发芽法与玻璃珠破碎法提取家蚕微孢子虫孢壳蛋白的双向电泳分析  

Two-dimensional Electrophoresis Analysis of Nosema bombycis Spore Coat Proteins Prepared by Germination Method and Glass Beads Breaking Method

作　　者：耿莉娜[1,2] 吴海晶[1] 刘婷[1] 田锐[1] 李田[1] 潘国庆[1] 周泽扬[1,3] 

机构地区：[1]西南大学,家蚕基因组生物学国家重点实验室,重庆400716 [2]重庆烟草科学研究所,重庆400715 [3]重庆师范大学,重庆400047

出　　处：《蚕业科学》2013年第5期928-934,共7页ACTA SERICOLOGICA SINICA

基　　金：国家自然科学基金重点项目(No.30930067);"十二五"农村领域国家科技计划课题(No.2012AA101301-3);重庆市蚕桑重大专项(No.CSTC2010AA1003)

摘　　要：采用不同方法获得家蚕微孢子虫孢壳,并提取孢壳蛋白进行双向电泳分析,确定适用于蛋白质组学研究的家蚕微孢子虫孢壳蛋白提取方法。通过密度梯度离心纯化家蚕微孢子虫孢子后,分别用发芽法和玻璃珠破碎法得到孢壳,再经密度梯度离心纯化孢壳。显微镜观察可见用发芽法得到的孢壳明显膨大趋于圆形,而用玻璃珠破碎法得到的孢壳形状变化不大,但有明显的缺口。使用蛋白质裂解液提取2种方法获取孢壳的蛋白质并进行双向电泳,在发芽法提取孢壳蛋白的双向电泳图谱中检测到205个蛋白点,在破碎法提取孢壳蛋白的双向电泳图谱中检测到413个蛋白点。采用发芽法提取过程中由于使用了碱性溶液,使孢壳表面的碱溶性蛋白脱落,导致提取的孢壳蛋白丰度降低;而玻璃珠破碎法避免了碱溶过程,提取的孢壳蛋白丰度较高,并且操作简单,有利于完整获取孢壳碱溶性蛋白和与内壁结合的蛋白。In order to establish a method of spore coat protein extraction for proteomic analysis, we prepared spore coats of Nosema bombycis with different methods and extracted their proteins for two-dimensional electrophoresis （2- DE）. After Nosema bombycis spores were purified by density gradient centrifugation method, spore coats were obtained by germination method and glass beads breaking method respectively. And then the spore coats were further purified by density gradient centrifugation method. Microscopic observation displayed that the spore coats from germination method swelled obviously and assumed round shape, while those from glass beads breaking method had little change in shape but had obvious gaps. The spore coat proteins were extracted with proteolysis solution and separated by 2-DE. As a re-suit, 205 protein spots were detected on 2-DE map of spore coat proteins extracted by germination method and 413 protein spots were detected on 2-DE map of spore coat proteins extracted by glass beads breaking method. Because an alkaline solution was used in the germination method, alkali-soluble proteins were lost from the surface of spore coats which led to the decrease of spore coat protein abundance. Glass beads breaking method avoi-ded alkaline dissolution process and thus had higher spore coat protein abundance. Nevertheless, the latter was easy to operate, being favorable for preparing alkali-soluble proteins and endospore-bound proteins completely.

关 键 词：家蚕微孢子虫 孢壳蛋白 蛋白质组 发芽法 玻璃珠破碎法 双向电泳 

分 类 号：S884[农业科学—特种经济动物饲养]