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目的 构建人高迁移率族蛋白 1(HMGB1)编码基因的表达载体 ,获得纯化的重组蛋白 ,研究其生物学功能。方法 通过RT PCR方法扩增出人HMGB1的编码基因 ,克隆于载体pGEM Teasy,再亚克隆于表达载体pGEX4T 2 ,经IPTG诱导可表达相对分子质量 (Mr)约 5 6× 10 3的融合蛋白GST HMGB1。经用GSTrapFF蛋白纯化柱和凝血酶行柱上酶切 ,得到纯化的重组蛋白HMGB1,用培养的人单核细胞系THP1检测蛋白活性。结果 构建了融合蛋白GST HMGB1的重组表达质粒 ,获得了Mr 约为 30× 10 3的纯化蛋白产物。该蛋白能刺激THP1细胞产生TNF α ,并能明显诱导THP1细胞的凋亡。结论 获得了纯化的人HMGB1原核表达产物 ,对研究其在脓毒症中的生物学功能具有重要的意义。
Objective To construct the expression vector of human high mobility group box 1 (HMGB1) gene and obtain the purified recombinant protein to study its biological function. Methods Human HMGB1 gene was amplified by RT PCR and cloned into vector pGEM Teasy. The recombinant plasmid was subcloned into expression vector pGEX4T 2 and induced by IPTG to express a fusion protein GST with molecular weight (Mr) of about 56 × 10 3 HMGB1. The purified recombinant protein HMGB1 was obtained by column digestion with GSTrapFF protein purification column and thrombin, and the activity of the protein was detected by cultured human monocytic cell line THP1. Results The recombinant expression plasmid of fusion protein GST HMGB1 was constructed and the purified protein product with Mr about 30 × 10 3 was obtained. The protein can stimulate THP1 cells to produce TNFα, and can significantly induce THP1 cell apoptosis. Conclusion The purified prokaryotic expression product of human HMGB1 is of great significance for the study of its biological function in sepsis.