Khan et al Table I. Primers Used for Fusion of Peptides to Enhanced Green Fluorescent Protein and Plasmid Circularization. Gb- GbI-F:5-GATCGCATGCATACCCAGTAACAAAGCCCGAAAGGAAGCTG-3 GbI-R: 5-ATGATCATAGTTCGGATGGCTGCCGCCGCCCTTGTACA-3 Gb.5-AGCCATCCGAACTATGATCATGATCGCATGCATACCCAG-3 Gb2-F: 5-GATATGCCGGGCGCGCATTAACAAAGCCCGAAAGGAAGCTG-3 Gb2-R: 5-ATGCGCGCCCGGCATATCGCTGCCGCCGCCCTTGTACA-3 Gb2-cir: 5-AGCTTTCGCGTGATTTATACCGATATGCCGGGCGCGCAT-3 Gb-3 Gb3-F: 5-CATCCGGTGAACACCGGCTAACAAAGCCCGAAAGGAAGCTG-3 Gb3-R: 5-TCCGCATACTGCATGCTGCTGCCGCCGCCCTTGTACA-3 Gb3-cir: 5-AGCAGCATGCAGTATGCGGATCATCCGGTGAACACCGGC-3 pRimers used for circularization of linear constructs blocked with 5% BSA in water for 2 h at 4C. After from overnight fasted mice and 0. 2 mg of recombinant wells were subjected to incubation with different co Ag-fused EGFPs or EGFP alone was injected into the ligated tions of FITC-conjugated peptides for I h, followed by segment. After l-h incubation at 37C, the ligated segment tion with HRP-conjugated anti-FITC antibody was excised, washed with phosphate-buffered saline(PBs)to dilution). The reaction was visualized with 100 HL HRP sub- remove the internal contents, and fixed with 4% PFA. [AQ: 4] strate and ODs was measured. Specific binding of each pep- For whole-mount staining, PPs were stained with anti-GP2 ide to GP-2 was calculated by subtracting the OD value of (Abcam, xXX, UK) followed by allophycocyanin-conjugated each peptide binding to BSA-coated wells from the OD value anti-rabbit IgG, whole mounted with antifade medium. Frozen that peptide binding to GP-2. Assays were performed three sections(10 um) were prepared after freezing PPs in OC times in duplicate. Binding affinity of each peptide was calcu-(Takara, XXX, Japan), blocked with 2.5% BSA and stained lated by nonlinear regression and transformed to Scatchard with anti-GP2, counterstained with 4, 6-diamidino-2-phenyl plot using the GraphPad Prism 6 program( GraphPad Software, indole ( DAPD), and analyzed by confocal laser scanning La Jolla, CA). microscopy(CLSM)(LSM 710: Carl Zeiss, Thornwood Next, we performed competition ELISA to investiga NY).[AQ: 5] the epitope binding sites on GP-2 using the above method. Production of Peptide-Conjugated Mice Immunization and Measurement of Ag- Specific Immune Responses We used enhanced green fluorescence protein(EGFP) as a Groups of five female BALB/c mice between 4 and 6 weeks model antigen and fused selected peptides to the C-terminal of of age were immunized with 100 ug of experimental anti EGFP. The EGFP gene was amplified by PCR from pEGFP- gen/PBS by oral gavage once every week for 6 weeks CI(Clontech, XXX) using primers F: 5'-GTGAGCAAGG Five days after the last immunization, serum and fecal GCGAGGAGCTG-3 and R: 5'-CTTGTACAGCTCGTCCA extracts were collected to monitor EGFP-specific systemic TGCCG-3. Fusion of DNA sequences of the selected peptides IgG and mucosal Ig A as described by Hackett et al. Since and insertion of the resulting construct into pET-28a was IgA in fecal extracts is rapidly degraded by the proteases achieved by our newly introduced single primer-mediated cir- from enteric bacteria, I mM phenylmethyl sulfonyl fluoride cular PCR using primers as shown in Table 1 Protein(PMFS) protease inhibitor was included in the extraction was expressed in BL21 (DE3)and purified on HisTrap FF col- cocktail. Ab titers were expressed as the reciprocal log2 of umns(GE Healthcare Bio-Sciences, xXX) according to the the highest sample dilutions that gave an ODsn of 0.08 manufacturer's instructions Purified protein was confirmed by which was the value of the PBS blank. sodium dodecyl sulfate polyacrylamide gel electrophoresis Lymphocytes from spleen(SPLs)and PPs(PPLs)were iso (SDS-PAGE)and Western blot using anti-His Tag and anti- lated, minced, and digested with 300 U/mL Collagenase D EGFP antibodies. [AQ: 3] (Roche, Mannheim, Germany) for 30 min at 37C. The digested mixture was passed through a nylon mesh to remove Ex Vivo Ag Uptake Assay undigested tissue and subjected to Percoll(Sigma-Aldrich, Louis, MO)density gradient centrifugation. Cells at the inter- Ex vivo Ag uptake of the selected ligands by M cells was face between 40% and 75% Percoll were collected as mono- assessed by gut loop assay as described previously. A gut nuclear cells and subjected to characterization for lymphocyte loop containing one or two Peyers patches(PPs)was prepared proliferation and cytokine secretion.Khan et al. 3 blocked with 5% BSA in water