SDS-PAGE and Western blot analysis of rOmpU expressed in <i>E</i>. <i>coli</i> BL21 (DE3).

<p>(<b>A</b>) M1, protein molecular weight standard; Lane 1, whole cell bacterial lysate after induction of pET-32a-OmpU/BL21 (DE3); Lane 2, soluble fraction part of the baterial lysate; Lane 3, purified rOmpU. (<b>B</b>) M2, protein molecular weight standard; Lane 1, Western blot of the purified rOmpU with OmpU-specific antibodies.</p

Amino acid sequenc of linear B-cell epitope peptide of OmpU protein.

<p>Amino acid sequenc of linear B-cell epitope peptide of OmpU protein.</p

Binding of OmpU derived epitope to EPC cells as determined by flow cytometry.

<p>EPC cells were co-incubated with (<b>A</b>) PBS, (<b>B</b>) an irrelevant FITC-labeled peptide, (<b>C</b>) unconjugated FITC dye, FITC-labeled epitope peptide 25–36 (<b>D</b>), 90–101(<b>E</b>), 115–126 (<b>F</b>), 173–192 (<b>G</b>), 211–222 (<b>H</b>), 239–250 (<b>I</b>), and cell surface binding was assessed by flow cytometry.</p

Inhibition of <i>V</i>.<i>mimicus</i> adhesion to EPC cells in the presence of OmpU-binding peptide determined by adhesion inhibition assay.

<p><b>(A)</b> Micrographs of control EPC cells stained with crystal violet; <b>(B)</b> Adhesion of <i>V</i>.<i>mimicus</i> 04–14 isolate to EPC cells, same staining as in (A). <b>(C)</b> Adhesion of 04–14 isolate to EPC cells after treatment with an irrelevant peptide same staining as in (A). <b>(D), (E) and (F)</b> Adhesion of 04–14 isolate to EPC cells after treatment with OmpU-binding peptide 1, 2, 3, respectively, same staining as in (A). Magnification 1000 ×. All scale bars are 5μm. Arrows show <i>V</i>. <i>mimicus</i> 04–14 isolate.</p

Binding reaction curve of adhesion antagonistic peptide—rOmpU protein interactions determined by solid phase non-competitive ELISA method.

<p><b>(A)</b> Binding reaction curve between P1 (from 2.5 to 40 μg/mL) and rOmpU fusion protein (from 0.2 to 12.8 μg/mL) at four different concentrations, with OD₄₉₂nm values as the ordinate and adhesion antagonistic peptide concentration as the abscissa. <b>(B)</b> Binding reaction curve between P2 and rOmpU fusion protein at the same concentrations as in (A).</p

Protective effects of the adhesion antagonistic peptides on grass carps challenged with <i>V</i>. <i>mimicus</i>.

<p>Protective effects of the adhesion antagonistic peptides on grass carps challenged with <i>V</i>. <i>mimicus</i>.</p

Deduced binding reaction curve equation of adhesion antagonistic peptide—rOmpU fusion protein and the corresponding peptide concentration at 50% OD_492nm value in the curve.

<p>Deduced binding reaction curve equation of adhesion antagonistic peptide—rOmpU fusion protein and the corresponding peptide concentration at 50% OD_492nm value in the curve.</p

OmpU-binding peptide sequences obtained by screening phage random 12-peptide library against rOmpU fusion protein.

<p>OmpU-binding peptide sequences obtained by screening phage random 12-peptide library against rOmpU fusion protein.</p

Phage ELISA OmpU-binding assay.

<p>Purified rOmpU fusion protein was served as coated antigen, bound phage was detected with HRP-conjugated anti-M13 antibody. Wild-type M13 phage was served as negative control. Data presented are the mean OD_490nm values (±SD) of triplicate samples, and the error bars indicate the standard deviations from the means. The asterisk represents the preliminary positive clones whose OD_490nm values are three times greater than that of M13 phage.</p

Inhibition of rOmpU fusion protein to EPC cells in the presence of OmpU-binding peptide determined by fluorescence-based adhesion assay.

<p><b>(A)</b> EPC cells were co-incubated with M199 medium. Nuclei was stained with DAPI (blue color). <b>(B)</b> EPC cells were co-incubated with rOmpU fusion protein, followed by washing and visualization of surface retained rOmpU by immunofluorescent staining with OmpU-specific antibodies (green color). Nuclei was counterstained with DAPI (blue color). (C) EPC cells were co-incubated in the mixture of rOmpU with an irrelevant control peptide, same staining as in (B). <b>(D), (E) and (F)</b> EPC cells were co-incubated in the mixture of rOmpU with an irrelevant control peptide or OmpU-binding peptide1,2,3, respectively, same staining as in (B). All scale bars are 100μm.</p