RESUMO
Objective To express and purify two domains GcⅠand GcⅡof Xinjiang hemorrhagic fever virus (XHFV) glycoprotein, and to study its immunogenicity and the effects on immune response in mice. Methods The prokaryotic expression plasmids of pET28a-GcⅠand pET32a-GcⅡwere constructed and transformed into E. coli BL21, respectively. The expression and purification conditions of rGcⅠand rGcⅡproteins were optimized. The antigenicity of the fusion protein was detected by Western Blot and enzyme-linked immunosorbent assay (ELISA). BALB/c mice were immunized by protein immunization and DNA priming-protein boosting. The mice were randomly divided into 5 groups, including pVAX1-GcⅠ+rGcⅠgroup, pVAX1-GcⅡ+rGcⅡgroup, rGcⅠgroup, rGcⅡgroup and saline group (control group) with 7 mice in each group. The serum antibody titer of mice was detected by indirect ELISA, and the immune effect was evaluated by spleen T lymphocyte proliferation assay and cytokine content determination. Results The fusion proteins rGcⅠand rGcⅡwere purified and obtained, which could react with positive serum of sheep and had good antigenicity. After three immunizations, the IgG levels in the serum of each experimental group were significantly higher than those in the control group (all P<0.001). The serum antibody titers of the experimental groups were reached above 1:12800. Among them, the concentration of Th2 type cytokine interleukin-4 (IL-4) in the spleen cell culture supernatant of rGcⅡ[(79.97±7.47) ng/L] and pVAX1-GcⅡ+rGcⅡgroup [(61.43±9.27) ng/L] was significantly higher than (24.29±3.81) ng/L of the control group, respectively (all P<0.01). The highest mass concentration [(42.46 ±2.60) ng/L] of Th1 type cytokine interferon-γ(IFN-γ) was observed in the pVAX1-GcⅡ+rGcⅡ group, which was significantly higher than (20.33±1.67) ng/L of the control group, and the difference was statistically significant (P<0.001). That showed a significant antigen-specific splenic T lymphocyte proliferation (P<0.001). Conclusions The purified recombinant proteins rGcⅠand rGcⅡhave good immunogenicity, which can make the immune system T lymphocytes tend to Th2 response, and pVAX1-GcⅡ combined with recombinant protein GcⅡ can induce better antigen-specific immune effect. And pVAX1-GcⅡ combined with recombinant protein GcⅡis expected to be used as vaccine candidates for the prevention and control of XHFV.
RESUMO
Objective To construct two DNA vaccines based on two glycoprotein antigen segments of Xinjiang hemorrhagic fever virus (XHFV) and to evaluate the immune responses in BALB/c mice following vaccination.Methods Two recombinant expression plasmids pVAX1-GcⅠand pVAX1-Gc Ⅱ were constructed by inserting XHFV YL04057 strain Gc Ⅰ (1 229-1 349 aa) and Gc Ⅱ (1 443-1 566 aa) fragments into the eukaryotic expression vector pVAX1 and then were identify by restriction enzyme digestion and sequencing analysis.The recombinant expression plasmids were transfected into mice by hydrodynamics-based transfection.Immune responses induced in mice were evaluated by testing the proliferation of T cells with MTT,measuring serum antibody level with ELISA and detecting cytokines in the supernatant of spleen cell culture with ELISA kit.Results The recombinant expression plasmids were successfully constructed as indicated by the results of restriction enzyme digestion and sequencing analysis.Expression of Gc Ⅰ and Gc Ⅱ genes in mice liver tissues was detected.Antibody titers in mice immunized with pVAX1-GcⅠor pVAX1-Gc Ⅱ were higher than those in mice immunized with pVAX1.Compared with pVAX1,pVAX1-Gc Ⅱ significantly enhanced the proliferation of splenic T lymphocytes and the expression of IFN-γ (P<0.01).Conclusion The constructed two DNA vaccines for XHFV can induce specific humoral and cellular immune responses in mice.pVAX1-Gc Ⅱ is better than pVAX1-GcⅠin immunogenicity and protective efficacy,suggesting that it can be used as a promising candidate for the development of DNA vaccine for XHFV.