Prevention of colon injury induced by Shiga toxin 1 by recombinant plant-derived IgA

reagents

Recombinant holotoxin Stx1 and Stx1B were prepared as previously described13,49; pGEM5zf/T cloning vectors.LHCB1.1pGEM5zf/T.LHCB1.3 and pGEM5zf/P.LHCB As described earlier18; Agrobacterium tumefaciens (radioactive roots) strain GV3101 as described previously50; and Stx1B-specific IgG1 mAb (D11C6) as previously described.13,29. A. thaliana The Col-0 ecotype was obtained from the Arabidopsis Biological Resource Center (Columbus, Ohio, USA). restriction enzymes NadaI, bagI-HF, noI-HF, bagsecond and NsiI-HF was purchased from New England BioLabs (Ipswich, MA, USA); SMI, KpnI, AskedI, pRI201-AN binary vector, Klenow fragment, DNA binding groupDNA binding groupfrom Takara Bio (Shiga, Japan); littledIII from Nippon Gene (Tokyo, Japan); A protease inhibitor cocktail for plant cells and tissue extracts, the myeloma proteins TEPC 15 (rat IgA), MOPC 21 (mouse IgG1, κ), and Alcian Blue 8GX from Sigma-Aldrich (St. Louis, MO, USA); 2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), Mayer’s Hematoxylin Solution, Murashige and Skoog (MS) vegetable salt mixture, and gellan gum from Wako Pure Chemical Industries, Ltd. (Osaka, Japan); Vero cells (a kidney-derived cell line of the African green monkey) from the American Culture Collection (Manassas, VA, USA); Fetal bovine serum (FBS) from Hyclone (South Logan, Utah, USA); Cell Counting Kit – 8 from DOJINDO (Kumamoto, Japan); 4,6-Diamidino-2-phenylindole dihydrochloride (DAPI) from Nacalai Tesque (Kyoto, Japan); Pierce™ BCA Protein Assay Kit, HiMark™ Stained Protein Standards, MagicMark™ XP Western Protein Standards, SuperSignal™ West Pico PLUS Chemiluminescent Substrate, Medium 199 and Alexa594-goat anti-rabbit IgG from Thermo Fisher Scientific (Waltham, MA, USA) ); Goat anti-mouse κ, horseradish peroxidase (HRP)-anti-mouse IgA, HRP-goat anti-mouse IgG, HRP-donkey anti-goat IgG and HRP-donkey anti-rabbit IgG from Southern Biotech (Birmingham, AL, USA) ; Rabbit anti-mouse series J from Proteintech (Wuhan, China); anti-human/mouse caspase-3 from BD Biosciences (Franklin Lakes, NJ, USA); and rabbit anti-mouse MUC2 from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

mice

Female BALB/c mice were purchased from SLC Japan (Shizuoka, Japan) and used at 5-8 weeks of age. Animal care and experiments were performed in accordance with the Animal Care and Management Act, with the Guidelines for the Care and Use of Laboratory Animals published by the Ministry of Education, Culture, Sports, Science and Technology of Japan, and those of Shizuoka University. All procedures and protocols were reviewed by the Institutional Animal Care and Use Committee of Shizuoka University and approved by the President of Shizuoka University (approval numbers: 186313, 206,454). The study was reported according to ARRIVE guidelines.

