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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小鼠后脾T淋巴细胞的增殖情况。方法热絮凝法提取转基因苜蓿疫苗叶蛋白,配成浓度为20μg/μL。88只BALB/c小鼠随机分为两组,用100μl(约含1μg融合抗原)口服灌胃和10μl(约含0.1 μg融合抗原)鼻腔黏膜接种分别免疫小鼠,每3天1次,连续免疫2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,取脾,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。结果口服灌胃组的脾T淋巴细胞增殖水平在末次免疫后4～10周升高,在末次免疫后6周达最高水平;鼻腔黏膜接种组的脾T淋巴细胞增殖水平在末次免疫后4～12周升高,在末次免疫后6周达最高水平;鼻腔黏膜接种组脾T淋巴细胞增殖水平高于口服灌胃组。EgAg或ConA刺激组脾T淋巴细胞增殖水平高于相应原液组,且ConA刺激组高于相应EgAg刺激组(P<0.01或P<0.05)。结论细粒棘球绦虫转Eg95-EgA31融合基因苜蓿疫苗能诱导免疫鼠产生脾T淋巴细胞增殖反应,鼻腔黏膜接种途径优于口服灌胃途径。
Objective To observe the proliferation of splenic T lymphocytes in mice immunized with Eg95-EgA31 fusion gene alfalfa vaccine. Methods The leaf protein of transgenic Alfalfa vaccine was extracted by hot-flocculation method, and the concentration was 20μg / μL. Eighty BALB / c mice were randomly divided into two groups. The mice were orally immunized with 100μl oral gavage (containing about 1μg fusion antigen) and 10μl nasal mucosa (containing about 0.1μg fusion antigen), once every 3 days Immunization February. Four mice were sacrificed at 0, 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 weeks after the last immunization. Spleens were harvested and spleen cells were isolated. ) Or concanavalin A (ConA). The proliferation of splenic T lymphocytes in immunized mice was detected by MTT assay. Results The proliferation of splenic T lymphocytes in oral gavage group increased from 4 to 10 weeks after the last immunization and reached the highest level at 6 weeks after the last immunization. The proliferation of splenic T lymphocytes in nasal mucosa inoculated group increased from 4 to 10 days after the last immunization, 12 weeks after the last immunization, reaching the highest level at 6 weeks after the last immunization. The proliferation of splenic T lymphocytes in nasal mucosa inoculation group was higher than that in oral gavage group. The proliferation of splenic T lymphocytes in EgAg or ConA group was higher than that in control group, and was higher in ConA-stimulated group than in the corresponding EgAg-stimulated group (P <0.01 or P <0.05). Conclusions The transgenic E. coli vaccine Eg95 fused with EgA31 could induce splenic T lymphocyte proliferation in immunized mice. The route of nasal mucosal inoculation was better than oral gavage.