日本血吸虫Calpain蛋白免疫原性研究

Studies on immune antigenicity of protein of schistosoma japanicum as a vaccine candidate molecule.

目的 探讨日本血吸虫疫苗候选分子Calpain蛋白的抗原性及其所诱导的免疫应答类型。 方法 用Genetyx -MAC9 0分析Calpain肽链的亲水性 ,在亲水性区域以 9个氨基酸为肽链单位的固相重叠肽链库 (每相邻两个肽链之间仅有一个氨基酸不同 )被Auto -spotRobot制备 ,用Dot-ELISA在合成的肽链库上筛选B细胞表位 ,含有B细胞表位的肽链免疫BALB/c小鼠 ,ELISA鉴别特异性IgG抗体亚类的产生。 结果 由 75 8个氨基酸组成的日本血吸虫Calpain蛋白含有两个主要的亲水区 ,其存在区域分别在氨基酸 2 2 9～ 3 75和 5 5 5～ 613 ;测定合成的肽链文库 ,在亲水区内筛选出 4个B细胞的表位 ,分别是氨基酸 2 3 4～ 2 5 1(YAHLSGGT) ;2 90～ 2 97(CLMGCSIH) ;3 3 8～ 3 46(PWGDSHEW ) ;3 5 8～ 3 64 (AWCDGAPQ) ;与对照组比较 ,免疫组鼠血清中IgGl、IgG2a和lgG2b特异性抗体有显著性意义的上升。 结论 日本血吸虫Calpain是一个具有免疫原性的蛋白 ,能够激发宿主产生高水平的免疫球蛋白 ,提示Calpain可以发展成为日本血吸虫病的诊断抗原和日本血吸虫疫苗候选分子。

Objective To explore immune antigenicity of calpain protein as a vaccine candidate molecular of schistosoma japonicum and immune response type which were induced by the B cell epitopes. Methods Hydrophilicity of predicted calpain amino acid sequence was analyzed using Genetyx-MAC 9.0. Overlapping 9-mer oligopeptides spanning hydrophilic portions of residues 229～375 and 555～613 were synthesized in solid phase by Auto-spot Robot. Dot-ELISA to detect liner epitopes for B cell epitopes in synthesizing peptides library. BALB/c mice was immunized by B cell epitope, IgG antibodies subcalss of immunized mice were detected by ELISA. Results Two major hydrophilic portions of Schistosoma japonicum(S.japonicum) calpain were predicted, which located at residues 229～375 and 555～613. When synthetic peptides were tested, there were four common B cell epitopes in schistosomal calpain, namely 234～251(YAHLSGGT);290～297(CLMGCSIH);338～346(PWGDSHEW);358～364(AWCDGAPQ); Four weeks after mice immunized with B cell epitopes, IgG1, IgG2a and IgG2b antibodies were significantly increased in sera of immunized mice as compared with control mice. Conclusion The results show that calpain of S.japonicum is of strong antigenicity, which seems to be not only a vaccine candidate, but also a target antigen for immunodiagnosis of human schistosomiasis.