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目的获得含有鼠疫F1和V抗原编码基因的重组质粒pcDNATE/F1-V,并测定其诱导特异性免疫应答的能力。方法PCR扩增鼠疫菌F1和V编码基因,分别与pGEM-T连接测序,构建pcDNATE/F1-V融合重组质粒,转染COS-7细胞,用Westernblot方法鉴定目的蛋白的表达,重组质粒pcDNATE/F1-V加集落刺激因子(GM-CSF)免疫Balb/c小鼠,观察免疫效果。结果pcDNATE/F1-V在COS-7细胞中表达,免疫鼠体内产生特异性抗体,抗体亚型分析、细胞因子等指标的测定结果表明所构建DNA疫苗以诱发Th1型免疫为主。结论成功构建重组真核表达质粒pcDNATE/F1-V,其具有诱导特异性细胞免疫和体液免疫应答的能力,为鼠疫菌新型疫苗研制奠定了基础。
Objective To obtain the recombinant plasmid pcDNATE / F1-V containing the gene encoding F1 and V plague and to determine its ability to induce specific immune responses. Methods The gene encoding F1 and V of Yersinia pestis was amplified by PCR and ligated with pGEM-T respectively. The recombinant plasmid pcDNATE / F1-V was constructed and transfected into COS-7 cells. The expression of target protein was identified by Western blot. The recombinant plasmid pcDNATE / F1-V plus colony stimulating factor (GM-CSF) Balb / c mice were immunized to observe the immune effect. Results pcDNATE / F1-V was expressed in COS-7 cells, and specific antibodies were produced in immunized mice. The analysis of antibody subtypes and cytokines showed that the constructed DNA vaccine induced Th1-type immunization. Conclusion The recombinant eukaryotic expression vector pcDNATE / F1-V was constructed successfully, which has the ability of inducing specific cellular and humoral immune responses, which lays the foundation for the development of a new vaccine against Pseudomonas.