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当前位置: 首页 > 文章 > 应用生物素标记和蛋白质组学方法分离鉴定柞蚕微孢子虫(Nosema pernyi)的表面蛋白 蚕业科学 2015 (4) 669-676
Position: Home > Articles > Separation and Identification of Nosem a pernyi Surface Proteins by Biotin Labeling and Proteomics Methods Science of Sericulture 2015 (4) 669-676

应用生物素标记和蛋白质组学方法分离鉴定柞蚕微孢子虫(Nosema pernyi)的表面蛋白

作  者：
赵唯希;王林玲;刘泽锋;周泽扬;李治
单  位：
西南大学家蚕基因组生物学国家重点实验室;重庆师范大学生命科学学院
关键词：
柞蚕微孢子虫;表面蛋白;生物素-亲和素系统;液相色谱-二级质谱连用;间接免疫荧光定位
摘  要：
建立灵敏、特异、稳定的柞蚕微孢子虫(Nosema pernyi)表面蛋白分离鉴定技术,对研究其侵染机制和早期检测技术非常重要。采用生物素Sulfo-NHS-SS-biotin能特异性标记柞蚕微孢子虫的表面蛋白;经生物素标记后的微孢子虫用0.01 mol/L NaOH和2%SDS的混合液裂解结合超声波破碎的方法能提取到更多的微孢子虫总蛋白质;用30%乙腈和70%甲酸配合沸水浴处理可有效洗脱微孢子虫表面蛋白。对得到的柞蚕微孢子虫表面蛋白检测样品进行LC-MS/MS质谱鉴定,共获得了8个假定的表面蛋白,其中分子质量为26.6 k D(Np SWP12)、25.7 k D(Np SWP26)、32.0 kD(Np SWP30)的3个假定蛋白经间接免疫荧光分析确证为定位在孢子表面的蛋白质。序列分析显示,NpSWP12、NpSWP26、NpSWP30与家蚕微孢子虫(Nosema bombycis)孢壁蛋白NbSWP12、NbSWP26、NbSWP30的氨基酸序列相似度分别为100%、93%、79%,其中Np SWP26无N-糖基化位点和O-糖基化位点,但C端具有介导孢子粘附宿主细胞的肝素结合基序。分离鉴定的3个表面蛋白可以作为柞蚕微孢子虫侵染机制研究和早期检测的靶标蛋白。
译  名：
Separation and Identification of Nosem a pernyi Surface Proteins by Biotin Labeling and Proteomics Methods
作  者：
Zhao Weixi;Wang Linling;Liu Zefeng;Zhou Zeyang;Li Zhi;College of Life Sciences,Chongqing Normal University;Southwest University,State Key Laboratory of Silkworm Genome Biology;
关键词：
Nosema pernyi;;Surface protein;;Biotin-avidin system;;Liquid chromatography-mass spectrometry / mass spectrometry(LC-MS/ MS);;Indirect immunofluorescence localization
摘  要：
Establishing an accurate,specific and stable method for separation and identification of Nosema pernyi surface proteins is of great significance to study the infection mechanism and early detection technology of N. pernyi. In this study,the surface proteins of N. pernyi were specifically labeled with Sulfo-NHS-SS-biotin. After being lysed in 0. 01 mol / L NaOH and 2%SDS combined with ultrasonic treatment,more total proteins of N. pernyi were extracted. The surface proteins of N. pernyi could be efficiently eluted with 30% acetonitrile and 70% formic acid in a boiling water bath. A total of 8 proteins were identified from the obtained surface proteins of N. pernyi by LC-MS/MS. Among them,three hypothetical proteins with molecular mass of 26. 6 k D( Np SWP12),25. 7 kD( NpSWP26) and32. 0 kD( NpSWP30) were localized on the surface of spores by indirect immunofluorescence assay. Sequence analysis showed that the sequence similarity of NpSWP12,Np SWP26 and Np SWP30 with spore wal proteins NbSWP12,NbSWP26 and NbSWP30 from Nosema bombycis was 100%,93% and79%,respectively. Of these three hypothetical proteins,Np SWP26 is predicted to have no potential N-glycosylationand O-glycosylation sites,but have a C-terminal heparin-binding motif which is known to potentially modulate the adherence of spores to host cells. These identified three surface proteins of N. pernyi can be used as the target proteins in studying the infection mechanism and early detection of N. pernyi.

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