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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)、Westernblot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MHCⅡ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MHCⅡ分子的表达,提示其可在一定程度上使巨噬细胞活化。
Objective To construct the fusion expression vector pGEX 6P 1 SjHSP40 (SjHSP40) and prokaryotic expression in Escherichia coli and purify the recombinant protein to explore the effect of SjHSP40 on macrophage activation. Methods The SjHSP40 gene fragment was amplified by PCR and cloned into prokaryotic expression vector pGEX 6P 1. After sequencing, it was transformed into E. coli BL21 (DE3). The recombinant protein was induced by isopropylthiogalactoside (IPTG) and purified by Glutathione Sepharose 4B affinity column. The obtained product GST SjHSP40 sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE), Westernblot identification. After the fusion protein was loaded on macrophages for 48 h, the surface molecules of macrophages were detected by flow cytometry. Results The sequencing results showed that the prokaryotic expression vector pGEX 6P 1 SjHSP40 was successfully constructed and the fusion protein was induced by IPTG. Western blot showed that the fusion protein can be specifically recognized by anti-GST antibody. Compared with control group such as GST, the expression of costimulatory molecules (CD40, CD80, CD86) and MHC II on macrophages loaded with the fusion protein were significantly up-regulated. Conclusion SjHSP40 can up-regulate the expression of costimulatory molecules (CD40, CD80, CD86) and MHC Ⅱ on macrophages, suggesting that it may activate macrophages to a certain extent.