SARS冠状病毒N蛋白单克隆抗体的制备及鉴定

目的 制备SARS冠状病毒(SARS-CoV)N蛋白特异性单克隆抗体(McAb)，为SARS的快速诊断及致病机制的研究提供实验材料。方法 用纯化的重组SARS-CoVN蛋白免疫BALB/c小鼠，经细胞融合和亚克隆后获得分泌针对N蛋白的杂交瘤细胞株，用Western blot和间接免疫荧光法检测这些细胞株分泌的单克隆抗体特异性，并将N蛋白分3段表达初步定位单克隆抗体识别表位所在区域。结果 通过细胞融合和3轮克隆化，筛选出分泌抗N蛋白的6个杂交瘤细胞株。Western blot及免疫荧光显示，获得的McAb可与SARS-CoVN蛋白及SARS-CoV发生特异性反应，有4个细胞株分泌的抗体的识别位点位于N蛋白N端，2个位于C端。结论 获得了SARS-CoV特异性单克隆抗体并进行了初步定位，可用于SARS的早期诊断及致病机制研究。

Preparation and characterization of monoclonal antibodies against SARS-associated coronavirus nucleocapsid protein

Objective To obtain monoclonal antibodies (McAbs) against severe acute respiratory syndrome (SARS) associated coronavirus (SARS-CoV) nucleocapsid (N) protein to develop diagnostic test for SARS and study the pathogenesis of the disease. Methods BALB/c mice were immunized with purified N protein of SARS-CoV. Hybridoma cell lines secreting monoclonal antibodies against SARS-associated coronavirus nucleocapsid were established after cell fusion with mouse splenic cells and SP2/0 cells. The specificity of the McAbs obtained was examined by Western blot and indirect fluorescence assay. Epitopes reacted with the McAbs were preliminarily located through Western blot by expressing truncated N proteins. Results After cell fusion and three rounds of cell cloning, six hybridoma cell lines secreting monoclonal antibodies specifically against SARS-CoV nucleocapsid were obtained. Western blot and indirect fluorescence assay showed that the McAbs reacted specifically with nucleocapsid protein and SARS-CoV. Among the six McAbs, three recognize the epitopes located in the N-terminus of the protein, whereas the others reacted with those located in the C-terminus. Conclusion The anti-SARS-CoV nucleocapsid McAbs were developed and these McAbs may be useful in the development of diagnosis assays and basic research of SARS.