Western blot analysis of fusion genes expression.

<p>Spore coat proteins were extracted and analyzed by western blotting with anti-influenza M2 mAb. Numbers of lanes refer to the strain used in experiment: 1-<i>B</i>. <i>subtilis</i> 168; 2-BTL20(CotC-M2eH-A-S-H, ~20 kDa); 3-BTL21(CotB-M2eH-A-S-H, ~43 kDa); 4-BTL23(CotB-LINKER-M2eH-A-S-H, ~44 kDa); 5-BTL22(CotZ-M2eH-A-S-H, ~28 kDa); 6-BTL24(CotZ-LINKER-M2eH-A-S-H, ~29 kDa); 7-BTL25(CgeA-M2eH-A-S-H, ~26 kDa); 8-BTL26(CgeA-LINKER-M2eH-A-S-H, ~27 kDa).</p

Strain list.

<p>Strain list.</p

List of PCR oligonucleotides.

<p>List of PCR oligonucleotides.</p

Antigen accessibility to antibodies in different constructs.

<p>Intact spores were incubated with mouse anti-M2e primary antibody followed by incubation with goat anti-mouse IgG peroxidase secondary antibodies. 10⁴ spores were incubated in TMB substrate solution to develop color reaction. Absorbance was measured at 450 nm and data are given as arithmetic mean with ±SD from three independent experiments.</p

ELISA of anti-M2e IgG isotyping.

<p>IgG1, IgG2a and IgG2b subtypes were tested using mouse sera from 61^st day of immunization. The titers were expressed as the endpoint dilutions that remained positively detectable. The data are presented as arithmetic mean ±SD of 3 mice per group.</p

Densitometric analysis of protein expression.

<p>Densitometric analysis of protein expression.</p

Germinantion of recombinant and parental strain induced by L-alanine and AGFK.

<p>Germination was followed by measuring the absorbance decrease at 600 nm of the spore suspension. Data are given as arithmetic mean from three independent experiments.</p

Schematic steps of building strains carrying fusion genes.

<p>Schematic steps of building strains carrying fusion genes.</p

Western blot analysis of sera from immunized mice.

<p>5 groups of 3 mice (A, B, C) were orally immunized with recombinant spores of strains <i>B</i>. <i>subtilis</i> 168; 2-BTL20(CotC-M2eH-A-S-H), BTL21(CotB-M2eH-A-S-H), BTL22(CotZ-M2eH-A-S-H) or BTL25(CgeA-M2eH-A-S-H). A 15 μg of recombinant M2eH-A-S-H-GST was separated in polyacrylamide gel and further electrotransferred on nitrocellulose which was further incubated with sera from immunized mice as a primary antibodies. Blots were developed using anti-mouse alkaline phosphatase conjugates. Figure is a merge of 20 different single-lane blot strips.</p

Immunofluorescence staining of recombinant spores.

<p>Purified spores were incubated with anti-influenza M2 mAb, followed by anti-mouse Cy3 conjugates. Spores were visualized by phase contrast (PC) and immunofluorescence (IF) microscopy. The same exposure time was used for all IF images.</p