Construction of a recombinant IgA expression vector

The cDNAs of the IgA heavy and light chains and the J chains of Stx1 were enhanced in Arabidopsis thaliana lettuce (Lactuca sativa), and then cloned into a cloning vector, pUC57, using GenScript (Piscataway, NJ, USA) to produce each construct (pUC57/opHc, pUC57/opLc, pUC57/opJc). A sequence corresponding to the endoplasmic reticulum retention signal peptide, KDEL (Lys-Asp-Glu-Leu), was added to the cDNA at the C-terminus of each protein. pUC57/opJc was digested using NadaI/bagThe resulting DNA fragment, opJc (cDNA), was ligated into NadaI/bagI-site of pRI201-AN with DNA-binding group, resulting in the production of pRI201/opJc. pGEM5zf/T.LHCB1.1 digested with littledIII, then the coherent ending of littledIII was terminated bluntly using the Klenow fragment. The resulting DNA fragment was digested by noI for DNA fragment TLHCB1.1. TLHCB1.1 A portion of the DNA has been linked to noI/SMI-digested pRI201/opJc, resulting in production of pRI201/opJc-TLHCB1.1. pUC57/opHc was digested with bagII /noI, in that order, to obtain (cDNA) from opHc. (cDNA) opHc has been linked to bagII /noI site pGEM5zf/T.LHCB1.3 With DNA ligation kit. The resulting plasmid, pGEM5zf/opHc-TLHCB1.3was digested with bagII /NsiI to get a piece of DNA, Hc-TLHCB1.3which was then linked to bagII /NsiI site pUC57/opLc, resulting in the production of pUC57/opLc-opHc-TLHCB1.3. DNA fragment of bagII-digestion pLHCB It was obtained from pGEM5zf/P.LHCB and inserted into bagSecond site of pUC57/opLc-opHc-TLHCB1.3. The resulting plasmid, pUC57/opLc-PLHCB-opHc-TLHCB1.3was digested with KpnI/AskedI. The resulting DNA fragment, opLc-PLHCB-opHc-TLHCB1.3with KpnI/AskedI-Digested pRI201/opJc-TLHCB1.1resulting in the production of pRI201/dimeric IgA.

Transformation Arabidopsis thaliana

transformers A. thaliana The plants were created by the floral dipping method as previously described20. A binary vector, pRI201/dimeric IgA, was introduced into A. Tumor Strain GV3101 by electroporation using Gene pulsar II (Bio-Rad, Hercules, CA, USA). The result A. Tumor It was used to injure A. thaliana To produce transgenic plants expressing Stx1-specific IgA. All experiments using plant materials have been approved with the national law (the Law on the Conservation and Sustainable Use of Biodiversity through Regulations on the Use of Living Modified Organisms) and approved by the Internal Review Committee (approval number: 618-2103). Recombinant plants were grown inside the Tier 1 Physical Containment (P1P) facility at Shizuoka University.

Protein extraction and concentration

Transgenic plants were grown on MS medium with gellan gum containing 50 mg/L kanamycin in a growth chamber, BioTRON NC220 (NK System, Osaka, Japan) at 20 °C, continuous white fluorescent light for 4 weeks. Leaves of transgenic plants were ground in liquid nitrogen and leaf proteins were extracted in protein extraction buffer (50 mM acetate (pH 5.0), 0.5 M NaCl, 0.5 mM EDTA and protease inhibitor cocktail). Extracted leaf proteins were concentrated with 50% precipitation of saturated ammonium sulfate (AmS) as previously described.19followed by ultrafiltration with Vivaspin 500 (100,000 MWCO, Sartorius, Goettingen, Germany).

Elisa

Total IgA and IgG concentrations in samples were determined by sandwich ELISA as previously described.19,29. For the IgA concentrations of leaf extracts, goat anti-mouse κ (1 μg/ml) was used as the capture antibody, and HRP-goat anti-mouse IgA (1:1000 dilution) was used as the detection antibody. IgA concentration was calculated from a standard curve generated with a commercially available mouse IgA myeloma protein, TEPC 15. For IgG1 concentrations from cultured supernatants, goat anti-mouse κ (1:1000) was used as the capture antibody, and HRP-goat IgG was used. Anti-mouse (1:1000) as a detection antibody. Mouse IgG1 myeloma MOPC 21 was used as a standard. Antigen recognition by antibodies was assessed by ELISA using inactivated Stx1B as the antigen19. Goat anti-mouse κ (1 μg/ml) plus HRP-donkey anti-goat IgG (1:1000 dilution) or HRP-donkey anti-mouse IgA (1:1000 dilution) were used as detection antibodies. ABTS was used as HRP substrate. The optical density was read at 405 nm using a microplate reader, SUNRISE Rainbow RC-R (Tecan, Salzburg, Austria).

SDS-PAGE and immunoglobulin

IgA proteins in leaf extracts were separated by SDS-PAGE using Mini-PROTEAN TXG Gel (Bio-Rad, Hercules, CA, USA) under reducing (12% gel) and non-reducing (4-15% gel) conditions. Immunoprecipitation was performed as previously described14. As detection antibodies, HRP-goat anti-mouse IgA (1:1000 dilution) was used to detect IgA heavy chain, goat anti-mouse κ (1 μg/ml) as well as HRP-donkey anti-goat IgG (1:1000 dilution) For light chain, rabbit anti-mouse J chain (0.4 μg/ml) plus anti-rabbit IGG HRP-goat (1:5000 dilution) for chain J. For visualization, HRP was reacted with SuperSignal™ West Pico PLUS Chemiluminescent Substrate and signals were detected Illuminated using the LAS-3000mini image analyzer (FUJIFILM, Tokyo, Japan). HiMark™ pre-stained high molecular weight protein standards and MagicMark™ XP Western protein standards were used as molecular weight indicators.

