Construction of anti-human integrin β3 ScFv gene and its expression in E. coli

Objective: To construct single chain antibody (ScFv) gene against human integrin β3 and express it in E. coli and get active ScFv protein. Methods:RNA was extracted from hybridoma cells secreting mAb 4F12 against human integrin β3 and the VH and VL genes were amplified by RT-PCR and connected to each other by a gentle linker peptide(Gly4Ser)3 to assemble scFv gene. The scFv gene was cloned into the prokaryotic expression vector pQE. The recombinant plasmid pQE-β3-ScFv was transformed into E.coli M15 and ScFv expression was induced by IPTG. The expressed ScFv was purified by affinity chromatography using Ni-NTA resin and renatured. Results: We obtained VH and VL genes of anti-human integrin β3 mAb and constructed pQE-β3-ScFv recombinant plasmid and expressed a fusion protein in E.coli. SDS-PAGE analysis showed that the fusion protein had a relative molecular weight of 26 kDa and existed in the form of inclusion bodies. ELISA analysis revealed that after purification and renaturation ScFv had good immunoreactivity to human interin β3. Conclusion:We successfully constructed and expressed ScFv gene against human integrin β3 and obtained active ScFv protein.