for 2 h at 4 °C. After washing, wells were subjected to incubation with different concentra￾tions of FITC-conjugated peptides for 1 h, followed by incuba￾tion with HRP-conjugated anti-FITC antibody (1:5000 dilution). The reaction was visualized with 100 µL HRP sub￾strate and OD450 was measured. Specific binding of each pep￾tide to GP-2 was calculated by subtracting the OD value of each peptide binding to BSA-coated wells from the OD value of that peptide binding to GP-2. Assays were performed three times in duplicate. Binding affinity of each peptide was calcu￾lated by nonlinear regression and transformed to Scatchard plot using the GraphPad Prism 6 program (GraphPad Software, La Jolla, CA). Next, we performed competition ELISA to investigate the epitope binding sites on GP-2 using the above method. Production of Peptide-Conjugated Recombinant Ag We used enhanced green fluorescence protein (EGFP) as a model antigen and fused selected peptides to the C-terminal of EGFP. The EGFP gene was amplified by PCR from pEGFP￾C1 (Clontech, XXX) using primers F: 5′-GTGAGCAAGG GCGAGGAGCTG-3′ and R: 5′-CTTGTACAGCTCGTCCA TGCCG-3′. Fusion of DNA sequences of the selected peptides and insertion of the resulting construct into pET-28a was achieved by our newly introduced single primer–mediated cir￾cular PCR method13 using primers as shown in Table 1. Protein was expressed in BL21 (DE3) and purified on HisTrap FF col￾umns (GE Healthcare Bio-Sciences, XXX) according to the manufacturer’s instructions. Purified protein was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot using anti-HisTag and anti￾EGFP antibodies.[AQ: 3] Ex Vivo Ag Uptake Assay Ex vivo Ag uptake of the selected ligands by M cells was assessed by gut loop assay as described previously.14 A gut loop containing one or two Peyer’s patches (PPs) was prepared from overnight fasted mice and 0.2 mg of recombinant Ag-fused EGFPs or EGFP alone was injected into the ligated segment. After 1-h incubation at 37 °C, the ligated segment was excised, washed with phosphate-buffered saline (PBS) to remove the internal contents, and fixed with 4% PFA.[AQ: 4] For whole-mount staining, PPs were stained with anti-GP2 (Abcam, XXX, UK) followed by allophycocyanin-conjugated anti-rabbit IgG, whole mounted with antifade medium. Frozen sections (10 µm) were prepared after freezing PPs in OCT (Takara, XXX, Japan), blocked with 2.5% BSA and stained with anti-GP2, counterstained with 4′,6-diamidino-2-phenyl￾indole (DAPI), and analyzed by confocal laser scanning microscopy (CLSM) (LSM 710; Carl Zeiss, Thornwood, NY).[AQ: 5] Mice Immunization and Measurement of Ag￾Specific Immune Responses Groups of five female BALB/c mice between 4 and 6 weeks of age were immunized with 100 µg of experimental anti￾gen/PBS by oral gavage once every week for 6 weeks.15 Five days after the last immunization, serum and fecal extracts were collected to monitor EGFP-specific systemic IgG and mucosal IgA as described by Hackett et al.16 Since IgA in fecal extracts is rapidly degraded by the proteases from enteric bacteria, 1 mM phenylmethyl sulfonyl fluoride (PMFS) protease inhibitor was included in the extraction cocktail.17 Ab titers were expressed as the reciprocal log2 of the highest sample dilutions that gave an OD450 of 0.08, which was the value of the PBS blank. Lymphocytes from spleen (SPLs) and PPs (PPLs) were iso￾lated, minced, and digested with 300 U/mL Collagenase D (Roche, Mannheim, Germany) for 30 min at 37 °C. The digested mixture was passed through a nylon mesh to remove undigested tissue and subjected to Percoll (Sigma-Aldrich, St. Louis, MO) density gradient centrifugation. Cells at the inter￾face between 40% and 75% Percoll were collected as mono￾nuclear cells and subjected to characterization for lymphocyte proliferation and cytokine secretion. Table 1. Primers Used for Fusion of Peptides to Enhanced Green Fluorescent Protein and Plasmid Circularization. Peptide Primer Gb-1 Gb1-F: 5-GATCGCATGCATACCCAGTAACAAAGCCCGAAAGGAAGCTG-3 Gb1-R: 5-ATGATCATAGTTCGGATGGCTGCCGCCGCCCTTGTACA-3 Gb1-cira : 5-AGCCATCCGAACTATGATCATGATCGCATGCATACCCAG-3 Gb-2 Gb2-F: 5-GATATGCCGGGCGCGCATTAACAAAGCCCGAAAGGAAGCTG-3 Gb2-R: 5-ATGCGCGCCCGGCATATCGCTGCCGCCGCCCTTGTACA-3 Gb2-cir: 5-AGCTTTCGCGTGATTTATACCGATATGCCGGGCGCGCAT-3 Gb-3 Gb3-F: 5-CATCCGGTGAACACCGGCTAACAAAGCCCGAAAGGAAGCTG-3 Gb3-R: 5-TCCGCATACTGCATGCTGCTGCCGCCGCCCTTGTACA-3 Gb3-cir: 5-AGCAGCATGCAGTATGCGGATCATCCGGTGAACACCGGC-3 a Primers used for circularization of linear constructs