Abstract:

Objective To clone and express heat?shock protein 40 gene of Schistosoma japonicum（SjHSP40）and analyze its effect on macrophage activation. Methods The fragment of gene encoding SjHSP40 was amplified by PCR. The gene was sub? cloned into the prokaryotic expression vector pGEX?6P?1. The recombinant plasmid pGEX?6P?1?SjHSP40 was transformed into E. coli BL21（DE3）and induced with IPTG. The recombinant protein was purified with Glutathione?Sepharose 4B resin and analyzed by SDS?PAGE and Western?blot. The fusion protein of GST?SjHSP40 was loaded to the macrophage cell?line RAW264.7 for 48 h. Following that，the surface molecules of the macrophages were analyzed by flow cytometry. Results DNA sequencing showed that the recombinant plasmid，pGEX?6P?1?SjHSP40，was successfully constructed. The fusion protein of GST?SjHSP40 was induced， purified and specifically recognized by anti?GST antibody. Compared to GST and medium control groups，this fusion protein signifi? cantly induced the expression of co?stimulatory molecules（CD40，CD80，and CD86）and MHC?II on the surface of the macro? phages. Conclusions SjHSP40 significantly up?regulates the expression of co?stimulatory molecules and MHC?II on the surface of the macrophages. These data indicate that SjHSP40 may initiate macrophage activation.

Key words: Schistosoma japonicum；Heat shock protein； Fusion protein； Prokaryotic expression； Macrophage activation

摘要：

目的 构建pGEX?6P?1?SjHSP40 （SjHSP40） 融合表达载体并在大肠埃希菌 （Escherich coli） 内进行原核表达及重组蛋白纯化， 以探讨SjHSP40对巨噬细胞活化的影响。方法 以PCR方法扩增SjHSP40基因片段， 将其克隆入原核表达质粒 pGEX?6P?1， 经测序验证后转化入E. coli BL21 （DE3） 中。重组蛋白经异丙基硫代半乳糖苷 （IPTG） 诱导表达后以Glutathione ?Sepharose 4B亲和柱进行纯化。所得产物GST?SjHSP40进行十二烷基磺酸钠?聚丙烯酰胺凝胶电泳 （SDS?PAGE）、 Western blot鉴定。将此融合蛋白负载巨噬细胞48 h后， 用流式细胞术检测巨噬细胞表面分子。结果 测序结果表明， 本研究成功构建了pGEX?6P?1?SjHSP40原核表达载体， 经IPTG诱导表达出融合蛋白。Western blot显示该融合蛋白能被抗GST抗体特异性识别。与GST等对照组相比， 此融合蛋白负载的巨噬细胞表面共刺激分子 （CD40、 CD80、 CD86） 及MHC Ⅱ分子表达显著上调。结论 SjHSP40可显著上调巨噬细胞表面共刺激分子 （CD40、 CD80、 CD86） 及MHC Ⅱ分子的表达， 提示其可在一定程度上使巨噬细胞活化。

关键词: 日本血吸虫； 热休克蛋白； 融合蛋白； 原核表达； 巨噬细胞活化