非洲猪瘟病毒pp62蛋白单克隆抗体的制备和鉴定

Generation and characterization of monoclonal antibodies against pp62 protein of African swine fever virus

pp62蛋白是非洲猪瘟病毒(ASFV)CP530R基因编码的多聚蛋白前体,可被蛋白水解酶切割形成成熟蛋白p35、p15和p8,与pp220多聚蛋白前体的加工产物组装成病毒核壳。本研究参考ASFV China/2018/AnhuiXCGQ毒株基因序列,分析后选取CP530R基因N端(1—612 bp)及C端(784—1593 bp),分别构建原核表达质粒pET-28a-pp62-N和pET-28a-pp62-C;转化Rosetta感受态细胞,IPTG诱导表达pp62蛋白;再用纯化蛋白免疫BALB/c小鼠,经细胞融合和阳性克隆筛选后,获得4株可稳定分泌pp62单克隆抗体的杂交瘤细胞。Western-blot检测ASFV感染PAM细胞中pp62剪切体的结果显示,1株仅识别pp62蛋白,1株仅识别p35蛋白,1株仅识别p15蛋白,1株可识别pp62和p8蛋白。IFA鉴定结果显示,4株均能与ASFV感染PAM细胞产生特异性绿色荧光。本研究为建立ASFV诊断方法和研究pp62的生物学功能奠定了生物材料基础。

pp62,a polyprotein precursor encoded by CP530R of African swine fever virus(ASFV),can be proteolyzed and generate mature proteins,p35 p15 and p8,which forms viral core shell with processed products of another polyprotein precursor pp220.In this study,the N-terminal(1—612 bp)and C-terminal(784—1593 bp)fragments of the CP530R gene of the ASFV strain China/2018/AnhuiXCGQ,were selected and cloned into the prokaryotic expression plasmid pET-28a-pp62-N and pET-28a-pp62-C,then transformed into E.coli Rosetta and induced by IPTG to express pp62 protein.Then BALB/c mice were immunized with purified proteins.After cell fusion and positive clone screening,four positive hybridoma cells that could stably secrete pp62 monoclonal antibodies were obtained.The detection of pp62 isoforms in PAM cells infected with ASFV by Western-blot showed that one strain can only recognize pp62 precursor,one strain can only recognize p15 protein,one strain can only recognize p35 protein,and one strain can recognize both pp62 and p8 proteins.Monoclonal antibodies were analyzed by IFA,and the results showed that all strains can recognize ASFV-infected PAM cells to produce specific green fluorescence.This study provides biological materials for the establishment of ASFV diagnostic methods and further exploring biological functions of ASFV pp62.