艾蒿花粉主要变应原的分离、纯化与鉴定

Purification and identification of major allergens in Artemisia argyi pollen

目的 对我国蒿属花粉中常见、重要的变应原艾蒿花粉进行分离、鉴定与纯化。方法采用不同的提取液得到艾蒿花粉粗浸液 ,经饱和 (NH4 ) 2 SO4 分级沉淀后用聚丙烯酰胺凝胶电泳 (SDS PAGE)分离蛋白质组分 ,并用凝胶成像系统测定各组分的相对分子质量 (Mr) ;采用Westernblot鉴定其主要及次要变应原 ;通过DEAE CelluloseDE 32离子交换层析 (ionexchangechromatography ,IEC)和SephadexG 75凝胶层析 (gelchromatography)对艾蒿花粉变应原进行纯化。结果 分离后得到 2 0多种蛋白质组分 ,其中Mr 为 5 8× 1 0 3、38× 1 0 3、2 5× 1 0 3、2 0× 1 0 3、1 6× 1 0 3等 5个条带蛋白含量最丰富 ;分离到的蛋白质组分中有 9种蛋白能与确诊的蒿属花粉过敏患者血清中蒿属花粉特异性IgE结合 ,其中Mr 为 6 2× 1 0 3、4 3× 1 0 3、38× 1 0 3的蛋白条带的结合率最高 ;经纯化后仅得到Mr 为 6 2× 1 0 3的主要变应原。结论 艾蒿花粉的主要变应原Mr 分别为 6 2× 1 0 3、4 3× 1 0 3和 38× 1 0 3,层析技术可以对Mr 为6 2× 1 0 3的主要变应原成分进行纯化。

Objective To purify and characterize the Artemisia argyi pollen, the mostly widespread and important pollen among the Artemisia pollens in China. Methods Artemisia argyi pollen extract was precipitated by saturated ammonium sulfate and then electrophoresed by SDS-PAGE. The molecular mass of each protein band was determined by gel media system. We identified the primary and secondary allergen proteins by Western blot using the sera from Artemisia pollen allergic patients as probe. Allergen proteins were purified by DEAE-cellulose DE-32 ion exchange chromatography (IEC) and Sephadex G-75 gel chromatography furthermore. Results We identified more than twenty protein bands from Artemisia argyi pollen extract, including the most abundant five bands whose molecular mass (M r) are 58×103, 38×103, 25×103, 20×103, 16×103 respectively. The results showed that 9 of the protein bands can be combined with the specific IgE from sera of Artemisia pollen allergic patients. The protein bands with M r are 62×103, 43×103, 38×103 have the highest binding capacity. When using the Sephadex G-75 Gel Chromatography to purify the proteins, only one of the primary allergen proteins was successfully isolated with M r as 62×103. Conclusion The primary allergens of Artemisia argyi include the allergen proteins whose M r are 62×103, 43×103 and 38×103, and the first one can be purified by Chromatography.