非洲猪瘟病毒p30蛋白单克隆抗体的制备与鉴定

Preparation and identification of monoclonal antibodies against p30 protein of the African swine fever virus

为制备非洲猪瘟病毒(ASFV)p30蛋白的单克隆抗体,以原核表达的重组p30蛋白为免疫原免疫6~8周龄雌性BALB/c小鼠,3次免疫后分离小鼠脾细胞并将其与SP2/0细胞进行融合,通过间接ELISA方法筛选,获得2株能稳定分泌抗ASFV p30蛋白单克隆抗体的杂交瘤细胞株。2株杂交瘤细胞从第10代开始均能稳定分泌抗体,细胞上清抗体效价均为1∶6400。亚型鉴定结果显示,2株单克隆抗体的重链均属于IgG1,轻链均为κ型。Western blot和间接免疫荧光试验(IFA)结果显示,2株单克隆抗体均能与ASFV感染的猪肺泡巨噬细胞发生特异性反应。试验结果为p30蛋白生物学和诊断方法研究提供了技术支持。

The purpose of this study was to prepare monoclonal antibodies against the p30 protein of the African swine fever(ASFV).6-8 week-old female BALB/c mice were immunized with recombinant p30 proteins expressed in a prokaryotic system.After booster immunization for three times,the spleen cells of the mice were obtained and fused with SP2/0 cells.By indirect ELISA,two hybridoma cell lines that steadily secreted monoclonal antibodies against ASFV p30 proteins were obtained.The two hybridoma cells were able to consistently secreted antibodies from the 10th generation,and the antibody titer of the cell supernatant was 1∶6400.The results of subtype identification showed that the heavy chains of the two monoclonal antibodies belonged to IgG1,and the light chains were of theκtype.Western blot and indirect immunofluorescence assay(IFA)showed that both monoclonal antibodies specifically reacted with ASFV-infected porcine alveolar macrophages,which indicated that the present study provided technological support for research on the biology of the viral p30 protein and on related diagnostic methods.