Upon inhibitor Nintedanib expression in COS-1 cells, we found that the hybrid-IgG/IgA inhibited Stx1B binding to Gb3/CD77-positive Ramos cells. In this study, we generated transgenic Arabidopsis thaliana expressing hybrid-IgG/IgA. In order to produce SIgA in plants, the production of functional dimeric IgA is an important step, because monomeric IgA without the J chain can not form SIgA. Therefore, we focused on the production of biologically active dimeric IgA in plants. The hybrid-IgG/IgA plantibody retained Stx1B binding activity and neutralized Stx1 holotoxin in vitro. These results indicate that edible plants containing hybrid-IgG/IgA against Stx1B can be created as a possible means of immunotherapy against Stx1-caused food poisoning.

Materials and Methods Vector construction As terminators, TCAB1 and TCAB2 (positions 15,424 to 16,075 and 19,906 to 20,768, respectively, derived from the FIN18 BAC clone, accession number “type”:”entrez-nucleotide”,”attrs”:”text”:”AC008030″,”term_id”:”9910052″,”term_text”:”AC008030″AC008030) were isolated from A. thaliana genomic DNA by PCR using specific primer sets, i.e., TCAB1 fd Not and TCAB1 rv Nsi or TCAB2 fd Sac and TCAB2 rv Nsi, which are listed in Table S1. Amplified DNA fragments were inserted into the pCR?4-TOPO? TA cloning vector (Invitrogen, Carlsbad, CA, USA). Following NotI/NsiI or SacI/NsiI digestion, the TCAB terminators were ligated into the pGEM5zf vector (Promega, Madison, WI, USA). Plasmids having one of the terminators were digested with NotI, and then ligated with NotI-digested DNA fragments of the Stx1B-specific hybrid-IgG/IgA Hc or Lc cDNA [32], resulting in the production of pGEM5zf/Hc-TCAB1 and pGEM5zf/Lc-TCAB2.

The Hc-TCAB1 and Lc-TCAB2 DNA fragments were excised from pGEM5zf/Hc-TCAB1 and pGEM5zf/Lc-TCAB2, respectively, with ApaI/HindIII, and then inserted into the HindIII site of pGEM3zf (Promega) by means of three-piece ligation, resulting in the production of pGEM3zf/TCAB1-Hc-Lc-TCAB2. As a promoter, PCAB (positions 16,915 to 19,092 in the FIN18 BAC clone) was isolated from A. thaliana genomic DNA by PCR using a primer set, i.e., PCAB2F2-SacII and PCAB1R2-SacII. The resulting DNA fragments were treated with SacII and inserted into SacII-digested pGEM3zf/TCAB1-Hc-Lc-TCAB2 to create the TCAB1-Hc-PCAB-Lc-TCAB2 DNA fragments termed the hybrid-IgG/IgA expression cassette (pGEM3zf/hybrid-IgG/IgA expression cassette). Mouse J chain cDNA (accession number “type”:”entrez-nucleotide”,”attrs”:”text”:”AB644392″,”term_id”:”359334445″,”term_text”:”AB644392″AB644392) AV-951 [32] was amplified by PCR using JCF-Xba and JCR-Xho. The amplified DNA fragments were treated with XbaI/XhoI and subcloned into the XbaI/XhoI-digested pBCH1 binary vector [33], resulting in the production of pBCH1/Jc.