【摘要】 目的筛选和鉴定日本血吸虫磷酸丙糖异构酶(SjTPI)的Th1型细胞表位,为构建短肽疫苗奠定基础。方法用BLAST比对并预测SjTPI的T细胞表位SjTPI-P18及其对照表位SjTPI-P9。设计并合成其编码DNA,重组入原核表达载体pET-32c(+)后进行表达,获得纯化的重组融合蛋白rSjTPI-P18和rSjTPI-P9。用rSjTPI、rSjTPI-P18及rSjT-PI-P9刺激经照射致弱尾蚴免疫、rSjTPI加强免疫的C57BL/6和C3H/HeJ小鼠淋巴细胞,3H-TdR掺入法检测淋巴细胞的增殖效果及细胞培养上清中IL-2水平;分别用重组rSjTPI-P18和合成SjTPI-P18刺激rSjTPI-P18、SjTPI-P18或PBS加弗氏佐剂两次免疫的C57BL/6小鼠淋巴细胞,并检测其细胞培养上清中IFN-γ及IL-4水平。结果参照曼氏血吸虫TPI T细胞表位预测的SjTPI-P18及SjTPI-P9,获得了与Trx融合表达的重组肽rSjTPI-P18及rSjTPI-P9。与rSjTPI-P9相比,rSjTPI及rSjTPI-P18均可刺激辐照致弱尾蚴免疫、rSjTPI加强免疫的C57BL/6或C3H/HeJ小鼠的淋巴细胞增殖且IL-2分泌量增加(P均<0.05);SjTPI-P18及rSjTPI-P18刺激经rSjTPI-P18或SjTPI-P18免疫两次的C57BL/6小鼠淋巴细胞分泌IFN-γ水平升高(P均<0.05),而IL-4水平较低。结论筛选和鉴定出的SjTPI-P18是C57BL/6小鼠特异的Th1型表位。更多还原

【Abstract】 Objective To screen and identify the Th1-type epitope in the triose-phosphate isomerase of Schistosoma japonicum(SjTPI) and facilitate the application of epitope-based vaccine.Methods The T cell epitopes SjTPI-P18 and SjTPI-P9 referenced from T cell epitopes of Schistosoma mansoni TPI(SmTPI-P18 and SmTPI-P9) were analyzed through NCBI BLAST.The encoding genes of the two epitopes were synthesized and cloned into pET-32c(+) and expressed in E.coli BL21(DE3).The recombinant fusion proteins,named rSjTPI-P18 and rSjTPI-P9,were purified using Ni-column.Groups of C57BL/6(H-2b)or C3H/HeJ(H-2k)mice were primed with irradiated S.japonicum cercariae and boosted with recombinant SjTPI(rSjTPI).The incorporation rate of 3H-thymidine of lymphocytes and interleukine-2 production in the cultured cell supernatant were measured after co-cultured with rSjTPI,rSjTPI-P18 and rSjTPI-P9.Mice C57BL/6(H-2b)were immunized twice with rSjTPI-P18,SjTPI-P18 or phosphate-buffered saline(PBS),emulsified with Freund’s adjuvant,and the levels of interferon-gamma and interleukine-4 in the lymphocytes supernatant were detected.Results In this study,the epitope fusion proteins rSjTPI-P18 and rSjTPI-P9 have been expressed successfully.Compared to the recombinant control epitope rSjTPI-P9,both rSjTPI and rSjTPI-P18 were efficient stimulators for lymphocytes proliferation and higher IL-2 secretion for C57BL/6 or C3H/HeJ mice immunized with the irradiated cercariae and boosted with rSjTPI(P<0.05).Both SjTPI-P18 and rSjTPI-P18 were able to effectively stimulate lymphocyts to secret higher level of interferon-gamma(P<0.05) and lower interleukine-4 in C57BL/6 mice immunized twice with rSjTPI-P18,SjTPI-P18 or PBS,emulsified with Freund’s adjuvant.Conclusion The SjTPI-P18 is an Th1-type epitope specific for C57BL/6 mouse.更多还原