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Immunological activity of recombinant L. agilis strains mediated via TLR5. (a) Genotypic and phenotypic characteristics of recombinant L. agilis strains. Partial genetic map of the flagellin gene of L. agilis strains is shown. The green and red arrowheads indicate epitope-replaced and fliC2 mutated sites, respectively. FliC2 expression was detected by either Western blot with 2F5 mAb or SDS-PAGE and CBB staining. Flagellar filaments were stained with <t>FLAGELLA</t> <t>STAIN</t> (Hardy Diagnostics) and observed using optical microscopy. The motility of L. agilis strains was observed using a semisolid MRS medium. (b and c) TLR5-reporter gene assay with whole L. agilis cells from different strains (b) and their cell surface proteins (c). Bacterial cells (1.0 × 10 7 CFU/well) or cell surface proteins extracted from 1.0 × 10 7 CFU of bacterial cells were added to HEK-Blue-hTLR5 cells (2.5 × 10 4 cells/well), and released secreted alkaline phosphatase (SEAP) was measured. Values are mean plus standard error ( n = 3). Statistical significance was determined by one-way ANOVA with Tukey multiple comparisons test. Different letters represent statistical significance.
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Immunological activity of recombinant L. agilis strains mediated via TLR5. (a) Genotypic and phenotypic characteristics of recombinant L. agilis strains. Partial genetic map of the flagellin gene of L. agilis strains is shown. The green and red arrowheads indicate epitope-replaced and fliC2 mutated sites, respectively. FliC2 expression was detected by either Western blot with 2F5 mAb or SDS-PAGE and CBB staining. Flagellar filaments were stained with <t>FLAGELLA</t> <t>STAIN</t> (Hardy Diagnostics) and observed using optical microscopy. The motility of L. agilis strains was observed using a semisolid MRS medium. (b and c) TLR5-reporter gene assay with whole L. agilis cells from different strains (b) and their cell surface proteins (c). Bacterial cells (1.0 × 10 7 CFU/well) or cell surface proteins extracted from 1.0 × 10 7 CFU of bacterial cells were added to HEK-Blue-hTLR5 cells (2.5 × 10 4 cells/well), and released secreted alkaline phosphatase (SEAP) was measured. Values are mean plus standard error ( n = 3). Statistical significance was determined by one-way ANOVA with Tukey multiple comparisons test. Different letters represent statistical significance.
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Immunological activity of recombinant L. agilis strains mediated via TLR5. (a) Genotypic and phenotypic characteristics of recombinant L. agilis strains. Partial genetic map of the flagellin gene of L. agilis strains is shown. The green and red arrowheads indicate epitope-replaced and fliC2 mutated sites, respectively. FliC2 expression was detected by either Western blot with 2F5 mAb or SDS-PAGE and CBB staining. Flagellar filaments were stained with <t>FLAGELLA</t> <t>STAIN</t> (Hardy Diagnostics) and observed using optical microscopy. The motility of L. agilis strains was observed using a semisolid MRS medium. (b and c) TLR5-reporter gene assay with whole L. agilis cells from different strains (b) and their cell surface proteins (c). Bacterial cells (1.0 × 10 7 CFU/well) or cell surface proteins extracted from 1.0 × 10 7 CFU of bacterial cells were added to HEK-Blue-hTLR5 cells (2.5 × 10 4 cells/well), and released secreted alkaline phosphatase (SEAP) was measured. Values are mean plus standard error ( n = 3). Statistical significance was determined by one-way ANOVA with Tukey multiple comparisons test. Different letters represent statistical significance.
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Immunological activity of recombinant L. agilis strains mediated via TLR5. (a) Genotypic and phenotypic characteristics of recombinant L. agilis strains. Partial genetic map of the flagellin gene of L. agilis strains is shown. The green and red arrowheads indicate epitope-replaced and fliC2 mutated sites, respectively. FliC2 expression was detected by either Western blot with 2F5 mAb or SDS-PAGE and CBB staining. Flagellar filaments were stained with <t>FLAGELLA</t> <t>STAIN</t> (Hardy Diagnostics) and observed using optical microscopy. The motility of L. agilis strains was observed using a semisolid MRS medium. (b and c) TLR5-reporter gene assay with whole L. agilis cells from different strains (b) and their cell surface proteins (c). Bacterial cells (1.0 × 10 7 CFU/well) or cell surface proteins extracted from 1.0 × 10 7 CFU of bacterial cells were added to HEK-Blue-hTLR5 cells (2.5 × 10 4 cells/well), and released secreted alkaline phosphatase (SEAP) was measured. Values are mean plus standard error ( n = 3). Statistical significance was determined by one-way ANOVA with Tukey multiple comparisons test. Different letters represent statistical significance.
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Immunological activity of recombinant L. agilis strains mediated via TLR5. (a) Genotypic and phenotypic characteristics of recombinant L. agilis strains. Partial genetic map of the flagellin gene of L. agilis strains is shown. The green and red arrowheads indicate epitope-replaced and fliC2 mutated sites, respectively. FliC2 expression was detected by either Western blot with 2F5 mAb or SDS-PAGE and CBB staining. Flagellar filaments were stained with <t>FLAGELLA</t> <t>STAIN</t> (Hardy Diagnostics) and observed using optical microscopy. The motility of L. agilis strains was observed using a semisolid MRS medium. (b and c) TLR5-reporter gene assay with whole L. agilis cells from different strains (b) and their cell surface proteins (c). Bacterial cells (1.0 × 10 7 CFU/well) or cell surface proteins extracted from 1.0 × 10 7 CFU of bacterial cells were added to HEK-Blue-hTLR5 cells (2.5 × 10 4 cells/well), and released secreted alkaline phosphatase (SEAP) was measured. Values are mean plus standard error ( n = 3). Statistical significance was determined by one-way ANOVA with Tukey multiple comparisons test. Different letters represent statistical significance.
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Immunological activity of recombinant L. agilis strains mediated via TLR5. (a) Genotypic and phenotypic characteristics of recombinant L. agilis strains. Partial genetic map of the flagellin gene of L. agilis strains is shown. The green and red arrowheads indicate epitope-replaced and fliC2 mutated sites, respectively. FliC2 expression was detected by either Western blot with 2F5 mAb or SDS-PAGE and CBB staining. Flagellar filaments were stained with <t>FLAGELLA</t> <t>STAIN</t> (Hardy Diagnostics) and observed using optical microscopy. The motility of L. agilis strains was observed using a semisolid MRS medium. (b and c) TLR5-reporter gene assay with whole L. agilis cells from different strains (b) and their cell surface proteins (c). Bacterial cells (1.0 × 10 7 CFU/well) or cell surface proteins extracted from 1.0 × 10 7 CFU of bacterial cells were added to HEK-Blue-hTLR5 cells (2.5 × 10 4 cells/well), and released secreted alkaline phosphatase (SEAP) was measured. Values are mean plus standard error ( n = 3). Statistical significance was determined by one-way ANOVA with Tukey multiple comparisons test. Different letters represent statistical significance.
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Immunological activity of recombinant L. agilis strains mediated via TLR5. (a) Genotypic and phenotypic characteristics of recombinant L. agilis strains. Partial genetic map of the flagellin gene of L. agilis strains is shown. The green and red arrowheads indicate epitope-replaced and fliC2 mutated sites, respectively. FliC2 expression was detected by either Western blot with 2F5 mAb or SDS-PAGE and CBB staining. Flagellar filaments were stained with <t>FLAGELLA</t> <t>STAIN</t> (Hardy Diagnostics) and observed using optical microscopy. The motility of L. agilis strains was observed using a semisolid MRS medium. (b and c) TLR5-reporter gene assay with whole L. agilis cells from different strains (b) and their cell surface proteins (c). Bacterial cells (1.0 × 10 7 CFU/well) or cell surface proteins extracted from 1.0 × 10 7 CFU of bacterial cells were added to HEK-Blue-hTLR5 cells (2.5 × 10 4 cells/well), and released secreted alkaline phosphatase (SEAP) was measured. Values are mean plus standard error ( n = 3). Statistical significance was determined by one-way ANOVA with Tukey multiple comparisons test. Different letters represent statistical significance.
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Immunological activity of recombinant L. agilis strains mediated via TLR5. (a) Genotypic and phenotypic characteristics of recombinant L. agilis strains. Partial genetic map of the flagellin gene of L. agilis strains is shown. The green and red arrowheads indicate epitope-replaced and fliC2 mutated sites, respectively. FliC2 expression was detected by either Western blot with 2F5 mAb or SDS-PAGE and CBB staining. Flagellar filaments were stained with FLAGELLA STAIN (Hardy Diagnostics) and observed using optical microscopy. The motility of L. agilis strains was observed using a semisolid MRS medium. (b and c) TLR5-reporter gene assay with whole L. agilis cells from different strains (b) and their cell surface proteins (c). Bacterial cells (1.0 × 10 7 CFU/well) or cell surface proteins extracted from 1.0 × 10 7 CFU of bacterial cells were added to HEK-Blue-hTLR5 cells (2.5 × 10 4 cells/well), and released secreted alkaline phosphatase (SEAP) was measured. Values are mean plus standard error ( n = 3). Statistical significance was determined by one-way ANOVA with Tukey multiple comparisons test. Different letters represent statistical significance.

