人CYP2C19.1野生型和CYP2C19.2突变体蛋白体外表达模型的构建、活性表征及抑制剂研究

发布时间：2018-01-12 22:18

本文关键词：人CYP2C19.1野生型和CYP2C19.2突变体蛋白体外表达模型的构建、活性表征及抑制剂研究　出处：《浙江大学》2012年硕士论文　论文类型：学位论文

【摘要】：本课题考察了慢病毒介导、pcDNA3.1(+)介导及杆状病毒表达系统等几个体外表达系统,成功通过Bac.to-BacTM系统异源表达了人CYP2C19.1野生型和CYP2C19.2突变体蛋白；并利用经典底物和抑制剂验证异源蛋白的活性；同时筛选了70个中药单体,首次发现多个对CYP2C19具有抑制作用的单体。目的体外异源表达人CYP2C19.1野生型和CYP2C19.2突变体蛋白,考察其代谢动力学参数,为药物I相代谢的研究提供单一性的酶源；同时进行抑制剂的体外筛选研究。方法分别使用慢病毒介导的稳定表达系统、pcDNA3.1(+)介导的稳定表达系统及杆状病毒表达系统在体外表达人CYP2C19.1野生型及CYP2C19.2突变体蛋白。以人肝组织RNA为模板进行RT-PCR扩增反应,获得CYP2C19基因的cDNA序列,然后通过定点突变获得突变体的cDNA序列。将这些基因分别连接至各自载体质粒上进行后续的质粒构建、转染及蛋白表达过程。其中在杆状病毒表达系统中,用构建好的重组CYP2C19s杆状病毒,与本实验室已构建好的重组CYPOR.CYPb5杆状病毒一起感染Sf9细胞,在优化后的最佳共表达条件下获得相应重组酶。以奥美拉唑为底物分别检测重组酶的活性,并比较CYP2C19野生型与突变体蛋白之间的代谢动力学参数差异。测定经典底物噻氯吡啶和氟伏沙明作用于CYP2C19的IC50值和K值,验证重组酶用于抑制剂筛选的可行性；将此重组酶用于实验室已有的70种中药单体的抑制剂筛选,并详细测定了其中三种单体异土木香内酯、莪术醇及五味子甲素作用于CYP2C19的IC50值和Ki值。结果对三种表达系统的考察结果表明,慢病毒介导的稳定表达系统、pcDNA3.1(+)介导的稳定表达系统均未能成功表达相应蛋白,杆状病毒表达系统仍是目前实验室用于异源表达P450酶的最有效的系统。Western blot实验结果显示CYP2C19蛋白获得了有效的表达。以奥美拉唑为代谢底物测得,CYP2C19.1的酶动力学参数如下：Km为(4.99±0.22)μmol·L-1,Vmax为(0.2539±0.0024)μmol·min-1·mg-1 protein,CLint为0.0509 L·min-1·mg-1 protein；CYP2C19.2的酶动力学参数如下：Km为(5.822±0.27)μmol·L-1,Vmax为(0.5481±0.0058)μmol·min-1·mg-1 protein,CLint为0.0941L·min-1·mg-1 protein.噻氯吡啶抑制CYP2C19.1和CYP2C19.2蛋白代谢奥美拉唑的IC50值分别为2.28μmol·L-1和2.47μmol·L-1,K值分别为(0.64±O.025)μmol·L-1和2.038μmol·L-1；氟伏沙明对CYP2C19.1和CYP2C19.2蛋白代谢奥美拉唑的IC50值均为0.19μmol·L-1,Ki值分别为(0.29±0.090)μmol·L-1和0.04μmol·L-1。同时发现白藜芦醇、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具有较强的抑制能力。结论实验成功建立CYP2C19.1野生型和CYP2C19.2突变体蛋白的体外表达模型并获得了具有良好活性的重组酶,该重组酶适用于CYP2C19的体外底物及抑制剂筛选,并为药物经过CYP2C19代谢和基因组学的体外研究提供了重组蛋白模型。
[Abstract]:In this study, lentivirus-mediated pcDNA3.1 () -mediated and baculovirus expression systems were investigated. Human CYP2C19.1 wild-type and CYP2C19.2 mutant proteins were successfully expressed by Bac.to-BacTM system. The activity of heterologous proteins was verified by classical substrates and inhibitors. At the same time, 70 traditional Chinese medicine monomers were screened, and several monomers with inhibitory effect on CYP2C19 were found for the first time. Objective to investigate the metabolic kinetics of human CYP2C19.1 wild type and CYP2C19.2 mutant proteins by heterologous expression in vitro, and to provide a unique enzyme source for the study of drug phase I metabolism. At the same time, the screening of inhibitors in vitro was carried out. Methods Lentivirus-mediated stable expression systems were used respectively. PcDNA3.1 (). Expression of human CYP2C19.1 wild-type and CYP2C19.2 mutant proteins in vitro by mediated stable expression system and baculovirus expression system. RT-PC using human liver RNA as template. R amplification reaction. The cDNA sequence of CYP2C19 gene was obtained, and then the cDNA sequence of the mutant was obtained by site-directed mutation. These genes were respectively linked to the respective vector plasmids for