Construction and expression of anti-SLT 2A scFv gene in E. coli

Objective To construct and express the gene of scFv against recombinant SLT 2A in E. coli. Methods The V_H and V_L genes cloned from mAb 5F3 were ligated with a flexible linker (Gly4Ser)_3 to construct scFv gene, and then corresponding restriction endonuc- lease digestion site was introduced into 5′and 3′ends of the constructed gene. The scFv gene was cloned into an expression vector pCANTAB6His and expressed in E. coli HB2151. Expressed protein was purified by affinity chromatography and detected by SDS-PAGE and Western blotting. Results DNA sequence analysis proved that scFv gene was correctly cloned into the expression vector. SDS-PAGE and Western blot analysis showed that scFv was successfully expressed in Ecoli HB2151 and the expressed protein had specific antigen-binding activity. The relative molecular mass (M_r) of the protein was 27 000, which was corresponding to prediction. Conclution The successful expression of anti-SLT2A scFv gene in E. coli lays a solid foundation for its further application in diagnosis and therapy of O157 infection.[