【作者】 张仁利； 黄达娜； 高世同； 耿艺介； 吴少庭； 胡章立； 张顺祥； 程锦泉；

【Author】 Zhang Renli,Huang Dana Gao Shitong,et al (Departement of Molecular Biology,Shenzhen CDC 518020,China)

【机构】 深圳市疾病预防控制中心； 深圳大学生命科学院；

【摘要】 目的探讨日本血吸虫疫苗侯选分子钙离子激活的中性蛋白激酶（Calpain）的保护性免疫力和免疫保护性机制。方法用重组纯化的Calpain抗原免疫BALB/c小鼠,制备抗Calpain血清,抗Calpain血清,机械转化的日本血吸虫童虫和来自于同种鼠激活的嗜酸性粒细胞共同培养,观察细胞对血吸虫童虫的粘附及对童虫的杀伤效果;RT-PCR分析Calpain抗原免疫BALB/c小鼠一氧化氮激酶（iNos.eNos.nNos）mRNA的表达;ELISA分析免疫小鼠体外培养脾细胞产生的细胞因子IFN-γ和IL-4。结果重组Calpain抗原免疫小鼠后,产生了一个极高的抗Calpain特异性抗体,此抗体介导了同种鼠的嗜酸性粒细胞对日本血吸虫童虫的粘附,在37℃,5%CO₂培养48小时后,33.8%的日本血吸虫童虫被杀死,与控制组14.5%的自然死亡率相比有一个显著性的增高（P<0.01）;RT-PCR测定重组Calpain抗原免疫一周小鼠iNos mRNA的表达显著性的增强,培养24小时脾细胞上清中IFN-γ浓度高于对照组。结论 Calpain特异性抗体介导了对日本血吸虫童虫的细胞毒作用,Calpain抗原能够诱导IFN-γ的产生,提示Calpain诱导Thl的免疫应答反应来抗日本血吸虫的感染。更多还原

【Abstract】 Objective Calpain,a calcium-activated neutral proteinase as a vaccine candidate molecule was cloned and expressed,so as to discuss its efficacy and mechanism of protective immunity.Methods Anti-calpain sera was prepared by immunizing BALB/c mice with recombinant calpain.Anti-calpain sera,schistosomula of Schistosoma japonicum and mouse eosinophils were culture at 37℃5%CO₂ for 48 hours,and observed adherence of eosinophils to schistosomula and schistosomulacidal efficacy of Schistosoma japonicum.mRNA expression of iNos.nNos and eNoswere decteced by RT-PCR in immunized BALB/c mice,and concentration of IFN- Y and IL-4 were analysized by ELISA in spleen cell of culture of immunizing mice.Results Mice immunized by recombinant calpain produeed a high level IgG antibodies speciafic to the immunizing antigen.Immunoblotting analysis showed murine anti-recombinant calpain sera binding specifically to recombinant calpain.Ninety six percent of schistosomula were covered by cell when incubated with mouse eosinophils,and A significant number of death of schistosomula was observed at 48 hours when incubated with mouse serum and eosinophils as comparing with control serum and eosinophils（P<0.01）.Both mRNA expression of iNos and concentration of IFN-γin immunized mice was significant increase.Conclusion Calpain antibodies induced antibody dependent cell-adherence and killing of the schistosomula,which suggested calpain of Schistosoma japonicum seems to be induce Th1 immune reaction due to IFN- Y increase in immunized mice.更多还原