中国生物工程杂志

Objective:Epidermal growth factor receptor (EGFR) is overexpressed in tumors and is associated with metastasis, invasion and poor prognosis of tumor cells. Therefore, EGFR has become an attractive target for the treatment of cancer. Several small molecule compounds that specifically inhibit EGFR have been clinically developed. Preparation of specific antibody to human EGFR with an identified biological activity is essential for this target-related immunotherapeutic study, such as a construction of single chain antibody fragment (scFv).Methods:Total RNA was extracted from the hybridoma cell line specific to EGFR. The light and heavy chain variable region genes V_L and V_H were obtained by 5' RACE technology. The constructed scFv antibody gene was cloned into the eukaryotic expression vector pcDNA3.1. ELISA was used to detect the specificity of expressed single chain antibody to human EGFR protein. The affinity between antigen and antibody was detected by Fortebio. Flow cytometry was used to detect the functional activity of single chain antibody by which binding to natural EGFR on lung cancer cell lines.Results:The V_L and V_H sequences of light and heavy chain variable regions were obtained, and assembled EGFR-scFv was successfully expressed. The EGFR-scFv was specifically bound to natural EGFR protein with an affinity of 3.22×10 ^-9mol/L, and the scFv was further indentified based on its binding to EGFR that expressed by lung cancer cell line H1975. Conclusion:The anti-human EGFR single chain antibody was successfully constructed, which lays the foundation for development of EGFR- targeted immunotherapy.