转Eg95-EgA31融合基因苜蓿疫苗诱导小鼠脾T淋巴细胞增殖的研究

Study on proliferation of splenocytes in mice resulting from immunization with transgenic Alfalfa(Medicago sativa) containing the 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 splenocyte proliferation in mice immunized with transgenic alfalfa（Medicago sativa） containing the Eg95-EgA31 fusion gene of Echinococcus granulosus（Eg） and challenged with Eg protoscoleces.Methods Leaf protein was extracted from transgenic alfalfa by heat coagulation and prepared at a concentration of 20 μg/μl.BALB/c mice were immunized intranasally or orally with leaf protein once every 3 days for 2 months.At the same time,leaf protein transfected with a pBI-121 blank vector and normal leaf protein without foreign antigens served as controls.The mice were challenged intraperitoneally with Eg protoscoleces（50 protoscoleces per mouse） on week 8 after the last vaccination and sacrificed on week 24 post-infection to count the rate of reduced hydatid cyst weight.Splenocytes were separated for culturing in vitro with EgAg or ConA stimulus.Splenocyte proliferation was determined by MTT method.Results The hydatid cyst weight in the oral immunization group was reduced 64.1% compared to that in the control group.Splenocyte proliferation in both immunized groups increased significantly in comparison to that in the control group,and the level of splenocyte proliferation in the oral group was obviously higher than that in the intranasal group.Conclusion The proliferation of splenocytes can be induced in mice by immunization with transgenic alfalfa.Oral immunization may be an effective route of immunization.