猪繁殖与呼吸综合征病毒GP5蛋白单克隆抗体的制备与鉴定

Development and Characterization of Monoclonal Antibody against GP5 Protein of Porcine Reproduction and Respiratory Syndrome Virus

为了制备猪繁殖与呼吸综合征病毒(PPRSV)的单克隆抗体,利用猪繁殖与呼吸综合征病毒(PPRSV)GP5重组蛋白免疫BALB/c小鼠,取脾细胞和NS0浆细胞瘤细胞进行融合,经间接ELISA和阻断ELISA筛选、有限稀释法克隆化获得3株能稳定分泌抗PRRSV GP5蛋白单克隆抗体的(mAb)杂交瘤细胞株,命名为4B7、2C5和2E4。间接免疫荧光(IFA)和免疫过氧化物酶单层试验(IPMA)结果显示,3株mAb均与感染PRRSV BJ 4株的Marc 145细胞特异性反应,表明其可识别天然病毒蛋白。mAb的Ig亚类均为IgG1,细胞培养上清和腹水的ELISA效价分别为1∶256和1∶51 200,Western blot分析表明,mAb均特异性识别PRRSV GP5蛋白。

In order to prepare monoclonal antibodies（mAbs） against porcine reproductive and respiratory syndrome virus（PPRSV）,the recombination GP5 protein of PRRSV was used to immunize BALB/c mouse and the spleen cells were fused with NS0 cells.Three hybridoma cell lines producing mAbs specific to the GP5 protein,designated as 4B7,2C5 and 2E4,were obtained after identified by indirect and inhibit ELISAs followed by limiting dilution subcloning.All of the mAbs were shown to react specifically with the Marc-145 cells infected with PRRSV BJ-4 strain in indirect immunofluorescence assay（IFA） andimmunoperoxidase monolayer assays（IPMA）.Ig subclass of the mAbs was determined as IgG1,and the ELISA titers of cell supernatant and ascites were 1∶256 and 1∶51 200,respectively.Western-blotanalysis showed that the mAbs were specific to the GP5 protein of PRRSV.