Objective    To express mouse-anti-human EpCAM single-chain antibody (ScFv) specifically targeting tumor cells or cancer stem cells of epithelial origin via phage display technology

and to identify its immunological activity.     Methods    Total gene synthesis method was used to synthesize mouse-anti-human EpCAM ScFv gene with a structure of VL+Linker+VH. The gene was cloned to pMD19-T Simple Vector and then was digested

after being purified

was cloned to phage vector pCANTAB 5E. Competent E. coli TG1 cells were transformed with pCANTAB 5E containing ScFv gene. Mouse-anti-human EpCAM ScFv was produced by helper phage M13K07 superinfection with 5 randomly selected clones

and the antigen binding activity was identified by ELISA and immunocytochemistry.     Results    Recombinant pCANTAB5E/EpCAM ScFv was constructed successfully

with a positive antigen-binding rate of 20%  proven by ELISA and immunocytochemistry. The selected ScFv could specifically bind to EpCAM antigen and human lung adenocarcinoma cell line A549.     Conclusion    Mouse-anti-human EpCAM ScFv produced via phage display technology has advantages such as simple

highly efficient

and time-saving

which is expected to be helpful in diagnosing tumor cells and cancer stem cells of epithelial origin.