Isolation and Purification of Lipopolysaccharide-binding Protein (LBP) from Grass Carp, Ctenopharyngodon idellus

Lipopolysaccharide binding protein (LBP) is a soluble acute phase protein that binds to bacterial LPS to elicit immune responses by presenting the LPS to important cell surface pattern recognition receptors, involved in the acute-phase immunologic response to gram-negative bacterial infections. In present study, fresh whole blood was collected from grass carp (Ctenopharyngodon idellus) challenged with Aeromonas hydrophila for 24h. The plasma was obtained by centrifuge. Integrated activity test of LBP and ammonium sulphate precipitation, CM Sephadex C-50 cation-exchange chromatography and DEAE Sephadex A-25 anion-exchange chromatography, lipopolysaccharide binding proteins (LBPs) were isolated and purified. The results indicated that the purified LBPs had relatively high affinity to Fluorescein isothiocyanate labeled lipopolysaccharide (FITC-LPS). SDS-PAGE stained with Coomassie Brilliant Blue, visualized that there were three distinct bands with molecular weight of 68kDa, 53 kDa and 48 kDa respectively. This research described a simple and maneuverable method for isolation and purification of LBPs and served the further study of LBP functions.