Journal: BioMed Research International

Article Title: Salamander-Derived, Human-Optimized nAG Protein Suppresses Collagen Synthesis and Increases Collagen Degradation in Primary Human Fibroblasts

doi: 10.1155/2013/384091

Figure Lengend Snippet: Western blot assay (a) and immunofluorescence (b) experiments showing nAG protein expression in primary human fibroblasts. (a) Western blot was performed after 24 h and 48 h of transfection. The cells were lysed by using RIPA cocktail; proteins were separated on 12% SDS polyacrylamide gel. The primary antibody V5 probe and HRP-conjugated secondary antibody were used for nAG protein detection in the following lanes: nontransfected fibroblasts (negative control 1), fibroblasts with nAG plasmid without lipofection (negative control 2), nAG transfected fibroblasts tested after 24 h and nAG transfected fibroblasts tested after 48 h. (b) Immunofluorescence assay was performed after 48 h of transfection and cells were fixed and permeabilized by 2% PFA/0.1% Triton x-100. V5 probe was used as primary antibody and green fluorescence was detected by using FITC-conjugated secondary antibody. Compare nAG protein expression in nontransfected fibroblasts (negative control) to nAG expression in transfected fibroblasts (magnification 40x).

Article Snippet: The cells were incubated with primary antibodies V5-probe (sc-58052, Santa Cruz) for nAG detection, COL1A1 (sc-28657, Santa Cruz) for collagen I detection, and COL3A1 (sc-8780-R, Santa Cruz) for collagen III detection using 1 : 50 dilution in blocking buffer for 1 hour at room temperature.

Techniques: Western Blot, Immunofluorescence, Expressing, Transfection, Negative Control, Plasmid Preparation, Fluorescence

Journal: NPJ Vaccines

Article Title: Selectively targeting haemagglutinin antigen to chicken CD83 receptor induces faster and stronger immunity against avian influenza

doi: 10.1038/s41541-021-00350-3

Figure Lengend Snippet: a Schematic representation of the full length HA protein of H9N2 (MsCon) virus. Precursor HA0 :1-560 amino acids (aa), HA1:19-338 aa, HA2: 339-560 aa, TM = transmembrane domain (525-547 aa) CT = cytosolic tail domain (548−560 aa). b Schematic representation of the soluble HA protein of H9N2 (MsCon) virus. The soluble H9HA was generated by removing the TM and CT domains (525-560 aa) and fusing the C-terminus of HA to 30 aa long trimerisation foldon sequence of the trimeric protein fibritin from T4 bacteriophage c His tag purification of the recombinant proteins. The expected sizes of the purified CD83 scFv antibody, rH9HA, and rH9HA-CD83 scFv are 30, 70, and 100 kDa respectively. Lane 1: control supernatant from the untransfected cells Lane 2: CD83 scFv Lane 3: rH9HA Lane 4: rH9HA-CD83 scFv. For the purification of the recombinant proteins, the harvested S2 cell culture supernatants containing recombinant protein bound to the metal ions (copper sulfate was used as an inducer of metallothionein promoter) were loaded onto uncharged “UNOsphere” resin derivatized with iminodiacetic acid functioning as a chelating ligand (Profinity ™ IMAC, Bio-Rad). Proteins were eluted with elution buffer containing 50 mM NaH 2 PO 4 , 300 mM NaCl and 50 mM imidazole. The purified proteins were analysed by 10% SDS-PAGE followed by Coomassie staining. All blots were derived from the same experiment and were processed in parallel.

Article Snippet: This was followed by further incubation with horseradish peroxidase-conjugated (HRP) anti-V5 secondary antibody (Bio-Rad Antibodies, primary antibody: CD83 scFv or rH9HA-CD83 scFv) or goat anti-mouse HRP secondary antibody (Bio-Rad Antibodies, primary antibody: CD83 mAb).

