Gadermaier Gabriele, Harrer Andrea, Girbl Tamara, Palazzo Paola, Himly Martin, Vogel Lothar, Briza Peter, Mari Adriano, Ferreira Fatima
Christian Doppler Laboratory for Allergy Diagnosis and Therapy, Department of Molecular Biology, University of Salzburg, Hellbrunnerstrasse 34, A-5020 Salzburg, Austria.
Mol Immunol. 2009 Jun;46(10):1919-24. doi: 10.1016/j.molimm.2009.03.021. Epub 2009 Apr 29.
Art v 3, the lipid-transfer protein (LTP) of Artemisia vulgaris pollen is a relevant allergen showing frequent cross-reactivity with homologues in other plants. Here we report the identification of four full-length Art v 3 sequences obtained by cDNA cloning using mass spectrometry-based sequencing. Two isoforms, Art v 3.0201 and Art v 3.0301 were expressed as soluble proteins in Escherichia coli Rosetta-gami B(DE3) pLysS using different expression systems. Purified natural and recombinant Art v 3 demonstrated similar secondary structures in circular dichroism analysis. All preparations showed high thermal stability but low resistance to gastric digestion with pepsin. Patient-specific IgE reactivity patterns to natural or recombinant isoallergens were observed among Art v 3-sensitized subjects. Using Immuno Solid-phase Allergen Chip (ISAC) assays, frequent cross-reactivity of Art v 3 with LTPs from peach and hazelnut was shown. The biological activity of both isoforms was comparable to the natural allergen in basophil release assays. The newly identified sequences provide the basis for recombinant mugwort LTP production enabling batch-to-batch reproducibility and thus ensuring high-quality products for diagnosis and therapy.
青蒿花粉的脂质转移蛋白（LTP）Art v 3是一种重要的变应原，与其他植物中的同源物常有交叉反应。在此，我们报告通过基于质谱测序的cDNA克隆获得了4个全长Art v 3序列。使用不同的表达系统，两种异构体Art v 3.0201和Art v 3.0301在大肠杆菌Rosetta-gami B(DE3) pLysS中表达为可溶性蛋白。在圆二色性分析中，纯化的天然和重组Art v 3表现出相似的二级结构。所有制剂均表现出高热稳定性，但对胃蛋白酶消化的抗性较低。在对Art v 3致敏的受试者中观察到针对天然或重组异变应原的患者特异性IgE反应模式。使用免疫固相变应原芯片（ISAC）检测，显示Art v 3与来自桃子和榛子的LTP常有交叉反应。在嗜碱性粒细胞释放试验中，两种异构体的生物活性与天然变应原相当。新鉴定的序列为重组艾蒿LTP的生产提供了基础，可以实现批次间的可重复性，从而确保用于诊断和治疗的高质量产品。
