Amylase activity analysis.

<p>Recombinant and nonrecombinant strains grew on the starch-containing LB plate before (A) and after (B) being stained by iodine. The integration of <i>cotC</i>::<i>ompC</i> gene fusion disrupts <i>amyE</i> gene and made the strain amylase deficient, transparent halo was produced around the <i>B</i>. <i>subtilis</i> 168, but in the recombinant <i>B</i>. <i>subtilis</i> SE2 clones, no transparent halo was produced.</p

Schematic representation of the construction of the recombinant spores.

<p>The <i>cotC</i>::<i>ompC</i> gene fusion and <i>cat</i> (chloramphenicol-resistance gene) gene carried by plasmid pDG364-<i>cotC</i>-<i>ompC</i> were integrated into the <i>amyE</i> gene locus of <i>B</i>. <i>subtilis</i> 168 chromosome by double cross-over recombination events. Arrows indicate direction of transcription.</p

Immunofluorescence microscopy analysis.

<p>Sporulation of <i>B</i>. <i>subtilis</i> strains was induced by the exhaustion method, and spores were collected in DSM after 48h. Spores were labeled with OmpC antiserum followed by Cy3-labeled goat anti-chicken IgG. Spores were visualized by phase-contrast (PC) and immunofluorescence (IF) microscopy.</p

Oligonucleotides list.

<p>Oligonucleotides list.</p

PCR analysis with different primer pairs.

<p>Site-directed PCR analysis of <i>cotC</i>::<i>ompC</i> gene fusion integrated into the chromosome of <i>B</i>. <i>subtilis</i> 168. lane WT, <i>B</i>. <i>subtilis</i> 168; lane RT, recombinant <i>B</i>. <i>subtilis</i> SE2; primer pairs used in PCR are labeled above.</p

Serum IgG and intestinal mucosal SIgA antibodies responses.

<p>PI, pre-immune samples. Samples obtained from the mice immunized with recombinant spores (A and C) and wild-type spores (B and D); E, samples obtained from a naïve, untreated control group. ** indicates <i>p</i><0.01.</p

Protection of mice against challenge with <i>S</i>. Typhimurium strain SL1344.

<p>Protection of mice against challenge with <i>S</i>. Typhimurium strain SL1344.</p

Minimum Distance Between Two Epitopes in Sandwich Immunoassays for Small Molecules

The pursuit of the limit between dimensionalities is a scientific goal with high applicability. Sandwich immunoassay, usually based on two antibodies binding two epitopes, is one of the most popular mainstay tools in both academic and industrial fields. Herein, we determined and evaluated the minimum distance of two epitopes in sandwich immunoassays for small molecules. Briefly, nine model analytes comprising two hapten epitopes, that is, melamine (MEL) and p-nitroaniline (NIA), were designed by increasing the linear chain linkers brick by brick. Two groups of monoclonal antibodies (mAbs) were produced with different recognition properties toward MEL and NIA using 12 new haptens with different spacer arms. The results indicated that two epitopes of the analyte with a distance of only 2.4 Å could be simultaneously bound by two mAbs, which is the known limit of epitope distance in sandwich immunoassays thus far. We further found that an epitope distance of below 8.8 Å for the analyte generally induces noticeable steric hindrance of antibodies, preventing a sandwich immunoassay with high probability. These observations were investigated and evaluated by molecular docking, molecular dynamics, and surface plasmon resonance and using model and real analytes. Altogether, we determined the minimum distance of two epitopes and explored the molecular mechanism of the antibody–analyte–antibody ternary complex in sandwich immunoassays, providing a theoretical basis for hapten design, antibody discovery and development, and sandwich immunoassay establishment for small molecules

DataSheet_1_Surface Display of porcine circovirus type 2 antigen protein cap on the spores of bacillus subtilis 168: An effective mucosal vaccine candidate.pdf

The oral mucosal vaccine has great potential in preventing a series of diseases caused by porcine circovirus type 2 (PCV2) infection. This study constructed a recombinant Bacillus subtilis RB with PCV2 Capsid protein (Cap) on its spore surface and cotB as a fusion partner. The immune properties of the recombinant strain were evaluated in a mouse model. IgA in intestinal contents and IgG in serum were detected by enzyme-linked immunosorbent assay (ELISA). The results demonstrated that recombinant spores could activate strong specific mucosal and humoral immune responses. In addition, spores showed good mucosal immune adjuvant function, promoting the proliferation of CD3+, CD4+ and CD8+ T cells and other immune cells. We also found that the relative expression of inflammatory cytokines such as IL-1β, IL-6, IL-10, TNF-α and IFN in the small intestinal mucosa was significantly up-regulated under the stimulation of recombinant bacteriophage. These effects are important for the balance of Th1/Th2-like responses. In summary, our results suggest that recombinant B. subtilis RB as a feed additive provides a new strategy for the development of novel and safe PCV2 mucosal subunit vaccines.</p