OBJECTIVETo clone the extracellular domain (ECD) of the type III variant of human epidermal growth factor receptor (EGFRvIII) and construct the recombinant expression plasmid.

METHODSA DNA fragment (vIII ECD) encoding the extracellular domain of human EGFRvIII was obtained by PCR, and its T-A was cloned and sequenced. The DNA fragment was then ligated into the GST fusion expression vector to construct the recombination plasmid. After identification with restriction digestion and DNA sequencing, the recombinant plasmid was transformed into E. coli BL21 (DE3) for expression of the recombinant protein. The target protein was identified by SDS-PAGE and Western blotting.

RESULTSThe results of restriction digestion and DNA sequencing confirmed the successful construction of the recombinant plasmid. SDS-PAGE showed that the fusion protein was expressed as inclusion bodies in E. coli BL21 (DE3), and the amount of the fusion protein expressed in the bacteria, after induction for 4 h, accounted for up to 15% of the total bacterial proteins. Western blotting demonstrated that the fusion protein could be recognized by the specific anti-EGFR antibody.

CONCLUSIONWe have successfully constructed the recombinant expression vector of vIII ECD and induced the expression of the fusion protein, which may facilitate functional and immunological studies of EGFRvIII.