秦川黄牛成熟朊蛋白单克隆抗体的制备及鉴定

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。

Preparation and characterization of monoclonal antibody against PrPc mature protein of Qinchuan yellow cattle

To prepare and identify monoclonal antibody(mAb) against the mature form(codons 25-242) of cellular(prion protein)(PrP~(C)) 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.