Expression and biological characterization of recombinant human HMGB1 B Box

Recently,researchers identified high mobility group box-1 protein(HMGB1),an intranuclear architectural protein,as a late mediator of endotoxemia and sepsis. The structural basis for the cytokine-inducing capacity of HMGB1 was mapped in B box (aa 88～162). To analyse the bioactivity of HMGB1 B box protein,we extracted the total RNA from human tonsil and amplified the HMGB1 B box cDNA using RT-PCR. Then the amplified products were inserted into pUC19. After sequenced,the target DNA fragment was cloned into pQE-80L/DHFR vector,which containing the gene encoding carrier protein DHFR. The recombinant plasmid was transformed into E.coli DH5α,and the target protein expressed in the presence of IPTG. Then analyzed by SDS-PAGE and Western blotting. The target protein was purified by Ni 2+-NTA and polymyxin B column chromatography. Then stimulated the PBMCs with the target protein,and detected the release of TNF-α and IL-6 by ELISA. The results showed that the recombinant HMGB1 B box could increase obviously the release of TNF-α and IL-6 from the PBMCs. This results provide a foundation for further study on the mechanism of HMGB1.