采用杂交瘤细胞融合技术,获得3株能稳定分泌抗GPV特异性抗体的杂交瘤细胞株,免疫印迹结果表明:这3株单抗均能与纯化的GPV病毒子及重组VP3蛋白发生特异性反应,而与鸭肝炎病毒、鸭细小病毒、鹅源新城疫病毒无交叉反应。采用真核表达载体pCAGGS对VP3基因进行分段克隆并转染COS-1细胞表达,用单抗介导的间接免疫荧光(IFA)进行检测,结果表明:3E8株单抗与VP3蛋白135~332氨基酸(aa)和322~535aa肽段均能发生特异反应,因此可以推定3E8株单抗识别的抗原表位在VP3的322~332aa上。
By using hybridoma cell fusion technology, we succeeded in developing three positive hybridoma cell lines secreting monoclonal antibodies (mAbs) against goose parvovirus (GPV). These prepared mAbs reacted specifically with the purified GPV particles and the VP3 proteins from prokaryotic expressed vector analysis, and had no cross reactivity between these mAbs and duck parvovirus, duck hepatitis virus and goose originated Newcastle Disease virus in Western bolt. The four overlapped subfragment DNA of the VP3 gene were cloned and expressed in the pCAGGS eukaryotic plasmid vector, and their reactivities with mAbs were characterized by the indirect immunofluoresce assay (IFA). The result revealed that the mAb 3E8 reacted with two subfragments harboring 135-332 amino acid(aa) or 322-535 aa, so it can be deduced that the linear epitope domain recognized by the 3E8 mAb was located between 322aa and 332aa of the VP3 protein.