Recombinant Human Anti-hbsag Single-chain Antibody Expression In E. Coli, Purification And Identification Of Its Activity

Hepatitis B is a world wide infectious disease, but up to now there has no effective methods to cure it, so prophylaxis of hepatitis B virus (HBV) is very important. The IgG anti-HBsAg derived from humam plasma can be used for the impassive immunization of HBV and prevention of HBV vertical transmission from mother to infant. But the IgG derived from human plasma is absent and has risk of transmisssion of infections. Therefore it is difficult to use it widely. Because human anti-HBsAg recombiant antibody can realize production of antibody in large scale and is very safe for clinical use, it will has good social effect and economic return that recombiant antibody takes place of the IgG derived from humam plasma. The single-chain antibody (scFv) gene that expresses anti-HBsAg preS2 epitope was transferered into E. coli. and was highly expressed in the host. The proportion of the protein of scFv to total protein of host can reach 30% and the recombinant scFv forms inclusion body in cells. The purity of 97% can be achieved after two step purification of scFv including Ni metal chelating chromatography (the first) and gel filtration (the second). And then the scFv in denaturant was renatured. By the optimized refolding method, the rate of recovery of protein is about 50% when the concentration of scFv is 0. 34mg/mL. The refolding scFv has strong ability to bind HBsAg, the affinity constant is 0.23 × 10~8, which is one of sixteenth of a murine Mab was detected at the same time. The result of competitive ELISA shows the scFv can repress the murine Mab and HBsAg to combine together, and the rate of repression can reach 40. 3%. A method to measure the relative potency of the scFv was established in the paper.We apply MALDI-TOF-MS to accurately measure the molecular weight of scFv, and there is only eight Dalton between the experimental result and theoretical volume. At the same time, we use DABITC/PITC method to confirm...