一种适用于家蚕微孢子虫膜蛋白提取的方法  

A Method Suitable for Extracting Membrane Protein from Nosemabombycis

作　　者：马强[1] 李田[1] 党晓群[2] 潘国庆[1] 许金山[2] 周泽扬[1,2] 

机构地区：[1]家蚕基因组生物学国家重点实验室,西南大学,重庆400716 [2]重庆师范大学动物生物学重点实验室,重庆400047

出　　处：《蚕业科学》2013年第2期319-323,共5页ACTA SERICOLOGICA SINICA

基　　金：重庆市自然科学基金项目(No.CSTC;2010BB1139);国家自然科学基金重点项目(No.30930067/C120110);国家自然科学基金青年基金项目(No.31001036/C1703);国家自然科学基金项目(No.31072089/C1703);中央高校基本科研业务费专项资金项目(No.XDJK2009C148);教育部科学技术重点项目(No.210180)

摘　　要：细胞膜蛋白在家蚕微孢子虫对宿主细胞的抵抗性、物质能量转运和信息传递过程中发挥着重要作用,也是家蚕微粒子病诊断防控的重要候选靶标。依次采用试剂盒直接抽提法、破碎抽提同步法、破碎抽提异步法,对家蚕微孢子虫膜蛋白提取过程中的抽提时间和孢壁处理方式进行优化,通过对提取蛋白质样品的质量检测,明确最佳提取方法。SDS-PAGE检测结果表明,采用破碎抽提异步法在Buffer 2B抽提40 min时能获得较高丰度的膜蛋白,与孢子总蛋白质相比,所得膜蛋白样品中含有丰富的大小为30～170 kD的差异蛋白,进一步利用已知家蚕微孢子虫膜蛋白——类枯草杆菌蛋白酶2的抗体(anti-Nb-SLP2)进行Western blotting分析发现,膜蛋白样品中含有丰富的NbSLP2,表明破碎抽提异步法适用于孢子膜蛋白的提取。此方法的建立将有助于对家蚕微孢子虫膜蛋白的鉴定和功能研究及检测防控候选膜蛋白靶标的筛选。Cellular membrane proteins play important roles in resistance against host cells,substance and energy transportation,and signal transmission in Nosema bombycis.They are also the important candidate targets for diagnosis and control of silkworm(Bombyx mori) pebrine disease.By using the methods of direct extraction with agent kit,synchronous breaking and extracting,and asynchronous breaking and extracting,extraction time and spore wall treatment condition were optimized to establish an optimal procedure for N.bombycis membrane protein extraction.Through quality inspection to the extracted protein samples,an optimum extraction method was obtained.SDS-PAGE detection revealed that membrane proteins with higher abundance could be obtained after extraction with buffer 2B for 40 min by using the method of asynchronous breaking and extracting.Compared with total protein of spores,the extracted membrane protein sample contained abundant differential proteins of 30～170 kD.Abundant subtilisin-like proteinase 2(NbSLP2),which is a known membrane protein of N.bombycis,was detected in the extracted membrane proteins by Western blotting analysis using the antibody of NbSLP2(anti-NbSLP2),indicating that asynchronous breaking and extracting method is applicable to membrane protein extraction from N.bombycis spores.The establishment of this method is conducive to identification,functional study and candidate target screening of N.bombycis membrane proteins for diagnosis and control of silkworm pebrine disease.

关 键 词：家蚕微孢子虫 膜蛋白 破碎抽提异步法 SDS-PAGE检测 WESTERN blotting分析 

分 类 号：S884[农业科学—特种经济动物饲养]