Utilizing cytotoxic proteins linked to tumor targeting molecules as anti-tumor drugs is a promising approach.However,most cytotoxins derived from bacteria or plants have inherent problems such as large molecular weights and they trigger a strong immune system reaction,which leads to drug failure and serious side effects.Amphinase (Amph) is a ribonuclease with a low molecular weight that is found in northe leopard frog oocytes.It has strong cytotoxicity against tumor cell lines in vitro and weak immunogenicity in vivo,and is a promising candidate in the development of targeted drugs.Transforming growth factor-α (TGF-α) that binds to the epidermal growth factor receptor (EGFR) is being used as a targeting molecule for the treatment of EGFR high-expressing tumors.In this study,we expressed and purified a recombinant amphinase and its TGF-α fusion protein (AGT) separately from Escherichia coli.AGT exhibited more significant cytotoxicity in vitro on EGFR high-expressing tumor cell lines,and stronger anti-tumor effects in vivo.This fusion protein also exhibited unusual thermostability,low in vivo immunogenicity,and side effects.Our results provide a new entry point for the development of novel,highly efficient anti-tumor targeting biological agents with low immunogenicity.