猪繁殖与呼吸综合征病毒N蛋白单克隆抗体的制备及生物学特性分析

分别用纯化的猪繁殖与呼吸综合征病毒(PRRSV)和纯化的重组N蛋白免疫BALB/c小鼠,采用杂交瘤技术制备抗PRRSV N蛋白的单克隆抗体(McAb)。用纯化的PRRSV免疫小鼠,经细胞融合获得2株可分泌特异性单抗的杂交瘤细胞株,分别命名为4B8、4D8。用重组N蛋白免疫的小鼠,经细胞融合获得3株可分泌特异性McAb的抗PRRSV N蛋白的杂交瘤细胞2F3、4D5、5D11。间接ELISA检测4D8、4B8、4D5和5D11杂交瘤上清效价为1∶32~1∶512,而2F3的腹水效价为1∶12 800。单抗2F3、4B8和4D8与纯化病毒的Western blotting反应都为阳性,而4D5和5D11为阴性。IFA检测结果5株单抗都有明显的荧光,与PRRSV呈阳性反应。2F3的Ig亚型为IgM。5株单抗杂交瘤细胞连续传代至20代,分泌相应McAb的效价基本一致。本研究为PRRSV生物学诊断和方法研究提供有用工具。

Development of monoclonal antibodies against N protein of porcine reproduction and respiratory syndrome virus

After the immunization of BALB/c mice with purified porcine reproduction and respiratory syndrome virus(PRRSV),the stimulated splenocytes were fused with SP2/0 myemomas to produce hybridomas.Two hybridoma lines of monoclonal antibody against N protein of PRRSV have been developed,designated as 4B8 and 4D8.Using recombinant N protein as immunogen,three hybridoma lines designated as 2F3,4D5 and 5D11,were obtained.McAb 2F3 was identified as subclass IgM and the light chain belongs κ chain.The ELISA titers of antibodies to PRRSV in cultural cell supernatant were 1∶32~1∶512,and the ELISA titer of the ascites of 2F3 was 1∶12800.The results of IFA showed the McAbs could react with PRRSV in MARC-145 cells.Results of western-blotting with purified PRRSV indicated that the McAbs 2F3,4B8 and 4D8 were against the lined epitopes in N protein of PRRSV.Together,it suggested that the McAbs would be useful reagents for diagnosis and epitope identification of PRRSV.