Objective: To construct a tumor-specific peptide vaccine P64k-EGFR(262-328); targeting the dimerization interface of EGFR and analyze its immunogenicity in BALB/c mice.
Methods: The fusion gene of P64k-EGFR(262-328); was amplified by splicing overlap extension-PCR (SOE-PCR) and cloned into the pMD18-T vector. After double-enzyme cleavage and sequence analysis, the fusion gene was cloned into the expression vector pET-21b by digestion with Nde I and Hind III and then transformed into the BL21(DE3). After induced by IPTG to express, the fusion protein P64k-EGFR(262-328); was purified by source Q and Ni-NTA chromatography. BALB/c mice were immunized with the purified protein dissolved in double-distilled water emulsified (1:1) in Freuds' adjuvant, and their sera were tested for antibody titers by ELISA.
Results: The P64k-EGFR(262-328); gene of 2031 bp was acquired. SDS-PAGE showed that the recombinant protein was 70 kDa, expressed mainly in a soluble form. After two steps of column chromatography, the purity was over 95%. The peptide vaccine could elicit a high titer of more than 1:16 000 in BALB/c mice.
Conclusion: The tumor-specific peptied vaccine P64k-EGFR(262-328); was constructed successfully, which could pay a good foundation for the further study on its function and application.