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目的:克隆人高迁移率族B1Abox(HMGB1 A box)基因,构建高效稳定的大肠杆菌(E.coli)表达菌株,并探讨其对免疫复合物(IC)刺激单核细胞活化的抑制作用。方法:根据本室优化合成的HMGB1基因序列设计引物,PCR扩增目的基因片段并插入克隆载体pMD19-T,菌落PCR、酶谱分析及DNA测序鉴定阳性克隆。重组克隆载体经NdeⅠ和XhoⅠ酶切,琼脂糖凝胶电泳分离目的基因,插入表达载体pQE-T7-2的相应位点,菌落PCR鉴定重组表达载体。重组菌株经IT-PG诱导,SDS-PAGE分析及Western blot鉴定重组蛋白。Ni2+-NTA层析柱纯化重组人HMGB1 A box,RT-PCR检测其对IC刺激单核细胞活化的抑制作用。结果:获得重组人HMGB1 A box的表达菌株,目的蛋白占菌体总蛋白的40%左右。Western blot显示重组蛋白能与抗人HMGB1多克隆抗体和抗His-Tag多克隆抗体特异反应。目的蛋白纯度高达90%以上,并能有效抑制IC诱导单核细胞分泌BAFF、IFN-γ及TNF-α。结论:成功构建了重组人HMGB1 A Box的表达载体,纯化的重组蛋白能有效抑制IC刺激单核细胞活化后细胞因子的分泌。
OBJECTIVE: To clone human high mobility group B1A box (HMGB1 A box) gene and construct a highly efficient and stable E. coli expression strain and investigate its inhibitory effect on immune complex (IC) stimulated monocyte activation. Methods: Primers were designed according to the HMGB1 gene sequence optimized and synthesized in our laboratory. The target gene fragment was amplified by PCR and inserted into the cloning vector pMD19-T. The positive clones were identified by colony PCR, zymography and DNA sequencing. The recombinant cloning vector was digested with Nde I and Xho I and the target gene was isolated by agarose gel electrophoresis and inserted into the corresponding site of the expression vector pQE-T7-2. The recombinant plasmid was identified by colony PCR. Recombinant strains were induced by IT-PG, identified by SDS-PAGE and Western blot. Recombinant human HMGB1 A box was purified by Ni2 + -NTA column and its inhibitory effect on IC-stimulated monocyte activation was detected by RT-PCR. Results: The recombinant protein of HMGB1 A box was obtained. The target protein accounted for about 40% of total bacterial protein. Western blot showed that the recombinant protein can specifically react with anti-human HMGB1 polyclonal antibody and anti-His-Tag polyclonal antibody. The purity of the target protein is over 90%, and can effectively inhibit IC-induced monocyte secretion of BAFF, IFN-γ and TNF-α. Conclusion: The expression vector of recombinant human HMGB1 A Box was successfully constructed. The purified recombinant protein can effectively inhibit the secretion of cytokines after IC stimulated monocyte activation.