高致病性猪繁殖与呼吸综合征病毒蛋白质组双向电泳方法的建立及优化

为建立高致病性猪繁殖与呼吸综合征病毒(HP-PRRSV)蛋白质组双向电泳方法,本研究通过对HP-PRRSV蛋白样品提取、裂解、一向等电聚程序、上样量、染色试剂、显色时间等条件优化,建立了有效的HP-PRRSV蛋白质组学双向电泳方法。优化结果表明采用冻融-超声-裂解提取样品,结合2-D clean-up试剂盒纯化蛋白,按上样量为200μg,采用硝酸银染色,显色6 min,选择适宜的一向等电聚焦参数,能获得分辨率高、重复性好的双向电泳图谱。HP-PRRSV蛋白质组双向电泳方法的建立,为开展该病毒蛋白质组和免疫蛋白质组研究奠定了基础。

Establishment and optimization of two-dimensional electrophoresis for proteome of highly pathogenic porcine reproductive and respiratory syndrome virus

To establish an efficient 2-DE protocol for proteomic method of highly pathogenic porcine reproductive and respiratory syndrome virus(PRRSV),the conditions were compared and optimized,including the sample preparation,IEF,appropriate strips,staining reagent and treatment.The results showed that the high resolving and repeatable 2-DE images were obtained with 200 μg of PRRSV protein samples which was treated consecutively with lysate buffer,frozen-thaw for four times,sonicated for 5 min,purified by 2-D Clean-up kit,and subjected to 2-DE separation under suitable electrophoresis parameters and visualized by silver-staining for 6 min.This study would facilitate future study on proteome and immunoprotein of highly pathogenic PRRSV.