AIM:To compare the efficiency of different plasmids asDNA vectors by cloning three HBsAg-encoded genes intotwo eukaryotic expression vectors,pRc/CMV and pSG5UTPL/Flag,and to express HBsAg S,MS,and LS proteins in SP2/0 cells,and to establish monoclone SP2/0 cell strains thatare capable of expressing S or S2S proteins stably.METHODS:Segments of S,preS2-S,preS1-preS2-S genesof Hepatitis B virus were amplified by routine PCR and preS1-S fragment was amplified by Over-Lap Extension PCR.Theamplified segments were cleaved with restrictedendonuclease Hind Ⅲ/Not Ⅰ followed by ligation with pRc/CMV,or BamHI/EcoR I followed by ligation with pSG5UTPL/Flag.After the plasmid vectors were cleaved with thecorrespond enzymes,the amplified segments were insertedinto pRc/CMV or pSG5UTPL/Flag plasmid vectors with T4DNA ligase.KOZAK sequence was added before the initialATG code of each fragment using specific primer.Theinserted segments in the recombinant plasmids weresequenced after subcloning.BALB/c mice myeloma cells(SP2/0 cell line) were transfected with the recombinantplasmids.The expressions of the different recombinants werecompared by Western-blot,using a monoclonal anti-HBsantibody as the primary antibody and peroxidase-labeledmulti-linker as the secondary.Stable SP2/0-pRc/CMV-S orSP2/0-pRc/CMV-MS clones were established through clonescreening with G418.RESULTS:Fragments with anticipated size were harvestedafter PCR.After recombination and screening,the sequencesof the inserted segments in the recombinants were confirmedto be S,preS2S,preS1-preS2S and preSiS encoding genes,determined by sequencing.The results of Western-blothybridization were positive for the anticipated proteins.Among them,pRc/CMV-S or pRc/CMV-MS demonstrated thehighest expressing their respective antigen. M,L or preS1S proteins are obtained.For hepatitis surfaceantigen expression in eukaryotic cells,the vector pRc/CMVis superior to pSG5UTPL/Flag,and pRc/CMV-S and pRc/CMV-MS are the most efficient in the pRc/CMV clones.SP2/0cells stably expressing HBsAg are established,and may beused as target cells for evaluating the CTL activity of a DNAvaccine in vitro. AIM: To compare the efficiency of different plasmids as DNA vectors by cloning three HBsAg-encoded genes intotwo eukaryotic expression vectors, pRc / CMV and pSG5UTPL / Flag, and to express HBsAg S, MS, and LS proteins in SP2 / 0 cells, and to establish monoclone SP2 / 0 cell strain that could capable of expressing S or S2S proteins stably. METHODS: Segments of S, preS2-S, preS1-preS2-S genes of Hepatitis B virus were amplified by routine PCR and preS1-S fragment was amplified by Over -Lap Extension PCR. The amplified fragments were cleaved with restrictendonuclease Hind III / Not I followed by ligation with pRc / CMV, or BamHI / EcoR I followed by ligation with pSG5UTPL / Flag. After the plasmid vectors were cleaved with thecorresponding enzymes, the amplification segments were inserted into pRc / CMV or pSG5UTPL / Flag plasmid vectors with T4 DNA ligase. KOZAK sequence was added before the initial ATG code of each fragment using specific primer. The inserted segments in the recombinant plasmids were sequenced after subcloning. BBB mice myeloma cells (SP2 / 0 cell line) were transfected with the recombinant plasmids.The expressions of the different recombinants werecompared by Western-blot, using a monoclonal anti-HBsantibody as the primary antibody and peroxidase-labeled multi-linker as the secondary.Stable SP2 / 0-pRc / CMV-S orSP2 / 0-pRc / CMV-MS clones were established through clonescreening with G418. RESULTS: Fragments with anticipated size were harvested after PCR. After recombination and screening, the sequences of the inserted segments in the recombinants were confirmedto beS, preS2S, preS1-preS2S and preSiS encoding genes, determined by sequencing. The results of Western-blothybridization were positive for the anticipated proteins. Among them, pRc / CMV-S or pRc / CMV-MS rendered thehighest expressing their respective antigen . M, L or preS1S proteins are obtained. For hepatitis surface antigen expression in eukaryotic cells, the vector pRc / CMVis superior to pSG5UTPL / Flag, and pRc / CMV-S and pRc / CMV-MS are the most efficient in the p Rc /CMV clones. SP2 / 0 cells stably expressed HBsAg are established, and may be used as target cells for evaluating the CTL activity of a DNA vaccines in vitro.