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目的在鼠模型中观察登革(DEN)病毒双价和四价重组质粒DNA的免疫原性,为登革多价DNA疫苗的研究奠定基础。方法采用可去除内毒素的试剂盒大量提取质粒DNA。将双价质粒DNA及将它们配伍后再与鼠源GM2CSF进行联合免疫,免疫途径采用肌肉注射,并加以电刺激,以提高质粒DNA的摄入效率。于初次免疫后第2和4周各加强免疫1次。然后在小鼠中分别测定针对登革1～4型病毒的体液和细胞免疫应答水平。采用间接免疫荧光法和中和试验测定血清抗体效价,细胞免疫应答水平通过测定脾淋巴细胞的增殖指数和其分泌的IFN2γ浓度进行评价。细胞浸润实验通过对免疫部位的肌肉进行HE染色确定。结果小鼠在初次免疫后第4周即开始产生针对DEN1～4型病毒的抗体,随着时间的延长,抗体效价逐渐上升。第14周中和抗体效价最高达1∶32。淋巴细胞的刺激指数及其分泌IFN2γ的浓度均显著高于对照组。GM2CSF对体液免疫应答有促进作用,但对细胞免疫应答无显著的促进作用。结论本研究所构建的双价和四价重组质粒DNA在鼠模型中具有较好的免疫原性,这为登革多价DNA疫苗的研究奠定了基础。
OBJECTIVE: To observe the immunogenicity of dendrimer (DEN) virus divalent and tetravalent recombinant plasmid DNA in rat model and lay the foundation for the study of dengue multivalent DNA vaccine. Methods Plasmid DNA was extracted extensively using a kit that removes endotoxin. The binary plasmid DNA and their compatibility with the mouse GM2CSF after co-immunization, the immune route by intramuscular injection, and be electrically stimulated to improve plasmid DNA uptake efficiency. After the first immunization 2 and 4 weeks each boost 1 times. The levels of humoral and cellular immune responses to dengue type 1 to 4 viruses were then determined in mice. Serum antibody titers were determined by indirect immunofluorescence and neutralization assays. The level of cellular immune response was assessed by measuring the proliferation index of splenic lymphocytes and the level of IFNγ secreted by them. Cell infiltration experiments were performed by HE staining of the muscles in the immunized area. Results The mice started to produce antibodies against DEN1 ~ 4 virus at the 4th week after the first immunization, and the antibody titers gradually increased with the passage of time. At week 14, the neutralizing antibody titers reached a maximum of 1:32. The stimulation index of lymphocytes and the concentration of IFN2γ secretion were significantly higher than that of the control group. GM2CSF has a promoting effect on humoral immune response but no significant promotion on cellular immune response. Conclusion The bivalent and tetravalent recombinant plasmid DNA constructed in this study has good immunogenicity in mouse model, which lays the foundation for the study of dengue multivalent DNA vaccine.