Journal: Materials Today Bio
Article Title: Inhalable engineered probiotic outer membrane vesicles co-expressing multiple mpox antigens induce potent specific systemic and mucosal immune responses
doi: 10.1016/j.mtbio.2026.103089
Figure Lengend Snippet: Design, Preparation, and Characterization of ABM@OMV. ( A ) Schematic illustrating the preparation of ABM@OMV. The pThioHisA plasmid, encoding Trx-A9R and ClyA-B6R-M1R fusion proteins, was transformed into Δ lpxM Escherichia coli Nissle 1917 (ΔEcN). OMVs were harvested via ultracentrifugation. ( B ) Bacterial lysates analyzed by SDS-PAGE and stained with Coomassie blue. Lanes: Marker; uninduced ΔEcN; ΔEcN + IPTG; uninduced BMΔEcN; BMΔEcN + IPTG; uninduced ABMΔEcN; ABMΔEcN + IPTG. Black boxes indicate ClyA-B6R-M1R and Trx-A9R fusion proteins. ( C ) Western blot analysis of proteins of interest expressed in ABMΔECN bacteria. Anti-His tag antibody detected Trx-A9R; anti-Flag tag antibody detected ClyA-B6R-M1R. Lane order is the same as in B . ( D ) Dynamic light scattering (DLS) size distribution profiles of Δ lpxM OMVs, BM@OMV, and ABM@OMV. ( E ) Zeta potential measurements of Δ lpxM OMVs, BM@OMV, and ABM@OMV ( n = 3). ( F ) Transmission electron microscopy (TEM) images of Δ lpxM OMVs, BM@OMV, and ABM@OMV. Scale bar: 100 nm. ( G ) Western blot analysis of proteins of interest expressed in ABM@OMV. Lanes: Δ lpxM OMVs, BM@OMV, ABM@OMV. ( H ) Endotoxin levels in Δ lpxM EcN-derived OMV and wild-type EcN-derived OMV, as measured by Limulus amebocyte lysate (LAL) assay ( n = 3). ( I ) Representative western blot showing the stability of proteins of interest in Δ lpxM OMVs, BM@OMV, and ABM@OMV following storage at 4 °C (left) or −80 °C (right) for the indicated time periods for the indicated time periods. ( J, K ) Changes in particle size ( J ) and zeta potential ( K ) of OMVs stored at 4 °C and −80 °C ( n = 3). All data were analyzed with GraphPad Prism 8 and are presented as mean ± SD. For panel H , statistical significance between two groups was determined by an unpaired two-tailed t -test. ∗∗∗ P < 0.001.
Article Snippet: The membrane was blocked with 5% BSA (BSA0020, Biosharp, China), then incubated with anti-His tag antibody (1:10,000, HY-P809476X, Med ChemExpress, USA) and anti-FLAG antibody (1:10,000, HY- P80111 , Med ChemExpress, USA), respectively for A9R and B6R-M1R, followed by HRP-conjugated secondary antibodies.
Techniques: Plasmid Preparation, Transformation Assay, SDS Page, Staining, Marker, Western Blot, Bacteria, FLAG-tag, Zeta Potential Analyzer, Transmission Assay, Electron Microscopy, Derivative Assay, LAL Assay, Two Tailed Test
