Gene sequence and translation of the synthesized <i>ltb/h</i>_<i>c</i><i>c/h</i>_<i>c</i><i>d</i> construct.

<p>Representation of the synthetic gene which encodes rLTB/H _CC/H _CD protein. The first codon of each subunit is in bold and identified by an <b>arrow</b>. The 3xGly linker codons are underlined. Stop codon is indicated by an asterisk (*).</p

SDS-PAGE and Western blot analysis of protein expression and purification.

<p>(A) SDS-PAGE of the whole <i>E. coli</i> cell extract after 3 h of induction of both constructs. Left, 1: Untransformed <i>E. coli</i> BL21 (DE3) Star; 2: <i>E. coli</i> BL21 (DE3) Star transformed with pAE/<i>h</i>_<i>c</i><i>c-h </i>_<i>c</i><i>d</i> before induction; 3: <i>E. coli</i> BL21 (DE3) Star transformed with pAE/<i>h</i>_<i>c</i><i>c-h </i>_<i>c</i><i>d</i> after induction. Right, 1: Untransformed <i>E. coli</i>BL21 (DE3) Star; 2: <i>E. coli</i> BL21 (DE3) Star transformed with pAE/<i>ltb-h </i>_<i>c</i><i>c-h </i>_<i>c</i><i>d</i> before induction; 3: <i>E. coli</i> BL21 (DE3) Star transformed with pAE/<i>ltb-h </i>_<i>c</i><i>c-h </i>_<i>c</i><i>d</i> after induction. (B) SDS-PAGE to evaluate the purity and integrity of purified proteins after Ni-affinity chromatography. Left, rH _CC. Right, rH _CD. All lanes designated “1” are untransformed <i>E. coli</i> BL21 (DE3) Star, and the lanes designated “2” are the purified antigens. (C) Anti-his Western blot to characterize the purified proteins. 1: Spectra Multicolor Broad Range Protein Ladder (Thermo Scientific), 2: Untransformed <i>E. coli</i> BL21 (DE3) Star, 3: rLTB/H _CC/H _CD, 4: rH _CC/H _CD.</p

Antigenicity evaluation of the recombinant chimeras by ELISA using standard anti-BoNT C and D sera.

<p>Anti-BoNT C serum (A) showed that the rLTB/H _CC/H _CD antigen has more antigenic epitopes than rH _CC/H _CD, while anti-BoNT D serum (B) shows no difference between these two antigens. Anti-cholera toxin serum (C) was used to evaluate the antigenicity of the rLTB domain. Crude protein extract from <i>E. coli</i> BL21 (DE3) Star and purified rLTB were used as controls for the specificity of the three sera. Different letters above the bars (A, B, or C) indicate significant differences (p < 0.001) according to Tukey’s test. Absorbances (Abs) shown were measured at 450 nm wavelength.</p

Schematic representations of the constructed gene and fusion proteins.

<p>(A) Structure of the gene designed to encode the fusion antigens. The <i>h_cc</i> (green) is flanked by <i>Kpn</i>I and <i>Xho</i>I sites, respectively, at the 5’ end. The coding sequence of <i>h_cd</i> (blue) is linked at the 3’ end of <i>h_cc</i> by three Glycine codons and is flanked by <i>Hind</i>III at the 3’ end. (B) Representation of the recombinant chimera region, followed by the adjuvant rLTB connected to rH _CC, a three-Glycine linker (3xGly) and the rH _CD at the C-terminal region. (C) Representation of the recombinant chimera rH _CC/rH _CD. The resulting protein has the same characteristics as shown in B, except for the absence of the rLTB sequence connected to the N-terminal region of HC _CC.</p

Schematic of the Lig proteins, expression and purification of rLigB(131–645).

<p>A) The full length amino acid sequences for LigA (1224 amino acids, 128.1 kDa) and LigB (1890 amino acids, 200.8 kDa) are indicated (black line), the square boxes indicate the BIDs and the LigB C-terminal domain is shown (rectangle). The recombinant proteins used as vaccine candidates are indicated: LigB(131–645) (green boxes) includes amino acids 131–645 (53.5 kDa) and is highly identical (97.9% pairwise identity) to the same region in LigA; the LigA(631–1224), also known as LigANI, (red boxes, amino acids 631–1224, 62.8 kDa) and LigB(625–1259), also known as LigBNI, (blues boxes, amino acids 625–1259, 66.2 kDa) fragments are not highly conserved (38.1% pairwise identity). B) Expression and purification of rLigB(131–645) analysed by 10% SDS-PAGE and Coomassie staining. Lanes 1: molecular mass marker (kDa); Expression of rLigB(131–645) in an <i>E</i>. <i>coli</i>(pLigB(131–645)) clone, lane 2: supernatant (soluble) fraction and lane 3: insoluble fraction; lane 4: IMAC purified rLigB(131–645), expected molecular mass of 57.2 kDa. C) Immunoblot analysis of rLigB(131–645), following transfer the nitrocellulose membrane was probed with an anti-His-HRP antibody, lane 1: molecular mass marker (kDa); lane 2: purified rLigB(131–645).</p

Protection conferred by immunization with rLigB(131–645) against lethal challenge in the hamster model of leptospirosis.

<p>Protection conferred by immunization with rLigB(131–645) against lethal challenge in the hamster model of leptospirosis.</p

Pathological findings in the hamster model.

<p>Animals vaccinated with rLigB(131–645) (A, C, E and G) or the PBS control group (B, D, F and H) were euthanized 10 days PC and tissue samples were collected. Vaccinated animals showed no gross pulmonary lesions (A) or microscopic pulmonary lesions (C). Liver (E) and kidney samples (G) showed no evidence of microscopic abnormalities. Unvaccinated animals showed gross pulmonary haemorrhaging (B) and they were confirmed to be alveolar haemorrhages by microscopic analysis (D). Dystrabeculaton (loss of cohesion) of hepatocytes (F) and swelling of kidney tubular epithelial cells (H) were prominent features. (C-F, haematoxylin-eosin, 100× magnification and G-H, haematoxylin-eosin, 200× magnification).</p

Le Peuple : organe quotidien du syndicalisme

21 août 19321932/08/21 (A12,N4236)-1932/08/21