食品科学 ›› 2017, Vol. 38 ›› Issue (2): 46-51.doi: 10.7506/spkx1002-6630-201702008

• 生物工程 • 上一篇    下一篇

单增李斯特菌nox基因的克隆、表达以及功能

吴 嫚,李 森,陈国薇,罗 勤,刘武康,丁承超,董庆利,刘 箐   

  1. 1.上海理工大学医疗器械与食品学院,上海 200093;2.华中师范大学生命科学学院,湖北 武汉 430079
  • 出版日期:2017-01-25 发布日期:2017-01-16
  • 基金资助:
    国家自然科学基金面上项目(31371776)

Gene Cloning, Expression and Functional Charaterization of NAD(P)H Oxidases Gene (nox) from Listeria monoeytogenes

WU Man, LI Sen, CHEN Guowei, LUO Qin, LIU Wukang, DING Chengchao, DONG Qingli, LIU Qing   

  1. 1. School of Medical Instrument and Food Engineering, University of Shanghai for Science and Technology, Shanghai 200093, China; 2. College of Life Sciences, Central China Normal University, Wuhan 430079, China
  • Online:2017-01-25 Published:2017-01-16

摘要: 以GenBank中报道的单增李斯特菌(Listeria monocytogenes,Lm)野生型菌株EGDe的nox基因(GenBankID:986631)为研究对象,探讨在高等动植物中普遍存在的烟酰胺腺嘌呤二核苷酸磷酸氧化酶在Lm中是否也可以介导产生活性氧(reactive oxygen species,ROS)。首先通过诱导nox基因在BL21中表达产生Nox蛋白,利用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和Western blot鉴定该蛋白质分子质量;然后构建过表达菌株EGDe-nox,测定ROS产生情况,并使用实时荧光定量-聚合酶链式反应检测nox基因的过表达对Lm毒力基因表达的影响。结果表明,经鉴定Nox蛋白分子质量约为33 kD,过表达菌株EGDe-nox与对照组EGDe的ROS产生量相比并无多大变化,nox基因的过表达会导致与侵袭相关的基因actA、inlA和inlB以及毒力基因prfA的表达上调。由此推测,该Nox蛋白不能独立主导ROS的产量,但其过表达却可以增强毒力基因表达上调。本研究为继续探讨细菌中Nox的作用提供一定的参考依据。

关键词: 单增李斯特菌, 烟酰胺腺嘌呤二核苷酸磷酸氧化酶, nox基因, 活性氧

Abstract: Nicotinamide adenine dinucleotide phosphate oxidases (Nox) is ubiquitous in higher animals and plants, and it is responsible for generating reactive oxygen species (ROS). The aim of this work was to explore whether the NOX mediates the generation of ROS in Listeria monocytogenes (Lm). The nox gene from the wild-type Listeria monocytogenes EGDe (GenBank ID: 986631) was tested. The gene was induced to express Nox in Escherichia coli BL21 and the molecular weight of the expressed enzyme was measured by using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. Then the over-expressed strain EGDe-nox was built to detect ROS production and examine the influence of nox gene over-expression on the expression of virulence genes by using quantitative real-time polymerase chain reaction (qRT-qPCR). The results showed that the molecular weight of the recombinant Nox was about 33 kD. Compared with EGDe, the ROS production of EGDe-nox was not changed significantly. The over-expression of nox resulted in up-regulated expression of the invasion-related genes actA, inlA and inlB and the virulence gene prfA. These results suggest that the Nox is unable to dominate ROS production independently but its over-expression can up-regulate the expression of virulence genes.

Key words: Listeria monocytogenes, NADH oxidase, nox, reactive oxygen species (ROS)

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