猪繁殖与呼吸综合征病毒N蛋白单克隆抗体制备及其抗原表位鉴定

Monoclonal antibodies preparation and antigenic epitope identification of N protein of porcine reproductive and respiratory syndrome virus

猪繁殖与呼吸综合征病毒(PRRSV)是影响世界养猪业的重要病原,为了解析该病毒N蛋白抗原表位,本研究制备获得纯化的重组N蛋白,经过BALB/c小鼠免疫、细胞融合、间接ELISA方法筛选和Western blot鉴定,获得了7株稳定分泌抗PRRSV N蛋白单克隆抗体的杂交瘤细胞株,细胞培养上清液抗体效价为1∶800~1∶25600,其中8F1、5E2、2B1、12E1的重链类型为IgG1,4F1、2H2、5A2的重链类型为IgG2b,轻链类型都为κ型。间接免疫荧光试验显示7株单克隆抗体均与PRRSV感染细胞产生反应,可识别3种不同的B细胞线性表位,分别位于47~57 aa、57~67 aa及67~77 aa区域,其中57~67 aa为新发现的抗原表位。本研究结果为PRRSV诊断试剂盒的开发和流行病学调查提供了有用工具。

Porcine reproductive and respiratory syndrome virus(PRRSV)is an important pathogen affecting the pig industry in the world.In order to analyze the antigenic epitopes of the N protein of the virus,the purified recombinant N protein was prepared in this study.Seven hybridoma cell lines stably secreting monoclonal antibodies against PRRSV N protein were obtained by immunizing BALB/c mice,cell fusion,indirect ELISA screening and by Western blot identification.The titer of the antibodies in the supernatant of the cell culture was 1∶800-1∶25600,among which the heavy chain type of 8F1,5E2,2B1 and 12E1 was IgG1.The heavy chain type of 4F1,2H2 and 5A2 was IgG2b,and the light chain type was κ type.All the 7 McAbs reacted with PRRSV infected cells by IFA,and were able to recognize three different B cell linear epitopes,which were located in the 47-57 aa,57-67 aa and 67-77 aa regions,of which 57-67 aa was a newly discovered antigen epitope.The present results would serve as a useful tool for development of a PRRSV diagnostic kit and for epidemiological investigation.