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目的制备马流感病毒(Equine influenza virus,EIV)2个亚型的单克隆抗体,并进行鉴定。方法将马流感病毒甲1型A/equine/布拉格/56 H7N7(简称H7N7)和马流感病毒甲2型A/equine/miami/63 H3N8(简称H3N8)分别接种SPF鸡胚,收获尿囊液,纯化后免疫BALB/c小鼠,取脾细胞,与SP2/0细胞进行融合,筛选阳性杂交瘤细胞,采用细胞培养法和体内诱生腹水法大量制备单克隆抗体,并对其生物学特性进行鉴定。结果筛选出能稳定分泌EIV H7N7单抗和H3N8单抗的杂交瘤细胞各2株,分别命名为3C2-B2、2B7-3A7和5G10-G12、5A10-2E3-2G4。3C2-B2分泌的单抗重链为IgM,轻链为κ链,细胞培养上清和腹水中抗体效价分别为1∶512和1∶262 144;5G10-G12分泌的单抗重链为IgG2a,轻链为κ链,细胞培养上清和腹水中抗体效价分别为1∶1 024和1∶1 048 576。2个亚型的单抗均具有良好的特异性。获得的杂交瘤细胞株连续培养2个月,培养上清中的抗体效价保持不变;冻存1个月复苏后,培养上清中的抗体效价接近原始值。结论成功制备了EIV 2个亚型的单克隆抗体,为进一步研制EIV快速分型特异性诊断试剂及治疗制剂奠定了基础。
Objective To prepare monoclonal antibodies against two subtypes of Equine influenza virus (EIV) and to identify them. Methods The SPF chicken embryos were inoculated with equine influenza A type A / equine / Bragg / 56 H7N7 (H7N7) and equine influenza A type 2A / equine / miami / 63 H3N8 (abbreviated as H3N8) The purified BALB / c mice were immunized with spleen cells, fused with SP2 / 0 cells and screened for positive hybridoma cells. The monoclonal antibodies were prepared in large quantities by cell culture method and in vivo ascites method, and the biological characteristics Identification. Results Two hybridoma cells that could stably secrete EIV H7N7 monoclonal antibody and H3N8 monoclonal antibody were screened. The monoclonal antibodies secreted by 3C2-B2, 2B7-3A7 and 5G10-G12, 5A10-2E3-2G4.3C2-B2 Heavy chain IgM, light chain κ chain, cell culture supernatant and ascites antibody titers were 1: 512 and 1:262 144; 5G10-G12 secretion of monoclonal antibody heavy chain IgG2a, light chain kappa chain, cells Antibodies in culture supernatants and ascites were 1: 1 024 and 1: 1 048 576.2 subtypes of monoclonal antibodies have good specificity. The obtained hybridoma cell lines were cultured continuously for 2 months, the antibody titers in the culture supernatant remained unchanged, and after 1 month of cryopreservation, the antibody titers in the culture supernatant were close to their original values. Conclusion The monoclonal antibodies against two subtypes of EIV were successfully prepared, which laid the foundation for the further development of EIV rapid typing diagnostic reagents and therapeutic preparations.