| Chronic hepatitis B threatens people's healthy seversely, but the treatment options for chronic HBV carriers is still limited and less effective. In chronic HBV infection, both weak HBV-specific cytotoxic T lymphocyte (CTL) and T helper cell responses may contribute to viral persistence. Hepatitis B DNA vaccination can induce both humoral and cellular immune responses and has been widely accepted as one of potential therapeutic candidates by researchers. Hepatitis B virus core antigen (HBcAg) represents an interesting target for DNA-based vaccine. The HBeAg sequence is preceded in-frame by pre-core-core sequence, Removal of part of the signal sequence in N-terminal and a C-terminal truncation from precursor HBeAg (P22, P20) yield the secreted 18 kDa HBeAg protein (P18). Although HBeAg (P18) contribute to immune tolerance, but precursor part of HBeAg (P22, 20) could induce stronger antigen-specific immune responses compared with HBcAg.In our project, we firstly got precore-core and enhancer II DNA sequences from an adr serotype HBV DNA complete genome by PCR methods, then the sequence were inserted into plasmid VR1012, and we succeeded in constructing a kind of HBV DNA vaccine named VEC. Secondly, based on VEC plasmid, we designed alternative DNA vaccines by introduction of substitution mutation to the protease cleavage site of HBeAg precursor and abolished the protease cleavage in C-terminal of precursor HBeAg protein. The expected mutation sites are located in amino acid position of 151,154,164 and 167 within HBeAg sequence, which are protease cleavage sites. In this part, we established a new method named "twice single primer PCR method" based on the traditional site-directed PCR mutagenesis and sequentially introduced 4 different kinds of substitution mutations into plasmid VEC. In this way we obtained 4 different kinds ofplasmids, which were named VE1 (amino acid position 151 mutation), VE2 (151+154 mutations), VE3 (151+154 +164 mutations) and VE4 (151+154+164+167 mutations) separately. Then these vaccines plus VEC as controls were separately transfected into HepG2 cells using the calcium phosphate method with appropriate plasmid DNA per dishes. After 48 hours, By ELISA methods, we measured the HBeAg concentration in cell culture medium and cell lysis separately. Our results showed that HBeAg concentration from cell lysis was higher in VE2 and VE4 transfected cells than VEC one, but HBeAg concentration in culture medium showed the contrary way. By Immunohistochemistry staining we found that mutated HBeAg was mainly located in cell cytosol, and the plasmid transfection efficiency seems higher in VE2 and VE4 compared with VEC. The Western blot showed that HBeAg from VEC, VE2 and VE4 transfected cells were detected at approximately molecular position of 18, 20 and 22 kDa separately. Three kinds of plasmid VEC, VE2 and VE4 were injected into the thigh muscles of different group of Balb/c mice. Blood and splenocytes from mice were isolated at 4 weeks after immunization. We also have mouse groups immunized with three of these plasmid combined with IFN-γ and S antigen of HBV gene plasmids. We found that after 2 weeks of DNA immunization, the anti-HBc and anti-HBe antibody could be detected in peripheral blood in mice and the titer reached the highest level at 4 weeks after immunization, anti-HBe titers of VE2 and VE4 immunizing groups are higher than VEC group. We also observed that VE2 and VE4 could induce stronger antigen-specific immune responses than VEC and when combined with IFN-γ plasmid, the antigen-specific immune responses are stronger than those without combination immunization in mice. Nevertheless combination of plasmid VAS showed neither cellular nor humoral responses against HBV. |
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