摘要
目的改进从兔胸腺中纯化能用于斑点印迹法检测抗-Jo-1抗体、Jo-1抗原的方法。方法用PBS提取兔胸腺匀浆中的ENA,经丙酮沉淀上清中的总蛋白制成兔胸腺丙酮粉,再经以特异性抗-Jo-1抗血清中的IgG为配体制备的亲和色谱柱对Jo-1抗原纯化,得到高比活的制品。结果100g兔胸腺可制得5～7g丙酮粉,其中的蛋白质含量为19%～24%。经亲和色谱分离的洗脱峰组分约可富集Jo-1抗原1900倍,抗原活性回收率为2.5%,并只对抗-Jo-1抗血清有特异性反应。虽然在SDS-PAGE上洗脱峰组分并非单一条带,但免疫印迹只发现50ku处有阳性条带。在含有MgCl2的水溶液中,高纯度的Jo-1抗原能长时间保持高活性。结论亲和色谱法可从兔胸腺中纯化出满足斑点印迹法检测抗-Jo-1抗体要求的抗原,并有可能成为大量获取高纯度Jo-1抗原的方法。
Objective To improve the purifying method of Jo-1 antigen from rabbit thymus used for detection of anti-Jo-1 antibody by dot-blotting immunoassay （DB）. Methods The rabbit thymus glands were cut into pieces, homogenized and extracted by PBS. Total protein was precipitated by acetone to get acetone powder （RTAP）. The RTAP was solved in PBS and separated by an by anti-Jo-1 IgG affinity column. Results 5 - 7 g RTAP was obtained from 100g rabbit thymus glands. There was 19% - 24% of protein in RTAP. Jo-1 antigen was enriched around 1900 folds through affinity chromatography, with 2.5% recovery of antigenic activity. In this preparation, there were several bands on SDS-PAGE, but only one band about 50 ku, reacted with anti-Jo-1 antisera on immunoblotting. Dot-blotting also showed that the antigen only reacted with Jo-1 antisera. The purified Jo-1 antigen was not stable for long time, but the antigenic activity could maintain for a long time when there was MgCl2 in the solution. Conclusion Affinity chromatography was a simple and easy method for purifying Jo-1 antigen from rabbit thymus. The antigen purified by affinity chromatography could meet the requirement for detecting Jo-1 antibody by dot-blotting.
出处
《基础医学与临床》
CSCD
北大核心
2007年第10期1146-1150,共5页
Basic and Clinical Medicine
基金
国家科技攻关计划(2002-BA711A11)
