Antigenicity and immunogenicity analysis of Xinjiang hemorrhagic fever virus Gc antigen fragment

Abstract

Objective To express and purify two domains GcⅠ and GcⅡ of 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-GcⅠ and pET32a-GcⅡ were constructed and transformed into E. coli BL21, respectively. The expression and purification conditions of rGcⅠ and rGcⅡ proteins 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+rGcⅠ group, pVAX1-GcⅡ+rGcⅡ group, rGcⅠ group, rGcⅡ group 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 rGcⅠ and rGcⅡ were 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∶12 800. 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+rGcⅡ group [(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 GcⅡ is expected to be used as vaccine candidates for the prevention and control of XHFV. Key words: Xinjiang hemorrhagic fever virus; Glycoprotein; Prokaryotic expression; Purification; Immunogenicity