人HMGB1基因的原核表达、产物纯化及活性鉴定

Expression,purification and identification of the human high mobility group-1 protein code gene in E.coli

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 recombinant vector, the expression of human HMGB1 code gene and to study its function. Methods Human HMGB1 code gene was amplified by RT-PCR and cloned into vector pGEM-T easy. Then it was subcloned into prokaryotic expressive vector pGEX4T-2. After four hours induction by IPTG, the expression of recombinant 56kDa fusion protein GST-HMGB1 was identified by SDS-PAGE. Purified HMGB1 was eluted from GSTrap FF affinity chromatography after thrombin cleavage. To identified the function of purified proteins, the product was co-cultured with THP1 cells. Results Recombinant expression plasmid pGEX-HMGB1 was constructed. The 30kDa purified proteins were obtained. The recombinant HMGB1 stimulated THP1 to secret TNF-α and enhanced the apoptosis of THP1 cells. Conclusion The purified prokaryotic expression human HMGB1 were obtained. It might be important for study of the function of human HMGB1.