【摘要】 目的构建葎草花粉变应原核酸疫苗pcDNA3.1-Hum,并探讨其是否通过诱导Foxp3+Treg细胞分化介导对哮喘模型小鼠的免疫保护作用。方法双酶切pTripIEx2-Hum质粒以获取目的基因,定向插入pcDNA3.1(-)载体以构建pcDNA3.1-Hum核酸疫苗并测序鉴定,转染至COS-7细胞以证实其表达。采用表达的目的蛋白刺激CD4+CD25-T细胞,验证其能否诱导Foxp3+Treg细胞分化。使用pcDNA3.1-Hum免疫正常小鼠,检测特异性IgE、IgG2a水平以探讨其免疫原性、变应原性;免疫哮喘模型小鼠以验证其免疫保护作用。结果测序证实该构建成功并可在真核细胞内表达;证实表达的目的蛋白可诱导CD4+CD25-T细胞向Foxp3+Treg细胞分化。动物实验提示pcDNA3.1-Hum可诱导特异性IgG2a,不能诱导特异性IgE;可明显降低哮喘模型小鼠气道炎症反应、气道高反应性;升高脾脏Foxp3+Treg细胞百分比;降低IL-4、升高IFN-γ水平。结论成功构建了pcDNA3.1-Hum核酸疫苗,表达的目的蛋白可介导Foxp3+Treg细胞分化。动物实验证实该疫苗具有免疫原性及免疫保护作用,并提示其通过诱导Foxp3+Treg细胞分化介导Th1倾向的免疫保护作用。更多还原

【Abstract】 Objective To construct a humulus pollen allergy DNA vaccine pcDNA3.1-Hum and investigate its effect for immune protection mediated by Foxp3+Treg cells in asthmatic mice. Methods The target humulus gene obtained from pTripIEx2-Hum plasmid by double enzyme digestion was inserted sequentially into pcDNA3.1(-) vector to generate the recombinant plasmid pcDNA3.1-Hum, which was validated by sequencing. The pcDNA3.1-Hum plasmid was transfected into COS-7 cells and the expression of the ectopic protein was analyzed using Western blotting. Co-cultured dendritic cells and CD4+CD25-T cells were stimulated with the expressed protein to test its efficacy in inducing Foxp3+Treg cells. The levels of humulus-specific IgE and IgG2a were assayed to evaluate the allergenicity and immunogenicity of pcDNA3.1-Hum in mice. The immunoprotective effect of pcDNA3.1-Hum was assessed in a mouse model of humulus-specific asthma. Results The constructed pcDNA3.1-Hum plasmid was validated by sequencing and Western blotting, and the expressed protein was shown to induce Foxp3+Treg cells in the co-culture. In normal mice, pcDNA3.1-Hum induced a significant increase of humulus-specific IgG2a but had no effect on IgE. In the asthmatic mice, pcDNA3.1-Hum significantly decreased inflammatory cell counts and eosinophil percentages in the BALF, ameliorated lung inflammation, and lowered AHR and IL-4 levels; immunization of the mice with pcDNA3.1-Hum reversed humulus-induced reduction of serum IFN-γ and prevented the humulus-triggered reduction of Foxp3+Treg cell percentage in the spleen. Conclusion We have successfully constructed a highly immunogenic pcDNA3.1-Hum DNA vaccine that can mediate immune protection by inducing Foxp3+Treg cells.更多还原