抗牛传染性鼻气管炎病毒gD蛋白中和活性单克隆抗体的制备及鉴定

Preparation and characterization of neutralizing monoclonal antibodies against gD protein of infectioous bovine rhinotracheities virus

为了制备牛传染性鼻气管炎病毒(IBRV)gD蛋白的单克隆抗体,本研究用纯化的IBRV免疫BALB/c小鼠,然后以纯化的昆虫细胞表达的gD蛋白作为ELISA包被抗原筛选杂交瘤细胞株。结果获得3株能够稳定分泌抗gD蛋白单克隆抗体的杂交瘤细胞株1B4、3E8和4F12。采用夹心ELISA鉴定单克隆抗体的Ig亚类,结果显示,1B4、3E8为IgG2b,4F12为IgG1。免疫印迹试验与间接免疫荧光试验表明,3株单克隆抗体均能与IBRV发生特异性反应。微量中和试验结果显示,1B4和3E8单克隆抗体具有中和IBRV的活性,中和效价均为1∶320。本研究成功制备了具备中和作用的抗牛传染性鼻气管炎病毒gD蛋白单克隆抗体,为建立特异性的IBRV诊断方法及致病机理的研究提供了工具。

To prepare the monoclonal antibodies(m Ab) against g D protein of infectious bovine rhinotracheities virus(IBRV), BALB/c mice were immunized with purified IBRV.The hybridoma cell lines were screened by an indirect ELISA coated with the purified IBRV g D protein expressed from insect cells sf9.Three hybridoma cell lines secreting specific mAb against gD were obtained,and designated 1B4,3E8 and 4F12,respectively.The sandwich ELISA for Ig subclass differentiated 1B4 and 3E8 mAbs as IgG2b,and 4F12 as IgG1.Western-blot analysis and indirect immunofluorescence assay revealed that three monoclonal antibodies could react with IBRV specifically.Micro-neutralization test demonstrated that 1B4 and 3E8 possessed neutralizing abilities to IBRV with titres of 1 ∶ 320 individually.The m Ab against g D presented in this report could be useful tools for further development of specific diagnostic method or investigation of IBRV pathogenesis.