objective:To construct the recombinant adenovirus containing pancreatic tumor associated MUC4 antigen and provide the basis for further research on pancreatic tumor vaccine. Methods:The HLA-A1 and HLA-A2 restricted epitopes of MUC4 was predicted by BIMAS and ProPred. The combined polyepitope gene with kozac sequence,sign peptide and PARDE was synthesized. The combined polyepitope was subcloned into pAdshuttle-CMV shuttle plasmid(pAdshuttle-CMV-PE),and then transformed into competent E.coli BJ5183 cells with backbone plasmid already. The homologous recombinant adenoviral plasmid pAdeasy-1-PE was validating with PacⅠ digesting and sequencing to verify their fidelity. Ad293 cells were transfected with PacⅠ linearized recombinant and lipofectamine reagent. Identified by green fluorescence protein(GFP) expression and PCR method,the virus was amplified by infection 293A cells repeatly. Viral particle concentration was determined by TCID50 plus flow cytometer. The virus was transduced into COS-7 cells,and expression of polyepitope protein was demonstrated by the GFP expression and SDS-Page analysis. Results:The recombined adenovirus containing combined polyepitope was constructed successfully and the titer was 10 pfu/ml. Conclusion:A recombinant adenovirus containing combined polyepitope was constructed successfully,a specific protein corresponding with the predicted mass was found in virus transduced COS-7 cells.