摘要
为了制备H5N6亚型禽流感病毒(AIV)的单克隆抗体(MAb),采用纯化的H5N6 AIV免疫BALB/c鼠,利用淋巴细胞杂交瘤技术制备杂交瘤细胞,通过间接ELISA方法进行筛选,获得2株能稳定分泌抗NP蛋白的杂交瘤细胞,分别命名为3D9和2B10。间接ELISA检测2株杂交瘤细胞培养上清液抗体效价分别为1∶1×10^3和1∶1×10^4,腹水的抗体效价分别达到1∶1×10^5和1∶1×10^6; 3D9和2B10亚类鉴定重链为IgM,轻链为κ链; Western blot分析表明,2株杂交瘤细胞上清均能与不同的AIV感染的尿囊液在56 kD处发生特异性反应,并能与原核表达的重组NP蛋白发生特异性反应;间接免疫荧光试验显示,2株单克隆抗体均能与不同的AIV感染的MDCK细胞产生特异性反应。以上结果表明,本研究针对H5N6 AIV的NP蛋白制备了2株单克隆抗体,且都具有较好的特异性、保守性和广谱性,为今后禽流感诊断方法的开发奠定了基础。
This study was to prepare monoclonal antibodies (MAb)against H5N6avian influenza virus (AIV).BALB/c mice were im- munized with the purified H5N6AIV,lymphocyte hybridoma technology was used to prepare hybridoma cells,and the positive hybridoma cells were screened by indirect ELISA.The hybridoma cells secreting anti-NP protein-were obtained,and were named 3D9and 2B10,re spectively.Indirect ELISA analysis showed that the antibody titers in the supernatants of the two hybridoma cells were 1 :l×10^3and 1 :1 × 10^4,respectively,and the titers in ascites reached 1 :l×l0^5and 1 :l×l0^6,respectively.The heavy chain subtypes of 3D9 and 2B10 were IgM,with a κ light chain.Western blot analysis showed that two the MAbs supernatant could specifically react with different AIV at 56 kD, and specifically react with the recombinant NP protein of prokaryotic expression.Indirect immunofluorescence assay showed that the two MAbs could produce green fluorescence reaction with MDCK cells infected with different AIV.These results indicated that the preparation of two MAbs here were targeted at the NP protein of H5N6 AIV,and they possessed good specificity,conservativeness and broad spectrum,which lay the foundation for future development of diagnostic methods.
作者
肖倩
姚露
毕振威
雷静
颜焰
闫丽萍
XIAO Qian;YAO Lu;BI Zhenwei;LEI Jing;YAN Yan;YAN Liping(MOE Joint International Research Laboratory of Animal Health and Food Safety,Jiangsu Engineering Laboratory of Animal Immunology,Jiangsu Detection Center of Terrestrial Wildlife Disease,Institute of Immunology,College of Veterinary Medieine,Nanjing Agrieultural University,Nanjing 210095,China;MOA Key Laboratory of Animal Virology,Department of Veterinary Medieine,Zhejiang University,Hangzhou 310058,China)
出处
《畜牧与兽医》
北大核心
2018年第12期53-58,共6页
Animal Husbandry & Veterinary Medicine
基金
国家重点研发计划项目(2016YFD0501600)
中央高校基本科研业务费(Y0201600146)
关键词
H5N6
禽流感病毒
单克隆抗体
NP蛋白
特异性
保守性
H5N6
avian influenza virus
monoclonal antibodies
NP protein
specificity
conservativeness
作者简介
肖倩(1994-),女,硕士研究生;通信作者:同丽萍,副研究员,博士,主要从事重要疫病病原分子流行病学、致病机理、诊断及基因工程疫苗研究,E-mail:yanliping@njau.edu.cn。
