BHK-21细胞蛋白样品双向凝胶电泳条件的优化  

Optimization of conditions of two-dimensional gel electrophoresis for detection of BHK-21 cellular protein sample

作　　者：卢国栋[1] 张克山[1] 刘永杰[1] 尚佑军[1] 郭建宏[1] 郑海学[1] 田宏[1] 刘湘涛[1] 

机构地区：[1]中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室农业部畜禽病毒学重点开放实验室国家口蹄疫参考实验室,甘肃兰州730046

出　　处：《中国兽医科学》2011年第12期1248-1252,共5页Chinese Veterinary Science

基　　金：国家现代肉羊产业技术项目(nycybx-39)

摘　　要：分别从BHK-21细胞处理方法、样品上样量、聚丙烯酰胺凝胶浓度和染色方法4个方面优化了BHK-21细胞蛋白双向凝胶电泳的技术条件,以为口蹄疫病毒感染细胞比较蛋白质组学研究奠定基础。结果表明,处理细胞时,采用超声破碎方法优于反复冻融方法;用考马斯亮蓝染色时,7cm胶条上样量为100μg时图谱的蛋白点较多且分离清晰;浓度为10%的聚丙烯酰胺凝胶所得的蛋白点较多;通过2种染色方法的比较,硝酸银染色方法优于考马斯亮蓝染色方法。优化后的双向凝胶电泳技术得到的图谱,蛋白点多且清晰,重复性好,拖尾现象减少,符合软件分析的要求,可用于口蹄疫病毒感染BHK-21细胞蛋白质组学研究。Conditions of two-dimensional gel electrophoresis for detection of BHK-21 cellular protein sample were optimized from four aspects including cell processing,sample loading quantity,concentration of polyacrylamide gel in SDS-PAGE and staining method,to provide comparative proteomics technical foundation for foot-and-mouth virus infected cell study.Results showed that compared with the method of freezing and thawing,ultrasonic method had a better effect on cells breaking.And 100 μg sample loading quantity was appropriate for the 7 cm IPG strip of pH3-10 with coomassie brilliant blue stain,while 10% polyacrylamide gel could separate more protein points than other concentrations of polyacrylamide gels.Silver stain was superior to coomassie brilliant blue staining.After optimization of the conditions,the acquired maps,which could be used for proteomics study of BHK-21 cells infected with FMDV,possessed many advantages,such as more and clearer protein spots,good reproducibility,reduced delay phenomenon,and complied with the requirements of the software analysis.

关 键 词：BHK-21细胞 口蹄疫病毒 双向凝胶电泳 条件优化 

分 类 号：S852.659.6[农业科学—基础兽医学] Q503[农业科学—兽医学]