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目的探讨细粒棘球绦虫(Eg)转Eg95-EgA31融合基因苜蓿疫苗免疫小鼠用Eg原头节攻击后脾T淋巴细胞增殖的变化。方法热絮凝法提取转基因苜蓿的叶蛋白,配成浓度为20μg/μl。分别用100μl(约含1μg融合抗原)口服灌胃和10μl(约含0.1μg融合抗原)滴鼻接种免疫BALB/c小鼠,每3 d 1次,连续免疫2个月。同时设转空质粒(pBI121)苜蓿叶蛋白及正常苜蓿叶蛋白对照组。末次免疫后8周,每只小鼠用50个Eg原头节腹腔注射感染。感染后24周杀鼠,检获包囊并称重,计算囊重减少率;分离脾细胞,体外经Eg粗抗原(EgAg)或刀豆素A(ConA)刺激培养,四甲基偶氮唑盐比色法(MTT法)检测免疫小鼠脾T淋巴细胞增殖情况。结果与正常蛋白对照组相比,疫苗口服灌胃组小鼠检获包囊质量明显降低,囊重减少率为64.1%。两免疫组小鼠的脾T细胞增殖水平显著高于正常蛋白对照组(P<0.01);口服灌胃组小鼠的脾T细胞增殖水平高于滴鼻接种组(P<0.01)。结论细粒棘球绦虫转Eg95-EgA31融合基因苜蓿疫苗接种可诱导小鼠脾脏T淋巴细胞增殖,口服接种是一种较好的免疫途径。
Objective To investigate the changes of splenic T lymphocyte proliferation in mice challenged with Eg protoscoleces from mice immunized with Eg95 Egg31 fusion gene of Eg. Methods The leaf protein of transgenic alfalfa was extracted by hot-flocculation method, and the concentration was 20μg / μl. BALB / c mice were inoculated intranasally with 100μl (containing about 1μg of fusion antigen) orally and 10μl (containing about 0.1μg of fusion antigen) intranasally to immunize BALB / c mice twice a day for 3 consecutive days. At the same time set the empty plasmid (pBI121) alfalfa leaf protein and normal alfalfa leaf protein control group. Eight weeks after the last immunization, each mouse was infected intraperitoneally with 50 Eg protoonoid sections. The mice were sacrificed at 24 weeks after infection, cysts were harvested and weighed to calculate the rate of reduction of cystic masses. Spleen cells were isolated and stimulated with EgAg or ConA in vitro. Tetramethylpyrazolium Colorimetry (MTT method) was used to detect the proliferation of splenic T lymphocytes in immunized mice. Results Compared with the normal protein control group, the quality of the seized cysts in the oral gavage group was significantly decreased, and the rate of reduction of cystic weight was 64.1%. The splenic T cell proliferation of the two immunized mice was significantly higher than that of the normal protein control group (P <0.01). The proliferation of splenic T cells in oral gavage mice was higher than that in the nasal inoculation group (P <0.01). Conclusions Vaccination with Eg95-EgA31 fusion gene of Echinococcus granulosus can induce spleen T lymphocyte proliferation in mice. Oral inoculation is a good immunization route.