副猪嗜血杆菌双向电泳方法的建立和优化

A2-DE Method for Detecting Haemophilus parasuis

为建立副猪嗜血杆菌蛋白质组学双向电泳方法,从裂解液配方、样品前处理、上样量以及聚焦时间对副猪嗜血杆菌临床分离株进行双向电泳体系的优化。通过超声-裂解(7mol·L-1尿素裂解液)-离心法提取全蛋白,结合2-D clean up试剂盒纯化样品,用银染方法确定了24cm胶条上样量为500μg,一向等电聚焦电压达到60 000Vh,可以获得重复性好、分辨率高的双向电泳图谱。

This study aimed to establish an efficient 2-DE proteomic method of H.parasuis.The 2-DE reaction suitable for the determination was established and optimized through the formulation of lysis buffer,modification on sample preparation,adjustment on sampling,and time needed for isoelectric focusing determination.Two clinical isolates of H.parasuis were used inthe comparative analysis.The results showed that high-resolution,highrepeatability 2-DE pages could be obtained with 500μg protein samples extracted by the ultrasound-lysis（7mol·L-1 ultra lysis buffer）-centrifugation method and purified by a 2-D clean up kit.They were separated by isoelectric focusing on the first dimension reaching 60 000 Vh,and visualized by silver-staining.