抗鸡传染性贫血病毒VP2蛋白单克隆抗体的制备及其应用研究

Development and Application of Monoclonal Antibody against VP2 Protein of Chicken Infectious Anemia Virus

为制备针对鸡传染性贫血病毒(Chicken infectious anemia virus,CIAV)的单克隆抗体,以CIAV的VP2原核表达蛋白作为抗原免疫小鼠,经杂交瘤技术获得3株能稳定分泌抗VP2蛋白特异性抗体的细胞株,进一步经IFA筛选出特异性和灵敏度俱佳的杂交瘤细胞Mab-VP2-4株,制备的小鼠腹水效价达到1∶2000。结果显示,制备的抗VP2蛋白单克隆抗体可与多株CIAV感染的MDCC-MSB1细胞发生特异性反应,而对EDSV、ALV、ILTV、ARV以及空白MDCC-MSB1细胞、CEF细胞等均无交叉反应,特异性良好,且应用于IFA方法时对CIAV感染的最低检出限为5 TCID 50。以该单克隆抗体对市场上10种不同来源的禽活疫苗进行病毒分离结合单克隆抗体IFA检测,同时与《中国兽药典》规定的鸡检查法进行对比检测,结果一致,均未检出CIAV污染。本研究制备了针对CIAV VP2蛋白的特异性单克隆抗体,为该病特异性诊断方法的建立奠定了基础。

To prepare chicken infectious anemia virus(CIAV)specific monoclonal antibody,codon-optimized VP2 protein was used as antigen and hybridoma technology to obtain three positive cell lines which could secrete specific antibody stably,the MAB-VP2-4 strain with high specificity and sensitivity was screened by IFA,and the ascites of mice was prepared with the titer of 1∶2000.The prepared monoclonal antibody could react specifically with CIAV infected MDCC-MSB1 cells,but had no cross-reaction with EDSV,AlV,ILTV,ARV,MDCC-MSB1 cells and CEF cells,and the minimum detection limit is 5TCID 50.The monoclonal antibody was used to detect 10 kinds of live poultry vaccines from different sources on the market,and the SPF chickens inoculated with the vaccine were tested by the method stipulated according to the Chinese Veterinary Pharmacopoeia,no CIAV contamination was detected.In this study,the monoclonal antibody against VP2 protein of CIAV was prepared,which laid a foundation for the establishment of the specific diagnostic method and the related research of CIAV.