单增李斯特菌nox基因的克

发布时间：2018-05-03 02:40

本文选题：单增李斯特菌 + 烟酰胺腺嘌呤二核苷酸磷酸氧化酶　；参考：《食品科学》2017年02期

【摘要】：以Gen Bank中报道的单增李斯特菌(Listeria monocytogenes,Lm)野生型菌株EGDe的nox基因(Gen Bank ID: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 k D,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act A、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。
[Abstract]:The nox gene, Gen ID986631, of wild-type Listeria monocytogenes strain EGDe, which was reported in Gen Bank, was used as the research object. To investigate whether nicotinamide adenine dinucleotide phosphate oxidase, which is common in higher plants and animals, can also mediate the production of reactive oxygen species (oxygen) in LM. Firstly, the nox gene was induced to express in BL21 to produce Nox protein, and the molecular weight of the protein was identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot, and then the overexpression strain EGDe-nox was constructed to determine the production of ROS. The effect of overexpression of nox gene on the expression of Lm virulence gene was detected by real-time fluorescence quantification-polymerase chain reaction. The results show that The molecular weight of Nox protein was about 33kD, and the overexpression of ROS gene of EGDe-nox did not change much compared with that of control group EGDe. The overexpression of ROS gene resulted in the up-regulation of the expression of invasion-related genes act Agnin A and inlB and virulence gene prfA. It is inferred that the Nox protein can not independently dominate the production of ROS, but its overexpression can increase the expression of virulence gene. This study provides some references for further study on the role of Nox in bacteria.
【作者单位】：上海理工大学医疗器械与食品学院;华中师范大学生命科学学院;
【基金】：国家自然科学基金面上项目(31371776)
【分类号】：R378;Q78

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