摘要
旨在制备牛冠状病毒(Bovine coronavirus,BCoV)单克隆抗体(MAb),为BCoV疫苗制备和建立免疫胶体金等特异性检测方法奠定基础。使用原核表达系统制备重组BCoV N蛋白,BCoV灭活全病毒液免疫BALB/c小鼠;以N蛋白和全病毒为抗原,通过间接ELISA法检测抗体效价。经细胞融合与5轮亚克隆,筛选出分泌目标抗体的单克隆细胞株。采用腹水法制备单克隆抗体并纯化,通过间接免疫荧光试验(IFA)和Western blot验证抗体反应性与特异性。结果:筛选出3株针对BCoV N蛋白的杂交瘤细胞株(2D12C、3D6E、4D1F),分泌的单克隆抗体轻链均为Kappa链,2D12C与4D1F重链为IgG2a亚型,3D6E重链为IgG1亚型;IFA试验表明3株单克隆抗体均能与BCoV发生特异性结合;Western blot证明3株单克隆抗体与BCoV和重组表达BCoV N蛋白均反应良好,且3株单克隆抗体与感染牛轮状病毒、牛传染性鼻气管炎病毒、牛病毒性腹泻病毒及牛副流感病毒3型的细胞均不发生特异性反应。综上,制备了3株特异性较高、生物学活性良好的BCoV单克隆抗体,为后续疫苗制备及检测方法研究奠定了基础。
This study aimed to prepare Bovine coronavirus(BCoV)monoclonal antibodies(MAbs)to lay a foundation for preparation of BCoV vaccines and for establishment of specific detection methods such as colloidal gold.Recombinant BCoV N protein was prepared using a prokaryotic expression system,and BALB/c mice were immunized with inactivated whole BCoV virus.The antibody titer was determined by indirect ELISA.After cell fusion and five rounds of sub-cloning,a monoclonal cell line secreting the target antibody was screened.Mono-clonal antibodies were produced via the ascites method and were purified.Finally,the reactivity and specificity of the antibodies were valida-ted by indirect immunofluorescence assay(IFA)and Western blot.The results were that three hybridoma cell lines targeting BCoV N protein were selected.The monoclonal antibodies secreted by these hybridomas had Kappa light chains.The heavy chains of 2D12C and 4D1F were of the IgG2a subtype,while 3D6E had an IgG1 subtype.The indirect immunofluorescence assay showed that all three monoclonal antibodies could specifically bind to BCoV.Western blot confirmed that the three monoclonal antibodies reacted well with both the BCoV and recombi-nant expressed BCoV N proteins.Furthermore,no specific reaction occurred with cells infected by bovine rotavirus,bovine infectious bovine rhinotracheitis virus,bovine viral diarrhea virus,and bovine parainfluenza virus type 3.To sum up,three monoclonal antibodies with high specificity and good biological activity against BCoV were successfully prepared here,laying a foundation for subsequent BCoV vaccine devel-opment and for research on specific detection methods,such as colloidal gold.
作者
范昊宇
蒋蔚
汪萍
宫震
韩翔舒
白航飞
刘姝悦
李月
张立雪
付修龙
杜玮
阿依江·木合塔尔
平继辉
朱启运
蒋松
夏俊
FAN Haoyu;JIANG Wei;WANG Ping;GONG Zhen;HAN Xiangshu;BAI Hangfei;LIU Shuyue;LI Yue;ZHANG Lixue;FU Xiulong;DU Wei;Ayijiang Muhetar;PING Jihui;ZHU Qiyun;JIANG Song;XIA Jun(College of Animal Science and Technology,Shihezi University,Shihezi 832003,China;Veterinary Research Institute,Xinjiang Academy of Animal Sciences,Urumqi 830000,China;Shanghai Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Shanghai 200241,China;Xinjiang Animal Disease Prevention and Control Engineering Technology Research Center,Urumqi 830000,China;College of Veterinary Medicine,Xinjiang Agricultural University,Urumqi 830000,China;College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210014,China;Animal Husbandry Research Institute,Xinjiang Academy of Animal Sciences,Urumqi 830000,China;College of Veterinary Medicine and Biosafety,Lanzhou University,Lanzhou 730000,China)
出处
《畜牧与兽医》
北大核心
2025年第12期116-123,共8页
Animal Husbandry & Veterinary Medicine
基金
新疆维吾尔自治区重大科技专项(2023A02007)
新疆维吾尔自治区天山英才青年科技拔尖人才项目(2022TSYCCX0046)
新疆维吾尔自治区肉牛产业技术体系项目资助项目(XJARS-10)
自治区西门塔尔(安格斯)牛群体改良体系经费资助项目(2025XM-14)。
