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目的获得鼠疫F1和V抗原的重组质粒pVAX1/F1和pVAX1/V,并比较其诱导的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pGEMT连接测序,构建pVAX1/F1和pVAX1/V重组质粒,转染COS7细胞。用细胞免疫化学方法鉴定目的蛋白的表达,重组质粒加GMCSF免疫Balb/c小鼠,观察免疫效果。结果F1和V均在COS7细胞中表达;免疫鼠体内产生特异性抗体,pVAX1/V所诱导的抗体水平比pVAX1/F1高;通过抗体亚型分析、细胞因子和CD分子等指标的测定表明所构建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pVAX1/F1和pVAX1/V,并且均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 plasmids pVAX1 / F1 and pVAX1 / V of plague F1 and V antigen and to compare their ability to induce specific immune response. Methods The F1 and V coding genes of Yersinia pestis were amplified by PCR and ligated with pGEMT respectively to construct pVAX1 / F1 and pVAX1 / V recombinant plasmids for transfection into COS7 cells. Immunocytochemistry was used to identify the expression of the target protein. Balb / c mice were immunized with recombinant plasmid plus GMCSF to observe the immune effect. Results Both F1 and V were expressed in COS7 cells. The specific antibodies were induced in mice immunized with pVAX1 / V, and the level of antibody induced by pVAX1 / V was higher than that of pVAX1 / F1. The analysis of cytokines and CD molecules showed that Construct DNA vaccine to induce Th1 type immunity. Conclusion The recombinant eukaryotic expression plasmids pVAX1 / F1 and pVAX1 / V were successfully constructed and their ability of inducing specific cellular and humoral immune responses was established, which laid the foundation for the development of a new vaccine against Pseudomonas.