Immunobiochemistry Lab, Immunology Research Center, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.
Immunol Lett. 2012 May 30;144(1-2):67-77. doi: 10.1016/j.imlet.2012.03.008. Epub 2012 Apr 4.
Allergen specific immunotherapy is the only remedy to prevent the progression of allergic diseases. Nowadays, using of recombinant allergens with reduced IgE-binding capacity is an ideal tool for allergen immunotherapy. Therefore, in this study we focused on a hybrid molecule (HM) production with reduced IgE reactivity from Chenopodium album pollen allergens. By means of genetic engineering, a head to tail structure of the three allergens of the C. album pollen was designed. The resulting DNA construct coding for a 46kDa HM was inserted into an expression vector and expressed as hexahistidine tagged fusion protein in Escherichia coli. IgE reactivity of the HM was evaluated by western blotting, inhibition ELISA and in vivo skin prick test and its immunogenic property was tested by proliferation assay. The recombinant HM was expressed and purified by nickel-affinity chromatography. Comparison of the recombinant HM with a mixture of three recombinant allergens, as well as natural allergens in the whole C. album pollen extract via immunological experiments revealed that it has a much lower potential of IgE reactivity. Furthermore, in vivo skin prick tests showed that it has a significantly lower potency to induce cutaneous reactions than the mixture of recombinant wild type allergens and whole extract while, it had been preserved immunogenic properties. Our results have demonstrated that assembling three allergens of C. album in a hybrid molecule can reduce its IgE reactivity.
变应原特异性免疫疗法是预防过敏疾病进展的唯一方法。目前，使用具有较低 IgE 结合能力的重组变应原是变应原免疫治疗的理想工具。因此，在这项研究中，我们专注于从藜花粉过敏原中生产具有较低 IgE 反应性的杂合分子（HM）。通过基因工程，设计了藜花粉三种过敏原的头尾结构。编码 46kDa HM 的所得 DNA 构建体被插入表达载体，并在大肠杆菌中表达为六组氨酸标记的融合蛋白。通过 Western blot、抑制 ELISA 和体内皮肤点刺试验评估 HM 的 IgE 反应性，并通过增殖试验测试其免疫原性。重组 HM 通过镍亲和层析进行表达和纯化。通过免疫学实验，将重组 HM 与三种重组过敏原的混合物以及整个藜花粉提取物中的天然过敏原进行比较，结果表明其具有较低的 IgE 反应性。此外，体内皮肤点刺试验表明，与重组野生型过敏原混合物和全提取物相比，它诱导皮肤反应的效力显著降低，而保留了免疫原性。我们的结果表明，将藜花粉中的三种过敏原组装成杂合分子可以降低其 IgE 反应性。
