Functional expression of human antibody fab fragment against hepatitis virus Bsurface antigen in Pichia pastoris

Objective To explor e the possibility of antibody production in Pichia pastoris.Methods A new type of Pichia pastoris expression vector,pD89,was constructed,which f aciliated to construct multiple copy inserts in vitro and screen multiple copy inserts in vivo .The Light chain and Fd fragment gene of the human antibody against Hepatitis Vi rus B Surface Antigen(HBsAg) were then inserted to pD89 under the control of AO X 1 promoter with a secretion signal in different expression cassette.After electr oporation and screening with G418,7 Mut+ clones with different G418 resistance were selected for expression.Results Results of SDS-PAGE showed that 3 clones expresse d a 50 KD protein.The protein was further confirmed to be a human Fab fragment b y western blot. Conclusion Binding activity to HBsAg was characterized by ELISA.