禽网状内皮增生症病毒gp90蛋白单克隆抗体的制备及鉴定

Development and Identification of the Monoclonal Antibodies Against Recombinant Reticuloendotheliosis Virus gp90 Protein

为获得分泌抗禽网状内皮增生症病毒单克隆抗体的杂交瘤细胞,通过表达pET32a-gp90重组蛋白,以纯化的gp90蛋白为抗原,免疫BALB/c鼠,筛选分泌抗REV McAb的杂交瘤细胞株。经过细胞的融合及筛选,共获得能稳定分泌抗REV gp90的McAb细胞系2株,分别命名为A9E8和B3F8。2株细胞培养上清的效价均在1∶4×102以上,腹水效价均在1∶8×103以上。亚型鉴定结果表明,2株皆为IgG型,轻链均为κ链;Western blot及间接免疫荧光结果显示,这2株杂交瘤细胞分泌的单克隆抗体均能识别REV全病毒抗原,与其发生特异性反应,并且不与禽类其他病毒发生反应;病毒中和试验结果表明,获得的单克隆抗体均能REV临床分离株发生中和反应,具有较好的中和活性。利用制备的单克隆抗体建立了REV的间接免疫荧光检测方法,临床病料检测结果表明,该方法与病毒分离及PCR的检测结果基本一致,阴、阳性符合率分别为100%和90%。作者制备了针对REV gp90的McAb,为REV的快速诊断及抗原表位分析与鉴定等奠定了基础。

In order to obtain hybridoma cells secreting monoclonal antibody（McAb） direct against Reticuloendotheliosis Virus（REV）,BALB/c mice was immunized with the purified gp90 as immunogen,then hybridoma cell strains were screened for the secretion of McAb against REV.Two hybridoma strains with the ability to stably secrete McAb against REV,designated as A9E8 and B3F8 respectively,were obtained after three-round screening.The titers of secreting antibody from cell culture supernatant were both above 1∶4×102,while above 1∶8×103 from ascites of the mice.The secreted McAbs were both IgG,and the light chains were both κ as indicated by antibody subtype identification assay.IFA results showed that secreted McAb could react specifically with the REV strain.Applying the property,an indirect immunofluorescent method for the REV antigen was established,and the actual application result showed the diagnosis was nearly in accordance with the result of virus isolation and PCR,and the positive according rate was 90%,the negative according rate was 100%.Hybridomas trains that secrete McAb specific against REV were produced,which will be useful tools for REV rapid diagnosis and immunogenic analysis of the protein such as epitope mapping.