| 利用细菌/杆状病毒系统在昆虫细胞中表达人CYP2E1 |
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| 引用本文： | 路珂,曾苏,姚彤炜. 利用细菌/杆状病毒系统在昆虫细胞中表达人CYP2E1[J]. 浙江大学学报(医学版), 2008, 37(2): 118-125. DOI: 10.3785/j.issn.1008-9292.2008.02.003 |
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| 作者姓名： | 路珂 曾苏 姚彤炜 |
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| 作者单位： | 浙江大学药学院,药物分析与药物代谢实验室,浙江,杭州,310058 |
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| 基金项目： | 浙江省中医药管理局资助项目 , 国家自然科学基金 , 浙江省科技厅资助项目 |
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| 摘 要： | 目的获得人CYP2E1重组酶,并用该重组酶的特征性探针底物对其进行代谢活性研究.方法以人肝组织RNA为模板,通过RT-PCR得到CYP2E1 cDNA片断,然后与pFastBac质粒连接,得到pFastBac-CYP2E1重组质粒,将其转化E.coli DH 10Bac大肠杆菌,通过转座作用,获得重组Bacmid-CYP2E1,将其转染草地夜蛾细胞(Sf9) 后,产生重组杆状病毒.将该病毒以及分别含有人CYPOR和人CYPb5的病毒共同感染Sf9细胞,收集共表达蛋白,以氯唑沙宗为底物鉴定重组酶的活性.结果利用细菌/杆状病毒系统得到重组人CYP2E1的表达,其对氯唑沙宗的Km值为(72.4±8.7)μmol·L-1,Vmax值为(2.41±0.10)μmol·min-11·g-1蛋白.结论利用杆状病毒系统成功表达了有催化活性的人CYP2E1重组酶,其活性与文献报道值相似.
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| 关 键 词： | 杆状病毒科 细胞色素P450酶系统 蛾/细胞学 大肠杆菌/遗传学 重组,遗传 细胞色素P450 杆状病毒 草地夜蛾细胞 氯唑沙宗 利用 细菌 病毒 系统 昆虫细胞 达人 system expression insect cells 相似 文献 催化活性 表达蛋白 Vmax 重组人 结果 酶的活性 氯唑沙宗 集共 共同感染 |
| 文章编号： | 1008-9292(2008)02-0118-08 |
| 修稿时间： | 2007-12-14 |
Expression of human CYP2E1 in insect cells using bac-to-bac expression system |
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| LU Ke,ZENG Su,YAO Tong-wei. Expression of human CYP2E1 in insect cells using bac-to-bac expression system[J]. Journal of Zhejiang University. Medical sciences, 2008, 37(2): 118-125. DOI: 10.3785/j.issn.1008-9292.2008.02.003 |
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| Authors: | LU Ke ZENG Su YAO Tong-wei |
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| Affiliation: | Department of Pharmaceutical Analysis and Drug Metabolism, College of Pharmaceutical Science, Zhejiang University, Hangzhou 310058, China. |
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| Abstract: | OBJECTIVE: To obtain recombinant human CYP2E1 and to determine its activity by using the specific probe substrate. METHODS: CYP2E1 cDNA was obtained by RT-PCR using human liver RNA as template. The cloned CYP2E1 cDNA was ligated with pFastBac vector to generate recombinant pFastBac-CYP2E1, which was then transformed into E. coli DH 10 Bac. Recombinant Bacmid-CYP2E1 was generated by transposition. Then Spodoptera frugiperda (Sf9) insect cells was infected with Bacmid-CYP2E1 to generate recombinant baculoviruses carrying human CYP2E1 cDNA. Finally, Sf9 insect cells were triinfected with recombinant baculoviruses carrying human CYP2E1, CYPOR and CYPb5. The activity of the recombinant enzymes was determined using chlorzoxazone as the substrate. RESULT: The Kmand Vmaxof recombinant CYP2E1 to chlorzoxazone was (72.4 +/-8.7) micromol. L(-1) and (2.41 +/-0.10) micromol.min(-1)?g(-1)protein, respectively. CONCLUSION: Active recombinant CYP2E1 has been obtained by bac-to-bac expression system and its activity is similar to previous reports. |
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| Keywords: | Baculoviridae Cytochrome P-450 Enzyme System Moths/cytol Escherichia coli/genet Recombination genetic Cytochrome P450 Baculovirus Spodoptera frugiperda(Sf9) insect cells Chlorzoxazone |
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