牛冠状病毒单克隆抗体的制备  

Preparation of Monoclonal Antibodies against Bovine Coronavirus

作　　者：项钰 周玉龙[1] Xiang Yu;Zhou Yulong(College of Animal Science and Technology,Heilongjiang Bayi Agricultural University,Daqing163319)

机构地区：[1]黑龙江八一农垦大学动物科技学院,大庆163319

出　　处：《黑龙江八一农垦大学学报》2025年第1期51-56,72,共7页journal of heilongjiang bayi agricultural university

基　　金：黑龙江省自然基联合引导项目(LH2020C083);博士后研究人员落户黑龙江科研启动金(LBH-Q21159)。

摘　　要：牛冠状病毒(Bovine coronavirus,BCoV)是引起犊牛肠胃炎的主要病原之一。为获得由天然免疫反应引起的单克隆抗体,采用纯化BCoV蛋白足垫法免疫BALB/c小鼠;采用间接ELISA方法筛选阳性杂交瘤细胞;经6~7次亚克隆获得了4株能稳定分泌抗体的杂交瘤细胞株,分别是5D6、5G5、1B9、1D9;采用腹水法制备大量单克隆抗体,并对腹水效价为1∶6.4×10~5的5D6纯化;最后,利用IFA、抗体亚类鉴定试验、杂交瘤细胞染色体数目检查、Western blot及特异性试验对4株单克隆抗体生物学活性鉴定。结果显示:IFA试验表明4株单克隆抗体均可以与BCoV反应呈特异性红光,制备的4株单克隆抗体轻链均为Kappa链,1D9、5D6、5G5重链为IgG2a,而1B9重链为IgG1;杂交瘤细胞染色体检查结果显示染色体数目在95~110个;Western blot结果显示,单克隆抗体与感染病毒的细胞存在55 kDa的特异性条带,且特异性试验结果表明4株单克隆抗体均不与感染牛传染性鼻气管炎病毒、牛副流感病毒3型及轮状病毒的细胞发生非特异性反应。综上,利用BCoV全病毒制备了4株单克隆抗体,能与BCoV发生特异性反应,生物学活性良好,为BCoV特异性诊断方法的建立及病毒的受体感染机制的研究奠定了基础。Bovine Coronavirus(BCoV)is one of the major pathogens causing gastroenteritis in calves.To obtain monoclonal antibodies induced by natural immune response,BCoV protein was purified and BALB/c mice were immunized by the footpad method.Positive hybridoma cells were screened using an indirect ELISA method.Four hybridoma cell lines,5D6,5G5,1B9,and 1D9,were obtained by 6-7 subclonations,and a large number of monoclonal antibodies were prepared by the ascites method,and 5D6 with an ascites potency of 1∶6.4×105 was purified.Finally,the biological activities of the four monoclonal antibodies were identified using IFA,antibody subclass identification,chromosome number check of hybridoma cells,Western Blot and specificity assay.The results showed that all four monoclonal antibodies could react with BCoV with specific red light in the IFA assay,the light chains of the four monoclonal antibodies prepared were Kappa chains,the heavy chains of 1D9,5D6 and 5G5 were IgG2a,and the heavy chain of 1B9 was IgG1.The chromosome examination of hybridoma cells showed that the chromosome number was 95-110.The results of Western Blot showed that the monoclonal antibodies had a specific band of 55 kDa with the infected cells,and the specificity assay showed that none of the four monoclonal antibodies reacted non-specifically with the cells infected with bovine infectious rhinotracheitis virus,bovine parainfluenza virus type 3 and rotavirus.In summary,four monoclonal antibodies were prepared using BCoV whole viruses,which could react with BCoV specifically and have good biological activity,laying the foundation for the establishment of BCoV-specific diagnostic methods and the study of the receptor infection mechanism of the virus.

关 键 词：牛冠状病毒 单克隆抗体 病毒浓缩 

分 类 号：S852.4[农业科学—基础兽医学]