Objective:To generate recombinant adenoviral vector containing CRT-HBsAg fusion gene for developing a safe, effective and HBsAg-specific therapeutic vaccine. Methods:The fusion of CRT and HBsAg gene was constructed by using polymerase chain reaction(PCR), endonuclease digestion and ligation methods, and then the fusion gene was cloned into pENTR/D-TOPO transfer vector after the base pairs of DNA(CACC) sequence was added to the 5′ end. Adenoviral expression vector(Ad-CRT/HBsAg)containing CRT-HBsAg fusion gene was constructed by homologous recombinantion. The linearized DNA plasmid of the recombinant adenoviral vector was transfected into human embryo kidney(HEK 293A) cells to package and amplify recombinant adenovirus. The recombinant adenovirus titer was characterized by using the End-dilution assay. The expression of the CRT/HBsAg fusion protein in Ad-CRT/HBsAg transfected 293A cells was detected by Western blot. Results:The CRT-HBsAg fusion gene was characterized by using PCR, and sequencing result revealed that the length and sequence were accurate. The recombinant adenoviral vector, Ad-CRT/HBsAg, was generated successfully. The titer of Ad-CRT/HBsAg was characterized as 2.68×1011 pfu/ml. The CRT-HBsAg fusion protein was expressed by HEK 293A cells correctly. Conclusion:Recombinant replication-defective adenovirus expression vector containing CRT/HBsAg fusion gene was constructed successfully, and this study has provided an experimental basis for further research of HBV gene therapy.