SARS冠状病毒N蛋白单克隆抗体的制备及鉴定  

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

作　　者：王彦斌[1] 常昭瑞[1] 王健伟[1] 计融[2] 韩春卉[2] 赵丽[1] 任丽丽[1] 晁彦公[3] 屈建国[1] 曲成毅[4] 洪涛[1] 

机构地区：[1]中国疾病预防控制中心病毒病预防控制所形态室,北京100052 [2]中国疾病预防控制中心营养与食品安全所 [3]清华大学附属北京酒仙桥医院 [4]山西医科大学

出　　处：《中华实验和临床病毒学杂志》2004年第4期316-320,共5页Chinese Journal of Experimental and Clinical Virology

基　　金：国家自然科学基金课题 (3 0 3 40 0 2 5;3 0 3 40 0 2 6);国家863计划SARS专项资助(2 0 0 3AA2 0 82 0 9;2 0 0 3AA2 0 840 3 )

摘　　要：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Westernblot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。Westernblot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 。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.

关 键 词：单克隆抗体 N蛋白 SARS-COV 分泌 SARS冠状病毒 杂交瘤细胞株 特异性 抗N 识别位点 细胞融合 

分 类 号：R392[医药卫生—免疫学] R373[医药卫生—基础医学]