本文關(guān)鍵詞：人CYP2C19.1野生型和CYP2C19.2突變體蛋白體外表達(dá)模型的構(gòu)建、活性表征及抑制劑研究　出處：《浙江大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： CYP2C19 重組酶 桿狀病毒表達(dá)系統(tǒng) 奧美拉唑 噻氯吡啶 氟伏沙明 天然產(chǎn)物 抑制劑

【摘要】：本課題考察了慢病毒介導(dǎo)、pcDNA3.1(+)介導(dǎo)及桿狀病毒表達(dá)系統(tǒng)等幾個體外表達(dá)系統(tǒng),成功通過Bac.to-BacTM系統(tǒng)異源表達(dá)了人CYP2C19.1野生型和CYP2C19.2突變體蛋白；并利用經(jīng)典底物和抑制劑驗證異源蛋白的活性；同時篩選了70個中藥單體,首次發(fā)現(xiàn)多個對CYP2C19具有抑制作用的單體。 目的體外異源表達(dá)人CYP2C19.1野生型和CYP2C19.2突變體蛋白,考察其代謝動力學(xué)參數(shù),為藥物I相代謝的研究提供單一性的酶源；同時進(jìn)行抑制劑的體外篩選研究。 方法分別使用慢病毒介導(dǎo)的穩(wěn)定表達(dá)系統(tǒng)、pcDNA3.1(+)介導(dǎo)的穩(wěn)定表達(dá)系統(tǒng)及桿狀病毒表達(dá)系統(tǒng)在體外表達(dá)人CYP2C19.1野生型及CYP2C19.2突變體蛋白。以人肝組織RNA為模板進(jìn)行RT-PCR擴(kuò)增反應(yīng),獲得CYP2C19基因的cDNA序列,然后通過定點突變獲得突變體的cDNA序列。將這些基因分別連接至各自載體質(zhì)粒上進(jìn)行后續(xù)的質(zhì)粒構(gòu)建、轉(zhuǎn)染及蛋白表達(dá)過程。其中在桿狀病毒表達(dá)系統(tǒng)中,用構(gòu)建好的重組CYP2C19s桿狀病毒,與本實驗室已構(gòu)建好的重組CYPOR.CYPb5桿狀病毒一起感染Sf9細(xì)胞,在優(yōu)化后的最佳共表達(dá)條件下獲得相應(yīng)重組酶。以奧美拉唑為底物分別檢測重組酶的活性,并比較CYP2C19野生型與突變體蛋白之間的代謝動力學(xué)參數(shù)差異。測定經(jīng)典底物噻氯吡啶和氟伏沙明作用于CYP2C19的IC50值和K值,驗證重組酶用于抑制劑篩選的可行性；將此重組酶用于實驗室已有的70種中藥單體的抑制劑篩選,并詳細(xì)測定了其中三種單體異土木香內(nèi)酯、莪術(shù)醇及五味子甲素作用于CYP2C19的IC50值和Ki值。 結(jié)果對三種表達(dá)系統(tǒng)的考察結(jié)果表明,慢病毒介導(dǎo)的穩(wěn)定表達(dá)系統(tǒng)、pcDNA3.1(+)介導(dǎo)的穩(wěn)定表達(dá)系統(tǒng)均未能成功表達(dá)相應(yīng)蛋白,桿狀病毒表達(dá)系統(tǒng)仍是目前實驗室用于異源表達(dá)P450酶的最有效的系統(tǒng)。Western blot實驗結(jié)果顯示CYP2C19蛋白獲得了有效的表達(dá)。以奧美拉唑為代謝底物測得,CYP2C19.1的酶動力學(xué)參數(shù)如下：Km為(4.99±0.22)μmol·L-1,Vmax為(0.2539±0.0024)μmol·min-1·mg-1 protein,CLint為0.0509 L·min-1·mg-1 protein；CYP2C19.2的酶動力學(xué)參數(shù)如下：Km為(5.822±0.27)μmol·L-1,Vmax為(0.5481±0.0058)μmol·min-1·mg-1 protein,CLint為0.0941L·min-1·mg-1 protein.噻氯吡啶抑制CYP2C19.1和CYP2C19.2蛋白代謝奧美拉唑的IC50值分別為2.28μmol·L-1和2.47μmol·L-1,K值分別為(0.64±O.025)μmol·L-1和2.038μmol·L-1；氟伏沙明對CYP2C19.1和CYP2C19.2蛋白代謝奧美拉唑的IC50值均為0.19μmol·L-1,Ki值分別為(0.29±0.090)μmol·L-1和0.04μmol·L-1。同時發(fā)現(xiàn)白藜蘆醇、大黃素、異土木香內(nèi)酯、莪術(shù)醇、薄荷腦、鬼臼毒素、補(bǔ)骨脂素、五味子甲素、五味子乙素、新狼毒素B等對人CYP2C19具有較強(qiáng)的抑制能力。 結(jié)論實驗成功建立CYP2C19.1野生型和CYP2C19.2突變體蛋白的體外表達(dá)模型并獲得了具有良好活性的重組酶,該重組酶適用于CYP2C19的體外底物及抑制劑篩選,并為藥物經(jīng)過CYP2C19代謝和基因組學(xué)的體外研究提供了重組蛋白模型。
[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 鹵0.22) 渭 mol 路L ~ (-1). Vmax was 0.2539 鹵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 鹵0.27) 渭 mol 路L ~ (-1). Vmax was 0.5481 鹵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 鹵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 鹵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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R373

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