秦川黄牛成熟朊蛋白单克隆抗体的制备及鉴定  

Preparation and characterization of monoclonal antibody against PrP^C mature protein of Qinchuan yellow cattle

作　　者：朱小玲[1] 刘永生[2] 张杰[2] 吴润[1] 陈豪泰[2] 姜海霞[1] 路伟[3] 谢庆阁[2] 

机构地区：[1]甘肃农业大学动物医学院,兰州730070 [2]中国农业科学院兰州兽医研究所,兰州730046 [3]沈阳农业大学,沈阳110161

出　　处：《中国人兽共患病学报》2006年第9期851-854,共4页Chinese Journal of Zoonoses

基　　金：甘肃省科技攻关重点项目(2GS042-A41-001-09);甘肃省自然科学基金暨中青年科技基金项目(3ZS051-A25-065);国家博士后科学基金(20040350585);中国农科院兰州兽医研究所所长基金

摘　　要：目的制备及鉴定抗秦川黄牛成熟朊蛋白的单抗。方法用重组成熟朊蛋白免疫BALB/c小鼠,取其脾细胞与SP2/0细胞融合,以间接ELISA方法筛选能够稳定分泌抗成熟朊蛋白的单抗的杂交瘤细胞株。以亲和层析法纯化腹水抗体,检测其Ig类和亚类、效价、相对亲和力以及特异性,并用基因片段表达作图法初步分析抗原表位。结果获得了能够稳定分泌抗牛成熟朊蛋白的杂交瘤细胞株N64,分泌的抗体属于IgG2a,腹水效价为1×105,相对亲和力为0.15μg/ml,Western blot表明该单抗具有较强的特异性。表位分析显示N64单抗仅与羧基端重组朊蛋白反应。结论抗牛成熟朊蛋白的腹水单抗N64效价高、亲和力和特异性强,可以作为疯牛病免疫诊断的核心试剂。To prepare and identify monoclonal antibody （mAb） against the mature form （codons 25-242） of cellular prion protein （PrPc） of Qinchuan yellow cattle, 8-week-old BALB/c female mice were immunized with purified recombinant bovine PrP, and the spleen cells of immunized mice were fused with myeloma cell line SP2/0. Then the hybridoma cell lines secreting mAb against mature PrP were screened by indirect ELISA test. The monoclonal antibody originated from ascetic fluid of hybridoma N64-inoculated mice was purified by Protein A Agarose chromatography. It was demonstrated that the mAb N64 was found to belong to IgG2a, and its titer and the relative affinity was 1 × 10^5 and 0.15 μg/ml respectively. Western blot analysis demonstrated that mAb N64 could specifically recognize bovine mature PrP, but did not react with the proteins of E. coli cells harboring pET30a（＋） vector. The epitope recognized by N64 was roughly identified through epitope mapping. Results showed mAb N64 only reacted with the carboxyl-terminated PrP. The prepared mAb N64 could be used as the reagent for the immunological diagnosis of bovine Spongiform ,encephalopathy.

关 键 词：牛海绵样脑病 成熟朊蛋白 单克隆抗体 间接ELISA 

分 类 号：R392[医药卫生—免疫学]