法国梧桐花粉的部分纯化及其主要变应原免疫活性分析

Partial purification and analysis of major allergenic components of Platanus acerifoli wild pollen allergen

目的 对法国梧桐花粉变应原进行部分纯化并对其主要变应原免疫活性进行分析。方法 采用凝胶层析法分离纯化法国梧桐花粉变应原 ,经SDS 聚丙烯酰胺凝胶电泳检测收集到的主要峰段的蛋白质分子质量 ,并对 7例法国梧桐花粉过敏支气管哮喘患者血清进行免疫印迹分析。结果 法国梧桐花粉变应原粗制液经SephadexG 10 0层析柱洗脱得两个洗脱峰 ,第一峰及第二峰升段富含蛋白 ;收集各段进行SDS PAGE电泳 ,第一峰升段、峰段、降段电泳图相似 ,主要含分子质量为 2 2～ 71ku之蛋白质 ,第二峰升段以分子质量为 14～ 16ku之蛋白质为主。电泳结果清晰显示 6条蛋白区带 ,分子质量分别为 71、5 0、35、39、2 2、16ku。免疫印迹分析可见 4条sIgG反应带 ,分子质量为 5 0、39、2 2、16ku。结论 法国梧桐花粉变应原含 6种主要蛋白成分 ,第一峰蛋白质含量高 ,为主要致敏组分 ;第二峰蛋白质含量少 ,为次要致敏组分。

Objective To purify and identif y Platanus acerifoli wild pollen. Methods We carried out intracutaneous test with Platanus pollen extract in 30 patients with allergic as thma who visited our hospital from March to May 2003. Seven subjects who had bee n diagnosed as having Platanus pollen-induced asthma were enrolled. Platanus po llen proteins were separated by gel filtration with Sephadex-G-100. To charact erize allergenic components, Platanus pollen extract was analyzed by means of so dium dodecylsulfate-polyacrylamide gel electrophoresis followed by immunoblotti ng. Results To purity the pollen we separated Platanus poll en extract in a first purification step by using gel filtration with Sephadex G -100. Two elution peaks were observed. Twelve percent SDS-PAGE analysis showed more than 10 protein bands whose molecular mass (Mr) ranged from 16 ku to 71 ku. Six bands abundant with protein at 71, 50, 35, 39, 22 and 16 ku were observed. On SDS-PAGE, the proteins of the first peak whose Mr we re 71, 50, 35, 39, and 22 ku and that of the second peak was 16 ku. SDS-PAGE and IgG-immunoblotting analysis with seven sera showed 4 IgG-binding component s whose Mr was 50, 39, 22 and 16 ku. The protein bands whose Mr was 50 ku and 22 ku had the highest binding capacity. Conclusion The strongest activity exists in the first peak which can be the major sensiti zing components and there is mild allergic activity in the second peak which is the minor sensitizing components.