|英文要約||Title:Technology Innovation Program for Small Business Innovation Research / The development of new measurement using the ability of galectin-8 carbohydrate recognition domain to capture endotoxin
Target: The final goal of R&D is to make a machine which is a compact size and is able to measure endotoxin of 10EU/L. But in the stage of F/S, our goal is to gain a proof of concept that galectin-8 carbohydrate recognition domain (shortly Gal-8) is able to capture endotoxin and to measure it at the level of 100EU/L.Concept: We have revealed that the reaction of phosphorylcholine derivertive and nano-bead with SH-function makes self assembled monolayers on the bead (PC/SAMs). And also have revealed that membrane anchoring peptide (MAP) in fucosyltransferase binds tightly to PC/SAMs and arranges an active site of enzyme outer side.As it is known that Gal-8 reacts to endotoxins of wide range of bacteria. So it would be possible to detect an endotoxin to use Gal-8/MAP fusion protein bound on PC/SAMs bead.Progress: 1. We succeed to reach an important step to produce a fusion protein of Gal-8 and MAP by E. coli. 2. The expression of Gal-8 is confirmed by western blot. 3. The function of Gal-8 is confirmed by the absorption of Gal-8 to lactose. 4.We succeeded to synthesize phosphorylcholine derivertive and to produce nano-particles coated with PC/SAMs both on a magnetic bead and quantum dots (shortly QDs) which emit fluorescence. 5. Gal-8/MAP fusion protein bound to a magnetic bead coated with PC/SAMs. 6. We revealed that PC/SAMs/QDs was able to bind endotoxin and lowered fluorescence intensity. At present 0.1 ng/ml (700EU/L) could be measurable.Future prospects: Endotoxin is composed of lipid A, core oligosaccharide and O-antigen. PC/SAMs/QDs would bind lipid A in endotoxin and would arrange polysaccharide including O-antigen outer side. On the other hand, Gal-8 would recognize polysaccharide which is located in outer side.So high sensitive measuring method of endotoxin would be obtained from the following way. First of all, the solution including endotoxin adds to PC/SAMs/QDs solution and then adds the solution of magnetic bead bound Gal-8/MAP to it. The complex formed from the reaction would be composed of PC/SAMs/QDs+endotoxin+ Gal-8/MAP/MAG-bead. If a magnet had accessed to the solution, the complex would be drawn to it and get rid of from fluorescent path. The removal of complex from light path becomes fluorescent intensity would be lowered compared from the existence of it.So this method would be able to measure lowered concentration of endotoxin. That means we would be obtained a high sensitive measurement of endotoxin by using this method.