Cloning And Expression Of Human High Mobility Group Box-1 A Box And B Box And Analysis Of Their Biological Functions

The innate immune system is rapidly activated and initiates an inflammatory cascade in response to infection, tissue injury and trauma. During inflammation, infectious factors, such as microbial components, and noninfectious factors, such as trauma, hypoxia, can stimulate many kinds of cells, such as monocytes, macrophage, neutrophil and endothelial cell, to release proinflammatory cytokines, including TNF, IL-1and IL-6, which trigger inflammatory reaction, and lead to severe sepsis. Numerous attempts have been made to treat sepsis by targeting well-known proinflammatory cytokines, including TNF and IL-1. Although these approaches have led to the development of successful therapies for rheumatoid arthritis (RA), they have not proven effective regarding sepsis. A major reason for inefficacy in sepsis is that proinflammatory cytokines are released early in the septic response, usually within a few hours. Recently, researchers identified high mobility group box-1 protein (HMGB1), an intranuclear architectural protein, as a late mediator of endotoxemia and sepsis. HMGB1 is released in response to proinflammatory stimuli, and itself can induce the production of inflammatory mediators by macrophages and neutrophils. Many reports have demonstrated that HMGB1 involves in many kinds of inflammation. This implicates HMGB1 as a central player in proinflammatory reactions. Human HMGB1 has two DNA-binding domains: the A box (aa 1-85) and the B box (aa 88-162). The structural basis for the cytokine-inducing capacity of HMGB1 was mapped in B box. Interestingly, it has been demonstrated that purified recombinant A box works as an antagonist to HMGB1-induced cytokine-production. The recent discovery of extracellular HMGB1 as a proinflammatory mediator has made HMGB1 become a target molecule for anti-inflammatory therapy. Therefore, we cloned the cDNAs encoding human HMGB1 A box and B box and constructed the prokaryotic expression vectors. Then the target proteins were induced by IPTG. Next, the target proteins were purified through Ni2+-NTA chromatography and polymyxin B column. Finally, their biological activitives were assayed. This study aims at providing a foundation for further study on the mechanism of HMGB1. Objectives: Cloning the cDNAs encoding human HMGB1 A box and B box. Preparation and functional analysis of recombinants in order to pave a way for further study on the mechanism of HMGB1. Materials and Methods: 1.Cloning the cDNAs encoding human HMGB1 A box and B box. Extracted the total RNA from human tonsil and amplified the HMGB1 A box and B box cDNAs using RT-PCR respectively. Then the amplified products were inserted into pUC19 seperately and sent to be sequenced. 2.Construction of the prokaryotic expression vectors. After sequenced, the target DNA fragments were separately cloned into pQE-80L/ DHFR vector, which containing the gene encoding carrier protein DHFR. The recombinant expression vectors were named as pQE-80L/ DHFR/ HMGB1 A box and pQE-80L/ DHFR/ HMGB1 B box. 3.Expression of the target proteins. The recombinant plasmids pQE-80L/ DHFR/ HMGB1 A box, pQE-80L/ DHFR/ HMGB1 B box and pQE-80L/ HMGB1, which has been constructed in our department, were transformed into E.coli DH5αrespectively, and the target proteins expressed in the presence of IPTG. Then analyzed by SDS-PAGE and Western Blotting. 4.Isolation and purification of the target proteins. Harvested the cells and identified the location of the target proteins expression. Collected the supernatant of the ultra sonicated E.coli, flowed through the Ni2+-NTA chromatography to purify the target proteins. Then the proteins were further purified through the polymyxin B column inorder to remove the LPS. 5.Functional analysis of the target proteins. Stimulated the PBMCs with the the target proteins, and deteced the release of TNF-αand IL-6 by ELISA. Results: 1.Cloning of the cDNAs encoding human HMGB1 A box and B box. After RT-PCR, an about 255 bps and 225bps fragment could be visible on agarose gel. The two fragments were sequenced after inserted into pUC19 respectively. The result show that the cloned cDNAs were consistent with the cDNA sequence encoding HMGB1 A box and B box reported in GenBank. 2.The recombinant prokaryotic expression vector pQE-80L/ DHFR/ HMGB1 A box and pQE-80L/ DHFR/ HMGB1 B box were successfully constructed respectively. 3.The recombinant plasmids pQE-80L/ DHFR/ HMGB1 A box, pQE-80L/ DHFR/HMGB1 B box and pQE-80L/ HMGB1were transformed into E.coli DH5αrespectively and the expression of the target proteins were induced under the optimal conditions. SDS-PAGE analysis showed that the molecular weight of the recombinant target protein was 36kDa, 35 kDa and 30 kDa respectively. Gray scale analysis indicated that all the target proteins were about 40% of total bacterial protein. The expressed proteins were identified by Western Blotting. 4.The target proteins were purified through Ni2+-NTA chromatography, and then LPS was removed through polymyxin B column. SDS-PAGE analysis showed that the target proteins were highly purified. 5.The biological activities of the target proteins were detected by ELISA. The result show that HMGB1 and HMGB1 B box could stimulate monocyte to secret TNF-αand IL-6, while HMGB1 A box could inhibit the cytokine-inducing function of HMGB1 and HMGB1 B box. Conclusions: In this experiment, the cDNAs encoding human HMGB1 A box and B box were amplified by RT-PCR and the recombinant prokaryotic expression vectors pQE-80L/ DHFR/ HMGB1 A box and pQE-80L/ DHFR/ HMGB1 B box were successfully constructed. And the corresponding proteins were prepared. Function analysis showed that HMGB1 and HMGB1 B box could stimulate the release of TNF-αand IL-6, while HMGB1 A box protein could inhibit the cytokine-inducing function of HMGB1 and HMGB1 B box.