Laboratory of Microbiology and Immunology and the Global COE program, University of Shizuoka School of Pharmaceutical Sciences, Suruga-ku, Shizuoka, Japan.
Scand J Immunol. 2011 Dec;74(6):574-84. doi: 10.1111/j.1365-3083.2011.02617.x.
Shiga toxin 1 produced by enterohaemorrhagic Escherichia coli is an AB(5) toxin that is involved in the life-threatening haemolytic-uraemic syndrome. The B subunits (Stx1B) are cell-binding subunits. We previously established mouse hybridoma cell line producing IgA and IgG monoclonal antibodies (mAbs) against Stx1B. Here, we cloned cDNAs encoding each of the heavy, light and joining (J) chains from the hybridoma cell lines by means of the 5' rapid amplification of cDNA ends (RACE) PCR method. Upon assignment of the variable regions of the heavy and light chains to known germline sequences, we found substantial somatic hypermutation in the complementarity-determining regions in both the IgA and IgG mAbs. We also established a hybrid-IgG/IgA heavy chain having variable regions of the IgG mAb by means of recombinant PCR methods. Upon transient expression of the hybrid-IgG/IgA heavy, IgG-associated light and J chains in COS-1 cells, the translated dimeric hybrid-IgG/IgA bound to immobilized Stx1B, as revealed on ELISA. The production of dimeric hybrid-IgG/IgA was revealed on immunoblot analysis. The dimeric hybrid-IgG/IgA inhibited the binding of digoxigenin-conjugated Stx1B to natural ligands (CD77) displayed on Burkitt's lymphoma cell line Ramos. These results indicate that the replacement of variable regions resulted in the production of more useful recombinant dimeric IgA against Stx1B.
产志贺毒素 1 型大肠杆菌是一种 AB(5)毒素，与危及生命的溶血性尿毒综合征有关。B 亚单位（Stx1B）是细胞结合亚单位。我们之前建立了产生针对 Stx1B 的 IgA 和 IgG 单克隆抗体（mAb）的小鼠杂交瘤细胞系。在这里，我们通过 5' 快速扩增 cDNA 末端 (RACE) PCR 方法从杂交瘤细胞系中克隆编码重链、轻链和连接（J）链的 cDNA。在将重链和轻链的可变区分配到已知的胚系序列后，我们发现 IgG 和 IgA mAb 的互补决定区都发生了大量体细胞超突变。我们还通过重组 PCR 方法建立了具有 IgG mAb 可变区的杂交-IgG/IgA 重链。在 COS-1 细胞中转瞬表达杂交-IgG/IgA 重链、IgG 相关轻链和 J 链后，通过 ELISA 显示，翻译的二聚体杂交-IgG/IgA 与固定化 Stx1B 结合。免疫印迹分析显示了二聚体杂交-IgG/IgA 的产生。二聚体杂交-IgG/IgA 抑制了地高辛标记的 Stx1B 与 Burkitt 淋巴瘤细胞系 Ramos 上显示的天然配体（CD77）的结合。这些结果表明，可变区的替换导致产生了针对 Stx1B 的更有用的重组二聚体 IgA。
