木麻黄花粉变应原蛋白质组分的双向电泳分析

Components of Total Proteins in Pollen of Australia Pine(Casuarina equisetifolia Linn) Revealed by Two-dimensional Electrophoresis

木麻黄(CasuarinaequisetifoliaLinn)为广东和华南沿海防护林及用材的优良树种。在最近的植物花粉过敏原调查中发现,木麻黄的花粉为中国东南沿海地区主要吸入性过敏原之一,开花季节会引起过敏人群产生哮喘、过敏性鼻炎等变态反应性疾病。目前,大约40%的过敏反应症状是由于接触特定的花粉后产生的,因此,当前有关花粉过敏原的研究都主要集中在花粉中特定致敏因子鉴定和分析上。为了解木麻黄花粉中的主要致敏因子,为今后花粉过敏反应的免疫治疗提供研究基础,本研究通过三氯乙酸(TCA)法提取木麻黄花粉总蛋白质,应用等电聚焦和第二向SDS-PAGE分析法获得完整的木麻黄花粉总蛋白图谱。结果表明:木麻黄花粉变应原中有85个不同的蛋白质组分,通过ImageMaster2D分析软件对获得的蛋白质图谱进行详细的全量蛋白质组分的量化分析,进一步确定木麻黄花粉蛋白质的等电点分布主要集中在4.5￣5.5之间,分子量分布集中在10￣30kD之间,各种蛋白质的相对含量差别很大,其中所有蛋白质种类等电点和各自所对应的分子量之间存在显著的相关性。相对国内外相关报道,本研究首次应用双向电泳技术获得木麻黄花粉高分辨率的蛋白质图谱,并通过相应软件获得详细的量化信息。本研究结果将为今后进一步确定木麻黄花粉变应原中的致敏蛋白并应用与相关临床研究奠定基础。

The pollen of Australia pine （Casuarina equisetifolia Linn）is one of the most popular aeroallergens in Guangdong and southeast of China. In recent allergen detection, pollen of Australia pine was identified as the main cause of allergenic disease in this area. The pollens can induce allergic diseases like rhinitis, asthma and hay fever in blossoming season. In recent years, research in this area has focused on the characterization of relevant plant pollen allergens because as many as approximately 40% of allergic individuals start their symptoms immediately after contacting with pollens of certain plants. In this research, the total proteins in the pollen were determined by a two-dimensional electrophoresis technique. The total proteins of the pollen were extracted by Trichloracetic acid （TCA） method, and then separated by isoelectric focusing （the first dimension） and SDS-PAGE （the second dimension）. The spots of proteins were visualized by staining with Coomassie Brilliant Blue. As the result, a total of 85 spots of proteins were observed with software ImageMaster 2D, and the majority of isoelectric point （pI） of the proteins was between 4.5-5.5, molecular weight （MW） were from 10 to 30kD and their relative volumes in the pollen were varied to a large extent. The Correlation between MW and pI of proteins is significant （analyzed by Spearman＇s）. According to the related reference, it is the first high-resolution two-dimension map of proteins in the pollen of Australia pine demonstrated in China. The information obtained in the current study will be a solid foundation for identification, characterization and gene cloning of allergenic proteins in the pollen of Australia pine in future work.