猪胸膜肺炎放线杆菌ApxⅠA蛋白的原核表达及单克隆抗体的制备

Preparation of Monoclonal Antibody Against Actinobacillus pleuropneumoniae ApxⅠA Protein

以猪胸膜肺炎放线杆菌(APP)血清10型标准株ApxⅠ天然外毒素为免疫抗原,接种Balb/c小鼠;设计编码ApxⅠN-端具有保护性抗原表位apxⅠA基因(1 149bp)的引物,构建原核表达载体pET-32a-apxⅠA,转化至E.coli BL21,获得分子质量约为41ku的rApxⅠA作为包被抗原,用间接ELISA方法筛选单克隆融合细胞上清,共获得2株稳定分泌抗ApxⅠA蛋白mAb的杂交瘤细胞系,亚类鉴定分别为IgG3、IgG2a,2株mAb能特异性地识别ApxⅠA蛋白,为ApxⅠA诊断试剂的研究奠定了基础。

Balb/c mice were immunized with purified APP serotype 10 out toxin ApxⅠ . The 1 149 bp Apx I A gene was cloned to construct recombinant plasmid pET-32a,then transformed into E. coli BL21. The expressed 41 ku fusion protein rApx Ⅰ A as coating antigen. Cell culture supernatant was checked for Apx I A antibody using indirected ELISA coated with rApx I A. Two strains secreting anti-Apx I A antigen monoclonal antibody hybridoma cell lines were successfully established, mAb Stypesub were IgG3 and IgG2a respectively. This study provided a foundation for the development of diagnostic reagent.