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目的家蝇是重要的卫生害虫,其抗药性问题突出,其中细胞色素P450介导的代谢解毒作用增强是其抗药性的重要机制。细胞色素P450还原酶(cytochrome P450 reductase,CPR)是细胞色素P450酶系的重要组分,建立家蝇细胞色素P450还原酶(Md CPR)表达体系有助于通过重组家蝇细胞色素P450酶系以鉴定家蝇抗药性的生物化学机制。方法将家蝇CPR基因的编码区克隆到表达载体(p B508)上构建重组质粒,在大肠埃希菌中重组质粒经异丙基-β-d-硫代半乳糖苷(IPTG)诱导表达,对表达的Md CPR蛋白进行细胞定位分析和活性测定。结果 Md CPR蛋白在大肠埃希菌中成功地得到表达,分子质量为76×103左右,表现出细胞色素C还原酶活性,主要定位在细胞的膜成分中。结论在大肠埃希菌中表达了具有还原酶活性的Md CPR蛋白,为进一步研究家蝇细胞色素P450的功能及其在抗药性中的作用奠定了基础。
Objective Housefly is an important health pest, its drug resistance problems prominent, including cytochrome P450-mediated metabolic detoxification is an important mechanism of its resistance. Cytochrome P450 reductase (CPR) is an important component of the cytochrome P450 enzyme system. The establishment of a housefly cytochrome P450 reductase (Md CPR) expression system is helpful for the expression of cytochrome P450 reductase Identification of biochemical mechanisms of housefly resistance. Methods The coding region of housefly CPR gene was cloned into the expression vector (pB508) to construct a recombinant plasmid. The recombinant plasmid was induced by IPTG in Escherichia coli, The expression of Md CPR protein was assayed for cell localization and activity. Results Md CPR protein was successfully expressed in Escherichia coli. The molecular weight of Md CPR protein was about 76 × 103, showing cytochrome C reductase activity, which mainly localized in the membrane components of cells. Conclusion Md CPR protein with reductase activity was expressed in Escherichia coli, which laid the foundation for further study on the function of housefly cytochrome P450 and its role in drug resistance.