新疆出血热病毒Gc抗原片段抗原性及免疫原性分析

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

目的表达纯化新疆出血热病毒(XHFV)糖蛋白的2个结构域GcⅠ和GcⅡ,研究其免疫原性及对小鼠免疫应答的影响.方法构建pET28a-GcⅠ和pET32a-GcⅡ原核表达质粒,分别转化大肠杆菌BL21,并优化rGcⅠ和rGcⅡ蛋白的表达和纯化条件.采用蛋白质印迹法(Western Blot)和酶联免疫吸附测定法(ELISA)检测融合蛋白的抗原性.通过蛋白免疫和核酸初免-蛋白加强免疫2种方式免疫BALB/c小鼠,将小鼠按随机数字表法分为5组:pVAX1-GcⅠ+rGcⅠ组、pVAX1-GcⅡ+rGcⅡ组、rGcⅠ组、rGcⅡ组和生理盐水组(对照组),每组7只.采用间接ELISA检测小鼠血清抗体效价,同时通过脾T淋巴细胞增殖实验和细胞因子含量测定对免疫效果进行评价.结果纯化获得融合蛋白rGcⅠ和rGcⅡ,两者能与绵羊阳性血清反应,具有较好的抗原性.3次免疫小鼠后,各实验组小鼠血清中的IgG水平明显高于对照组(均P<0.001),血清抗体效价均达到1:12800以上,其中rGcⅡ和pVAX1-GcⅡ+rGcⅡ组脾细胞培养上清中Th2型细胞因子白细胞介素-4(IL-4)的质量浓度明显高于对照组[(79.97±7.47)ng/L比(24.29±3.81)ng/L,(61.43±9.27)ng/L比(24.29±3.81)ng/L,均P<0.01];pVAX1-GcⅡ+rGcⅡ组Th1型细胞因子干扰素-γ(IFN-γ)的质量浓度最高,与对照组比较差异具有统计学意义[(42.46±2.60)ng/L比(20.33±1.67)ng/L,P<0.001],并显示出明显的抗原特异性脾T淋巴细胞增殖(P<0.001).结论纯化后的重组蛋白rGcⅠ和rGcⅡ具有较好的免疫原性,可使被免疫机体T淋巴细胞趋向于Th2效应,且pVAX1-GcⅡ联合重组蛋白GcⅡ的抗原特异性免疫效果较好,有望作为防治XHFV的候选疫苗.

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+rGcⅠgroup,pVAX1-GcⅡ+rGcⅡ group,rGcⅠgroup,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.