Ferreira F D, Hoffmann-Sommergruber K, Breiteneder H, Pettenburger K, Ebner C, Sommergruber W, Steiner R, Bohle B, Sperr W R, Valent P
Institut für Allgemeine und Experimentelle Pathologie, Universität Wien, Vienna, Austria.
J Biol Chem. 1993 Sep 15;268(26):19574-80.
Pollen from trees of the order Fagales (e.g. birch, alder, hazel, oak, and hornbeam) are a major cause of Type I allergies observed in early spring. Previously, we reported the cloning and sequencing of Bet v I, the major birch pollen allergen, which showed high sequence similarities to a family of plant pathogen-activated genes (Breiteneder, H., Pettenburger, K., Bito, A., Valenta, R., Kraft, D., Rumpold, H., Scheiner, O., and Breitenbach, M. (1989) EMBO J. 8, 1935-1938). Here, we present the results on the expression, purification, and characterization of recombinant Bet v I produced in Escherichia coli as fusion and non-fusion protein, respectively. The purified recombinant proteins were analyzed to verify purity and structural integrity, and their immunological properties were compared to those of Bet v I isolated from birch pollen (natural Bet v I). Immunoblot analyses showed that the recombinant proteins are specifically recognized by monoclonal antibodies raised against natural Bet v I as well as by IgE from birch pollen-allergic patients. However, enzyme-linked immunosorbent assays revealed a decreased IgE-binding activity of the recombinant fusion Bet v I compared to the non-fusion and natural Bet v I proteins, which probably results from conformational changes due to the fusion tail. Recombinant non-fusion Bet v I was equivalent to natural Bet v I with respect to IgE-binding properties, the ability to induce in vitro proliferation of allergen-specific T-cell clones, and the ability to release histamine from basophils derived from birch pollen-allergic patients.
壳斗目树木（如桦树、桤木、榛树、橡树和鹅耳枥）的花粉是早春时节I型过敏的主要诱因。此前，我们报道了主要桦树花粉过敏原Bet v I的克隆和测序，它与一类植物病原体激活基因显示出高度的序列相似性（Breiteneder, H., Pettenburger, K., Bito, A., Valenta, R., Kraft, D., Rumpold, H., Scheiner, O., and Breitenbach, M. (1989) EMBO J. 8, 1935 - 1938）。在此，我们分别展示了在大肠杆菌中作为融合蛋白和非融合蛋白产生的重组Bet v I的表达、纯化及特性分析结果。对纯化后的重组蛋白进行分析以验证其纯度和结构完整性，并将它们的免疫学特性与从桦树花粉中分离出的Bet v I（天然Bet v I）进行比较。免疫印迹分析表明，重组蛋白能被针对天然Bet v I产生的单克隆抗体以及桦树花粉过敏患者的IgE特异性识别。然而，酶联免疫吸附测定显示，与非融合和天然Bet v I蛋白相比，重组融合Bet v I的IgE结合活性有所降低，这可能是由于融合尾导致的构象变化所致。重组非融合Bet v I在IgE结合特性、诱导过敏原特异性T细胞克隆体外增殖的能力以及从桦树花粉过敏患者的嗜碱性粒细胞中释放组胺的能力方面与天然Bet v I相当。
