CAJ | 학술논문

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。
목적：구건률초화분변응원핵산역묘pcDNA3.1-Hum，병탐토기시부통과유도Foxp3+Treg세포분화개도대효천모형소서적면역보호작용。방법쌍매절pTripIEx2-Hum질립이획취목적기인，정향삽입pcDNA3.1(-)재체이구건pcDNA3.1-Hum핵산역묘병측서감정，전염지COS-7세포이증실기표체。채용표체적목적단백자격CD4+CD25-T세포，험증기능부유도Foxp3+Treg세포분화。사용pcDNA3.1-Hum면역정상소서，검측특이성IgE、IgG2a수평이탐토기면역원성、변응원성；면역효천모형소서이험증기면역보호작용。결과측서증실해구건성공병가재진핵세포내표체；증실표체적목적단백가유도CD4+CD25-T세포향Foxp3+Treg세포분화。동물실험제시pcDNA3.1-Hum가유도특이성IgG2a，불능유도특이성IgE；가명현강저효천모형소서기도염증반응、기도고반응성；승고비장Foxp3+Treg세포백분비；강저IL-4、승고IFN-γ수평。결론성공구건료pcDNA3.1-Hum핵산역묘，표체적목적단백가개도Foxp3+Treg세포분화。동물실험증실해역묘구유면역원성급면역보호작용，병제시기통과유도Foxp3+Treg세포분화개도Th1경향적면역보호작용。
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.