【摘要】：約90%的傳染性疾病是病原體通過入侵機(jī)體局部黏膜引起的,黏膜成為機(jī)體抵抗病原微生物入侵的第一道防線。黏膜疫苗不僅能在接種部位誘導(dǎo)分泌特異性抗體,還能同時引起系統(tǒng)性免疫應(yīng)答,因此接種黏膜疫苗是預(yù)防和控制這類疾病的有效手段之一。沙門菌是常見的黏膜病原體,主要經(jīng)污染的食物或水感染人和動物。為預(yù)防和控制沙門菌引起的人獸共患病,對沙門菌疫苗的研究已持續(xù)了幾十年。其中,基于鼠傷寒沙門菌的疫苗研究得最多,目前已鑒定的200多種鼠傷寒沙門菌血清亞型中,又以鼠傷寒沙門菌UK-1的減毒衍生物的免疫原性為佳。常用的用于致弱細(xì)菌毒力的方法是利用基因工程方法調(diào)控其重要基因的表達(dá)或?qū)⑦@些基因直接缺失。全局調(diào)控基因就屬于沙門菌的一類非常重要的基因。以前的研究表明,將UK-1的某一全局調(diào)控基因缺失后,能使其毒力大大減弱,且保留著較強(qiáng)的免疫原性,能抵抗對UK-1的致死性感染。但UK-1各全局調(diào)控調(diào)控基因減毒株誘導(dǎo)系統(tǒng)性體液免疫應(yīng)答和黏膜免疫應(yīng)答的能力尚未見研究進(jìn)行綜合性的比較�；诖�,本研究構(gòu)建了△crp UK-1、 △envZ UK-1、△fis UK-1、△fur UK-1、△hilA UK-1、△mlc UK-1和△phoP UK-1等7株鼠傷寒沙門菌UK-1全局調(diào)控基因突變株,然后檢測經(jīng)口免疫小鼠后各突變株激發(fā)的血清IgG抗體水平以及唾液、陰道洗脫液和糞便中的分泌型IgA抗體水平,并分析比較了各突變株在小鼠體內(nèi)誘導(dǎo)系統(tǒng)性體液免疫應(yīng)答和局部黏膜免疫應(yīng)答的能力。結(jié)果表明,不同的全局調(diào)控基因突變株誘導(dǎo)系統(tǒng)性體液免疫應(yīng)答的能力及在不同黏膜部位誘導(dǎo)局部免疫應(yīng)答的能力是不同的。在血液中,△fur、△envZ、 △mlc突變株能誘導(dǎo)較高水平的OMP特異性IgG抗體；△crp、△fur、△envZ、△phoP則能誘導(dǎo)產(chǎn)生較高水平的LPS特異性IgG抗體。在口腔內(nèi),以△envZ、△fur、△phoP誘導(dǎo)分泌的OMP特異性sIgA抗體水平為高；LPS特異性sIgA抗體水平則以△crp、 △fur、△phoP刺激分泌的較高。在陰道內(nèi),△crp、△fur誘導(dǎo)產(chǎn)生OMP特異性sIgA的能力較強(qiáng)；△crp、△fur、△phoP誘導(dǎo)產(chǎn)生LPS特異性sIgA抗體的能力較強(qiáng)。在直腸內(nèi),△crp、△envZ、△mlc突變株誘導(dǎo)分泌OMP特異性sIgA的能力較強(qiáng)；△crp、△envZ、 △mlc、△phoP刺激分泌LPS特異性sIgA的能力較強(qiáng)。盡管在不同部位誘導(dǎo)免疫應(yīng)答的能力不同,但所有的突變株對野生型UK-1的致死性感染都提供了完全的保護(hù)作用且存活小鼠未表現(xiàn)出不健康的表觀癥狀�？偟膩碚f,本研究為哪些基因突變應(yīng)用到減毒沙門菌疫苗的構(gòu)建以提高其在小鼠各部位尤其是局部黏膜部位誘導(dǎo)的免疫反應(yīng)提供了基本的參考數(shù)據(jù)。
[Abstract]:About 90% of infectious diseases are caused by pathogens invading local mucosa, which becomes the first line of defense against the invasion of pathogenic microorganisms. Mucosal vaccine can not only induce the secretion of specific antibody but also induce systemic immune response at the same time. Therefore mucosal vaccine is one of the effective methods to prevent and control this kind of disease. Salmonella is a common mucosal pathogen that infects humans and animals mainly through contaminated food or water. In order to prevent and control zoonosis caused by Salmonella, Salmonella vaccine has been studied for decades. The vaccine based on Salmonella typhimurium has been studied most. Among the more than 200 serotypes of Salmonella typhimurium identified at present, the immunogenicity of attenuated derivatives of Salmonella typhimurium UK-1 is better than that of Salmonella typhimurium. The commonly used methods for the virulence of weak bacteria are to regulate the expression of important genes or to delete them directly by means of genetic engineering. Global regulatory genes are one of the most important genes in Salmonella. Previous studies have shown that the deletion of a global regulatory gene of UK-1 can greatly weaken its virulence and retain a strong immunogenicity. It can resist lethal infection of UK-1. However, the ability of the attenuated UK-1 genes to induce systemic humoral immune response and mucosal immune response has not been comprehensively compared. Based on this, 7 strains of Salmonella typhimurium, including crp UK-1, envZ UK-1, fis UK-1, fur UK-1, hilA UK-1, mlc UK-1 and phoP UK-1, were constructed in this study. Then the levels of serum IgG antibodies and secretory IgA antibodies in saliva, vaginal elusions and feces were detected after oral immunization of mice with various mutants. The ability of each mutant to induce systemic humoral immune response and local mucosal immune response in mice was analyzed and compared. The results showed that the ability of inducing systemic humoral immune response and the ability to induce local immune response in different parts of mucous membrane were different in different mutants of global regulatory gene. A high level of OMP-specific IgG antibody; crp, fur, envZ, phoP could be induced by, fur, envZ, mlc mutants in the blood, but a higher level of LPS-specific IgG antibody could be induced. In oral cavity, the level of OMP-specific sIgA antibody induced by envZ, fur, phoP was higher than that of LPS-specific sIgA antibody, and that of LPS-specific sIgA antibody was higher than that of crp, fur, phoP. The ability of, crp, fur to induce OMP specific sIgA in vagina was stronger than that of; crp, fur, phoP to induce LPS specific sIgA antibody. The ability to induce the secretion of OMP-specific sIgA by, crp, envZ, mlc mutants was stronger than that by; crp, envZ, mlc, phoP to stimulate the secretion of LPS-specific sIgA. Although the ability to induce immune responses varied at different sites, all the mutants provided complete protection against fatal infections of wild-type UK-1 and the survival mice showed no symptoms of unhealthy appearance. In general, this study provides basic data on which gene mutations are used in the construction of attenuated Salmonella vaccine in order to improve the immune response induced by attenuated Salmonella vaccine in mice, especially in the part of local mucosa.
【學(xué)位授予單位】：四川農(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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