【摘要】 探讨了巨型艾美耳球虫抗原Em8体外对鸡外周血单核细胞(PBMCs)功能的影响。选取抗原基因Em TFP250中具有较好免疫保护性的Em8基因片段,并对其进行原核表达纯化出重组蛋白。无菌采集未被鸡球虫感染的鸡的外周血,分离出单核细胞,加入终浓度为5μg/m L的Em8重组蛋白进行体外培养,通过免疫荧光抗体技术观察重组蛋白与单核细胞的结合情况;Em8重组蛋白不同浓度体外刺激鸡外周血单核细胞,用q PCR技术测定各实验组单核细胞中IL-4、IL-17D、IFN-γ、TGF-β4的mRNA转录水平;采用CCK-8法测定细胞的增殖情况;Em8重组蛋白不同浓度(低、中、高浓度)体外与巨噬细胞共培养,测定巨噬细胞吞噬和NO分泌功能。结果表明:重组蛋白Em8是巨型艾美耳球虫的重要抗原,发挥广泛而重要的免疫作用。其中,重组蛋白Em8能够与外周血单核细胞结合并能刺激细胞因子IL-17D和IFN-γ的表达水平显著提高(P<0.01),能够引起单核细胞增值(P<0.01),巨噬细胞吞噬作用增强(P<0.01)且NO分泌增加(P<0.01)。更多还原

【Abstract】 Effects of Em8 of Eimeria maxima on the functions of peripheral blood mononuclear cells( PBMCs) from chickens were investigated. A gene fragment of Em8 which has good immunogenicity was expressed and the recombinant protein was purified. Blood samples were collected from the chickens non- infected with Eimeria. PBMCs were cultured in vitro with the recombinant protein Em8 at a final concentration of 5 μg / m L. The binding of the recombinant protein to PBMCs was observed by immunofluorescence antibody technique. After stimulated with recombinant protein,cell proliferations and the mRNA levels of IL- 17 D,IFN- γ and TGF- β were tested by CCK- 8 method and real- time quantitative PCR( q PCR),respectively. The phagocytosis and NO release of macrophage were also examined. The results showed that the recombinant protein Em8 was an important antigen in E. maxima and mainly induce Th1 type immune response. Em8 could result in significant production of IL- 4,IL- 17 D and IFN- γ( P < 0. 05) and cell proliferation( P < 0. 01). It also strengthened macrophage phagocytosis( P < 0. 01) and boosted NO production( P < 0. 01).更多还原