结核分枝杆菌菌体蛋白双向电泳技术的优化

目的：提取结核分枝杆菌菌体蛋白并建立一种利用双向电泳分离结核分枝杆菌蛋白质组的方法。方法：分离提取结核分枝杆菌菌体蛋白。样品采用不同pH梯度的鹏胶条进行第一向等电聚焦，12％SDS—PAGE凝胶进行二向电泳。银染后双向电泳图谱用Molecular Image Fx激光图像扫描仪扫描，PDQuest6．0软件完成配比分析。结果：优化了结核分枝杆菌菌体蛋白的提取方法，用裂解液8mol／L尿素结合2mol／L硫脲，140mmol／LDTT，0．5％biolyte，4％CHAPs，400mg／m1lOG处理，成功提取了蛋白，并通过结核分枝杆菌双向电泳技术体系的优化，建立了结核分枝杆菌菌体蛋白的分解图谱。pH4—7及pH7—10两胶面上共1387个点，占所检测到的蛋白总数的86％，绝大部分（1194个）蛋白位于pH4—7范围内。结论：为进一步开展结核分枝杆菌的比较蛋白质组学研究提供了方法学参考。

Optimizing of two dimensional chromatography technology about cytosolic protein of Mycobacterium tuberculosis

Background and Objective:To extract and develop an approach for fractionating the cytosolic proteins of Mycobacterium tu- berculosis by two dimensional polyacrylamide gel electrophoresis(2-DE).Methods:After extracting,cytosolic proteins were separated by 2- DE with different pH IPG and 12% SDS-PAGE.2-DE maps were scanned with Molecular Image Fx scanner and then compared with PDQuest 6.0 software after silve stained.Results:We successfully optimized lytic buffer and technologic systems of 2-DE for sample preparating,built 2 -DE maps of cytosolic proteins of Mycobacterium tuberculosis.There are total 1387 spots on gels of pH4-7 and pH7-10,86% of all proteins detecting on 2-DE maps,and many proteins(1194)appeared on pH4-7 gel.Conclusion:Our study laid a foundation for further studies of My- cobacterium tuberculosis cytosolic proteins with comparative proteome.