Objective    To replace the  endogenous  CTL epitope LLD in HBsAg with EYILSLEEL of glypican (GPC3) in order to prepare a GPC3-HBsAg multiple peptides vaccine for HBV infection.     Methods    The HBsAg/GPC3 DNA was amplified by gene splicing by overlap extension (SOEing) PCR from pcHBsAg plasmid and then inserted into pBSSK+ vector to construct a pBSSK/GPC3 vector. The vector was then identified by PCR

double digesting and sequencing. The fragment encoding GPC3 CTL epitope EYILSLEEL was obtained by double digesting the plasmid pBSSK/GPC3 and then inserted into pcDNA3.1+ vector.     Results    Sequencing and restrict endonulease digestion indicated that eukaryotic expression vector pcDNA-HBsAg/GPC3 was constructed successfully.     Conclusion    The endogenous CTL epitope LLD of HBsAg is replaced by the EYILSLEEL

and an eukaryotic expression vector pcDNA-HBsAg/GPC3 is constructed.