Construction And Expression Of Eukaryotic Expression Vectors Of HBV Antigen Genes

ObjectivesHepatitis B Virus(HBV) can not be cultivated in vitro,which has limit range of host,thus interrupt the developing of HBV research.While transforming plasmid containing HBV genes into target cells,establishing cell model expressing HBV genes in vitro can be helpful to further research of pathogenesis of HBV related disease.We investigated the expression and optimization of HBV antigen gene in eukaryotic cells in vitro,the construction of the eukaryotic expression vectors and their target gene expression in vitro provide a solid foundation for optimization of HBV antigen gene expression and further experimental research on the biological function of HBV antigens.Methods1.Construction of eukaryotic expression vector containing HBV antigen genes(1)The fragments of HBV antigen genes were amplified by PCR(2)Construction of eukaryotic expression vector pCMV-tag2B-HBs,pCMV-tag2B-HBe, pCMV-tag2B-HBc , pCMV-tag2B-HBeAg(P22) via intermediate cloning vector pEASY-T1 Simple. (3)Identification of recombinant plasmids by double enzymic digestion and DNA sequencing.2.Transient transfection of eukaryotic cells in vitro(1)Transiently transfect recombinant plasmids into HepG2 cells and LO2 cells as a control using lipofectamineTM 2000,establishing eukaryotic cell model expressing HBV antigen genes in vitro. HepG2 cells were divided into three groups,transfection group (transfected recombinant plamid), control group (transfected empty plamid), blank control group (mormal cells). There are three duplicated wells in each group.(2)Identification of transfection efficiency Recombinant plasmids were co-transfected with pEGFP-C1 vector containing EGFP marker gene into HepG2 cells. The expression of green fluorescence protein(GFP) was monitored 24,48 and 72 hours post transfection,and transfection efficiency was calculated accordingly.(3)Analysis of antigen gene expression in transfected cells1)Western Blot and Immunofluorescence were used to analyze the expression of HBV gene products in the transfected cells.Total protein of HepG2 cells 48,72 hours after transfection with recombinant plamid were extracted, Total protein of HepG2 cells 48 hours after transfection with empty plamid and no plsmids were extracted,for Western Blot analysis. HepG2 cells 48 hours after transfection with recombinant plamid, empty plamid and no plsmids were used for immunofluorescence analysis. 2)Quantification of antigen gene expression by Electrochemi- luminescence Immunoassay(ECLIA) Supernatant of HepG2 cells 24,48,72 hours after transfection with recombinant plamid were collected,Supernatant of HepG2 cells 48 hours after transfection with empty plamid and no plsmids were collected for ECLIA analysis.Each experiment was repeated three times.Resluts1. Construction of eukaryotic expression vector Fragments of HBV antigen genes were obtained and recombinant eukaryotic expression vector containing HBV antigen genes were constructed successfully,named pCMV-tag2B-HBs,pCMV-tag2B-HBe, pCMV-tag2B-HBc,pCMV-tag2B- HBeAg(P22),double enzymic digestion and DNA sequencing showed that inserted fragments were correct.2.Establishment of eukaryotic cell model expressing HBV antigen genes in vitro(1)Identification of transfection efficiency HepG2 cells were co- transfected with pEGFP-C1 vector containing EGFP marker gene. The expression of GFP was monitered 24,48,72hours post transfection. The transfection efficiency is high. There was almost the same transfection efficiency among different time point.(2)The relative quantification of antigen gene expression different time post transfection 48 and 72 hours after transfection, antigen protein can be detected in HepG2 cells in transfection group.There's no significant difference between the two time point by Western Blot(WB) analysis, P>0.05;While antigen protein cannot be detected in the control and black control group,and in the transfection group,antigen protein mainly locates in cytoplasm,minor in cell nucleus and membrane by immunofluo- rescent,which was consistent with results from WB.L02 cells transfected with recombinant plasmid can also express corresponding antigen.(3) Quantification of antigen protein in the supernatant of the transfection group The quantification of HBsAg,HBeAg was measured by ECLIA. HBsAg,HBeAg can be detected in the supernatant of HepG2 cells 24h after transfection with pCMV-tag2B-HBs,pCMV-tag2B-HBe accordingly,and reach their peak 48h after transfection,then HBsAg decreases 72h after transfection, P<0.05.While HBeAg cannot be detected 72h after transfection; In pCMV-tag2B-HBeAg(P22) transfection group, HBeAg can only be detected 48h post transfection.Conclusion1. The eukaryotic expression plasmid pCMV-tag2B-HBs ,pCMV-tag2B–HBe, pCMV-tag2B-HBc, pCMV-tag2B-HBeAg(P22), and the control plasmid pCMV-tag2B were transfected into HepG2 cells with lipofectamineTM 2000,respectively.The transfection efficiency is high.2.Antigen protein can be detected in HepG2 cells in transfection group rather than in the control group and blank control group.There's no significant difference of the relative quantity of antigen between 48 and 72 hours after transfection.3.There's significant difference of the quantity of secreted antigen in the supernatant of HepG2 cells among different hours after transfection.