Cloning, Expression And Preliminary Studying On Biological Functions Of The Mouse HMGB1 Domains, HMGB1A Box And LBPK95A Fusion Gene

Lipopolysaccharides (LPS) is an integral part of the outer membrane of all Gram-negative bacteria (GNB) and is considered to be the primary trigger of pathophysiologic events in sepsis. GNB sepsis or septic shock, characterized as a catastrophic and frequently fatal systemic syndrome, causes an estimated mortality rate of 10-15% in children and 40% in adults. High mobility group 1 protein (HMGBl) was recently identified as a potential late pro-inflammatory cytokine of delayed endotoxin lethality. Inhibition of HMGBl activity may contribute to treatment of sepsis. HMGBl B box alone is sufficient to recapitulate the cytokine-stimulating effects including the TNF-stimulating effects of full-length HMGBl in sepsis. HMGBl A box is a competitiveantagonist of HMGBl. A box could inhibit HMGB1-mediated TNF release by macrophage dose-dependently in vitro and reduce the endotoxin and sepsis lethality. LPS-binding protein (LBP) had been shown to greatly potentiate cell responses to LPS and contribute largely to LPS toxicity in sepsis. It has been reported recently that LBP-derived peptides(LBPK95A) could block the high-affinity interaction between LPS and LBP, efficiently inhibit the TNF- α response to LPS challenge. Therefore, we postulate that HMGB1 A box and LBPK95A fusion protein could contribute to treatment of sepsis by inhibiting early cytokine (LPS) and late cytokine (HMGBl) in the septic inflammatory cascade.Part I: Cloning, expression, purification and identification of the mouse high mobility group 1 protein A box and B box in E.coli: Total RNA was extracted from the lung tissue of a new born mouse, and the HMGBl A box and HMGB1 B box coding sequence was obtained by RT-PCR using specific primers. Then the A box and B box coding sequence was cloned into BamHI and EcoRI site of pGEX-4T-2 expression vector and confirmed by sequencing, respectively. After transforming E.coli BL21(DE3), the recombinant bacteria was induced at 30℃, IPTG 0.3mM for 4h, the expression of recombinant Mr 36kD fusion protein GST-A and GST-B was confirmed by SDS-PAGE. Recombinant GST-A and GST-B protein was purified by GSTrap FF affinity chromatography column. GST-A and GST B concentration are 1885μg/ml and 2288μg/ml. After GST-B was added to the culture medium of THP1 cells, TNF-α secretion was detected by ELISA. GST-B caused the release of TNF-α in a dose-dependent way(from 5μg/ml to 80μg/ml ) and raised the level of apoptosis. GST-A was co-cultered with THP1 cells include GST-B 20μg/ml, GST-A dose-dependently(from 20μg/ml to 160μg/ml) inhibited...