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Introduction: Glycoprotein B (gB) is the major antigen for induction of humoral responses against human cytomegalovirus (HCMV) making it an attractive candidate for immune prophylaxis. In the present study, the humoral immune response of BALB/c mice to a truncated HCMV gB protein fused with GFP was evaluated. Methods: The truncated gB coding sequence was synthesized and cloned in pEGFPN1 eukaryotic expression vector and expressed in HEK 293T cell line. After optimization, expression of the recombinant truncated HCMV gB was verified using HRP-conjugated polyclonal antibody specific for HCMV gB.The level of humoral immune responses was assessed in mice using DNA/DNA, peptide/peptide, and DNA/ peptide (prime-boost) immunization strategies. Results: Cloning of the truncated gB coding sequence in the pEGFPN1 was verified by restriction enzyme analysis and sequencing. After optimizing the transfection procedure the number of the GFP positive cells reached 32%. Western blot analysis confirmed the in vitro expression of the truncated HCMV gB protein with an apparent molecular weight of approximately 70 kDa. In vivo prime-boost immunization using HCMV gB DNA/peptide regimen showed significantly higher humoral immune responses compared to the control groups. Conclusion: This study demonstrated that the pEGFPN1 eukaryotic expression vector could be used to optimize and evaluate the expression of this truncated protein.The results also showed that the DNA/peptide vaccination could induce a significant antibody response in animal model.