Functional expression of NADPH-cytochrome P450 reductase of the house fly (Musca domestica) in Escherichia coli

doi:10.11853/j.issn.1003.8280.2016.02.007

Chines Journal of Vector Biology and Control ›› 2016, Vol. 27 ›› Issue (2): 121-123.DOI: 10.11853/j.issn.1003.8280.2016.02.007

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Functional expression of NADPH-cytochrome P450 reductase of the house fly (Musca domestica) in Escherichia coli

FENG Jing^1,2, TIAN Kai^2,3, HOU Xin¹, QIU Xing-hui^2,3

1 Inner Mongolia University, College of Life Sciences, Hohhot 010021, Inner Mongolia Autonomous Region, China;
2 State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences;
3 University of Chinese Academy of Sciences

Received:2015-11-12 Online:2016-04-20 Published:2016-04-20
Supported by:
Supported by the Municipal Natural Science Foundation of Beijing of China (No. 5142014)

家蝇细胞色素P450还原酶的原核表达与活性鉴定

丰婧^1,2, 田凯^2,3, 侯鑫¹, 邱星辉^2,3

1 内蒙古大学生命科学学院, 呼和浩特 010021;
2 中国科学院动物研究所, 农业虫害鼠害综合治理研究国家重点实验室, 北京 100101;
3 中国科学院大学, 北京 100049

通讯作者: 侯鑫,Email:houxin2005@hotmail.com;邱星辉,Email:qiuxh@ioz.ac.cn
作者简介:丰婧,女,在读硕士,从事生物化学与分子生物学研究。
基金资助:
北京市自然科学基金(5142014)

Abstract

Abstract:

Objective House fly (Musca domestica) is a mechanical vector of many diseases which can cause severe consequences for human and animal health. Cytochrome P450 monooxygenases play an important role in resistance to many insecticides in the house fly. Establishment of functional expression system of NADPH-cytochrome P450 reductase will be helpful for the characterization of P450 mediated insecticide resistance. Methods In this study, we cloned the open reading frame of MdCPR gene into the expression vector pB508. The constructed plasmid MdCPR-pB508 was transformed into Escherichia coli (DH5α). The production of MdCPR protein in E. coli was induced by isopropyl β-thiogalactoside (IPTG). Results MdCPR was expressed in E. coli with an expected molecule weight of around 76×10³. The E. coli-produced MdCPR protein was mainly present in the membrane fraction, showing the catalytic activity of NADPH-dependent reduction of cytochrome C. Conclusion The successful functional expression of MdCPR can facilitate further investigation of the role of various cytochrome P450s in insecticide resistance in house flies.

Key words: Musca domestica, NADPH-cytochrome P450 reductase, Functional expression, Functional analysis

摘要：

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(MdCPR)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(pB508)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MdCPR蛋白进行细胞定位分析和活性测定。结果 MdCPR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MdCPR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。

关键词: 家蝇, 细胞色素P450还原酶, 功能表达, 活性检测

CLC Number:

FENG Jing, TIAN Kai, HOU Xin, QIU Xing-hui. Functional expression of NADPH-cytochrome P450 reductase of the house fly (Musca domestica) in Escherichia coli[J]. Chines Journal of Vector Biology and Control, 2016, 27(2): 121-123.

丰婧, 田凯, 侯鑫, 邱星辉. 家蝇细胞色素P450还原酶的原核表达与活性鉴定[J]. 中国媒介生物学及控制杂志, 2016, 27(2): 121-123.

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Functional expression of NADPH-cytochrome P450 reductase of the house fly (Musca domestica) in Escherichia coli

家蝇细胞色素P450还原酶的原核表达与活性鉴定

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