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目的动态观察细粒棘球绦虫转Eg95-EgA31融合基因苜蓿疫苗免疫Balb/c小鼠后诱导的免疫应答。方法热絮凝法提取转基因苜蓿的叶蛋白,再用无菌双蒸水将叶蛋白提取液的浓度配制成20μg/μl。88只Balb/c小鼠随机分为2组,分别用100μl灌胃和10μl滴鼻免疫小鼠,每3天1次,连续免疫2个月。在末次免疫后0、2、4、6、8、10、12、14、16、18和20周各组随机剖杀4只小鼠,眼球取血,常规酶联免疫吸附法(enzyme-linked immunosorbent assay,ELISA)检测血清中IgG及其亚类和IgE水平;取脾,分离脾细胞,流式细胞仪(flow cytometr,FCM)检测脾CD4+和CD8+T淋巴细胞亚群的百分比,体外经脾细胞悬液或加入Eg粗抗原(EgAg)、伴刀豆球蛋白A(ConA)或脂多糖(LPS)刺激培养,四甲基偶氮唑盐比色法(MTT法)检测免疫鼠脾T淋巴细胞增殖情况,ELISA法检测脾细胞培养上清液中IL-12、IL-10、IFN-γ和TNF-α水平。结果在末次免疫后4～6周,2组免疫小鼠的血清IgG及其亚类和IgE水平升高,脾T淋巴细胞增殖水平升高,CD4+和CD8+T细胞亚群百分比分别升高,脾细胞培养上清液中IL-12、IFN-γ、TNF-α和IL-10水平分别升高。结论细粒棘球绦虫转Eg95-EgA31融合基因苜蓿疫苗在免疫早期(4～6周)可诱导免疫鼠脾T淋巴细胞增殖,产生Th1和Th2混合型免疫应答,CD4+和CD8+T细胞亚群在细粒棘球绦虫转Eg95-EgA31融合基因苜蓿疫苗诱导的保护性免疫机制中起重要作用。
Objective To observe the immune response induced by Balb / c mice immunized with Eg95-EgA31 fusion gene alfalfa vaccine. Methods The leaf protein of transgenic alfalfa was extracted by hot-flocculation method and the concentration of leaf protein extract was prepared to 20μg / μl with sterile double-distilled water. Eighty Balb / c mice were randomly divided into 2 groups. Mice were immunized with 100μl intragastric administration and 10μl intranasal instillation. The mice were immunized once every 3 days for 2 months. Four mice were randomly sacrificed at 0, 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 weeks after the last immunization. Blood was collected from the eyeballs and routine enzyme-linked immunosorbent assay The percentage of splenic CD4 + and CD8 + T lymphocyte subsets were detected by flow cytometry (FCM). The spleen, Spleen cells were stimulated with EgAg, ConA or LPS, and splenic T cells were detected by MTT assay The proliferation of lymphocytes and the levels of IL-12, IL-10, IFN-γ and TNF-α in supernatant of splenocytes were detected by ELISA. Results At 4 to 6 weeks after the last immunization, the serum IgG and its subclasses and IgE levels of the two immunized mice increased, the splenic T lymphocyte proliferation level increased, the percentage of CD4 + and CD8 + T cell subsets increased, The levels of IL-12, IFN-γ, TNF-α and IL-10 in spleen cell culture supernatant were increased respectively. Conclusions The transgenic E. coli vaccine Eg95 with EgA31 fusion gene can induce the proliferation of splenic T lymphocytes in mice at 4 ~ 6 weeks after immunization, resulting in a combination of Th1 and Th2 immune responses. The CD4 + and CD8 + T cell subsets Plays an important role in protective immunity mechanism induced by Eg95-EgA31 fusion gene alfalfa vaccine of Echinococcus granulosus.