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目的在克隆人HMGB1(highmobilitygroupbox-1)全长cDNA的基础上,用大肠杆菌进行表达并鉴定其生物学活性。方法用RT-PCR方法扩增人HMGB1cDNA,克隆至载体pUC19行序列测定后,再构建于原核表达载体pQE-80L并转化大肠杆菌DH5α,以IPTG诱导HMGB1蛋白表达,Ni2+-NTA和多粘菌素B亲和层析柱进行纯化后,用培养的人单核细胞系THP1检测重组蛋白活性。结果构建了人HMGB1蛋白的重组表达质粒pQE-80L/HMGB1,获得了纯度约96%的纯化蛋白产物,该蛋白能刺激THP1细胞产生TNF-α。结论成功制备了具有生物学活性的人HMGB1蛋白纯品,为进一步的功能研究奠定了基础。
Objective To clone and express the full-length cDNA of human HMGB1 (highmobilitygroupbox-1) and express it in Escherichia coli and identify its biological activity. Methods Human HMGB1 cDNA was amplified by RT-PCR and cloned into pUC19 vector. The recombinant plasmid was then constructed into prokaryotic expression vector pQE-80L and transformed into E. coli DH5α. The expression of HMGB1 was induced by IPTG. The expression of Ni2 + -NTA and polymyxin B affinity chromatography column, the recombinant human monocyte cell line THP1 was used to test the activity of the recombinant protein. Results The recombinant plasmid pQE-80L / HMGB1 containing human HMGB1 was constructed. The purified protein product with about 96% purity was obtained, which stimulated THP1 cells to produce TNF-α. Conclusion The preparation of biologically active human HMGB1 protein has laid the foundation for further functional studies.