细粒棘球绦虫转Eg95-EgA31融合基因苜蓿疫苗免疫BALB/c小鼠后脾T淋巴细胞亚群的动态观察

Dynamic observation on splenocytes subsets in mice immunized with the transgenic alfalfa vaccine containing Eg95-EgA31 fusion gene of Echinococcus granulosus

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诱导的脾T淋巴细胞亚群的变化。方法热絮凝法提取转基因苜蓿疫苗叶蛋白,将其浓度配制成20μg/μL,132只BALB/c小鼠随机分为3组,用100μL（约含1μg融合抗原）灌胃接种和10μL（约含0.1μg融合抗原）滴鼻接种分别免疫小鼠,每3 d 1次,连续免疫2个月,同时设非转基因苜蓿叶蛋白滴鼻免疫对照组。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,流式细胞仪（FCM）检测脾CD4＋和CD8＋T淋巴细胞亚群的百分比。结果灌胃接种组小鼠脾CD4＋和CD8＋T细胞亚群分别在免疫后6-10周和4-12周升高,分别在免疫后6周和8周达高峰,百分比分别为0.294±0.002和0.168±0.027;滴鼻接种组小鼠脾CD4＋和CD8＋T细胞亚群分别在免疫后4-6周和4-10周升高,分别在免疫后6周和8周达高峰,百分比分别为0.318±0.051和0.197±0.008。结论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。

To observe changes on subsets of splenocytes in mice immunized with the transgenic alfalfa（Medicago sativa） vaccine containing Eg95-EgA31 fusion gene of Echinococcus granulosus dynamically,the leaf protein was extracted from the transgenic alfalfa by heat-coagulation method and prepared for a solution with a concentration of 20 g/ L.The 132 BALB/c mice were divided into 3 groups randomly and immunized intranasally or orally with the leaf protein solution once per 3 days for 2 months.At the same time,the group of intranasal immunization with leaf protein from the non-transgenic alfalfa was set as control group.4 mice randomized from each group were killed to get the spleens at Week 0,2,4,6,8,10,12,14,16,18 and 20 after last immunization,and the splenocytes were separated to measure CD＋4and CD＋8T cell subsets by FCM.In the oral group,CD＋4subset increased significantly from Week 6 to Week 10 after the last immunization and reached the peak at Week 6.While CD＋ 8 subset increased obviously from Week 4 to Week 12 and reached the highest level at Week 8.In the intranasal group,the significant increase of the CD＋4subset was observed from Week 4 to Week 6 and also reached the peak at Week 6.The similar trend of CD＋8 subset was observed from Week 4 to Week 10 and reached the highest level at Week 8.It was suggested that CD＋ 4and CD＋ 8subsets played an important role in the protection induced in mice immunized with the transgenic alfalfa vaccine.