Secretion Expression of Extracellular Region of EGFR Using Mam malian Cell and its Identification

Objective: To obtain epidermal growth factor receptor(EGFR) extracellular region,which will be used as the target antigens for selection anti-EGFR specific antibody.Methods: EGFR extracellular region gene was amplified by PCR and cloned into the eukaryotic expression vector pABhFc,and the recombinant plasmids were transfected transiently into HEK293T cells.The recombinant proteins were expressed and secreted,then they were purified respectively.The purified protein were identified by ELISA,Western blot and Biacore3000 system.Results: The gene sequence of EGFR extracellular domain was confirmed by DNA sequencing,then the result of SDS-PAGE exhibited that the monomer and dimer EGFR extracellular domain successfully expressed.ELISA dem onstrated the specific binding of dimer recombinant hFc-EGFR and commercial EGF,and Western blot proved the specific binding of monomer recombinant His-EGFR and the commercial specific antibody.Affinity of commercial antibody Erbitux and dimer recombinant hFc-EGFR achieved to 0.5 nmol/L calculated by the Biacore3000 protein interactions system.Conclusion: Monomer and dimer two different recombinant EGFR extracellular domain with correct structure were obtained by using mammalian cell expression system,which found a good basis for the selec tion of anti-EGFR specific antibodies.