| Lipopolysaccharide binding protein(LBP), produced by hepatocytes under trauma, burn and Gram-negative bacteria infection, is one kind of emergency reaction proteins that conduct the binding between lipopolysaccharide and the target cells' receptor. LBP can promote the binding between lipopolysaccharide and membrane CD14 (mCD14) of mononuclear/macrophage cells, to form the complex of LPS-LBP-mCD14 and to activate mononuclear/macrophage cells by TLR4-MD2-MYD88 and NF-kappa B. LBP can accelerate the occurrence and development of inflammation. LBP has quite important effect on inflammatory reaction induced by LPS, Leading the damage of the liver, lung and kidneys.Burn injury with endotoxemia is a common clinic disorder, easily causing systemic inflammatory response syndrome(SIRS) and multiple organs dysfunction syndrome(MODS),whose mortality rate is quite high. What roles LBP play in the occurrence and development of burn injury with endotoxemia is still unclear today, so it's quite important for us to investigate the biological function of LBP in the earlier period of burn injury and endotoxemia, to explore the way that restrain the biological effects of LBP. In this study, we reproduce burn injury with endotoxemia rabbit model, our aims are: to purify and extract LBP, to investigate the biological effects of LBP, to explore the function of LBP in inflammation, in the other way, to express the human N terminal LBP protein (NH-LBP) by insect cell, to make monoclonal antibody to NH-LBP and to explore the way to restrain the biological effects of LBP.The main results are as following:1. To reproduce burn injury with endotoxemia rabbit model by 20% total body surface area(TBSA) Ⅲand LPS abdominal injection( 0.5mg/Kg weight). 6h after infliction, the concentration of ALT and TNFα increased markedly. Microscopically, we found spotty necrosis, infiltration of inflammatory cells in liver and congestion and dilation of capillaries in alveolar walls in the lung. 2. LBP was purified by two-step ion-exchange chromatography( Bio-Rex 70 Resin, Mono-Q ) and gel chromatography ( Sephadex G-100 ), molecular weight 60KDa, the NH2-terminal amino acid sequence was TNPGLITRIT. Certified by FACS, ELISA,agglutination test of sheep erythrocyte, LBP had the function to promote the binding between lipopolysaccharide and mononuclear/macrophage cells. The binding rate of LBP is 8.55 times as much as the binding rate of control group. LBP could promote the U937 cells to excrete TNFα with very low concentration of LPS( 0.5ng/mL ). It was proved that LBP induced the inflammation in the earlier period of burn injury with endotoxemia, and the effects was quite powerful and played an important roles in the damages of the liver and lung. 3. Based on extraction and purification of LBP, an unknown protein was obtained, with molecular weight 48KDa, the NH2-terminal amino acid sequence was GSQGTFTSEE, there has not been seen the same sequence in the web protein bank ( NCBI, PDB, and so on). The protein also could promote the binding between lipopolysaccharide and mononuclear/macrophage cell certified by FACS, ELISA, agglutination test of sheep erythrocyte. The binding rate of the protein is 8.07 times as much as the binding rate of control group. The protein could promote the U937 cells to excrete TNFα with very low concentration of LPS( 0.5ng/mL )too, there was no difference between LBP and the protein on biological effects ( P>0.05), the protein had the similar function with LBP. Maybe, the protein was a new kind of lipopolysaccharide binding protein, tentatively named it P48.4. Amplified baculovirus encoding human N terminal LBP protein (NH-LBP) by insect cell sf9 or sf21, then expressed and excreted NH-LBP by sf21. NH-LBP was purified by TALON metal affinity resin chromatography. The total product 8.0mg was got. During the experiment, we found the sf9 cell was more potent to amplified baculovirus, but only sf21 cell could express and excrete NH-LBP.5. Screened the human bacteriophage antibody bank with NH-LBP a...
|
PDF Full Text Request