PDF(Pubmed)
Abstract：
Common ragweed pollen allergy has become a health burden worldwide. One of the major allergens in ragweed allergy is Amb a 1, which is responsible for over 90% of the IgE response in ragweed-allergic patients. The major allergen isoform Amb a 1.01 is the most allergenic isoform in ragweed pollen. So far, no recombinant Amb a 1.01 with similar allergenic properties to its natural counterpart (nAmb a 1.01) has been produced. Hence, this study aimed to produce a recombinant Amb a 1.01 with similar properties to the natural isoform for improved ragweed allergy management. Amb a 1.01 was expressed in insect cells using a codon-optimized DNA construct with a removable N-terminal His-Tag (rAmb a 1.01). The recombinant protein was purified by affinity chromatography and physicochemically characterized. The rAmb a 1.01 was compared to nAmb a 1.01 in terms of the IgE binding (enzyme-linked immunosorbent assay (ELISA), immunoblot) and allergenic activity (mediator release assay) in well-characterized ragweed-allergic patients. The rAmb a 1.01 exhibited similar IgE reactivity to nAmb a 1.01 in different IgE-binding assays (i.e., IgE immunoblot, ELISA, quantitative ImmunoCAP inhibition measurements). Furthermore, the rAmb a 1.01 showed comparable dose-dependent allergenic activity to nAmb a 1.01 regarding basophil activation. Overall, the results showed the successful expression of an rAmb a 1.01 with comparable characteristics to the corresponding natural isoform. Our findings provide the basis for an improvement in ragweed allergy research, diagnosis, and immunotherapy.
摘要：
常见的参草花粉过敏已成为全球范围内的健康负担。斑草变态反应中的主要变应原之一是Ambα1,其负责斑草变态反应患者中超过90%的IgE应答。主要的变应原同工型Amba1.01是参草花粉中最具致敏性的同工型。到目前为止,没有产生具有与其天然对应物（nAmba1.01）相似的致敏特性的重组Amba1.01。因此，这项研究旨在生产具有与天然同工型相似特性的重组Amba1.01，以改善参草过敏管理。使用具有可去除的N-末端His-Tag(rAmbα1.01)的密码子优化的DNA构建体在昆虫细胞中表达Ambα1.01。通过亲和色谱纯化重组蛋白并进行物理化学表征。在IgE结合方面，rAmba1.01与nAmba1.01进行比较(酶联免疫吸附测定(ELISA)，免疫印迹）和过敏性活性（介体释放测定）在特征明确的参草过敏患者中。在不同的IgE结合测定中，rAmba1.01表现出与nAmba1.01相似的IgE反应性(即，IgE免疫印迹,ELISA,定量免疫CAP抑制测量)。此外，rAmba1.01在嗜碱性粒细胞活化方面显示出与nAmba1.01相当的剂量依赖性过敏活性。总的来说,结果表明rAmba1.01的成功表达，其特征与相应的天然同工型相当。我们的发现提供了改进的基础上，在斑驳变态反应的研究，诊断,和免疫疗法。
