3株鸭疫里默氏杆菌OmpA单克隆抗体的制备及特性鉴定  

Preparation and Characterization of Three Monoclonal Antibodies against OmpA of Riemerella anatipestifer

作　　者：韩文龙 张雪梅 王小兰[1] 于圣青[1] HAN Wenlong;ZHANG Xuemei;WANG Xiaolan;YU Shengqing(Shanghai Veterinary Research Institute,CAAS,Shanghai 200241,China;College of Animal Science and Technology,Anhui Agricultural University,Hefei 230036,China)

机构地区：[1]中国农业科学院上海兽医研究所,上海200241 [2]安徽农业大学动物科技学院,安徽230036

出　　处：《中国动物传染病学报》2022年第4期144-149,共6页Chinese Journal of Animal Infectious Diseases

基　　金：国家重点研发计划项目(2016YFD0500805);中国农业科学院科技创新工程协同创新任务(CAAS-XTCX2016011-04-8)。

摘　　要：鸭疫里默氏杆菌(RA)病,是家禽中主要的细菌性传染病之一,血清型复杂且各血清型间缺少交叉免疫保护,其中血清1型、2型、10型是国内优势血清型。外膜蛋白A(OmpA)是RA的毒力因子之一,在不同血清型RA中保守性良好。用OmpA蛋白作为抗原免疫BALB/c小鼠,与骨髓瘤细胞SP2/0融合后用间接ELISA法筛选阳性杂交瘤细胞株,腹腔注射BALB/c小鼠制备单克隆抗体。通过间接ELISA检测单克隆抗体效价及稳定性,秋水仙素法进行染色体分析,Western blot和玻片凝集试验鉴定单克隆抗体特异性。获得3株稳定分泌抗RA OmpA的单克隆抗体,分别命名为2D5、2A6、4H8;3株单克隆抗体的亚型均为IgG2a,腹水效价均为1∶2048000,均与鸭疫里默氏杆菌血清1型RA-CH3、血清2型RA-NJ3、血清10型RA-HXb2发生特异性反应,而与其他禽源致病菌不发生交叉反应。本研究成功研制的3株单克隆抗体具有良好的菌种特异性,有助于RA致病机理和RA快速诊断试剂盒的研制。Riemerella anatipestifer(RA)infection is one of the major bacterial infectious diseases in poultry.R.anatipestifer consists of complex serotypes and lacks cross-protection among serotypes.Serotypes 1,2 and 10 are the dominant domestic serotypes.Outer membrane protein A(OmpA)is one of the virulence factors of RA and well conserved in different serotypes.Preparation and characterization of monoclonal antibodies(mAbs)to OmpA was performed in the present study.BALB/c mice were immunized with OmpA protein.The spleen cells were fused with murine myeloma cells SP2/O and positive hybridomas were screened by indirect ELISA.The ascitic fl uid containing mAbs was prepared by intraperitoneal injection of hybridoma cells into BALB/c mice.The mAb titers were titrated by indirect ELISA and the specifi city was determined by Western blot and slide agglutination.In addition,colchicine method was used for chromosome analysis of monoclonal cells.As a result,3 mAbs against RA OmpA were obtained and designated as 2D5,2A6,and 4H8. The subtypes of these mAbs were determined as IgG2a. The titers of 3 mAbs were 1: 2 048 000. All 3 mAbs reacted with RA serotype 1 strain CH3, serotype 2 strain NJ3, and serotype 10 strain HXb2 but not with other avian pathogens. The availability of specifi c mAbs would be benefi cial to the research of RA pathogenesis and diagnosis.

关 键 词：鸭疫里默氏杆菌 单克隆抗体 外膜蛋白 特异性 

分 类 号：S852.61[农业科学—基础兽医学]