三裂叶豚草花粉致敏蛋白组分的分离、纯化及鉴定

Isolation,Purification,and Identification of Allergens of Ambrosia trifida Pollen

目的分离、纯化及鉴定三裂叶豚草花粉致敏蛋白组分。方法提取三裂叶豚草花粉粗提液,用十二烷基硫酸钠-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量。收集存在变态反应患者血清,通过免疫印迹法(Western-blotting)对花粉致敏蛋白组分进行鉴定,用离子交换层析初步纯化其致敏蛋白组分,并通过Western-blotting鉴定。结果三裂叶豚草花粉有30余条蛋白条带,其中有14条主要条带,其特异性致敏蛋白组分的相对分子质量为63 000、56 000、40 000和36 500,主要为63 000、40 000和36 500。三裂叶豚草花粉通过离子交换层析纯化出相对分子质量分别为63 000、40 000和36 500的致敏蛋白组分主要集中在Ⅳ、Ⅲ、Ⅱ、Ⅰ峰。结论初步分离、纯化及鉴定了三裂叶豚草花粉致敏蛋白组分。

Objective To characterize and purify the allergens isolated from Ambrosia trifida （giant ragweed） pollen. Methods Crude extracts of giant ragweed pollen were prepared and analyzed by SDS-PAGE and proteins bands were visualized by staining the gel with Coomassis blue. Antigenic properties of the separated proteins were analyzed by immuno-blotting. Ion-exchange chromatography with DE-52 was used for the purification of the antigens. The purified proteins were characterized by Western-blotting. Results Thirty protein bands were separated from the extract of giant ragweed pollen by SDS-PAGE. Four protein bands showed immuno-reactivity with serum slgE of patients with giant ragweed pollen allergy, whose molecular weight were 63, 56, 40, and 36.5 kDa respectively. The 63,40, and 36.5 kDa proteins were identified as the major allergen. Result of the ion-exchange chromatography indicated that the three giant ragweed pollen allergens （63, 40, and 36. 5 kDa） were eluted in the fourth, third, second and first oeak.Conclusions Allergens from giant ragweed pollen were purified and characterized.