分泌抗猪流行性腹泻病毒中国变异株S蛋白单克隆抗体杂交瘤细胞系的建立

The development of the hybridoma cell line secreting monoclonal antibody against S protein of a PEDV variant

以纯化的猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)中国变异株(CH/ZMDZY/11)S蛋白N端高变区(22-380aa)重组蛋白为抗原免疫BALB/C小鼠,以纯化CH/ZMDZY/11全病毒及带His标签的重组蛋白分别为筛选抗原,利用淋巴瘤杂交技术获得了一株稳定分泌抗S蛋白特异性单克隆抗体细胞株,命名为2D1。该株杂交瘤细胞诱生小鼠产生的腹水抗体效价为1∶3 200,免疫球蛋白类型为IgM。ELISA检测该单抗与PEDV流行株及PEDV CV777疫苗株全病毒的反应显著差异,可区分流行毒株和疫苗毒株;但该单抗用于Western blot不能区分PEDV流行株和疫苗株。ELISA及Western blot试验结果显示,该单抗有较好的PEDV特异性,可用于检测PEDV。

Gene encoding 22-380 aa of spike protein of porcine epidemic diarrhea virus （PEDV） isolate in China was cloned and expressed as a recombinant protein in Escherichia coli BL21（DE3）. Then,female BALB/c mice were immunized with the purified recombiant S protein, and a monoclonal antibody （MAb） against S protein named 2D1 was achieved by hybridoma technique. The antibody titer of ascites induced by this strain was 1：3 200,and the type of immune globulin was IgM. The monoclonal antibodies against PEDV and CV777 PEDV vaccine strains were significantly different from the whole strain with the ELISA detection,which could be used to distinguise the epidemic strains from vaccine strains, but there has no different between PEDV epidemic strains and vaccine strains in the Western detect. The monoclonal antibody had a good specificity for PEDV, which could be used for the detection of PEDV. The research provide a useful tool as a specific diagnostic reagent for detecting PEDV and for further study in the virus pathogenic mechanism.