Supplementary Material for: Recombinant Fusion Proteins Assembling Der p 1 and Der p 2 Allergens from <i>Dermatophagoides pteronyssinus</i>

<i>Background:</i> Fusion proteins assembling multiple allergens can be engineered by recombinant DNA technologies in order to produce tools for diagnostic and immunotherapeutic purposes. Herein, we developed and characterized chimeras assembling Der p 1 and Der p 2 allergens as potential candidate vaccines against house dust mite allergy. <i>Methods:</i> Fusion proteins encompassing Der p 2 with either mature or proDer p 1 were expressed in <i>Escherichia coli</i> or <i>Pichia pastoris</i>. Forms with mutation in Der p 1 catalytic site were also engineered. Purified chimeras were characterized by immunoblotting, circular dichroism, disulfide bond mapping, basophil and T lymphocyte stimulation assays. <i>Results:</i> Four fusion proteins were expressed in <i>E. coli </i>as inclusion bodies, whereas only chimeras comprising proDer p 1 were obtained in yeast. All such hybrids formed polymers and aggregates, and yeast-expressed chimeras were unstable. Circular dichroism analysis performed after refolding of bacteria expressed chimeras encompassing mature Der p 1 confirmed partial folding, consistent with the occurrence of both correct and inappropriate intramolecular disulfide bonds. All fusion molecules were recognized by Der p 1- and Der p 2-specific human IgEs, monoclonal and polyclonal antibodies. Fusion proteins activate basophils from mite-allergic patients and trigger the proliferation of specific CD4+ T cells, albeit to a lower level when compared to individual allergens. <i>Conclusions:</i> Production of multiple Der p 1-Der p 2 fusion proteins exhibiting partial folding and proper antigenic properties has been achieved. Nonetheless, significant solubility and stability issues currently limit the application of such chimeras for immunotherapy or diagnostic.**背景：** 借助重组DNA技术（recombinant DNA technologies）可构建整合多种变应原的融合蛋白，以制备用于诊断及免疫治疗的工具。本研究构建并表征了整合屋尘螨主要变应原Der p 1与Der p 2的嵌合蛋白，作为针对屋尘螨过敏的潜在候选疫苗。 **方法：** 将分别携带成熟Der p 1（mature Der p 1）或前体Der p 1（proDer p 1）的Der p 2融合蛋白，在大肠杆菌（Escherichia coli）与毕赤酵母（Pichia pastoris）中表达；此外还构建了Der p 1催化位点发生突变的融合蛋白变体。通过免疫印迹（immunoblotting）、圆二色谱（circular dichroism）、二硫键定位分析（disulfide bond mapping）、嗜碱性粒细胞（basophil）与T淋巴细胞（T lymphocyte）刺激实验，对纯化后的嵌合蛋白进行表征。 **结果：** 四种融合蛋白均以包涵体（inclusion bodies）形式在大肠杆菌中表达，而仅携带前体Der p 1的嵌合蛋白可在毕赤酵母中成功获得。所有上述融合蛋白均形成聚合物与聚集体，且酵母表达的嵌合蛋白稳定性较差。对大肠杆菌表达的、携带成熟Der p 1的嵌合蛋白进行复性（refolding）后开展的圆二色谱分析证实，其存在部分折叠结构，这与正确及不恰当的分子内二硫键（intramolecular disulfide bonds）同时存在的情况相符。所有融合分子均可被Der p 1和Der p 2特异性人免疫球蛋白E（IgE）、单克隆抗体（monoclonal antibody）及多克隆抗体（polyclonal antibody）识别。融合蛋白可激活尘螨过敏患者的嗜碱性粒细胞，并触发特异性CD4阳性T细胞（CD4+ T cells）增殖，不过其活性相较于单个变应原更低。 **结论：** 本研究成功获得了具备部分折叠结构与良好抗原特性的多种Der p 1-Der p 2融合蛋白。但目前存在的显著溶解性与稳定性问题，限制了此类嵌合蛋白在免疫治疗或诊断领域的应用。