牛支原体NM001株单克隆抗体的制备与鉴定

Preparation and Identification of the Monoclonal Antibodies against Mycoplasma bovis Strain NM001

为获得抗牛支原体NM001株单克隆抗体,并评价其特性,以牛支原体分离株NM001作为抗原免疫6周龄BALB/c小鼠,利用杂交瘤技术和间接ELISA方法筛选到2株能稳定分泌抗牛支原体的单克隆抗体细胞,命名为2C5和7G3。其细胞上清的间接ELISA抗体效价分别为6.4×10^(3)和1.2×10^(4)。经亚型测定,单抗2C5和7G3均属于IgG1类,轻链均是λ型。制备腹水并对单抗进行纯化和特性鉴定,两株单抗的间接ELISA抗体效价分别为1.02×10^(5)和4.09×10^(5),且两株单抗与无乳支原体、山羊支原体山羊肺炎亚种、丝状支原体山羊亚种、绵羊肺炎支原体、牛巴氏杆菌均无交叉反应。Western Blot结果显示,2株单抗均能特异性识别牛支原体全菌蛋白中的相应蛋白。试验表明,单抗2C5和7G3能够与牛支原体发生特异性反应,从而为牛支原体血清学检测提供一定的物质基础。

This study was conducted to obtain the monoclonal antibodies against Mycoplasma bovis strain NM001 and to evaluate the characterization of the monoclonal antibodies.Six-week-old BALB/c mice were immunized with Mycoplasma bovis strain NM001,two hybridoma cell strains stably secreting antibodies against NM001,separately named as 2C5 and 7G3,were obtained by hybridoma technology and indirect ELISA.The antibody titers in cell supernatant of 2C5 and 7G3 were 6.4×10^(3) and 1.2×10^(4),respectively.Both monoclonal antibodies 2C5 and 7G3 were determined to be IgG1 category,and all light chains of 2C5 and 7G3 were belonged toλchain.The monoclonal antibodies 2C5 and 7G3 were purified with antibody titers of 1.02×10^(5) and 4.09×10^(5),respectively.Moreover,results of indirect ELISA showed that both 2C5 and 7G3 can react with Mycoplasma bovis,but not react with the Mycoplasma agalactia,Mycoplasma capricolum subsp.capripneumoniae,Mycoplasma mycoides subsp.capri,Mycoplasma ovipneumoniae or Pasteurella multocida.The result of Western Blot showed that both monoclonal antibodies 2C5 and 7G3 can specifically recognize proteins of Mycoplasma bovis.These data suggest that monoclonal antibodies 2C5 and 7G3 can react specifically with Mycoplasma bovis,which would provide a basis for serological detection of Mycoplasma bovis.