禽脑脊髓炎病毒单克隆抗体的制备、鉴定与初步应用

Preparation,Identification and Preliminary Application of Monoclonal Antibody of Avian Encephalomyelitis Virus

为获得禽脑脊髓炎病毒(Avian Encephalomyelitis virus,AEV)VP1蛋白的单克隆抗体,通过原核表达AEV VP1蛋白,纯化后作为免疫原免疫BALB/c小鼠,并按常规方法制备杂交瘤细胞。经ELISA方法筛选阳性杂交瘤细胞,经过3次亚克隆获得两株杂交瘤细胞株,命名为4#、19#,并进行了抗体亚类的鉴定、Western-blot和IFA检测。结果显示:制备的两株单克隆抗体亚型分别为IgG2b、IgG2a,Western-blot和IFA试验结果表明单克隆抗体均能与AEV发生特异性反应而与其他家禽常见病毒均无交叉反应。采用建立的IFA方法对单抗进行了初步运用,在疫苗外源病毒检测AEV时,与经典方法的符合率100%。本研究成功制备了AEV单克隆抗体,为进一步建立AEV检测方法和深入研究AEV的生物学特性奠定了基础。

In order to obtain the monoclonal antibody against VP1 protein of avian encephalomyelitis virus(AEV),the VP1 protein of AEV was expressed in prokaryotic cells,purified and used as immunogen to immunize BALB/c mice,and the hybridoma cells were prepared according to the conventional method.The positive hybridoma cells were screened by ELISA,and two hybridoma cell lines were obtained after three times of subcloning,named 4#and 19#,and the antibody subclasses were identified,Western-blot and IFA were detected.The results showed that the two monoclonal antibodies were IgG2b and IgG2a,respectively.The results of Western-blot and IFA showed that the monoclonal antibodies could specifically react with AEV and had no cross-reaction with other common viruses in poultry.The established IFA method was used to preliminarily apply the monoclonal antibody,and the coincidence rate with the classical method was 100%when AEV was detected by vaccine exogenous virus.In this study,AEV monoclonal antibody was successfully prepared,which laid a foundation for further establishment of AEV detection methods and in-depth study of the biological characteristics of AEV.