【摘要】 目的 制备抗猪流行性腹泻病毒的单克隆抗体。方法 本研究以哺乳动物细胞293T表达的PEDV S1重组蛋白免疫BALB/c小鼠；利用细胞融合技术，经间接ELISA方法筛选和细胞克隆，得到5株能稳定分泌抗PEDV S1单克隆抗体的杂交瘤细胞株。筛选效价最高的杂交次瘤细胞株制备单克隆抗体并进行特性鉴定。结果 免疫小鼠的脾细胞和SP2/0骨髓瘤细胞融合后，经多次克隆及亚克隆筛选，共得到5株能与PEDV S1蛋白产生特异性反应的单抗表达细胞株。分别命名为：12D14H4、13F5B9、10D2B10、11A7C9和14E6F5，其分泌的抗体效价分别为：1∶51 200、1∶25 600、1∶12800、1∶12 800、1∶3 200。5株单抗与PEDV S1蛋白反应后，均在130 KDa附近出现清晰条带。所得单克隆抗体为IgGⅠ亚型，轻链为κ链；杂交瘤细胞用无血清培养基培养，上清中抗体效价最高可达到1∶1 024 000，且经连续传代20代效价没有显著降低。Western blot及间接免疫染色检测结果显示，单克隆抗体能识别PEDV S1蛋白，并与Vero细胞、PPRV、PRRSV、PCV2、PP和CSFV等均无交叉反应，具有良好的特异性，可用于PEDV感染检测。结论 PEDV单克隆抗体的成功研制，为PEDV免疫诊断、表位识别及蛋白研究奠定了良好基础。更多还原

【Abstract】 Objective To generate monoclonal antibodies against porcine epidemic diarrhea virus.Methods Monoclonal antibodies against porcine epidemic diarrhea virus(PEDV)S1 protein were generated and an indirect immuno-fluorescence assay method was developed for PEDV diagnosis. BALB/c mice were immunized with recombinant S1 protein expressed from eukaryotic cells. Spleen cells from mice with high antibody titer were isolated and fused with SP2/0 cells. The hybridoma cell lines secreting monoclonal antibodies against PEDV S1 protein were screened out and cultured with serum-free medium. Results After fusion of spleen cells from immunized mice and SP2/0 myeloma cells,5 hybridomas were established to secrete monoclonal antibodies anginst PEDV S1 protein. They were named as:12D14H4,13F5B9,10D2B10,11A7C9 and 14E6F5 with the secreted antibody titers of 1∶51200,1∶25600,1∶12800,1∶12800,1∶3200. All of 5 monoclonal antibodies can reacted with PEDV S1 protein with a clear band around 130 k Da. The obtained monoclonal antibody is Ig GⅠ subtype,and the light chain is κ chain;the hybridoma cells are cultured in serumfree medium,and the antibody titer in the supernatant can reach up to 1:1024000,the titer did not decrease significantly after 20 generations of continuous passage. Western blot and indirect immunostaining showed that the monoclonal antibody could specifically recognize PEDV S1 protein,and had no cross-reaction with porcine pseudorabies virus(PPRV),porcine reproductive and respiratory syndrome virus(PRRSV),porcine circovirus 2(PCV2),porcine parvovirus(PP)and transmissible gastroenteritis virus(TGEV). Conclusion The development of PEDV monoclonal antibody and indirect immuno-staining assay has provided an effective method for PEDV detection.更多还原