非洲猪瘟病毒p54蛋白单抗制备及B细胞线性抗原表位鉴定

Development and B cell linear mapping of monoclonal antibodies against African swine fever virus protein p54

以原核系统表达的非洲猪瘟病毒p54重组蛋白为免疫原接种BALB/c小鼠,经过细胞融合、间接ELISA筛选和多次亚克隆获得了3株能够稳定分泌抗p54蛋白单克隆抗体的杂交瘤细胞系,分别命名为2A4、5F6和RH15。3株单克隆抗体的重链均属于IgG1亚类,轻链均为Kappa型。Western blot和免疫荧光试验(IFA)证明3株单克隆抗体具有良好的反应性,可以用于靶抗原的特异性检测。通过噬菌体展示肽库筛选,确定了单克隆抗体2A4、5F6识别的抗原表位为^(157)NTASQ^(161),RH15识别的抗原表位为^(170)RQRNTYTHKDL^(180),进一步完善了靶抗原的B细胞线性抗原表位信息。

In this study,the recombinant p54 protein of African swine fever virus was expressed in a prokaryotic system and was used as an immunogen to vaccinate BALB/c mice.Then,three hybridoma cell lines that could stably secrete mouse anti-p54 monoclonal antibodies(mAbs)were prepared through cell fusion,indirect ELISA screening,and multiple sub-cloning.These cell lines were named 2A4,5F6,and RH15,respectively.The heavy chains of the three mAbs belonged to the IgG1,and the light chains were all of the Kappa type.Western blot and indirect immunofluorescence assay showed that the three mAbs exhibited good reactivity and were capable of detecting the target antigen specifically.Epitopes were identified by screening of phage-displayed peptide libraries,which showed that anti-p54 mAbs 2A4 and 5F6 recognized the same epitope ^(157)NTASQ^(161),and RH15 recognized ^(170)RQRNTYTHKDL^(180),and further improved the B cell linear epitope mapping of the target antigen.