摘要
目的筛选识别呼吸道合胞病毒（respiratory syncytial virus，RSV）核蛋白（N）的单克隆抗体，并对其性质进行鉴定。方法使用大肠杆菌表达系统原核表达重组N蛋白，蛋白质纯化后免疫BALB/c小鼠。通过脾脏免疫、融合获得杂交瘤细胞，结合有限稀释法亚克隆细胞株，间接酶联免疫吸附试验（ELISA）对单克隆抗体株进行筛选，并通过蛋白质免疫印迹法（Western blot）、ELISA和间接免疫荧光技术对最终筛选的单克隆抗体进行性质鉴定，根据正交试验的结果挑选捕获能力和检测效果较好的抗体进行诊断试剂的初步调试。结果成功构建N-pTO-T7原核表达载体，并成功表达、纯化得到高纯度重组N蛋白用于小鼠免疫，最终筛选获得24株N蛋白的单克隆抗体，24株抗体均具有较好的ELISA反应性，5A2等8株抗体有较好的Western blot检测特性，11H8等13株抗体可用于间接免疫荧光检测，并获得能够实现在病毒上检测N蛋白的优选配对12F2/11H8-HRP和12F2/15A8-HRP。结论本研究成功筛选出可用于N蛋白特异性检测的单克隆抗体及抗体配对，为RSV感染机制的研究及诊断试剂的研发提供实验资料。
ObjectiveTo prepare and identify the monoclonal antibodies against respiratory syncytial virus nucleoprotein （RSV N protein）.MethodsA prokaryotic expression system was used to express recombinant RSV N protein in Escherichia coli （E.coli）. BALB/c mice were immunized with the recombinant N protein after purification. Monoclonal antibodies （McAbs） against the N protein were sorted from these BALB/c mice and then were further characterized by Western blot, ELISA and immunofluorescence. Furthermore, this study used orthogonal experiment to identify McAbs pairs, which could be used for diagnosis.ResultsThis study succeeded in obtaining 24 hybridoma cells that stably secreted monoclonal antibodies against RSV N protein. These antibodies showed good reactivity in ELISA, of which eight had strong specificity in Western blot and 13 could be used in immunofluorescence. This study obtained two McAbs pairs （12F2/11H8-HRP and 12F2/15A8-HRP） that could be used in RSV detection.ConclusionThis study succeeded in screening and preparation of McAbs against RSV N protein and obtaining two potential McAbs pairs for rapid detection of RSV.
作者
林雪
张伟
赵敏
郑子峥
夏宁邵
Lin Xue, Zhang Wei, Zhao Min, Zheng Zizheng, Xia Ningshao(State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, National Institute of Diagnostics and Vaccine Development in Infectious Diseases, Xiamen University, Xiamen 361102, China ; School of Public Health, Xiamen University, Xiamen 361102, China ; School of Life Science, Xiamen University, Xiamen 361102, Chin)
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2018年第3期161-168,共8页
Chinese Journal of Microbiology and Immunology
基金
国家自然科学基金（81401668）
关键词
呼吸道合胞病毒
N蛋白
单克隆抗体
Respiratory syneytial virus
Nueleoprotein
Monoelonal antibodies
