白花鬼针草花粉过敏原的分离鉴定与纯化

Isolation,Identification and Purification of Allergens in Bidens Alba Pollen

目的提取和分离纯化白花鬼针草花粉主要过敏原,并鉴定主要过敏原的致敏性。方法提取白花鬼针草花粉蛋白粗提液,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分离粗提液蛋白组分并测定其分子质量,经离子交换层析法分离纯化几类主要的蛋白,Western blotting分别检测其与花粉过敏患者血清IgE结合情况及10位正常人阴性混合血清结合情况,对比鉴定每种主要蛋白质致敏原的致敏性。结果 SDS-PAGE结果示：在12.5-120ku之间广泛分布了20余条蛋白条带,其中主带7条,分子质量在35-70ku和10-15ku的区带蛋白含量最为丰富,余下的范围内还有约10余条次带。Westen-blotting检测示：白花鬼针草花粉变应原的致敏性强,与花粉过敏患者血清特异性IgE结合大。离子交换层析出6类主要变应原蛋白,其分子质量分别为70、65、54、49、39及33ku;阴性对照组Westen-blotting在70ku处有弱显色。结论白花鬼针草花粉的主要过敏原为70、65、54、49、39及33ku;70ku变应原致敏性最强。

Objective To isolate and purify the allergens from Bidens alba pollen,and to identify the allergenicity of major allergens.Methods Pollen extracts from Bidens alba were prepared and analyzed by SDS-PAGE.The antigens were purified by ion-exchange chromatography.The binding of allergens to serum IgE was detected and the allergenicity of major allergens was identified in patients with pollen allergy and 10normal controls.Results SDS-PAGE analysis showed more than 20protein bands at 12.5-120ku,including 7major bands.The 35-70ku and 10-15ku proteins were the most abundant proteins.Western blotting showed a strong allergenicity of Bidens alba pollen allergens with a extraordinary binding to serum IgE in patients with pollen allergy.Ion-exchange chromatography indicated that the 70,65,54,49,39and 33kDu proteins were the major allergens.In normal controls,Western blotting showed a weak immunoreaction at 70kDu.ConclusionThe 70,65,54,49,39and 33ku proteins are the major allergens and the 70ku protein is the strongest allergen in Bidens alba pollen.