Techniques: Generated, Sequencing, Purification, Recombinant, Cell Culture, SDS Page, Staining, Derivative Assay

Journal: NPJ Vaccines

Article Title: Selectively targeting haemagglutinin antigen to chicken CD83 receptor induces faster and stronger immunity against avian influenza

doi: 10.1038/s41541-021-00350-3

Figure Lengend Snippet: a BS3 cross-linking experiment for determining the native structure of H9HA ectodomain with foldon. Lane 1: rH9HA without BS3 Lane 2: rH9HA with 10 mM BS3 Lane 3: rH9HA-CD83 scFv without BS3 Lane 4: rH9HA-CD83 scFv with 10 mM BS3. About 15 µg of the recombinant protein was mixed with BS3 to a 10 mM final concentration and incubated for 1 h at room temperature. The cross-linking reaction was stopped by the addition of 1 M Tris-HCl pH 8.0 to a final concentration of 50 mM and incubated for 15 min at room temperature. After cross-linking, proteins were separated on 8% SDS-PAGE under reducing conditions, blotted, and analysed by western blot using anti-H9HA monoclonal antibody. M: monomer (70 kDa* 100 kDa^) D: dimer (140 kDa* 200 kDa^) T: Trimer (210 kDa* 300 kDa^) *rH9HA ^rH9HA-CD83 scFv. All blots were derived from the same experiment and were processed in parallel. b Haemagglutination assay to test the activity of recombinant H9HA with foldon to agglutinate chicken red blood cells. 1. rH9HA 2. rH9HA–CD83 scFv 3. Negative control (PBS). For the haemagglutination assay, a two-fold serial dilution of 35 μg of the recombinant HA proteins was carried out in 96-well plates. About 50 μl of 1% chicken RBCs was added. The plates were incubated at 4 °C for 1 h and the highest dilution of the protein causing the agglutination of the RBCs was noted.

Article Snippet: This was followed by further incubation with horseradish peroxidase-conjugated (HRP) anti-V5 secondary antibody (Bio-Rad Antibodies, primary antibody: CD83 scFv or rH9HA-CD83 scFv) or goat anti-mouse HRP secondary antibody (Bio-Rad Antibodies, primary antibody: CD83 mAb).

Techniques: Recombinant, Concentration Assay, Incubation, SDS Page, Western Blot, Derivative Assay, Hemagglutination Assay, Activity Assay, Negative Control, Serial Dilution, Agglutination

Journal: NPJ Vaccines

Article Title: Selectively targeting haemagglutinin antigen to chicken CD83 receptor induces faster and stronger immunity against avian influenza

doi: 10.1038/s41541-021-00350-3

Figure Lengend Snippet: a Indirect ELISA for testing the activity of CD83 mAb b Indirect ELISA for testing the activity of CD83 scFv and rH9HA-CD83 scFv. Purified 8 μg of chicken CD83 ectodomain was coated onto each well of the ELISA plate, a two-fold serial dilution was carried out and the plate was incubated overnight for 4 °C. For detection, the plates were incubated with an equimolar concentration of purified CD83 scFv and rH9HA-CD83 scFv or 1 μg/ml of CD83 mAb. This was followed by incubation with goat anti-mouse HRP secondary antibody for ( a ) and HRP-conjugated anti-V5 secondary antibody for ( b ). The colorimetric detection was carried out by adding TMB substrate and absorbance at 450 nm was recorded.

Article Snippet: This was followed by further incubation with horseradish peroxidase-conjugated (HRP) anti-V5 secondary antibody (Bio-Rad Antibodies, primary antibody: CD83 scFv or rH9HA-CD83 scFv) or goat anti-mouse HRP secondary antibody (Bio-Rad Antibodies, primary antibody: CD83 mAb).

Techniques: Indirect ELISA, Activity Assay, Purification, Enzyme-linked Immunosorbent Assay, Serial Dilution, Incubation, Concentration Assay

Journal: NPJ Vaccines

Article Title: Selectively targeting haemagglutinin antigen to chicken CD83 receptor induces faster and stronger immunity against avian influenza

doi: 10.1038/s41541-021-00350-3

Figure Lengend Snippet: a Cytokines and chemokine mRNA levels in splenocytes analysed using qRT-PCR. b IFNγ protein level analysed using ELISA. For a : splenocytes were isolated from the spleen of 3-week-old SPF chickens using Histopaque 1083 and stimulated with 10 μg of CD83 scFv/rH9HA/rH9HA-CD83 scFv for 5, 22, and 30 h in vitro. Stimulated splenocytes were harvested for RNA extraction and expression levels of the respective cytokines and chemokine were measured by qRT-PCR. Data were calculated using 2 −∆∆CT approach ( n -fold change compared to the media only control group) and reported as values normalised to the expression level of a housekeeping gene RPLPO1. For b : supernatants from the stimulated splenocytes were analysed for the presence of IFNγ by ELISA. Data are represented as mean ± SD and analysed by one-way ANOVA followed by Tukey’s multiple comparison test. *** p < 0.001 ** p < 0.01 * p < 0.05. The data represent three independent experiments.

