Abstract: OBJECTIVE To prepare a highly active recombinant immunotoxin TP25 for human epidermal growth factor receptor 2(HER2) positive breast cancer. METHODS The recombinant region of the trastuzumab variable fragment and cytotoxin was recombined by gene engineering technology, and a highly active fusion protein TP25 with intact structure was obtained by prokaryotic expression. The binding ability of TP25 to different breast cancer cell lines was detected by immunofluorescence and flow cytometry. The killing activity of TP25 was detected by ³H-Thymidin incorporation. The apoptosis of tumor cells induced by TP25 was studied by measuring the production of single stranded DNA. RESULTS The recombinant expression plasmid of TP25 was successfully constructed and verified by restriction enzyme digestion and PCR experiment. The TP25 product with a molecular weight of 52 kDa was prepared by prokaryotic expression. It was proved that TP25 could bind to breast cancer cells with high expression of HER2 and had an active spatial conformation. Cell proliferation assay showed that the cytotoxicity of TP25 was positively correlated with dosage and could induce apoptosis of HER2 positive breast cancer cell. CONCLUSION HER2 positive breast cancer targeting protein TP25 is prepared.