[Cloning and expression of human single-chain Fv antibody against A beta(1-42) peptide involved in Alzheimer disease]

Abstract

Objective: Screening of antibody clones specific for beta amyloid peptide 1-42 from human phage-display single-chain Fv (scFv) antibody library, and to clone the antibody gene and to express it in a bacterial system, with an ultimate intention to obtain human anti-A beta(1-42) antibody for Alzheimer disease (AD) therapy.

Methods: beta amyloid peptide 142 was bound on the solid surface of 96 wells plate as the antigen for the binding antibody clones from a human phage-display scFv antibody library. After four rounds of biopanning, random, well-separated colonies were identified by ELISA test. The specific positive phage clones were transfected into the host E. coli HB2151 to express soluble scFv antibodies. These antibodies were identified by SDS-PAGE and Western blot and their antigen-binding activities were determined by ELISA. Genes of the positive scFv antibodies were then sequenced.

Results: ELISA test showed that 7 clones could bind A beta(1-42). The soluble scFv antibody from clone A10 was expressed successfully to produce a 33000 protein present mainly in the whole cell extract which was five folds in amount to that of the control as determined by A490 nm. DNA sequencing demonstrated that the gene of the positive antibody was the scFv gene and the deduced amino acids sequence confirmed its typical antibody V domain structure.

Conclusions: The specific antibody against A beta(1-42) was successfully identified from human phage-display scFv antibody library. The soluble scFv antibody specific to A beta(1-42) was expressed by E. coli HB2151 in a significant quantity. This cloned antibody promises to provide a solid basis for future studies of the pathogenesis and development of therapeutic agents for Alzheimer's disease.