摘要
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 the eDNA encoding human HMGB1, express it in E. coli, and identify its biological activity. Methods Human HMGB1 eDNA was amplified by RT-PCR and cloned into vector pUC19. Alter sequence analysis, the eDNA was ligated into prokaryotic expression vector pQE-80L and induced by IPTG to express HMGB1. The protein was purified with Ni^2＋-NTA chromatography and polymyxin B affinity column. To identify the function of purified protein, the product was co-cultured with THP1 cells. Results Recombinant expression plasmid pQE-80L/HMGB1 was constructed successfully. After purification, the protein purity reached 96%. The recombinant HMGB1 stimulated THP1 to secrete TNF-α . Conclusion The highly purified HMGB1 was obtained successfully, which showed biological activity. These results lay the foundation for further research on the function of human HMGB1.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2005年第19期1931-1934,共4页
Journal of Third Military Medical University
