Abstract: In order to delete the immune tolerance of self-protein HER2 and produce an efficient immune response, the genetic code expansion technique was employed to introduce the immunogenic amino acid p-nitrophenylalanine into the position 5, 26 and 79 of the HER2 fragment and HER2 mutants containing p-nitrophenylalanine were obtained. Prototype and mutant HER2 molecules with a purity of more than 95% were obtained through a nickel affinity column. Immunogenicity analysis of the HER2 vaccine showed that pNO₂Phe⁷⁹HER2 was able to produce high titer cross-reactive antibodies in C57BL/6 mice and that the mouse antisera could recognize SKBR-3 cells with intact HER2 receptor extracellular domain and cleave HER2 highly expressed HER2+B16F10 cells through ADCC effect. These findings suggested that pNO₂Phe⁷⁹HER2 which incorporates p-nitrophenylalanine could be used as a tumor vaccine candidate targeting HER2.