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目的:探讨细粒棘球绦虫(Eg)转Eg95-EgA31融合基因苜蓿疫苗诱导BALB/c小鼠产生的免疫应答及其对Eg原头节攻击感染的保护性作用。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20g/L。分别用100μL灌胃和10μL滴鼻免疫小鼠,每3d免疫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,用Eg原头节腹腔注射进行攻击感染(50个Eg原头节/每鼠),感染后24周剖杀小鼠,分离并称重细粒棘球蚴组织,计算囊重减少率;采眼球血,常规ELISA检测血清中IgG及其亚类和IgE水平;取脾,分离脾细胞,流式细胞术(FCM)检测脾CD4+和CD8+T淋巴细胞亚群的百分比;脾细胞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)刺激培养后,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,AnnexinV-FITC和碘化丙啶(PI)双染色法检测脾细胞的凋亡发生率,常规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:与正常蛋白对照组相比,疫苗口服接种组小鼠检获包囊质量明显降低,囊重减少率为64.1%,脾细胞凋亡发生率明显降低,脾T细胞增殖水平、CD4+T细胞亚群的百分比和CD4+/CD8+比值显著升高,血清中IgG、IgG2b和IgE水平显著升高,脾细胞培养上清液中IFN-γ、1L-12和TNF-α水平显著增高,IL-10水平明显降低。结论:细粒棘球绦虫转Eg95-EgA31融合基因苜蓿疫苗口服接种可抑制免疫鼠脾细胞发生凋亡,诱导免疫鼠脾T细胞增殖,产生Th1型细胞免疫应答以对抗Eg原头节的攻击感染,CD4+ T细胞亚群、IgG、IgG2b和IgE在疫苗诱导的保护力中起重要作用。
OBJECTIVE: To investigate the immune response induced by the Eg95-EgA31 fusion gene of Echinococcus granulosus (Eg) -transfected BALB / c mice and its protective effect on the Eg-infected nodose infection. Methods: The leaf protein of transgenic alfalfa was extracted by hot flocculation method, and the concentration of leaf protein extract was prepared into 20g / L with sterile double distilled water. The mice were respectively immunized with 100μL gavage and 10μL intranasal instillation. The mice were immunized once every 3 days and continuously for 2 months. 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, challenge infection (50 Eg progenitor cells / per mouse) was performed by intraperitoneal injection of Eg protoscoleces and the mice were sacrificed at 24 weeks post infection and the Echinococcus granulosus tissues were isolated and weighed, The percentage of splenic CD4 + and CD8 + T lymphocyte subsets were detected by flow cytometry (FCM). The percentage of splenic CD4 + The splenocytes were stimulated with Splenocyte suspension or EgAg, ConA or LPS in vitro. After MTT assay, The proliferation of spleen T lymphocytes was detected by immunofluorescence staining. The apoptosis rate of splenocytes was detected by Annexin V-FITC and PI staining. The levels of IL-12, IL -10, IFN-γ and TNF-α levels. Results: Compared with the normal protein control group, the quality of the cysts in the oral vaccination group was significantly decreased, the cyst weight loss rate was 64.1%, the splenocyte apoptosis rate was significantly decreased, the splenic T cell proliferation level, CD4 + T cell The percentage of CD4 + / CD8 + and the ratio of CD4 + / CD8 + were significantly increased, the levels of IgG, IgG2b and IgE in serum were significantly increased, and the levels of IFN-γ, IL-12 and TNF- Significantly lower the level. CONCLUSION: Oral administration of Eg95-EgA31 fusion gene alfalfa vaccine can inhibit the apoptosis of splenocytes in immunized mice and induce the proliferation of splenic T lymphocytes in mice, resulting in a Th1-type cellular immune response against the challenge of Eg protoscoleces , CD4 + T cell subsets, IgG, IgG2b and IgE play important roles in vaccine-induced protection.