源表达与活性  

Heterogenous expression and activity analysis of human cytochrome P450 3A4 mutants CYP3A4.3,CYP3A4.4,CYP3A4.5 and CYP3A4.18

作　　者：王晓雯[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,浙江杭州310058

出　　处：《中国药理学与毒理学杂志》2009年第6期456-463,共8页Chinese Journal of Pharmacology and Toxicology

基　　金：国家"重大新药创制"科技重大专项资助项目(2009ZX09304-003)~~

摘　　要：目的重组表达人细胞色素P450(CYP)3A4突变体CYP3A4.3,CYP3A4.4,CYP3A4.5和CYP3A4.18蛋白,为CYP3A4代谢活性的体外研究提供单一酶源。方法应用杆状病毒表达系统构建含有上述各CYP3A4突变体基因序列的重组病毒,将其连同含人源还原型烟酰胺腺嘌呤二核苷磷酸-P450氧化还原酶(POR)和细胞色素b5基因的重组病毒共同感染昆虫草地夜蛾细胞Sf9得到CYP3A4突变体与POR和细胞色素b5共表达的重组蛋白,分别以高效液相色谱法和荧光分析法测定各重组酶对睾酮和7-甲氧基-4-三氟甲基香豆素的代谢活性。结果在mRNA分子水平上验证了CYP3A4突变体CYP3A4*3,CYP3A4*4,CYP3A4*5和CYP3A4*18基因在Sf9细胞中的转录。感染了各重组病毒的Sf9细胞裂解液对睾酮和7-苄氧基-4-三氟甲基香豆素有明显代谢。结论应用杆状病毒-昆虫细胞表达系统在体外成功表达了具有催化活性的人CYP3A4突变体CYP3A4.3,CYP3A4.4,CYP3A4.5和CYP3A4.18蛋白,其中CYP3A4.5活性显著低于野生型蛋白,CYP3A4.18活性显著高于野生型蛋白,而CYP3A4.3和CYP3A4.4与野生型蛋白活性近似。AIM To express recombinant human cytochrome P450 3A4 mutants CYP3A4.3,CYP3A4.4,CYP3A4.5 and CYP3A4.18,and to employ them for in vitro metabolism studies of CYP3A4.METHODS Use Bac-to-Bac baculovirus expression system to recombinant baculovirus carrying cDNA of CYP3A4 mutants CYP3A4.3,CYP3A4.4,CYP3A4.5 and CYP3A4.18.Spodoptera frugiperda 9（Sf9）,cells were co-infected by recombinant viruses of CYP3A4 mutants,human NADPH-P450 oxidoreductase and cytochrome b5 to obtain recombinant proteins CYP3A4.3,CYP3A4.4,CYP3A4.5 and CYP3A4.18 with metabolic activity.RESULTS The mRNA transcription of CYP3A4 mutants in Sf9 cells were validated by RT-PCR.Testosterone and 7-benzyloxy-4-（trifluoromethyl） coumarin were metabolized by the lysates of Sf9 cells infected by the recombinant viruses.CONCLUSION CYP3A4 mutants CYP3A4.3,CYP3A4.4,CYP3A4.5 and CYP3A4.18 with metabolic activity were successfully expressed by baculovirus-insect cell expression system.The results indicated that recombinant CYP3A4.5 showed lower activity comparing to the wild type protein towards testosterone,while CYP3A4.18 with higher activity,and for CYP3A4.3 and CYP3A4.4 showing similar activity to the wild type protein.

关 键 词：细胞色素P450CYP3A4 突变 杆状病毒科 睾酮 

分 类 号：R965.1[医药卫生—药理学]