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目的制备抗猪呼吸与生殖综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)Nsp9蛋白单克隆抗体,并进行初步鉴定。方法用纯化的重组Nsp9蛋白免疫BALB/c小鼠,采用杂交瘤技术制备抗PRRSV Nsp9蛋白单克隆抗体,采用Western blot和间接免疫荧光试验分析单抗的特异性;使用免疫球蛋白标准亚类鉴定试剂盒鉴定单抗的亚类;将杂交瘤细胞分别保存1、2、3、4个月后复苏,采用间接ELISA法检测细胞分泌抗体的能力。结果共获得3株可稳定分泌特异性单抗的杂交瘤细胞株,分别命名为2D6、3C11和4E6,其细胞培养上清的ELISA效价分别为1∶6 400、1∶3 200和1∶1 600,腹水的ELISA效价分别为1∶640 000、1∶512 000和1∶256 000。3株单抗均可与Nsp9蛋白和PRRSV特异性结合;2D6和3C11株单抗的Ig亚型为IgG1,4E6株单抗为IgM亚类抗体;3株杂交瘤细胞保存4个月均能稳定分泌单抗。结论已制备抗PRRSVNsp9蛋白单克隆抗体,其特异性高,稳定性良好,为建立鉴别自然感染与注射疫苗感染PRRSV的诊断方法提供了材料。
Objective To prepare monoclonal antibodies against Porcine reproductive and respiratory syndrome virus (PRRSV) Nsp9 protein and preliminary identification. Methods BALB / c mice were immunized with purified recombinant Nsp9 protein and hybridoma technology was used to prepare anti-PRRSV Nsp9 monoclonal antibody. The specificity of monoclonal antibodies was analyzed by Western blot and indirect immunofluorescence assay. Kits were used to identify the subclasses of monoclonal antibodies. The hybridomas were resuscitated in 1, 2, 3 and 4 months after resuscitation respectively. The ability of the cells to secrete antibodies was tested by indirect ELISA. Results Three hybridoma cell lines secreting specific monoclonal antibodies were obtained, which were named as 2D6, 3C11 and 4E6, respectively. The ELISA titers of cell culture supernatants were 1: 400, 1: 3 and 200 respectively 1 600, the ELISA titers of ascites were 1: 640 000, 1: 512 000 and 1: 256 000. The monoclonal antibodies of MNCs were specifically bound to Nsp9 protein and PRRSV. The Ig subtypes of 2D6 and 3C11 monoclonal antibodies IgG1 and 4E6 monoclonal antibodies were IgM subclass antibodies; all three hybridoma cells could stably secrete monoclonal antibodies after being stored for 4 months. CONCLUSION Monoclonal antibodies against PRRSVNsp9 protein have been prepared with high specificity and good stability. This study provides a useful tool for the identification of natural and PRRSV vaccines.