Article Snippet: This was followed by further incubation with horseradish peroxidase-conjugated (HRP) anti-V5 secondary antibody (Bio-Rad Antibodies, primary antibody: CD83 scFv or rH9HA-CD83 scFv) or goat anti-mouse HRP secondary antibody (Bio-Rad Antibodies, primary antibody: CD83 mAb).

Techniques: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Isolation, In Vitro, RNA Extraction, Expressing

Journal: NPJ Vaccines

Article Title: Selectively targeting haemagglutinin antigen to chicken CD83 receptor induces faster and stronger immunity against avian influenza

doi: 10.1038/s41541-021-00350-3

Figure Lengend Snippet: Groups of 7 days old chickens ( n = 8) were immunised with 2.8, 28, and 49 µg of recombinant rH9HA-CD83 scFv equivalent to 2, 20, and 35 µg of recombinant rH9HA (equimolar concentration). Boost vaccination was given after 7 days post primary vaccination. The chickens were bled on day 6, 14, 21, and 28 posts primary vaccination. Antibody titres in sera were measured by haemagglutinin inhibition (HI) assay. The highest dilution of serum inhibiting the agglutination of RBCs by H9N2 virus (UDL01/08) was recorded. Data are presented as mean ± SD and analysed by one-way ANOVA followed by Tukey’s multiple comparison test. **** p < 0.0001 *** p < 0.001 ** p < 0.01 * p < 0.05. ppv post primary vaccination.

Article Snippet: This was followed by further incubation with horseradish peroxidase-conjugated (HRP) anti-V5 secondary antibody (Bio-Rad Antibodies, primary antibody: CD83 scFv or rH9HA-CD83 scFv) or goat anti-mouse HRP secondary antibody (Bio-Rad Antibodies, primary antibody: CD83 mAb).

Techniques: Recombinant, Concentration Assay, Inhibition, HI Assay, Agglutination

Journal: NPJ Vaccines

Article Title: Selectively targeting haemagglutinin antigen to chicken CD83 receptor induces faster and stronger immunity against avian influenza

doi: 10.1038/s41541-021-00350-3

Figure Lengend Snippet: Groups of 7-day-old chickens ( n = 7) were immunised with 35 µg of recombinant rH9HA-CD83 scFv equivalent to 25 µg of recombinant rH9HA (equimolar concentration). The second vaccine dose as boost was given on day 7 post primary vaccination. The chickens were challenged with 100 μl of 1 × 10 7 pfu/ml H9N2 virus (UDL 01/2008) after 7 days post boost vaccination. Some of the vaccinated and control birds were left unchallenged to serve as contacts. Swabs were taken from the buccal cavity until day 7 post infection and the amount of live virus particles recovered from swabs was titrated via plaque assays and presented as plaque-forming units (PFU)/ml. a Shedding profiles of each chicken from the buccal cavity at different days post infection are indicated by a single coloured dot. b The average shedding profile from the buccal cavity per group for directly infected birds. c The average shedding profile from buccal cavity per group for contact birds. Data are presented as mean ± SD for ( a ) and analysed by one-way ANOVA followed by Tukey’s multiple comparison test for ( a ) and by unpaired t-test for ( b, c ). For ( b ) the asterisks represent significant difference between rH9HA and rH9HA-CD83 scFv (direct) groups. **** p < 0.0001 *** p < 0.001 ** p < 0.01 * p < 0.05. pi post infection.

Article Snippet: This was followed by further incubation with horseradish peroxidase-conjugated (HRP) anti-V5 secondary antibody (Bio-Rad Antibodies, primary antibody: CD83 scFv or rH9HA-CD83 scFv) or goat anti-mouse HRP secondary antibody (Bio-Rad Antibodies, primary antibody: CD83 mAb).

Techniques: Recombinant, Concentration Assay, Infection