【摘要】 应用杆状病毒真核表达系统体外表达并通过Ni柱亲和纯化猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus, PRRSV)全长NSP9蛋白(NSP9编码PRRSV RNA依赖的RNA聚合酶，RdRp),然后用其免疫BALB/c小鼠。取免疫后小鼠脾细胞与骨髓瘤SP20细胞进行融合，通过间接ELISA方法筛选抗体阳性杂交瘤细胞，经过筛选和亚克隆，获得1株阳性杂交瘤细胞株，制备腹水并进行效价测定，最后应用免疫印迹(Western blot)、间接免疫荧光(IFA)和免疫共沉淀(Co-IP)试验验证其反应性、特异性及应用性能。结果显示，成功获得可溶性全长NSP9蛋白；并得到1株能稳定分泌抗NSP9单克隆抗体的细胞株，命名为3F6;所制备的腹水效价可达到1∶1 280 000;经鉴定该抗体重链属于Ig-1型，轻链为κ链，仅特异性识别PRRSVⅡ型毒株。Western blot、IFA和Co-IP试验结果显示，该抗体具有良好的反应性、特异性和应用性能。结果表明，成功制备了PRRSV NSP9单克隆抗体，为PRRSV特异性检测、免疫学血清诊断试剂的研发及研究PRRSV RdRp决定的病毒复制功能奠定了重要的基础。更多还原

【Abstract】 To generate the monoclonal antibodies against full-length non-structural protein 9(NSP9),the RNA-dependent RNA polymerase of porcine reproductive and respiratory syndrome virus(PRRSV),the full-length NSP9 protein of PRRSV was expressed and purified using a baculovirus eukaryotic expression system.BALB/c mice were immunized with the purified protein,and their splenocytes were subsequently fused with SP2/0 myeloma cells.Hybridoma cells secreting specific antibodies were screened by indirect ELISA.After subcloning,one positive hybridoma cell line was obtained,and ascites was prepared for antibody titer determination.The reactivity,specificity,and applicability of the monoclonal antibody(mAb) were further validated by Western blot,indirect immunofluorescence assay(IFA),and co-immunoprecipitation(Co-IP).The results showed that a stable hybridoma cell line(designated 3F6) secreting anti-NSP9 mAb were successfully obtained with ascites antibody titer of 1:1 280 000.The mAb 3F6 was identified as IgGl for the heavy chain and κ chain for the light chain.Specificity assay demonstrated the antibody specifically recognized PRRSV genotype Ⅱ strains with no cross-reactivity with genotype Ⅰ.Western blot,IF A,and Co-IP confirmed that the mAb exhibited high reactivity,specificity,and applicability.In summary,a PRRSV NSP9-specific monoclonal antibody was successfully generated in this study,which provides a valuable tool for PRRSV-specific detection,serological diagnostics,and further investigation into the viral replication functions of PRRSV RdRp(NSP9).更多还原