本文關(guān)鍵詞： 羊種布魯氏菌16M 16M△AsnC 胞內(nèi)生存 AsnC家族蛋白　出處：《山西農(nóng)業(yè)大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：布魯氏菌病(簡稱布病)是一種危害嚴重的人獸共患傳染病,在全球范圍內(nèi)廣泛流行,給人類的健康和畜牧業(yè)的發(fā)展帶來很大的危害。目前,免疫是控制動物布病最為有效的手段。受到現(xiàn)有疫苗的限制,亞單位疫苗由于組份簡單清楚,毒副作用小,安全性高成為研究者的首選。研究了布魯氏菌的一個非常有潛力的保護性抗原AsnC,為布普氏菌亞單位疫苗和治療藥物的開發(fā)奠定基礎(chǔ)。通過同源重組抗性替換的方法,成功構(gòu)建了突變株16M△AsnC,通過同源重組的方法,將AsnC基因整合到突變株中進而構(gòu)建了互補株16M△AsnC-C.胞內(nèi)存活實驗和小鼠毒力實驗顯示,突變株的胞內(nèi)存活能力下降,毒力減弱,說明AsnC與布魯氏菌的毒力相關(guān)。在體外模擬了巨噬細胞胞內(nèi)的多種刺激條件(高鹽、高滲、氧壓力、低pH值和熱休克),突變株在這些刺激條件下生存能力均降低,說明AsnC基因缺失后,布魯氏菌對這些刺激條件的抵抗能力下降。提示AsnC基因能夠使布魯氏菌適應(yīng)機體防御機制。利用定量RT-PCR分析了AsnC在小鼠毒力實驗、胞內(nèi)存活實驗和刺激條件下的轉(zhuǎn)錄水平變化,結(jié)果顯示AsnC基因的轉(zhuǎn)錄水平都上調(diào),AsnC的表達是布魯氏菌胞內(nèi)生存必需的,在動物感染的后期AsnC基因轉(zhuǎn)錄水平的上調(diào),促使布魯氏菌能夠在胞內(nèi)長期生存,建立慢性感染。同時AsnC失活后,與胞內(nèi)生存相關(guān)的毒力蛋白vjbR、dnaK、htrA、gntR的表達量下降,提示AsnC可能通過調(diào)控毒力相關(guān)蛋白的表達來表達布魯氏菌胞內(nèi)的生存�？寺”磉_了7個AsnC家族的蛋白,鎳柱親和層析純化后,免疫動物,對免疫后的動物進行攻毒實驗,顯示AsnC家族蛋白具有明顯的保護性。綜上所述,我們對AsnC蛋白在布魯氏菌胞內(nèi)生存和適應(yīng)內(nèi)環(huán)境壓力中的作用及其調(diào)控的毒力基因進行研究,得出AsnC是一個毒力相關(guān)的調(diào)控蛋白,調(diào)控了布魯氏菌胞內(nèi)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.
【學(xué)位授予單位】：山西農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S852.61

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本文編號：1471591