live cell measurement

Plant body IgA neutralizing activity was assessed by a previously described WST-8-based live cell assay .19. Vero cells were seeded at 1 × 104 Cells/well on a 96-well cell culture plate were then cultured overnight in 10% medium containing FBS 199 at 37 °C in a humidified 5% CO atmosphere.2/ 95% air. Vero cells were subjected to aliquot of the Stx1/antibody mixture prepared by pretreating 100 pg/ml of Stx1 with transgenic plant leaf extract or culture supernatant of the D11C6 hybridoma for 1 h. After 45 h culturing, the supernatants were removed, and then live cells were detected by WST-8 assay using the 8-cell counting kit following the manufacturer’s instructions. After incubation for 1.5 h with the WST-8 reagent, the optical density at 450 nm (OD450) was read using a microplate reader, SUNRISE Rainbow RC-R. To determine the number of live cells remaining after Stx1 exposure, the amount of formazan product (OD450) in each culture was compared with that without exposure to toxins (control).

Intra-rectal administration of Stx1

PBS (100 μl total) containing 0.1-1.0 μg/mL (excluding Figure 3; 5.0 μg/mL) of Stx1 holotoxin was intravaginally introduced into mice using a plastic feeding tube (20G, 38 mm; Instech Laboratories, Plymouth Meeting, PA). , USA) under anaesthesia via IP injection of a ketamine/xylazine cocktail (100 mg/kg ketamine and 10 mg/kg xylazine). Sixteen hours later, mice were sacrificed by cervical dislocation, and the distal colon (more than 5 cm below the cecum) was collected and fixed in methanol-carnoyal fixative (60% methanol, 30% chloroform and 10% acetic acid) or 10% formalin- PBS. The methanol-fixed colon was Carnoyi embedded in paraffin and the formalin-fixed colon was embedded in an OCT composite (Sakura Finetech, Tokyo, Japan). For electron microscopy analysis, the collected colon samples were opened longitudinally, fixed in 2.5% glutaraldehyde-0.1M phosphate buffer (PB) and subsequently fixed in 1% osmium tetroxide (OsO4)-0.1 M PB. For transmission electron microscopy (TEM), they were dehydrated through a gradient series of ethanol and embedding in Epon 812. Ultrathin sections were cut, stained with uranyl acetate and lead citrate, and observed under a transmission electron microscope (JEM-1010C; JEOL, Tokyo, Japan) . For scanning electron microscopy (SEM), after dehydration, samples were dried at a critical point and covered with gold using spray coating, and then observed under a scanning electron microscope (JSM-5600 LV SEM, JEOL).

histological analysis

For Alcian blue staining, 4 μM paraffin sections were obtained using an RX-860 microtome (Yamato Koki, Tokyo, Japan). Paraffin sections were dewaxed and rehydrated. Sections were fixed with 0.5% (vol/vol) glutaraldehyde in PBS for 15 min. Fixed sections were stained with 1% Alcian blue in 3% (v/v) acetic acid for 10 min and then cores were counterstained with Mayer’s hematoxylin. For immunofluorescence staining, 10 μm frozen sections were obtained using a Leica CM3050 S cryostat (Leica Biosystems, Wetzlar, Germany). The cut sections were blocked at 3% of the body surface area in PBS for 10 min at room temperature. Sections were stained with rabbit anti-mouse activated caspase 3 (2 μg/ml) plus Alexa594-goat anti-rabbit IgG (5 μg/ml) or rabbit anti-mouse MUC2 (2 μg/ml) plus Alexa594-goat anti-rabbit IgG. IgG (5 μg/ml). Nuclei were stained with DAPI (0.1 μg/ml). Immunofluorescence images were acquired using a BZ-810 fluorescence microscope (KEYECE, Osaka, Japan). The percentage of the MUC2-stained area divided by the total area of ​​the mucosal epithelium was determined using software in the microscope.

statistical analysis

Multiple comparisons were made using Tukey’s test using GraphPad Prism 7 (GraphPad Software, San Diego, CA, USA).

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