鼠伤寒沙门氏菌多种全局调控基因突变株的构建及其免疫原性的比较

发布时间：2019-04-16 21:14

【摘要】：约90%的传染性疾病是病原体通过入侵机体局部黏膜引起的,黏膜成为机体抵抗病原微生物入侵的第一道防线。黏膜疫苗不仅能在接种部位诱导分泌特异性抗体,还能同时引起系统性免疫应答,因此接种黏膜疫苗是预防和控制这类疾病的有效手段之一。沙门菌是常见的黏膜病原体,主要经污染的食物或水感染人和动物。为预防和控制沙门菌引起的人兽共患病,对沙门菌疫苗的研究已持续了几十年。其中,基于鼠伤寒沙门菌的疫苗研究得最多,目前已鉴定的200多种鼠伤寒沙门菌血清亚型中,又以鼠伤寒沙门菌UK-1的减毒衍生物的免疫原性为佳。常用的用于致弱细菌毒力的方法是利用基因工程方法调控其重要基因的表达或将这些基因直接缺失。全局调控基因就属于沙门菌的一类非常重要的基因。以前的研究表明,将UK-1的某一全局调控基因缺失后,能使其毒力大大减弱,且保留着较强的免疫原性,能抵抗对UK-1的致死性感染。但UK-1各全局调控调控基因减毒株诱导系统性体液免疫应答和黏膜免疫应答的能力尚未见研究进行综合性的比较。基于此,本研究构建了△crp UK-1、 △envZ UK-1、△fis UK-1、△fur UK-1、△hilA UK-1、△mlc UK-1和△phoP UK-1等7株鼠伤寒沙门菌UK-1全局调控基因突变株,然后检测经口免疫小鼠后各突变株激发的血清IgG抗体水平以及唾液、阴道洗脱液和粪便中的分泌型IgA抗体水平,并分析比较了各突变株在小鼠体内诱导系统性体液免疫应答和局部黏膜免疫应答的能力。结果表明,不同的全局调控基因突变株诱导系统性体液免疫应答的能力及在不同黏膜部位诱导局部免疫应答的能力是不同的。在血液中,△fur、△envZ、 △mlc突变株能诱导较高水平的OMP特异性IgG抗体；△crp、△fur、△envZ、△phoP则能诱导产生较高水平的LPS特异性IgG抗体。在口腔内,以△envZ、△fur、△phoP诱导分泌的OMP特异性sIgA抗体水平为高；LPS特异性sIgA抗体水平则以△crp、 △fur、△phoP刺激分泌的较高。在阴道内,△crp、△fur诱导产生OMP特异性sIgA的能力较强；△crp、△fur、△phoP诱导产生LPS特异性sIgA抗体的能力较强。在直肠内,△crp、△envZ、△mlc突变株诱导分泌OMP特异性sIgA的能力较强；△crp、△envZ、 △mlc、△phoP刺激分泌LPS特异性sIgA的能力较强。尽管在不同部位诱导免疫应答的能力不同,但所有的突变株对野生型UK-1的致死性感染都提供了完全的保护作用且存活小鼠未表现出不健康的表观症状。总的来说,本研究为哪些基因突变应用到减毒沙门菌疫苗的构建以提高其在小鼠各部位尤其是局部黏膜部位诱导的免疫反应提供了基本的参考数据。
[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.
【学位授予单位】：四川农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.61

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