用protein G纯化口蹄疫病毒单克隆抗体的特性分析  

Monoclonal antibody against foot and mouth disease virus with high purity and high titer by purification through affinity column with protein G

作　　者：宋帅[1] 林彤[1] 邵军军[1] 丛国正[1] 独军政[1] 高闪电[1] 常惠芸[1] 

机构地区：[1]中国农业科学院兰州兽医研究所,国家口蹄疫参考实验室,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,兰州730046

出　　处：《中国人兽共患病学报》2009年第2期139-142,共4页Chinese Journal of Zoonoses

基　　金：国家科技支撑计划(No.2006BAD06A14)和(No.2006BAD06A10)联合资助

摘　　要：目的为了获得高效价和高纯度的O型口蹄疫病毒单克隆抗体。方法用O型口蹄疫乳鼠毒(LD50=10-8.5)感染免疫小鼠,按有限稀释法和ELISA方法筛选出阳性杂交瘤细胞株,制备单抗腹水,所收腹水用50%和45%的饱和硫酸铵沉淀,再用proteinG亲和层析柱进行亲和层析纯化单体,并用SDS-PAGE和单抗检测方法分析其纯化抗体的纯度和活性。结果经3次亚克隆获得了一株杂交瘤细胞株(10E6),抗体亚类鉴定为IgG2a;SDS-PAGE电泳显示,纯化的抗体纯度较高,只有IgG2a的重链和轻链,而没有其它杂蛋白带;单抗ELISA检测方法显示,纯化的抗体具有良好的活性,其效价为1∶102400,高于腹水效价。结论用proteinG亲和层析法获得了高纯度和高效价的单克隆抗体,将为O型口蹄疫病毒的基础研究和应用研究奠定基础。The aim of this study is to prepare a monoclonal antibody against foot and mouth disease virus （FMDV）. BALB/c mice was immunized with FMDV O type with LD50 of 10^-8.5 and the positive clones were screened by indirect ELISA and limiting dilution assay. The monoclonal antibody produced in the ascitic fluids of mice was purified with 45% and 50% saturated ammonia sulfate and affinity column with protein G and the purity and titer of the monoclonal antibody were assayed with indirect ELISA and SDS-PAGE. The result showed that one strain of hybridoma cell designated as 10E6 was obtained and its monoclonal antibody secreted was proved to be IgG2a subclass. As demonstrated by SDS-PAGE, the purity of this antibody was rather high, and only heavy and light chain of IgG2a were present without any other protein band in the SDS-PAGE analy sis. The titer of this antibody present in the ascitic fluids of mice revealed by indirect ELISA was 1：102,400. It is evident that the monoclonal antibody against FMDV with high purity and high titer can be obtained by purification through affinity column with protein G. And the monoclonal antibody would provide a basis for the basic studies and clinical use in foot-and mouth virus infection.

关 键 词：O型口蹄疫病毒 单克隆抗体 链球菌G蛋白 亲和层析 

分 类 号：S852.65[农业科学—基础兽医学]