桔小实蝇幼虫总蛋白双向电泳技术体系的建立和优化
Establishment and optimization of a two-dimensional gel electrophoresis system for Bactrocera dorsalis (Hendel)larvae crude protein
白蓉蓉1, 2** 金 涛2 金启安2 林玉英2 温海波2 彭正强2***
点击：1602次 下载：21次
DOI：10.7679/j.issn.2095-1353.2015.060
作者单位：1. 海南大学环境与植物保护学院，海口 570228；2. 中国热带农业科学院环境与植物保护研究所，农业部热带农林有害生物入侵监测与控制重点开放实验室，海口 571101
中文关键词： 桔小实蝇，蛋白质提取方法，双向电泳
英文关键词：Bactrocera dorsalis (Hendel), crude protein extraction methods, two-dimensional gel electrophoresis
中文摘要：
【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 pH 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 pH 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)。
英文摘要： [Objectives] To establish and optimize 2-DE profiles of crude proteins from the 3th instar larvae of Bactrocera dorsalis (Hendel). [Methods] The effectiveness of the BPP protocol and three TCA/acetone precipitation procedures (TCA/acetone A: ground sample with lysis buffer directly, TCA/acetone B: extraction buffer of 10% TCA in acetone containing 40 mmol/LDTT, TCA/acetone C; extraction buffer of 10% TCA in acetone containing 0.07% β-mercaptoethanol), were assessed with respect to crude protein extraction from 3th instar larvae of Bactrocera dorsalis (Hendel). Linear gradient 13 and 24 cm IPG strips with pH 4-7 were used to separate the crude protein. The separation gels were visualized by coomassie brilliant blue and silver staining, respectively. Mass spectra of the specific proteins obtained by the BPP method were read on a 5800 MALDI-TOF-TOF MS/MS mass spectrometer and the database of Metazoa (Animals) and Drosophila (Fruit flies) was indexed for matches. [Results] The TCA/acetone C precipitation procedure produced the best resolved protein spots among the three TCA/acetone methods, but the BPP method gave the best distinction of proteins on 2-DE gels. Coomassie brilliant blue staining and silver staining showed similar resolutions. The average protein yield of 24 cm strips was higher than that of 13 cm strips, lots of small proteins were enriched in 24 cm strips. The Metazoa database provided more comprehensive information than that of Drosophila (Fruit flies). [Conclusion] The BPP protocol revealed the most protein spots with linear gradient 24 cm IPG strips, pH 4-7. Coomassie brilliant blue was both sensitive and compatible with MS. Database retrieval should give priority to the Metazoa.