双向电泳结合免疫印迹技术分析蜉蝣变应原

Analysis of Allergenic Components of Ephemeroptera with Two Dimensional Electrophoresis Followed by Immunoblot

目的用双向电泳和免疫印迹技术(Western blot)对蜉蝣提取液蛋白质组分进行分析,寻找蜉蝣主要致敏蛋白质。方法提取蜉蝣总蛋白,应用双向电泳结合免疫印迹技术,获得相应的电泳图谱和免疫印迹图谱,免疫印迹一抗为蜉蝣过敏患者血清,二抗为鼠抗人IgE抗体。结果蜉蝣蛋白质提取液经双向电泳后,考马斯亮蓝染色,电泳图谱显示约805个主要蛋白点。相对分子质量约为20 000~130 000;等电点主要集中在4.0~9.5。免疫印迹结果显示,阳性患者组与对照组的差异蛋白点数约为27个。结论双向电泳联合免疫印迹技术对蜉蝣提取液蛋白质组分分析具有实用价值,并可进一步用于变应原致敏组分的研究。

Objective To investigate the major allergens of Ephemeroptera based on the analysis of allergenic components of its protein extract by using two dimensional electrophoresis （2-DE） and im- munoblot （Western blot）. Methods Ephemeroptera protein extract was prepared by routine method. Allergenic components of Ephemeroptera were analyzed by using 2-DE and immunoblot. The Ephemer- optera allergic patients＇ serum was used as first antibody, and mouse anti-human [gE antibody as second antibody. Results About 805 soluble proteins spots were identified by using two dimensional electro- phoresis with Coomassie brilliant blue staining. The calculated molecular weight of the most identified proteins ranged from 20 000 - 130 000, and the calculated pI ranged from 4.0 - 9.5. Twenty-seven al- lergenic spots were identified using 2-DE and immunoblot compared with the control group. Conclusion 2-DE is a good method for analysis of Ephemeroptera protein components and further investigation on its allergenic components.