subsequent plasmid construction. In the baculovirus expression system, recombinant CYP2C19s baculovirus was constructed. Sf9 cells were infected with recombinant CYPOR.CYPb5 baculovirus constructed in our laboratory. The recombinant enzyme was obtained under the optimized co-expression conditions, and the activity of the recombinant enzyme was detected using omeprazole as the substrate. The metabolic kinetic parameters of CYP2C19 wild-type and mutant proteins were compared. The IC50 and K values of CYP2C19 treated by classical substrates ticlopyridine and fluvoxamine were determined. To verify the feasibility of the recombinant enzyme used in the screening of inhibitors; The recombinant enzyme was used to screen the inhibitors of 70 traditional Chinese medicine monomers in laboratory, and three of them were determined in detail. Zedoary curcumol and Schisandrin A acted on IC50 and Ki values of CYP2C19. Results the results showed that the stable expression system mediated by lentivirus, pcDNA3.1 () -mediated stable expression system, could not express the corresponding protein successfully. Baculovirus expression system is still the most effective system for heterologous expression of P450 enzyme in laboratory. Western. Blot results showed that CYP2C19 protein was expressed effectively. Omeprazole was used as the metabolic substrate. The enzyme kinetic parameters of CYP2C19.1 were as follows: 1: km: 4.99 卤0.22) 渭 mol 路L ~ (-1). Vmax was 0.2539 卤0.0024 渭 mol 路min-1 路mg-1 protein. CLint was 0.0509L 路min-1 路mg-1 protein; The enzyme kinetic parameters of CYP2C19.2 were as follows: 1: km = 5.822 卤0.27) 渭 mol 路L ~ (-1). Vmax was 0.5481 卤0.0058 渭 mol 路min-1 路mg-1 protein. CLint 0.0941L 路min-1 路mg-1. The IC50 values of ticlopyridine inhibited the metabolism of omeprazole in CYP2C19.1 and CYP2C19.2 proteins were 2.28 渭 mol 路L -1 and 2.47 渭 mo, respectively. L 路L-1. K values were 0.64 卤0.025 渭 mol 路L -1 and 2.038 渭 mol 路L -1, respectively. The IC50 values of floroxamine on the metabolism of CYP2C19.1 and CYP2C19.2 protein were 0.19 渭 mol 路L ~ (-1), and those of omeprazole were 0.19 渭 mol 路L ~ (-1). The Ki values were 0.29 卤0.090 渭 mol 路L -1 and 0.04 渭 mol 路L -1, respectively. Resveratrol, emodin, isophora lactone, zedoary alcohol and menthol were also found. Podophyllotoxin, psoralen, Schisandrin A, Schisandrin B and New Wolf toxin B have strong inhibitory effect on human CYP2C19. Conclusion the in vitro expression model of CYP2C19.1 wild-type and CYP2C19.2 mutant proteins was successfully established and the recombinant enzyme with good activity was obtained. The recombinant enzyme is suitable for the screening of substrates and inhibitors of CYP2C19 in vitro and provides a recombinant protein model for the in vitro study of drug metabolism and genomics of CYP2C19.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R373

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