Preparation and functional identification of human high mobility group box-1 protein / 第三军医大学学报

ABSTRACT

Objective To clone the cDNA encoding human HMGB1, express it in E. coli, and identify its biological activity. Methods Human HMGB1 cDNA was amplified by RT-PCR and cloned into vector pUC19. After sequence analysis, the cDNA 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-? . 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.