Objective: To investigate the immunological functions of heat shock protein 40 kDa of Schistosoma japonicum (SjHSP40).
Methods: The homology of the SjHSP40 protein sequence was analyzed and the B and T cell epitopes of SjHSP40 were predicted using bioinformatics tools. The full-length SjHSP40 gene was amplified using a PCR assay, and cloned into the prokaryotic expression vector pGEX-6P-1, which was transformed into Escherichia coli BL-21. The protein expression was induced with isopropyl β-D-thiogalactoside (IPDG), and then, the recombinant protein was purified with glutathione-sepharose 4B resin to yield the fusion protein GST-SjHSP40, which was checked with SDS-PAGE and Western blotting. Following immunization with GST-SjHSP40, the serum levels of anti-SjHSP40 IgG antibody and IgG1 and IgG2a subtypes were detected in BALB/c mice using ELISA. In addition, the effect of SjHSP40 on CD4+ T-cell subset differentiation was examined using flow cytometry.
Results: SjHSP40 contained 7 potential B cell epitopes and multiple T cell epitopes (CTL epitopes and Th epitopes). The prokaryotic expression plasmid pGEX-6p-1-SjSHP40 was successfully constructed, and the fusion protein GST-SjHSP40 was obtained following IPDG induction and protein purification. Significantly higher serum levels of anti-SjHSP40 IgG, IgG1 and IgG2a antibodies were detected in mice immunized with GST-SjHSP40 than in other groups; however, SjHSP40 showed no remarkable effects on CD4+ T-cell subset differentiation.
Conclusions: SjHSP40 may induce specific humoral immune responses in mice; however, it does not affect the balance of Th immune responses. It is suggested that SjHSP40 may be a potential vaccine candidate.
［摘要］ 目的 探索日本血吸虫热休克蛋白 40 kDa (SjHSP40) 免疫学功能。方法 采用生物信息学方法分析 SjHSP40 蛋白序列同源性, 并预测其 B 细胞及 T 细胞表位。应用 PCR 技术扩增全长SjHSP40 基因, 将其克隆入原核表达质粒pGEX-6P-1, 再转入大肠埃希菌BL21中; 以异丙基-β-D-硫代半乳糖苷 (IPTG) 诱导表达, 再经亲和柱分离纯化获得谷胱甘肽巯基转移酶 (GST) -SjHSP40融合蛋白, 分别采用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳 (SDS-PAGE) 及 Western blotting 进行鉴定。以 GST-SjHSP40 免疫BALB/c小鼠后, 以酶联免疫吸附试验 (ELISA) 检测小鼠血清中抗SjHSP40特异性 IgG 抗体及其亚型 IgG1、IgG2a 抗体, 采用流式检测术观察 SjHSP40 对 CD4+ T 细胞亚群分化的影响。结果 SjHSP40 含有 7 个潜在 B 细胞表位及多个 T 细胞表位 (CTL 表位和 Th 表位)。成功构建了原核表达重组质粒 pGEX-6P-1-SjHSP40, 并通过诱导表达和蛋白纯化获得 GST-SjHSP40 融合蛋白。与其他组相比, GST-SjHSP40 免疫小鼠血清中抗SjHSP40特异性 IgG 抗体及其亚型 IgG1 和 IgG2a 水平均显著升高, 但 SjHSP40 对CD4+ T淋巴细胞分化无显著影响。结论 SjHSP40 可诱导小鼠产生特异性体液免疫应答, 但不影响小鼠体内各类 Th 免疫反应平衡, 可作为潜在疫苗候选分子。.
Keywords: Heat shock protein 40 kDa; Humoral immunity; Immunological function; Schistosoma japonicum.