Barderas Rodrigo, Villalba Mayte, Rodríguez Rosalía
Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas, Universidad Complutense de Madrid, Madrid, Spain.
Int Arch Allergy Immunol. 2004 Dec;135(4):284-92. doi: 10.1159/000082321. Epub 2004 Nov 24.
Pollinosis to chenopods is one of the main causes of allergy in desertic regions and it is increasing in the South of Europe and Western USA. Che a 1 is a major allergen for chenopod-allergic subjects and belongs to the Ole-e-1-like family of proteins.
Pichia pastoris yeast has been used as expression system to produce the recombinant form of Che a 1 (rChe a 1). The allergen was isolated using a gel permeation column and reverse-phase/high-performance liquid chromatography. Molecular characterization was performed using Edman degradation, mass spectrometry and concanavalin A staining. Sera from patients allergic to chenopod pollen, as well as polyclonal and monoclonal antibodies raised against Ole e 1, were used in immunoblotting, ELISA and inhibition assays for immunological characterization of rChe a 1.
The allergen was purified to homogeneity with a final yield of 15 mg/l of cell culture and showed a glycosylated character. N-terminal amino acid sequence of rChe a 1 and molecular mass were according to those of the protein isolated from chenopod pollen. The recombinant allergen maintained the IgG and IgE epitopes of the natural allergen deduced from the immunological assays.
Structural and in vitro immunological properties of rChe a 1 produced in P. pastoris were equivalent to those of the natural form of the allergen and, thus, it could be used in testing patients allergic to chenopods.
藜科植物花粉症是沙漠地区过敏的主要原因之一，在欧洲南部和美国西部呈上升趋势。Che a 1是藜科植物过敏患者的主要过敏原，属于Ole-e-1样蛋白家族。
利用毕赤酵母作为表达系统生产重组形式的Che a 1（rChe a 1）。使用凝胶渗透柱和反相/高效液相色谱法分离过敏原。采用埃德曼降解法、质谱法和伴刀豆球蛋白A染色法进行分子表征。来自藜科植物花粉过敏患者的血清，以及针对Ole e 1产生的多克隆和单克隆抗体，用于免疫印迹、ELISA和抑制试验，以对rChe a 1进行免疫学表征。
过敏原被纯化至同质，细胞培养的最终产量为15 mg/l，显示出糖基化特征。rChe a 1的N端氨基酸序列和分子量与从藜科植物花粉中分离的蛋白质一致。重组过敏原保留了免疫试验推断的天然过敏原的IgG和IgE表位。
在毕赤酵母中产生的rChe a 1的结构和体外免疫学特性与过敏原的天然形式相当，因此可用于检测藜科植物过敏患者。
