| Although the relationship between mutations in HBV/C region and disease severity has been demonstrated in many epidemiological studies, the causality between them remains unclear. In this study, the adenovirus was employed as vector to transfer 1.2 copies of HBV genome containing L60V and I97L mutations into HepG2 cells and BALB/C mice, to investigate the biological implications of the two mutations in vitro and in vivo. We intended to determine whether the two mutations could change the immunogenity of HBcAg and had influence on the humoral and cellular immune response in mice.1. Construction of L60V and I97L mutants and recombinant adenovirusesBy site-directed mutagenesis technique, p3.8llplasmid, which contains 1.2 copies of HBV genome, was used as template to produce the two desired mutations. The HBV genome was cloned into the shuttle vector pAdTrack; the resultant constructs were cotransformed intoE.coli strain BJ5183, and the recombinant adenoviral plasmids were cleaved with Pac I to expose their inverted terminal repeats and transfected into 293 packaging cell to generate viruses. Three strains of recombinant adenoviruses carrying wild type or mutant HBV genomes were obtained.2. Biological characteristics of recombinant adenovirusesThe resultant recombinant adenoviruses showed typical adenovirus morphology with a diameter about 70 nm under electron microscope. The integration of HBV DNA into the adenovirus genome was identified by PCR and Southern blot assay. The viral genomes were stable when tested by PCR during the passage in 293 cells. HBV/C specific mRNA in 293 cells infected by recombinant adenoviruses was detected by RT-PCR and HBeAg in its culture medium by ELISA.3. Expression of HBV genes in HepG2 cellsBy ELISA and Western blot analysis, the expressions of HBV antigens were detected in cell lysate or culture medium of HepG2 cells infected by each of three strains of recombinant adenoviruses, respectively. When HepG2 cells were infected with AdHBV-WT at various multiplicity of infection (MOD , the expressions of HBsAg and HBeAg increased with MOI. Additionally, we infected HepG2 cells with the three strains of recombinant adenoviruses at same MOI. The results showed that the expression of HBeAg in HepG2 cells infected with AdHBV-WT was stronger than that of AdHBV-L60V and AdHBV- I97L during the whole test period. As for expression of HBsAg, AdHBV-WT was similar to AdHBV-L60V, however, the expression of HBsAg by AdHBV-I97L was so weak that it can be seldom detected.4. The immune response of BALB/C mice induced by recombinant adenoviruses Three of five groups of BALB/C mice were inrranasally inoculated withAdHBV-WT, AdHBV-L60V and AdHBV-I97L, and the other two groups with wild Ad5 and saline as control, respectively. Samples of mice serum were taken and tested for antibodies to HBV antigens by ELISA. The mice in experimental groups producedanti-HBc efficiently, and the levels had no significant difference, indicating that L60V3and I97L mutation may have no influence on the humoral immune response to HBcAg. All samples of mice serum were tested negative for anti-HBs. IgGl and IgG2a specific for HBcAg were positive in serum samples, but the level of IgG2a predominated in all samples, which reflected the type of immune response to HBcAg was inclined to Th1-like. The result implicated that L60V and I97L mutation might not change Th1/Th2 polarity. Lymphocytes were isolated from spleen of mice and stimulated with recombinant HBcAg in vitro. The proliferation response was assessed by 3H-TdR uptake. We found that T cell proliferation response in mice infected by AdHBV-WT was stronger than AdHBV-L60V and AdHBV-I97L. The results suggested that L60V and I97L mutation might downregulate T response to HBcAg.In this study, HBV genome was transferred and expressed successfully in HepG2 cells and BALB/C mice. Based on these models, the biological effects of the two hot-spot mutations L60V and I97L were studied. The results suggested that these mutations m...
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