【作者】 刘文丽； 刘怀然； 刘培欣； 孔宪刚； 陈维多；

【Author】 Wen-li LIU~(1,2) Huai-ran LIU~1 Pei-xin LIU~1 Xian-gang KONG~(1*) Wei-duo CHEN~2 (1.Division of Avian Infectious Diseases,State key Laboratory of Veterinary Biotechnology,Harbin Veterinary Research Institute,CAAS,Harbin 150001,China;2.College of Life Science North-east Agricultural University,Harbin 150030,China)

【机构】 中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室禽传染病研究室； 东北农业大学生命科学学院；

【摘要】 【目的】制备针对新城疫病毒F48E9株单克隆抗体,以用于NDV抗原变异研究及检测方法建立。【方法】用新城疫病毒F48E9株全病毒及原核表达的重组F48E9-NP蛋白免疫BALB/e小鼠,全病毒间接ELISA筛选杂交瘤细胞株。用免疫印迹和间接免疫荧光试验鉴定单抗反应原性,间接ELISA试验鉴定单抗与其他NDV毒株的交叉反应,并采用NP蛋白分段融合表达技术对单抗识别区域初步定位。【结果】共筛选到3株单抗杂交瘤细胞株3E7、2D11和4B12,免疫印迹和间接免疫荧光试验分析表明,3株单抗均针对病毒NP蛋白的线性表位;单抗3E7、4B12与基因I、Ⅱ、Ⅲ、Ⅵ、Ⅸ、Ⅶ型共11株NDV均有反应性,单抗2D11仅与基因Ⅶ型中QY97、Mallard/CH/HLJ001/06和基因Ⅲ型中Mutkaswar株反应。单抗2D11识别NP基因开放阅读框C端的1303-1413处(37个aa);单抗3E7、4B12识别NP基因ORF C端的1372-1470处(共33个aa)。【结论】获得了针对NDVF48E9株NP蛋白的3株识别线性表位单抗,将在NDV抗原变异研究及检测方法建立方面进行应用。更多还原

【Abstract】 [Objective]To prepare Monoclonal antibodies(McAbs) against F48E9 strain of Newcastle disease virus(NDV),so that they could be used in study of NDV antigen variation and establishment of detection methods.[Methods]BALB/c mice were immunized with purified F48E9 or recombinant NP protein expressed in E.coli,hybridoma cells were selected by indirect F48E9-ELISA.The reactivity of McAbs was identified by Western blot and indirect immunofluorescence assay,and the cross-reaction of McAbs with other NDV strains were determined by indirect ELISA.The McAbs recognition locations were determined by Western blot using recombinant NP protein fragments in E.coli.[Results]There are three strains of hybridoma cells were screened.Western blot and indirect immunofluorescence assay showed that three McAbs recognize linear epitopes located on NP protein,McAb 2D11 recognize 1303-1413 located on -COOH end of NP gene open reading frame(ORF),McAbs 3E7 and 4B12 recognize 1372-1470 near -COOH end of NP gene.In cross reaction tests,it showed that McAb 3E7 and 4B12 can react with 11 strains of NDV belonged to genotypeⅠ,Ⅱ,Ⅲ,Ⅵ,ⅨandⅦrespectively,McAb 2D11 can react with QY97(Ⅶ),Mallard/CH/HLJ001/06 strain(Ⅶ) and Mutkaswar strain(Ⅲ).[Conclusion]Three hybridoma cell lines secreting McAbs recognize linear epitopes locating on NP protein of F48E9 were Obtained,and they would be applied to the research in NDV antigen variation and establishment of detection methods.更多还原