新城疫病毒基因型差异性单克隆抗体的制备及鉴定  

Preparation and Identification of Monoclonal Antibody in Response to Different Genotypes of Newcastle Disease Virus

作　　者：任亭亭[1] 李仕超[1] 詹媛[1] 孟春春[1] 孙英杰[1] 宋翠萍[1] 谭磊[1] 封振[1] 仇旭升[1] 丁铲[1] 

机构地区：[1]中国农业科学院上海兽医研究所,上海200241

出　　处：《中国家禽》2015年第22期24-28,共5页China Poultry

基　　金：国家高技术研究发展计划(863)项目(2011AA10A209);中央级公益性科研院所基本科研业务费专项资金项目(2013JB03);公益性行业(农业)科研专项(201303033)

摘　　要：目前新城疫病毒(NDV)弱毒株的混杂对禽病实验室病原诊断工作造成很大困扰,NDV强弱毒株以及基因型的快速鉴别是亟待解决的重要问题。研究选用一株与疫苗株La Sota存在显著抗原性差异的强毒株PI/CHINA/SD/2012/167制备油乳剂疫苗免疫小鼠制备单克隆抗体。采用间接ELISA检测抗体对该强毒株及La Sota株的反应性,筛选广谱型和强弱毒株差异型的单克隆抗体,最终获得2株存在基因型差异的单克隆抗体。其中,3F10株不与ClassⅠ以及ClassⅡ基因Ⅰ型毒株反应,而与其他毒株都有很好的反应性,而7E11株不仅与ClassⅠ以及ClassⅡ基因Ⅰ型毒株反应性弱,而且与La Sota株和Mukteswar株的反应性也远远低于ClassⅠ基因Ⅵ、Ⅶ和Ⅸ型的强毒株。为了确定两株克隆抗体靶向的病毒蛋白,构建了NDV蛋白真核表达质粒转染DF1细胞,并利用Western blot和间接免疫荧光试验进行检测。结果显示,3F10针对NDV M蛋白,而7E11针对NDV F蛋白。两株单克隆抗体与NDV不同基因型毒株反应的差异性为NDV的鉴别诊断技术研究奠定了基础。There is always a big trouble for rapid identification of Newcashe disease virus （NDV） genotypes, and corresponding technology is urgently needed. A virulent NDV,PI/CHINA/SD/2012/167,which is quite different from LaSota strain in antigenicity, was immunized to BALB/C mice as oil emulsion vaccine for preparation of monoclonal antibodies （McAbs）. According to the reactivity to Pi/China/SD/2012/167 and LaSota in indirect ELISA assay,the hybridoma cell lines were screened for specificity. Finally, two hybridoma cell lines steadily secreting McAb 3F10 and 7E11 were obtained,which showed higher reactivity with virulent NDV strains. 3F10 did not reacted with non-virulent Class Ⅰ and genotype Ⅰ in Class Ⅱ strain;while 7E11 showed obvious higher reacivity with virulent NDV strains,including genotype Ⅵ, Ⅶ and IX in Class Ⅱ. Further indirect immunofluorescence assay （IFA）and Western blot assay revealed that 3F10 and 7E11 reacted with cells transfected with plasmids expressing NDV M protein and F protein respectively.In conclusion, 7Ell and 3F10 could contributed to the development of rapid diagonosis technique for NDV of different genotypes.

关 键 词：新城疫病毒 单克隆抗体 鉴别诊断 

分 类 号：S855.3[农业科学—临床兽医学]