人CYP3A4与POR和cyt b_5的三重共表达及其条件优化  

Optimization of tri-expression of human CYP3A4 with POR and cyt b_5 in Sf 9 cells

作　　者：谢章明[1] 刘文慧[1] 徐迎春[1] 陈枢青[1] 

机构地区：[1]浙江大学药学院药理毒理与生化药学研究所,浙江杭州310058

出　　处：《浙江大学学报（医学版）》2013年第1期38-44,共7页Journal of Zhejiang University(Medical Sciences)

基　　金：国家科技重大专项基金(重大新药专项课题;No:2012ZX09506001-004)

摘　　要：目的:研究人CYP3A4与POR和cyt b5用悬浮细胞共表达时的最佳条件。方法:悬浮培养Sf 9细胞,分别摸索摇床的温度、转速、摇瓶中细胞液的量、表面活性剂的加量、细胞的培养时间,以及3种病毒的加量比例等条件,并进行优化;然后,将表达产物制成微粒体后用于睾酮的代谢,并分别测其代谢活性。结果:当恒温摇床的温度为27℃,转速为90 r/min,每250 ml摇瓶中加入80～120 ml细胞密度为5×105个/ml的细胞液,并加入0.1%的PluronicF-68,细胞培养72 h时,最适合细胞的生长和目的蛋白的表达。此时,当3种病毒的加量比例为1∶1∶1时,表达条件最佳,其对底物睾酮代谢的Km值、Vmax和CLint分别为119.6μmol/L、0.52μmol/(min.g蛋白)和4.34 ml/(min.g蛋白)。结论:通过三重共表达CYP3A4、POR、cyt b5,以及对表达条件的优化,提高了目的蛋白的表达量,得到了较高代谢活性的CYP3A4,从而为新药代谢及药物-药物相互作用研究奠定了基础,为药物代谢酶的大量表达提供了可行的方案。Objective： To investigate the optimal conditions of tri-expression of CYP3A4, POR and eyt b5 in Sf9 cells. Methods： The Sf 9 cells expressing CYP3A4, POR and cyt b5 were cultured in shaker flasks. The optimized conditions, including the temperature and rotation speed, the culture volume, the amount of surfaetant and the culture time were studied. The expressed products in microsomes were used to metabolize the testosterone and their metabolic activity was determined. Results： When the temperature and rotation speed of the shaker were 27℃ and 90 r/min, the cell density and culture volume were 5 × 10^5 cells/ml and 80 - 120 ml per 250 ml shaker flasks,respectively. When Pluronic F-68 was 0.1% and the culture time was 72 h, the condition was most suitable for culture of Sf9 cells and expression of targeted proteins. When the ratio of the volume of three added viruses was 1 ： 1 ： 1, the expression condition was optimal, under which the K,n, Vmax, and GLint for testosterone metabolism were 119. 6 μmol/L, 0. 52 μmol/（ min · g protein） and 4. 34 ml/（ min · g protein ）, respectively. Conclusions： The conditions of tri-expressing of CYP3A4, POR and cyt bshave been optimized in the study and the product CYP3A4 is obtained with higher metabolic activity.

关 键 词：细胞色素b5类 细胞色素P450酶系统 氧化还原酶类 药物相互作用 药物设计 昆虫 细胞学 基因 病毒 细胞 培养的 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]