抗鹅细小病毒NS1蛋白单克隆抗体的制备及对应抗原表位区的分析

Preparation of monoclonal antibodies against NS1 protein of goose parvovirus and analysis of their antigenic epitope domains

以鹅细小病毒非结构蛋白NS1(GPV-NS1)的重组质粒pcDNA3.1-GPV-NS1及纯化的重组蛋白GPV-NS1为抗原,分组免疫4～6周龄的BALB/c鼠,免疫3次后,取其脾细胞与SP2/0骨髓瘤细胞融合,利用间接ELISA方法筛选阳性杂交瘤细胞株,并研究其部分生物学特性。结果共获得6株能稳定分泌特异性抗体的阳性杂交瘤细胞株。单克隆抗体亚型鉴定结果显示,2株为IgG2a型,4株为IgM型,轻链均为κ链。用制备的单克隆抗体对分段表达的NS1蛋白进行免疫印迹分析,结果鉴定出3个抗原表位区,分别位于NS1蛋白的第453～514 aa、485～542 aa、533～598 aa区段。证实,6株杂交瘤细胞在体外长期培养能稳定地分泌抗GPV-NS1单克隆抗体。

The monoclonal antibodies（McAbs） against goose parvovirus NS1 protein（GPV-NS1） were obtained by fusing myeloma cells SP2/0 and spleen cells of BALB/c mice which were immunized with the recombinant plasmid pcDNA3.1-GPV-NS1 or the purified recombinant protein GPV-NS1 three times.Six hybridoma cell lines against GPV-NS1 were obtained by screening with indirect ELISA.The subtypes of two McAbs were IgG2a,and the others were IgM.Their light chain were κ.GPV-NS1 was dissected into 15 overlapping epitopes,which were used to react with McAbs by Western-blot.Western-blot analysis showed that the six McAbs could react with the recombinant GPV-NS1.It indicated that the non-structural protein linear B-cell epitopes located at the C-terminus 453-514 aa,485-542 aa and 533-598 aa.The results revealed that the six hybridoma cell lines could secret stably in vitro the McAbs against GPV-NS1 protein.