基于哺乳动物细胞表达S1蛋白的猪流行性腹泻病毒单克隆抗体  

Monoclonal antibodies against porcine epidemic diarrhea virus based on S1 protein expressed in mammalian cells

作　　者：李虎林 颜仁和 陈泽典 仇珍珍 李堪贺 马曼欣 毛莹莹 李建军[1] 吕宗吉[1] 李红卫 LI Hulin;YAN Renhe;CHEN Zedian;QIU Zhenzhen;LI Kanhe;MA Manxin;MAO Yingying;LI Jianjun;LV Zongji;LI Hongwei(Foshan University,Foshan,Guangdong,China,528225;Guangzhou Bioneeds Biotechnology Co.Ltd.,Guangzhou,Guangdong,China,510000;Southern Medical University,Guangzhou,China,510000)

机构地区：[1]佛山科学技术学院,广东佛山528225 [2]广州伯尼兹生物科技有限公司,广东广州510000 [3]南方医科大学,广东广州510000

出　　处：《分子诊断与治疗杂志》2023年第4期694-698,共5页Journal of Molecular Diagnostics and Therapy

基　　金：国家重点研发计划项目(2017YFD0500601)。

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。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 kDa.The obtained monoclonal antibody is IgGⅠsubtype,and the light chain isκchain;the hybridoma cells are cultured in serum-free 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.

关 键 词：猪流行性腹泻病毒 S1蛋白 单克隆抗体 免疫染色 免疫诊断 

分 类 号：S858.28[农业科学—临床兽医学]