Abstract: Objective To study GAGE-1 gene cloning, in vitro prokaryotic expression and protein separation and purification for future clinical application to obtain specific anti-tumor immune response induced by cancer-testis antigens GAGE-1. Methods GAGE-1 segment from QGY-7701 was amplified by RT-PCR and cloned into the expression vector pGEX-6p-1 to construct the expression plasmid pGEX-6p-1-GAGE-1. The recombinant vector was transformed to E.coli BL21 and GST fusion protein expressed was induced by IPTG. The protein was purifi ed by GST affi nity chromatography and was analyzed by SDS-PAGE. Results The sequence of 5 'terminal 251bp of GAGE-1 segment was amplified and identical with the published counterpart in GeneBank. The BL21(DE3) containing the PGEX-6p-1-GAGE-1 expressed a Mr 35.7kD GST-GAGE-1fusion protein. The purity of the protein was more than 90%. Conclusion The recombinant expression vector PGEX-6p-1-GAGE-1 was constructed successfully, and the fusion protein was expressed and purifi cated,which is the foundation for further study of GAGE protein.