短穗鱼尾葵花粉总蛋白的双向电泳及分析

目的对短穗鱼尾葵花粉过敏原蛋白质组分进行双向电泳及其结果分析,构建短穗鱼尾葵花粉蛋白双向电泳图谱,明确短穗鱼尾葵花粉的过敏原蛋白组分的分布和构建蛋白质组学数据库。方法提取短穗鱼尾葵花粉的总蛋白,取上清液进行Bradford法定量总蛋白浓度,采用13 cm IPG胶条(p H 3~10)和12%的SDS-PAGE凝胶作为二项分离胶进行双向电泳(2D)分离,银染法分析其总蛋白组成,利用Image Master2D软件对2D胶进行检测和图像分析。结果双向电泳图谱共检测到516个短穗鱼尾葵花粉蛋白点,其蛋白相对分子质量在12~174 k D,主要在10~50 k D;等电点在p H 3~10,主要分布在p H 5~8。含量最多的蛋白质其相对分子质量和等电点分别为13 k D和p H 6.258 37。含量最低的蛋白质其相对分子质量和等电点分别为29 k D和p H 3.615 79。结论成功构建较为完整的短穗鱼尾葵花粉蛋白双向电泳图谱,为建立短穗鱼尾葵花粉蛋白质组学数据库及其过敏的诊断和治疗提供了依据。

Two-dimensional gel electrophoresis and analysis of total proteins from Carvota mitis pollen

Objective To carry out the two-dimensional gel electrophoresis (2D),analyze the results of total proteins from Carvota mitis pollen,structure 2D of Carvota mitis pollen protein,identify Carvota mitis pollen allergen protein distribution,and structure the proteomics database.Methods The extract of Carvota mitis pollen protein was carried on.The supernatant of Carvota mitis pollen protein was taken for quantitative detection by the Bradford method.By using 13 cm IPG strip(pH3-10)and 12％ SDS-PAGE gels as the two separation gel,the separation was performed by 2D,and the total proteins were detected with silver staining.The detection and image analysis of 2D were performed by the ImageMaster2D software.Results About 516 protein spots were visualized on the 2D map by staining.Their relative molecular weights of proteins were between 12 and 174 kD and most of them were in the range of 10-50 kD.The isoelectric points were found to lie between 3 and 10,and most of them were in the range of pH 5-8.The relative molecular weight and isoelectric point of the most content protein were 13 kD and 6.258 37 respectively.The relative molecular weight and isoelectric point of the lowest content protein were 29 kD and 3.615 79 respectively.Conclusion The 2D image pattern of Carvota mitis pollen is built up,not only which will be used as a base for the establishment of proteomics database from Carvota mitis pollen,but also which will be used for further study on Carvota mitis pollen related allergy.