二维凝胶电泳分离结核分枝杆菌胞外蛋白和膜蛋白  

Separation of extracellular and membrane proteins in mycobacterium tuberculosis using two-dimensional gels electrophoresis

作　　者：何秀云[1] 庄玉辉[1] 张小刚[1] 李国利[1] 丁勤学[2] 

机构地区：[1]解放军第三○九医院结核病研究室,北京100091 [2]军事医学科学院基础医学研究所神经生物学研究室,北京100850

出　　处：《医学研究生学报》2002年第2期102-104,共3页Journal of Medical Postgraduates

基　　金："973"资助课题 (No .G19990 5 410 4) ;解放军第三○九医院基金资助课题 ( 2 0 0 1~ 2 0 0 3)

摘　　要：目的 :建立二维凝胶电泳分离结核分枝杆菌 (M .TB)胞外蛋白和膜蛋白的方法。　方法 :M .TBH37Rv菌株在苏通液体培养基培养 3周后获得胞外蛋白和膜蛋白 ,第一相电泳用 pH 3～ 10线性IPG预制胶条进行等电聚焦 ,第二相电泳用SDS PAGE凝胶再分离蛋白。银染PAGE凝胶、干燥 ,用ImageScanner扫描凝胶 ,ImageMaster 2DElite 3 .10 (图像分析软件 )分析 2D图像。　结果 :胞外蛋白和膜蛋白的蛋白质斑点数分别为 90 7和 681,两组蛋白的斑点大部分在酸性区域 (pH <7.0 )。在极碱性区域 ( pH >9.0 ) ,胞外蛋白只有 10个蛋白质斑点 (占1.1% ) ,而膜蛋白有 5 2个蛋白质斑点 (占 7.6% ) ;在极酸性区域 ( pH <4 .0 ) ,膜蛋白只有 15个蛋白质斑点 (占2 .2 % ) ,而胞外蛋白有 99个蛋白质斑点 (占 10 9% )。约 5 0 %的胞外蛋白和膜蛋白相对分子质量 <3 0 0 0 0。　结论 :二维凝胶电泳分离M .TB蛋白是研究M .TB蛋白质组学的重要途径之一。Objectives:To establish a method for separating extracellular proteins and membrane proteins in mycobacterium tuberculosis using two dimensional gel electrophoresis. Methods:Extracellular proteins and membrane proteins were extracted from H 37 Rv which were inoculated and cultured on Suton's medium for three weeks. The first dimensional electrophoresis was performed on immunobilized pH gradient rod gels (pH 3～10).Then proteins in the rod gels were separated using SDS PAGE gels. The silver stained gels were dried and scanned. The image manipulation was performed with Image Master 20 Elite 3.10. Results:Extracellular proteins and membrane proteins contained 907 and 681 protein spots, respectively, and most protein spots deriving from extracellular proteins and membrane proteins were in acidic range. In the basic range (pH more than 9.0),the number of protein spots belonging to extracellular proteins was only 10(1.1%),however, that of protein spots belonging to membrane proteins was 52(7.6%).In the acidic range (pH less than 4.0),the number of protein spots belonging to membrane proteins was only 15(2.2%),while that of protein spots belonging to extracellular proteins was 99( 10.9 %).The relative molecular weights of about 50% extracellular proteins and membrane proteins were less than 30 000. Conclusions:Two dimensional gel electrophoresis is useful to separate proteins in mycobacterium tuberculosis.

关 键 词：结核分枝杆菌 二维凝胶电泳 胞外蛋白 膜蛋白 

分 类 号：R378.911[医药卫生—病原生物学]