Expression Vector Construction and Prodoct Purification of Single-Chain Antibody with Specificity for Carcinoembryonic Antigen in E.coli

Objective Research in construction of anti-carcinoembryonic antigen (CEA) antibody gene, expression in E.coli and purification of the expressed product. Methods The heavy and light chain variable region (V H and V k) genes encoding the murine monoclonal antibody E 7B 10 with specificity for CEA were linked into single-chain antibody gene with 45 oligenucleotide (Gly 4Ser) 3 by using recombinant PCR, and was cloned into the expression vector pET-22b(+). Results The construction was expressed in E.coli with high level soluble fusion protein tailed with six additional histidine residues at its C-terminus, reaching 10% of the total bacteria protein, and characterized by SDS-PAGE, and Western blot. The histidine-tagged ScFv was purified with nickeliminodiacetic acid affinity chromatography. 100ml induction culture had 0.8mg ScFv-(His) 6 fusion proteins. Its purifity reached 90%. Conclusion The purified ScFv's affinity constant binding to CEA reached 5.4× 10 7/M , lower its parental monoclonal antibody E 7B 10 2.7×10 9/M.