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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。
Objective To screen and identify the Th1-type epitopes of SjTPI in Schistosoma japonicum, and lay the foundation for the construction of short peptide vaccine. Methods BLAST was used to compare and predict SjTPI T cell epitope SjTPI-P18 and its control epitope SjTPI-P9. The coding DNA was designed and synthesized, and then expressed in prokaryotic expression vector pET-32c (+). The recombinant fusion proteins rSjTPI-P18 and rSjTPI-P9 were obtained. The lymphocytes of C57BL / 6 and C3H / HeJ mice immunized with rSjTPI, rSjTPI-P18 and rSjT-PI-P9 were stimulated with cercarial immunization and rSjTPI, and the proliferation of lymphocytes was detected by 3H-TdR incorporation assay. C57BL / 6 mice were immunized twice with rSjTPI-P18, SjTPI-P18 or PBS plus Freund’s adjuvant with recombinant rSjTPI-P18 and synthetic SjTPI-P18, respectively, IFN-γ and IL-4 levels in cell culture supernatants. Results The recombinant peptides rSjTPI-P18 and rSjTPI-P9 fused with Trx were obtained according to the predicted SjTPI-P18 and SjTPI-P9 of Schistosoma mansoni TPI T cell epitopes. Compared with rSjTPI-P9, both rSjTPI and rSjTPI-P18 could stimulate irradiation-induced cercariae immunization. The lymphocyte proliferation and IL-2 secretion of C57BL / 6 or C3H / HeJ mice immunized with rSjTPI enhanced (P < 0.05). SjTPI-P18 and rSjTPI-P18 stimulated IFN-γ secretion in lymphocytes of C57BL / 6 mice immunized twice with rSjTPI-P18 or SjTPI-P18 (all P <0.05) low. Conclusion SjTPI-P18 was selected and identified as a specific Th1 epitope in C57BL / 6 mice.