The Study Of The Immuneprotective Responses Induced By The HBcAg Fusion Protein Expressing DNA Vaccine In The Mice Model

Objective1. To construct the eukaryotic expression plasmids expressing the extra cellular part of woodchuck CTLA-4, mouse CD27 or mouse CD40L and HBV core protein: pwCTLA-4-HBc,pmCD27-HBc和pmCD40L-HBc; and test the expression secretion level of the encode fusion protein2. Inspect the immunological response of mice after vaccination with electroporation3. Inspect the replication and expression of pAAV/HBV 1.2 in the preimmunized miceMethods1. To construct the plasmids pwCTLA-4-HBc,pmCD27-HBc and pmCD40L-HBc with molecular cloning technology2. To immunized the mice with elecroporation using plasmids pwCTLA-4-HBc, pmCD27-HBc, pmCD40L-HBc, pHcex, pHeex, test the HBcAb and IgG2a/IgG1 level with ELISA, IFN-γproducing cell number in the spleen with ELISPOT.3. pwCTLA-4-HBc and pHcex immunized mice pAAV/HBV 1.2 hydrodynamic injection, test the HBsAg, HBcAb and HBsAb in the serum with ELISA and the IFN-γproducing cell number in the spleen with ELISPOT, test the HBV DNA in the serum with realtime PCRResults1. Three plasmids named pwCTLA-4-HBc, pmCD27-HBc and pmCD40L-HBc are successfully constructed. After transfected into BHK cells, the expression of the fusion protein can be detected by HBc specific IF in these vectors. The HBcAg expression vector pHcex and HBeAg expression vector pHeex served as a control. In addition, after transfected into Hela cells, we can detect different level of HBeAg expression by ELISA in cell lysis and supernatant as we predicted.2. We immunized mice with pwCTLA-4-HBc, pmCD27-HBc, pmCD40L-HBc and control plasmid pHcex, pHeex. After 3 times immunization, the mice can develop different level of HBcAg specific antibody response, respectively. The subtype and dynamic of the antibody are different. The pwCTLA-4-HBc vector can induce a fast and stronger antibody response, a relative Thl/Th2 balancing response and keep this status after second immunization. By using ELISPOT, We can detect more IFN-gamma producing cells in 3(?)vectors immunized mice. Especially, the pwCTLA-4-HBc and pHcex immunized group are much higher than other group.3. After immunization, we challenge mouse with an adenovirus based HBV expressing plasmid pAAV/HBV1.2 by hydrodynamic tail vein injection. As detected by realtime PCR, The HBV DNA is significant lower than control group. For immune response, the IFN-gamma producing cells, the total anti-HBC antibody production are higher than control group. The pwCTLA4-HBc immunized group can produce more Th2 type antibody and less Thl antibody than pHcex group. The Th1 type anti-HBs and total IgG amount are not significant between different groups. Two DNA immunized groups can induce more Th2 type antibody response than mock group.Conclusion1. Three plasmids named pwCTLA-4-HBc, pmCD27-HBc and pmCD40L-HBc are successfully generated.2. All HBc or HBe expressing plasmid can induce humeral and cellular response in immunized mice. The response induced by pwCTLA-4-HBc is the strongest.3. Pre-immunization with pwCTLA-4-HBc and pHcex can inhibit the replication and reduce the HBsAg in the serum after pAAV/HBV1.2 hydrodynamic injection.