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Abstract
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Background: Epidermal growth factor receptor (EGFR) overexpression is a characteristic of several malignancies and could be considered
as an excellent target for designing specific inhibitors such as anti-EGFR monoclonal antibodies for cancer therapy. Drawbacks exerted by
large sizes of full-length antibodies have lead to the development of single chain antibodies, which benefit from having smaller sizes and
short circulation half-lives. Objectives: The aim of this study was cloning, expression and purification of variable regions of anti-EGFR monoclonal antibody in E. coli
for production of single chain antibodies. Materials and Methods: The RNA, extracted from the C225 hybridoma cells, was reverse transcribed into cDNA and used for PCR
amplification of genes encoding light and heavy chains from the variable regions. The PCR products were cloned and expressed in E. coli BL21 for production of a single chain antibody. The expressed protein was analyzed by SDS-PAGE and purified by Ni-NTA affinity
chromatography. The reactivity of purified C225-scFv with EGFR-expressing A431 tumor cell line was tested by Western blotting and
enzyme-linked immunosorbent assays. Results: The results indicated that C225-scFv was highly expressed in E. coli and appeared as a protein with a mass of 27 kDa in the sodium
dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the induced cell lysate. Reactivity analysis of the purified C225-
scFv with A431 tumor cell line by western blotting and enzyme linked immunosorbent assay (ELISA) revealed high binding affinity of the
recombinant C225-scFv to the target cells. Conclusions: The results of this study indicated that C225-scFv is capable of binding to EGFR and could be considered as a useful tool for
diagnosis and treatment of EGFR-overexpressing tumor cells.
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