The Construction And Eukaryotic Expression Of The DNA Vaccine Encoding The Fusion Of Woodchuck CTLA-4 Extracellular Segment And Hepatitis B Virus Core Protein And Mice Immunization

Objective To construct the plasmid pwCTLA-4-HBc encoding the fusion of woodchuck cytotoxic-T-lymphocyte-associated antigen-4 (wCTLA-4) extracellular segment and hepatitis B virus core protein (HBc), and express the fusion protein in eukaryotic cells. To test the level of the fusion protein in the culture medium, and identify the ability of the constructed plasimd inducing antibody-response in mice.Methods The sequence encoding the fusion of wCTLA-4 extracellular segment and WHc first 149 amino acids was amplified by PCR from the plasmid pCTLA-4-C, and cloned into pcDNA3.1/V5-His?TOPO?TA expression vector. The constructed plasmid pwCTLA-4-WHc was identified by enzyme restriction and its expression level was tested by indirect immunofluorescence assay (IF) in baby hamster kidney (BHK) cells. The WHc fragment in plasmid pwCTLA-4-WHc was replaced with HBc fragment by enzyme digestion and T4 ligation,and the constructed plasmid pwCTLA-4-HBc was identified by restriction enzyme digestion. BHK and Hela cells were transfected with the plasmid pwCTLA-4-HBc, then the fusion protein expression was detected by indirect immunofluorescence assay (IF) and Chemiluminescent Microparticle Immunoassay (CMIA) respectively. 4 BALB/cJ (H-2d) mice were immunized 3 times with the plasmid pwCTLA-4-HBc, and the anti--HBc/anti-HBe antibody in the mice sera was tested by Enzygonost? anti-HBc/anti-HBe monoclonal kit respectively.Results The fragments of expected sizes were obtained after restriction enzyme digestion of the constructed plasmid pwCTLA-4-HBc. HBc antigen expression in the transfected BHK cells was detected by IF, the supernatant and cell lysate of the transfected Hela cells were both tested to be HBeAg positive. The relative concentration of the fusion protein in the culture medium was 10-fold as in the cell lysate. 3 weeks after immunization, the anti-HBc antibody was shown in 2 mice and one of them also tested to be anti-HBe positive.Conclusion The expression plasmid pwCTLA-4-HBc of woodchuck CTLA-4 extracellular segment and hepatitis B virus core fusion protein was successfully constructed, and the fusion protein could be produced in eukaryotic cells and secreted into the cell culture. Fusion protein expressing plasmid pwCTLA-4-HBc immunization could induce anti--HBc/anti-HBe antibody response in mice.