AsnC蛋白在布鲁氏菌胞内生存作用及其机制研究

发布时间：2018-01-28 21:28

  本文关键词： 羊种布鲁氏菌16M 16M△AsnC 胞内生存 AsnC家族蛋白　出处：《山西农业大学》2015年硕士论文　论文类型：学位论文

【摘要】：布鲁氏菌病(简称布病)是一种危害严重的人兽共患传染病,在全球范围内广泛流行,给人类的健康和畜牧业的发展带来很大的危害。目前,免疫是控制动物布病最为有效的手段。受到现有疫苗的限制,亚单位疫苗由于组份简单清楚,毒副作用小,安全性高成为研究者的首选。研究了布鲁氏菌的一个非常有潜力的保护性抗原AsnC,为布普氏菌亚单位疫苗和治疗药物的开发奠定基础。通过同源重组抗性替换的方法,成功构建了突变株16M△AsnC,通过同源重组的方法,将AsnC基因整合到突变株中进而构建了互补株16M△AsnC-C.胞内存活实验和小鼠毒力实验显示,突变株的胞内存活能力下降,毒力减弱,说明AsnC与布鲁氏菌的毒力相关。在体外模拟了巨噬细胞胞内的多种刺激条件(高盐、高渗、氧压力、低pH值和热休克),突变株在这些刺激条件下生存能力均降低,说明AsnC基因缺失后,布鲁氏菌对这些刺激条件的抵抗能力下降。提示AsnC基因能够使布鲁氏菌适应机体防御机制。利用定量RT-PCR分析了AsnC在小鼠毒力实验、胞内存活实验和刺激条件下的转录水平变化,结果显示AsnC基因的转录水平都上调,AsnC的表达是布鲁氏菌胞内生存必需的,在动物感染的后期AsnC基因转录水平的上调,促使布鲁氏菌能够在胞内长期生存,建立慢性感染。同时AsnC失活后,与胞内生存相关的毒力蛋白vjbR、dnaK、htrA、gntR的表达量下降,提示AsnC可能通过调控毒力相关蛋白的表达来表达布鲁氏菌胞内的生存。克隆表达了7个AsnC家族的蛋白,镍柱亲和层析纯化后,免疫动物,对免疫后的动物进行攻毒实验,显示AsnC家族蛋白具有明显的保护性。综上所述,我们对AsnC蛋白在布鲁氏菌胞内生存和适应内环境压力中的作用及其调控的毒力基因进行研究,得出AsnC是一个毒力相关的调控蛋白,调控了布鲁氏菌胞内生存。
[Abstract]:Brucellosis (brucellosis) is a serious zoonotic infectious disease, which is widespread in the world and brings great harm to human health and the development of animal husbandry. Immunization is the most effective means of controlling animal brucellosis. Due to the limitation of existing vaccines, subunit vaccines have simple components and little side effects. High safety has become the first choice of researchers. A highly potential protective antigen AsnC of Brucella has been studied. In order to lay a foundation for the development of brucellosis subunit vaccine and therapeutic drugs, a mutant 16M AsnC was successfully constructed by homologous recombination resistance replacement method. The AsnC gene was integrated into the mutant strain and the complementary strain 16M AsnC-C.Cytotoxicity test and murine virulence test showed that the viability of the mutant was decreased and the virulence was weakened. AsnC was related to the virulence of Brucella. In vitro, many stimulation conditions (high salt, high permeability, oxygen pressure, low pH and heat shock) were simulated in macrophages. The survival ability of the mutant was decreased under these stimuli, indicating that the AsnC gene was deleted. The resistance of Brucella to these stimuli was decreased. It was suggested that AsnC gene could make Brucella adapt to the mechanism of organism defense. The virulence of AsnC in mice was analyzed by quantitative RT-PCR. The results of intracellular survival test and stimulation showed that the transcription level of AsnC gene up-regulated the expression of AsnC, which was necessary for the survival of Brucella. At the late stage of animal infection, the up-regulation of AsnC gene transcription level promoted the long-term survival of Brucella and the establishment of chronic infection. At the same time, AsnC was inactivated. The expression of virulence protein vjbRndnahtrAgntR, which is related to intracellular survival, was decreased. These results suggest that AsnC may express the intracellular survival of Brucella by regulating the expression of virulence related proteins. Seven AsnC family proteins were cloned and purified by nickel column affinity chromatography to immunize animals. The AsnC family proteins were shown to have obvious protective effects on the immunized animals. In conclusion, the results were as follows: 1. We studied the role of AsnC protein in the intracellular survival and adaptation of Brucella and its virulence gene, and concluded that AsnC is a virulence related regulatory protein. It regulates the intracellular survival of Brucella.
【学位授予单位】：山西农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.61

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