麇鹿重组朊蛋白单克隆抗体的制备及初步鉴定

摘要

为研制鹿慢性消耗性疾病(chronic wasting disease,CWD)单克隆抗体,本研究以原核表达后经纯化的麋鹿重组成熟朊蛋白(PrPc)为免疫原免疫PrP基因敲除小鼠(PrPc-null mice).两次加强免疫后,利用淋巴细胞杂交瘤技术,取脾细胞与SP2/0骨髓瘤细胞进行细胞融合,间接ELISA方法筛选阳性杂交瘤细胞,采用3次有限稀释法实现杂交瘤细胞的亚克隆,筛选出5株能稳定分泌针对麋鹿重组成熟朊蛋白特异性单克隆杭体(McAbs)的杂交瘤细胞株5A5、3B2、6D12、5E3、1F5,其腹水ELISA效价均达到1:10000以上.经鉴定,5株单抗均为IgG抗体,5A5、6D12、5E3株为IgG1亚类,3B2、1F5株为IgG2a亚类.Western blotting鉴定结果表明,获得的McAbs均能特异性识别糜鹿重组成熟朊蛋白和健康麋鹿脑组织匀浆中的PrPc.本研究制备了CWD腹水McAbs,同时也为CWD的研究及其诊断方法的建立奠定了基础.%To develop the monoclonal antibody against chronic wasting disease, in this study, the PrP￡-null mice were immunized with elk purified recombinant prion protein(PrPC). After twice booster immunizations, mouse spleen cells were fused with myelomas SP2/0 by lymphocyte hybridoma technique. Five hybridoma cell strains which could stably excrete specific monoclonal antibodies against elk PrPC were obtained by ELISA and three times subclone. The five hybridoma cell strains were named 5A5.3B2, 6D12, 5E3 and 1F5 respectively, whose ELISA titer were all more than 1 ' 10000. By evaluating, three strains belonged to IgG1 subset and other two were IgG2a subset. These McAbs could identify both elk recombinant PrP and PrPC from elk healthy brain homogenate by Western blotting. This study filled the void of monoclonal antibody against CWD in China. Furthermore, the monoclonal antibodies laid a foundation for the research and detection of CWD.