细粒棘球绦虫感染犬及EgM9蛋白免疫犬引起的免疫应答研究

发布时间：2018-11-16 11:15

【摘要】： 目前,国内外对宿主抗细粒棘球绦虫免疫的研究主要集中在宿主体内的免疫保护、免疫逃避与免疫损伤机制。有很多关于细粒棘球绦虫病的中间宿主的免疫抗原Eg95的研究报道,但对终末宿主犬的免疫保护抗原还在研究当中。本研究通过免疫组织化学方法与分子生物学方法,探索宿主感染细粒棘球绦虫和EgM9蛋白免疫后,机体外周免疫系统、肠道的粘膜免疫系统产生免疫应答的情况,以期为临床研制相关疫苗提供实验依据。 本研究首先经pET-41b重组质粒转化的大肠埃希菌(BL-21)对EgM9蛋白进行诱导表达,通过SDS-PAGE对表达产物进行分析,证明所表达的确实为EgM9蛋白,再对其进行纯化,得到符合免疫抗原要求的EgM9重组目的蛋白。 将实验犬分为四组(Ⅰ组:EgM9蛋白免疫组;Ⅱ组:EgM9蛋白免疫后感染原头蚴组;Ⅲ组:直接感染原头蚴组;Ⅳ组:空白对照组),用纯化后的EgM9蛋白对Ⅰ组和Ⅱ组进行免疫试验,再分别对Ⅱ组和Ⅲ组人工感染原头蚴。将实验犬剖检后进行荷虫数计算,结果表明用EgM9蛋白免疫犬后,可以使犬取得一定的免疫保护效果。 采集实验犬的肠系膜淋巴结和小肠,依据免疫酶组织化学和免疫荧光组织化学原理,运用直接法和抗原~抗体~抗体标记物法对其进行检测。直接法结果表明Ⅰ组、Ⅱ组和Ⅲ组犬的肠系膜淋巴结和小肠上存在IgG抗体,反应都呈阳性;抗原~抗体~抗体标记物法结果表明Ⅰ组、Ⅱ组表现阳性,而Ⅲ组呈阴性,荧光显微镜下有明显的荧光位点,说明该方法可以对免疫犬的肠系膜淋巴结和小肠上的IgG进行定位检测。 通过Real-Time PCR对上述实验犬的肠系膜淋巴结和小肠组织中的三种细胞因子(IL-4、TGF-β和INF-γ)检测,表明I组和Ⅲ组实验犬的肠系膜淋巴结和小肠中IL-4与INF-γ均有明显变化,IL-4的数值远高于INF-γ,而在II组中,IL-4的数值虽然也高于INF-γ,但IL-4与INF-γ的数值同时也高于其他两组。结果表明不论对犬进行免疫还是感染,都能引起Th1型和Th2型反应,但以Th2型反应为主。
[Abstract]:At present, the studies on host immunity against Echinococcus granulosus mainly focus on the immune protection, immune evasion and immune damage mechanism. There are many studies on the immune antigen Eg95 of the intermediate host of Echinococcus granulosus, but the immune protective antigen of the terminal host dog is still being studied. The aim of this study was to investigate the immune response of the host infected with Echinococcus granulosus and EgM9 protein on peripheral immune system and intestinal mucosal immune system by means of immunohistochemical and molecular biological methods. In order to provide experimental basis for clinical development of relevant vaccines. In this study, the expression of EgM9 protein was induced by Escherichia coli (BL-21) transformed by pET-41b recombinant plasmid. The expression product was proved to be EgM9 protein by SDS-PAGE analysis, and then purified. The recombinant EgM9 target protein was obtained according to the requirements of immune antigen. The experimental dogs were divided into four groups (group 鈪，

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