重组牛朊蛋白特异性抗体的制备及鉴定  

Characterization of monoclonal antibodies against recombinant bovine prion protein

作　　者：赵文杰[1] 杨利峰[2] 王平利[1] 赵琼[2] 梁宏德[1] 赵德明[2] 

机构地区：[1]河南农业大学牧医工程学院,河南郑州450000 [2]中国农业大学国家海绵状脑病实验室,北京100193

出　　处：《中国兽医学报》2016年第9期1531-1536,共6页Chinese Journal of Veterinary Science

摘　　要：以原核表达后经纯化的重组牛朊蛋白为免疫原,免疫prnp-/-基因敲除鼠。4次免疫后,利用淋巴细胞杂交瘤技术,取脾细胞和SP2/0骨髓瘤细胞进行细胞融合。间接ELISA方法筛选出阳性杂交瘤细胞,采用有限稀释法对阳性杂交瘤细胞进行3次克隆,用间接ELISA筛选出了稳定分泌针对牛重组朊蛋白特异性单克隆抗体的杂交瘤细胞株,命名为5C9D6。Western blotting鉴定结果表明,5C9D6均能特异性识别重组牛朊蛋白、健康牛、BALB/c脑组织匀浆中的PrPc,不识别prnp-/-基因敲除鼠脑组织匀浆液。本试验制备了可与牛、BALB/c鼠反应的单克隆抗体,同时也为牛海绵状脑病的研究及其诊断方法的建立奠定了基础。Bovine spongiform encephalopathy （BSE）, commomly called mad cow disease,is one type of transmissible spongiform encephalopathies （TSEs） and is kind of chronic, debilitating, fatal communicable disease. To develop the monoclonal antibody against BSE in this study, the prnp null mice were immunized with bovine purified recombinant prion protein. After the fourth immunizations,mouse spleen cells were fused with myelomas SP2/0 by lymphocyte hybridoma technique. The positive hybridoma were screening by indirect ELISA, and then one hybridoma cell strain which could stably excrete specific monoclonal antibodies against bovine PrP was obtained by ELISA and three times subclone,which was named 5C9D6. The 5C9D6 could identify recombi- nant bovine PrP,and PrPCfrom bovine, mouse healthy brain homogenate by Western blotting. One McAb reacting to bovine, BALB/c mouse was successfully generated. Furthermore, the monoclonal antibodies laid a foundation for the research and detection of BSE.

关 键 词：牛海绵状脑病 重组牛朊蛋白 单克隆抗体 间接ELISA WESTERN BLOTTING 

分 类 号：S852.659.7[农业科学—基础兽医学]