猪流行性腹泻病毒M蛋白单克隆抗体的制备及鉴定

Preparation of the monoclonal antibody against membrane protein of porcine epidemic diarrhea virus

为制备猪流行性腹泻病毒(PEDV)抗M蛋白单克隆抗体(MAb),本研究以截短表达的His-M重组蛋白免疫BALB/c小鼠;以截短表达的GST-M重组蛋白作为包被抗原,采用常规的淋巴细胞杂交瘤技术制备杂交瘤细胞,通过间接ELISA进行筛选,得到一株稳定分泌抗M蛋白MAb。MAb亚类鉴定为IgG2b型,轻链为κ链,杂交瘤细胞培养上清和诱导的小鼠腹水抗体效价分别为1∶3000和1∶2×105。Westernblot试验表明该MAb能够识别重组及天然的PEDVM蛋白。间接免疫荧光试验表明该MAb能够与PEDV感染的VeroE6细胞产生特异性免疫荧光。

To prepare the monoclonal antibody （MAb） against of porcine epidemic diarrhea virus （PEDV）, hybridomas were produced by fusing SP2/0 cells with spleen cells from mouse immunized with PEDV His-M recombinant protein. A hybridoma stable secreting MAb against M protein was identified by indirect ELISA detection with GST-M as coating antigen. The MAb was IgG2b subtype with K chain. The titers in cell culture medium of the hybridoma and the ascetic fluids were l：3,000 and 1：200,000, respectively. Western blot analysis showed that the MAb could recognize the recombinant and authentic M protein of PEDV, and the specific immlmoflurescence was detected in PEDV infected Vero E6 cells by in direct immunoflurescence assay.