發(fā)布時間：2018-11-16 11:15

【摘要】： 目前,國內(nèi)外對宿主抗細(xì)粒棘球絳蟲免疫的研究主要集中在宿主體內(nèi)的免疫保護(hù)、免疫逃避與免疫損傷機(jī)制。有很多關(guān)于細(xì)粒棘球絳蟲病的中間宿主的免疫抗原Eg95的研究報道,但對終末宿主犬的免疫保護(hù)抗原還在研究當(dāng)中。本研究通過免疫組織化學(xué)方法與分子生物學(xué)方法,探索宿主感染細(xì)粒棘球絳蟲和EgM9蛋白免疫后,機(jī)體外周免疫系統(tǒng)、腸道的粘膜免疫系統(tǒng)產(chǎn)生免疫應(yīng)答的情況,以期為臨床研制相關(guān)疫苗提供實(shí)驗(yàn)依據(jù)。 本研究首先經(jīng)pET-41b重組質(zhì)粒轉(zhuǎn)化的大腸埃希菌(BL-21)對EgM9蛋白進(jìn)行誘導(dǎo)表達(dá),通過SDS-PAGE對表達(dá)產(chǎn)物進(jìn)行分析,證明所表達(dá)的確實(shí)為EgM9蛋白,再對其進(jìn)行純化,得到符合免疫抗原要求的EgM9重組目的蛋白。 將實(shí)驗(yàn)犬分為四組(Ⅰ組:EgM9蛋白免疫組;Ⅱ組:EgM9蛋白免疫后感染原頭蚴組;Ⅲ組:直接感染原頭蚴組;Ⅳ組:空白對照組),用純化后的EgM9蛋白對Ⅰ組和Ⅱ組進(jìn)行免疫試驗(yàn),再分別對Ⅱ組和Ⅲ組人工感染原頭蚴。將實(shí)驗(yàn)犬剖檢后進(jìn)行荷蟲數(shù)計算,結(jié)果表明用EgM9蛋白免疫犬后,可以使犬取得一定的免疫保護(hù)效果。 采集實(shí)驗(yàn)犬的腸系膜淋巴結(jié)和小腸,依據(jù)免疫酶組織化學(xué)和免疫熒光組織化學(xué)原理,運(yùn)用直接法和抗原~抗體~抗體標(biāo)記物法對其進(jìn)行檢測。直接法結(jié)果表明Ⅰ組、Ⅱ組和Ⅲ組犬的腸系膜淋巴結(jié)和小腸上存在IgG抗體,反應(yīng)都呈陽性;抗原~抗體~抗體標(biāo)記物法結(jié)果表明Ⅰ組、Ⅱ組表現(xiàn)陽性,而Ⅲ組呈陰性,熒光顯微鏡下有明顯的熒光位點(diǎn),說明該方法可以對免疫犬的腸系膜淋巴結(jié)和小腸上的IgG進(jìn)行定位檢測。 通過Real-Time PCR對上述實(shí)驗(yàn)犬的腸系膜淋巴結(jié)和小腸組織中的三種細(xì)胞因子(IL-4、TGF-β和INF-γ)檢測,表明I組和Ⅲ組實(shí)驗(yàn)犬的腸系膜淋巴結(jié)和小腸中IL-4與INF-γ均有明顯變化,IL-4的數(shù)值遠(yuǎn)高于INF-γ,而在II組中,IL-4的數(shù)值雖然也高于INF-γ,但I(xiàn)L-4與INF-γ的數(shù)值同時也高于其他兩組。結(jié)果表明不論對犬進(jìn)行免疫還是感染,都能引起Th1型和Th2型反應(yīng),但以Th2型反應(yīng)為主。
[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，

本文編號：2335361