Construction of anti-Stx2B ScFv gene and its secretory expression in Ecoli

Objective To construct a gene of ScFv against recombinant Shiga like toxin 2B and express it in Ecoli.Methods The V_H and V_L genes cloned from McAb 1A5 hybridoma cell were ligated with a flexible linker(Gly4Ser)_3 to construct ScFv gene.The corresponding restriction endonuclease digestion sites were introduced into 5′ and 3′ ends of the ScFv gene,and then the ScFv gene was cloned into expression vector pCANTAB6His and expressed in Ecoli HB2151.Expression proteins were purified by affinity chromatography and detected by SDS-PAGE and ELISA.Resaults Restriction endonuclease digestion and DNA sequencing proved that the ScFv gene was correctly cloned into expression vector.SDS-PAGE and ELISA analysis showed that ScFv was successfully expressed in Ecoli HB2151 and the expression protein had specific antigen binding activity.The relative molecular mass(M_r) of the expression protein was 27 000,according with the predicted M_r(value.) Conclution The successful expression of anti-Stx2B ScFv gene in Ecoli lays a foundation for its further application in diagnoss and therapy of human O157 infection.