Expression of Human Single-chain Antibody Against A?40 in Pichia Pastoris / 医学研究杂志

ABSTRACT

Objective To express human single-chain antibody against amyloid?peptide 40 in pichia pastoris for passive immuniza- tion to Alzheimer's disease patients.Methods Human single-chain antibody gene from a phage display library was mutated to obliterate BamHI site and cloned into pAO815 plasmid for pT-scFvA?40 construction which was identified by PCR amplification and endonuclease digestion.Vector pT-scFvA?40 was cut by EcoRI and NotI endonucleases and the antibody gene was cloned into pPICgK plasmid to con- struct pPIC9K-scFvA?40 expression vector which was confirmed by gene sequencing.Linearized pPIC9K-scFvA?40 was used to trans- form pichia pastoris GS115 and the recombinant was induced by 0.5% methanol to express human single - chain antibody against amyloid?peptide 40.Results Gene sequencing confirmed pPIC9K-scFvA?40 orientation.Recombinants were obtained by linearized pPICgK - scFvA?40 transformation.After inducing with 0.5% methanol,the recombinant secreted proteins with 33 kD size.Conclusions Expression vector pPIC9K-scFvA?40 was constructed successfully.Human single-chain antibody against amyloid?peptide 40 was recombinantly expressed in pichia pastoris.