Objective: To investigate the enhanced role of B(7-1) in gene immunization of eukaryotic vector expressing HBsAg, and further to eliminate the immunotolerance to hepatitis B virus.
Methods: B(7-1) and IRES-HBs with linker and promoter sequence prior to HBs gene were amplified with high fidelity PCR, then subcloned into plasmid pBluescriptks+, which was used to sequence. The eukaryotic expressing vector was constructed by inserting B(7-1) and IRES-HBs into plxsn. Recombinant vector was transfected into PA317 by means of lipid mixtures. Titre of pseudovirus packaged in PA317 cells was detected by infecting NIH3T3 cells. B(7-1) and HBsAg expression in PA317, NIH3T3, HepG(2), 293, EL4 cell lines was detected by RT-PCR, immunohistochemistry and ELISA, respectively.
Results: No change was found in the sequences of target genes amplified by high fidelity PCR compared with the known sequences. Plxsn-B(7-1)-HBs was constructed by first inserting B(7-1) into plxsn with enzyme EcoRI/XhoI, then IRES-HBs was inserted in the same way with enzyme XhoI/BamHI. 43 and 90 G418-resistant clones were obtained two weeks later after 1:10 and 1:5 passages at 24 hours after transfection. Titre of the peudovirus was 5.4x10(5) CFU/ml. B(7-1) was expressed in all the cell lines above by RT-PCR test. The contents of HBsAg in the supernatant of the PA317, NIH3T3, HepG(2), and 293 cells at 24, 48, 72 hours intervals were 0.15, 0.11, 0.20, 0.13; 0.63, 0.47, 0.76, 0.58; 0.77, 0.61, 0.89, 0.69, respectively.
Conclusions: The retroviral vector expressing B(7-1) and HBsAg simultaneously is constructed successfully. The target antigens are expressed in above cell lines in vitro after transfection with lipids mixtures. It may be used to conduct gene immunization and to investigate the role of B(7-1) on HBsAg immune response, which may be further used to the prevention and therapy of hepatitis B.