Laboratory of Microbiology and Immunology, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka City, Shizuoka, Japan.
PLoS One. 2013 Nov 28;8(11):e80712. doi: 10.1371/journal.pone.0080712. eCollection 2013.
Shiga toxin 1 (Stx1) is a virulence factor of enterohemorrhagic Escherichia coli, such as the O157:H7 strain. In the intestines, secretory IgA (SIgA) is a major component of the immune defense against pathogens and toxins. To form SIgA, the production of dimeric IgA that retains biological activity is an important step. We previously established hybrid-IgG/IgA having variable regions of the IgG specific for the binding subunit of Stx1 (Stx1B) and the heavy chain constant region of IgA. If hybrid-IgG/IgA cDNAs can be expressed in plants, therapeutic or preventive effects may be expected in people eating those plants containing a "plantibody". Here, we established transgenic Arabidopsis thaliana expressing dimeric hybrid-IgG/IgA. The heavy and light chain genes were placed under the control of a bidirectional promoter and terminator of the chlorophyll a/b-binding protein of Arabidopsis thaliana (expression cassette). This expression cassette and the J chain gene were subcloned into a single binary vector, which was then introduced into A. thaliana by means of the Agrobacterium method. Expression and assembly of the dimeric hybrid-IgG/IgA in plants were revealed by ELISA and immunoblotting. The hybrid-IgG/IgA bound to Stx1B and inhibited Stx1B binding to Gb3, as demonstrated by ELISA. When Stx1 holotoxin was pre-treated with the resulting plantibody, the cytotoxicity of Stx1 was inhibited. The toxin neutralization was also demonstrated by means of several assays including Stx1-induced phosphatidylserine translocation on the plasma membrane, caspase-3 activation and 180 base-pair DNA ladder formation due to inter-nucleosomal cleavage. These results indicate that edible plants containing hybrid-IgG/IgA against Stx1B have the potential to be used for immunotherapy against Stx1-caused food poisoning.
志贺毒素 1（Stx1）是肠出血性大肠杆菌，如 O157：H7 株的一种毒力因子。在肠道中，分泌型免疫球蛋白 A（SIgA）是针对病原体和毒素的主要免疫防御成分。为了形成 SIgA，产生保留生物活性的二聚体 IgA 是一个重要步骤。我们之前建立了具有针对 Stx1 结合亚基（Stx1B）的 IgG 特异性可变区和 IgA 重链恒定区的杂交-IgG/IgA。如果杂交-IgG/IgA cDNA 可以在植物中表达，则有望在食用含有“植物抗体”的植物的人群中产生治疗或预防效果。在这里，我们建立了表达二聚体杂交-IgG/IgA 的转基因拟南芥。重链和轻链基因置于拟南芥叶绿素 a/b 结合蛋白的双向启动子和终止子（表达盒）的控制下。该表达盒和 J 链基因被亚克隆到单个二元载体中，然后通过农杆菌方法引入拟南芥。通过 ELISA 和免疫印迹法揭示了植物中二聚体杂交-IgG/IgA 的表达和组装。杂交-IgG/IgA 与 Stx1B 结合，并抑制 Stx1B 与 Gb3 的结合，如 ELISA 所示。当 Stx1 全毒素用由此产生的植物抗体预处理时，Stx1 的细胞毒性被抑制。通过几种测定法，包括 Stx1 诱导质膜上的磷脂酰丝氨酸易位、半胱天冬酶-3 激活和由于核小体间切割导致的 180 碱基对 DNA 梯形成，也证明了毒素中和作用。这些结果表明，含有针对 Stx1B 的杂交-IgG/IgA 的可食用植物具有用于针对 Stx1 引起的食物中毒的免疫治疗的潜力。
