非洲猪瘟病毒H240R蛋白单克隆抗体的筛选与鉴定

Screening and Identification of Monoclonal Antibodies against African Swine Fever Virus H240R Protein

为制备抗非洲猪瘟病毒(ASFV)H240R蛋白单克隆抗体,首先构建原核表达质粒pET-29b-H240R,再进行蛋白表达与纯化,随后免疫Balb/c小鼠,通过杂交瘤细胞技术制备、间接ELISA方法筛选获得单克隆抗体。结果显示,共获得4株杂交瘤细胞株,腹水效价均高于1∶40000;亚型鉴定结果显示,3株单克隆抗体(12D6、21G3、8G7)重链亚类为IgG2b,13D2重链亚类为IgG1,轻链亚类均为k。Western blot和Dot blot鉴定结果表明,21G3、13D2株单克隆抗体识别的抗原表位为线性表位,而12D6和8G7识别的是构象表位。间接免疫荧光试验(IFA)结果显示,4株单克隆抗体均能特异性识别HEK293T细胞中表达的H240R蛋白。制备了ASFV H240R蛋白单克隆抗体,为H240R蛋白的表位和功能研究奠定了基础。

To develop monoclonal antibodies(mAbs)against African swine fever virus(ASFV)H240 R protein,a prokaryotic expression plasmid pET-29 b-H240 R was first constructed,followed by recombinant protein expression and purification,followed by immunization of Balb/c mice.The monoclonal antibodies were obtained by hybridoma cell technology preparation and indirect ELISA screening method.The results showed that four hybridoma cell lines were obtained,and the ascites potency was higher than 1:40000;the subtype identification results showed that the heavy chain subclass of three monoclonal antibodies(12 D6,21 G3 and 8 G7)was IgG2 b,the heavy chain subclass of 13 D2 was IgG1,and the light chain subclass was k.Western blot and Dot blot identification showed that the monoclonal antibodies of strains 21 G3 and 13 D2 recognized linear epitopes of antigen,while 12 D6 and 8 G7 recognized conformational epitopes.The indirect immunofluorescence assay(IFA)showed that all four monoclonal antibodies specifically recognized the H240 R protein expressed in HEK293 T cells.In this study,mAbs against ASFV H240 R protein were successfully prepared,providing a powerful tool for epitope and function studies of H240 R protein.