Abstract: Objective: To construct a DNA vaccine containing heterological genetic sequence encoding most cytotoxic lymphocyte epitopes of human prostate stem cell antigen（PSCA）——pVAX1-PSCA₃-Fc-GPI-IRES-GM/B7（pVAX1-PSCA₃FcGB）,and detect its expression in a eukaryotic cell Cos7. Methods: The heterological PSCA genetic fragment was synthesized by overlapping extending-PCR,and then 3 copies of the fragment（PSCA₃） were linked up by co-adhesive end restriction and ligation strategy.The PSCA₃ fragment was inserted into a eukaryotic expression vector pCI-Fc-GPI including the gene of the signal peptide of human Igк,human IgG-Fc and glycosyl phosphatidyl inositol（GPI）.At last,the fusion genetic fragment PSCA₃-Fc-GPI was cloned into the final eukaryotic expression vector-pVAX1-IRES-GM/B7 which includes several genetic fragments encoding internal ribozyme entry site（IRES）,human granulocyte-macrophage colony-stimulating factor（GM-CSF） and costimulatory molecules-B7.1 at the downstream of the inserted site.The final recombinant plasmid pVAX1-PSCA₃FcGB DNA vaccine was liposomally transfected into Cos7 cells.And the expression of this DNA vaccine was detected by immunofluorescence and flow cytometry. Results: DNA sequencing result confirmed that the sequence of heterological PSCA genetic fragment was consistent with the design.Enzyme digestion analysis showed that the recombinant plasmid-pVAX1-PSCA₃FcGB DNA vaccine was successfully constructed.The expression of the DNA vaccine in eukaryotic cells was demonstrated by immunofluorescence and flow cytometry. Conclusion: The DNA vaccine-pVAX1-PSCA₃FcGB has been successfully constructed and well expressed in Cos7 cells.These results have provided necessary bases for the study of the anti-prostate cancer effect of this vaccine in the future.