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目的:一种适用于双向电泳体系的松材线虫全蛋白提取方法的建立及其双向电泳体系的优化。方法:以松材线虫为实验材料,比较2种不同的蛋白提取方法,并对双向电泳中的IPG胶条长度、IPG胶条最适pH范围、上样量等3个方面的条件进行优化。结果:采用TCA-丙酮法提取的蛋白质浓度较高,达到2.18μg/μl。使用pH 5～8、24cm的IPG干胶条,上样量为120μg,经双向电泳分离可得到背景清晰、分辨率较高的2-DE图谱,能检测到2 000个左右清晰的蛋白点,含有相对丰富的蛋白信息量。结论:该实验所建立的松材线虫提取方法和优化体系可以为今后松材线虫蛋白质组学的研究奠定技术基础。
OBJECTIVE: To establish a method for extraction of whole protein of Bursaphelenchus xylophilus which is suitable for two-dimensional electrophoresis system and to optimize its two-dimensional electrophoresis system. Methods: Pine wood nematode was used as experimental material to compare two different protein extraction methods. The conditions of IPG strip length, IPG strip pH range and sample loading capacity in two-dimensional electrophoresis were optimized. Results: The protein extracted by TCA-acetone method had a high concentration of 2.18μg / μl. Using IPG strips with pH 5 ~ 8 and 24cm, the sample volume was 120μg. The 2-DE pattern with clear background and high resolution could be obtained by two-dimensional electrophoresis. About 2 000 clear protein spots could be detected, Contains a relatively rich amount of protein information. Conclusion: The extraction method and optimization system of pine wood nematode established in this experiment can lay the technical foundation for the future research on the pine wood nematode proteomics.