Journal: Applied and Environmental Microbiology

Article Title: Harnessing flagellin of Ligilactobacillus agilis as a surface display scaffold for an HIV-1 epitope

doi: 10.1128/aem.00674-25

Figure Lengend Snippet: Immunological activity of recombinant L. agilis strains mediated via TLR5. (a) Genotypic and phenotypic characteristics of recombinant L. agilis strains. Partial genetic map of the flagellin gene of L. agilis strains is shown. The green and red arrowheads indicate epitope-replaced and fliC2 mutated sites, respectively. FliC2 expression was detected by either Western blot with 2F5 mAb or SDS-PAGE and CBB staining. Flagellar filaments were stained with FLAGELLA STAIN (Hardy Diagnostics) and observed using optical microscopy. The motility of L. agilis strains was observed using a semisolid MRS medium. (b and c) TLR5-reporter gene assay with whole L. agilis cells from different strains (b) and their cell surface proteins (c). Bacterial cells (1.0 × 10 7 CFU/well) or cell surface proteins extracted from 1.0 × 10 7 CFU of bacterial cells were added to HEK-Blue-hTLR5 cells (2.5 × 10 4 cells/well), and released secreted alkaline phosphatase (SEAP) was measured. Values are mean plus standard error ( n = 3). Statistical significance was determined by one-way ANOVA with Tukey multiple comparisons test. Different letters represent statistical significance.

Article Snippet: The flagellar filaments of L. agilis strains were stained with FLAGELLA STAIN (Hardy Diagnostics) according to the manufacturer’s protocol.

Techniques: Activity Assay, Recombinant, Expressing, Western Blot, SDS Page, Staining, Microscopy, Reporter Gene Assay