细粒棘球绦虫转Eg95-EgA31融合基因苜蓿疫苗诱导小鼠免疫应答的研究

The study on immune responses in BALB/c mice by immunization with the transgenic Alfalfa(Medicago sativa) containing Eg95-EgA31 fusion gene of Echinococcus granulosus

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10灿滴鼻免疫小鼠，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殴粗抗原（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瞻原头节的攻击感染，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。

AIM： To investigate the immune responses and the protection induced by the transgenic Alfalfa （ Medicago sativa） containing Eg95-EgA31 fusion gene of Echinococcus granulosus against Eg protoscoleces. METHODS： The leaf protein was extracted from the transgenic alfalfa by heat-coagulation method, its concentration was prepared for 20 g/L. BALB/c mice were immunized intranasally or orally with the leaf protein once per 3 days for 2 months. At the same time, the leaf protein transfected with pBI-121 blank vector and the normal leaf protein without foreign antigen was served as control. The mice were then challenged intraperitoneally with Eg protoscoleces （50 protoscoleces per mouce） on week 8 after the last vaccination and sacrified on week 24 postinfection to count the rate of reduced hydatid cyst. The specific antibody （IgE, IgG and its subclasses）in the sera collected from the eyeballs was evaluated by IELISA. Splenocytes were separated and cultured in vitro with EgAg, ConA or LPS stimulus. The substes of CD4^＋ and CD8 ^＋ T cells were measured by FCM. The splenocytes＇ proliferation was determined by MTT method. Then the cells were collected, stained by PI and Annexin V-FITC, and analyzed by FCM to get the splenocytes＇ apoptotic rate. The supematant was collected to measure the level of IL-12, IL-10, IFN-γ and TNF-α by ELISA. RESULTS： Compared with the control group, The hydatid cyst weight in the oral immunization group decreased by 64.1% ; the splenocytes＇ apoptotic rate got obviously lower than that in the control group; the splenocytes＇ proliferation increased significantly, the CD4^＋ subsets and the ratio of the CD4^＋/CD8^＋ did so, and the similar trend about the specific antibody titer and the level of cytokines could be seen in this group. CONCLUSION： Apoptosis of splenocytes may be inhibited in mice by immunization with the transgenic alfalfa, splenocytes＇ proliferation and Thl response can be induced in the mice against the challenge of Eg protoscoleces. CD4^＋ T cell and the specific antibody （IgG, IgG2b and IgE） may play important roles in the protection induced by the transgenic alfalfa vaccine.