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【目的】建立和优化桔小实蝇幼虫Bactrocera dorsalis(Hendel)总蛋白的双向电泳条件。【方法】使用BPP法和3种TCA-丙酮法(TCA-丙酮-A法:直接加入裂解液磨样;TCA-丙酮-B法:样品提取液中加入40 mmol/L DTT;TCA-丙酮-C法:样品提取液中加入0.07%β-巯基乙醇)提取桔小实蝇幼虫总蛋白;使用13 cm和24 cm p H 4~7的IPG胶条分离桔小实蝇幼虫总蛋白;使用考马斯亮蓝法及硝酸银染色法对双向电泳凝胶进行染色;使用5800 MALDI-TOF-TOF MS/MS质谱分析仪对BPP法获得的特异蛋白进行质谱鉴定,并将检索数据库物种分别设为Metazoa(Animals)与Drosophila(Fruit flies)进行数据库检索。【结果】TCA-丙酮法中,TCA-丙酮-C法提取效果最好,BPP法优于所有TCA-丙酮法;使用考马斯亮蓝染色与硝酸银染色效果相当;使用24 cm胶条的蛋白分辨率明显高于13 cm胶条;检索数据库物种设为Metazoa(Animals)可获得比Drosophila(Fruit flies)更加全面的信息。【结论】使用24 cm p H 4~7的IPG胶条对BPP法提取的桔小实蝇幼虫总蛋白进行双向电泳,采用考马斯亮蓝法对双向电泳凝胶进行染色,可得到更好的双向电泳图谱,检索数据库时检索物种可优先设为Metazoa(Animals)。
【Objective】 To establish and optimize the two-dimensional electrophoresis conditions of the total protein of Bactrocera dorsalis (Hendel) larvae. 【Method】 BPA method and three kinds of TCA-acetone method (TCA-acetone-A method: direct adding lysate grinding method; TCA-acetone-B method: adding 40 mmol / L DTT to the sample extract; TCA- Method C: 0.07% β-mercaptoethanol was added to the sample extract to extract the total protein of larvae of Drosophila melanogaster; the total protein of larvae of Drosophila melanogaster was isolated using IPG strips of 13 cm and 24 cm p H 4 ~ 7; MALDI-TOF-TOF-TOF MS / MS mass spectrometry was used to identify the specific proteins obtained by BPP method, and the search database was set to Metazoa ( Animals) and Drosophila (Fruit flies) for database search. 【Result】 TCA-acetone method showed the best extraction efficiency with TCA-acetone-C method, and BPP method was superior to all TCA-acetone methods. The staining with Coomassie Brilliant Blue was equivalent to that with silver nitrate staining. The rate was significantly higher than 13 cm strips; the search database species set to Metazoa (Animals) gives more comprehensive information than Drosophila (Fruit flies). 【Conclusion】 Two-dimensional gel electrophoresis was performed on the total protein of Bipolaris citrinopileatus larvae extracted by BPP using IPG strips of 24 cm p H 4 ~ 7, and the two-dimensional electrophoresis gels were stained by Coomassie brilliant blue method to obtain better bi-directional Electrophoresis patterns, retrieved species search database can be set to Metazoa (Animals).