Preparation And Application Of Cellulose Membrane Immobilized Fc Fragment Of Antibody

Protein A is a cell wall protein of Staphylococcus aureus. It has been applied in purification of antibodies and treatment of autoimmune disease since protein A can interact with Fc of antibodies. It is one of importants to get plenty of highly purified protein A with high bioactivity or small molecules with function of protein A. In order to get the idea, cellulose membrane immobilized Fc fragment was prepared and applied in purification of protein A and selection of biomimetic small affinity ligands with structures of molecular clusters. The main results are followed.1. Fc fragment was got after digestion of IgG. The best condition of IgG digestion by papain was that the ratio of papain and IgG was 480 U/mg and reacted for 2h, pH 7.5, at 37℃.Then Fc fragment was purified with column of Protein A chromatography.2. After cellulouse membrane was oxidized by 0.2 mol/L NaIO4 for 15 min at pH 5 and couped hexamethylenediamine and glutaraldehyde in turn, Fc fragment was immobilized with a concentation of 11.75 mg/g membrane.3. The modified cellulouse membrane could adsorb 8.22 mg/g Protein A theoretically and 6.84 mg/g Protein A in dynamic adsorption condition. Moreover, a high puritied rProtein A was acquried from E.coil disruption by the affinity membrane. The modified cellulouse membrane could adsorb 5.38 mg/g rProtein A in dynamic adsorption condition.After repeating 8 times, the adsorption capacity of membrane did not changed significantly. So the membrane can be used in isolation and purification of rProtein A.4. With the example of small affinity ligands with structures of molecular clusters and mimic ligand, chitosan and tyramine was selected in this study. The results showed the cellulouse membrane imobilized with Fc fragment showed the higest adsorption to chitosan-tyramine, compared with that of chitosan or tyramine. 31.1 chitosan-tyramines were adsorbed by each Fc fragment.In conclusion, the celloluse membrane imobilized Fc fragment could be used in isolation and purification of rProtein A, and selection of biomimetic affinity ligands with structures of molecular clusters.