a1 dna fragment  (New England Biolabs)

Journal: Applied and Environmental Microbiology

Article Title: Application of a Novel Epitope- and Structure-Based Vaccinology-Assisted Fimbria-Toxin Multiepitope Fusion Antigen of Enterotoxigenic Escherichia coli for Development of Multivalent Vaccines against Porcine Postweaning Diarrhea

doi: 10.1128/AEM.00274-20

Figure Lengend Snippet: Illustration of PWD MEFA A1 domain gene construction and PyMOL protein modeling of the A1 domain of PWD MEFA and PWD MEFA proteins. (A) Schematic illustration of the A1 domain of the PWD fimbria-toxoid MEFA gene. Nucleotides encoding two K88 epitopes (GRTKEAFATP and PMKNAGGTKVGSVKVN), two F18 epitopes (IPSSSGTLTCQAGT and QPDATGSWYD), the Stx2e A subunit epitope (QSYVSSLN), and two copies of the STa toxoid STaN12S domain (CCELCCSPACAGCY) and STb epitope (KKDLCEHY) were embedded in the A1 segment of LT toxoid LTR192G by replacing LTA1 epitope nucleotides. (B, C) 3D modeling of the A1 domain of PWD MEFA protein (front and back views). (D, E) Secondary structure of the A1 domain of PWD MEFA protein (front and back views). (F, G) 3D modeling of the protein structure of PWD MEFA (A1 domain with epitope substitution, A2 domain, and LTB peptide in a single peptide [front and back views]). (H, I) Secondary structure of the PWD MEFA protein (A1 domain, A2 domain, and LTB peptide in a single peptide [front and back views]). Epitopes, as well as the A2 domain and LTB peptide, are in different colors.

Article Snippet: The optimized chimeric PWD MEFA A1 DNA nucleotide fragment (with substitutions of epitope-coded nucleotides) was synthesized by GenScript (Piscataway, NJ) and initially cloned in vector pUC57 and then pET28α.

Techniques:

Journal: Applied and Environmental Microbiology

Article Title: Application of a Novel Epitope- and Structure-Based Vaccinology-Assisted Fimbria-Toxin Multiepitope Fusion Antigen of Enterotoxigenic Escherichia coli for Development of Multivalent Vaccines against Porcine Postweaning Diarrhea

doi: 10.1128/AEM.00274-20

Figure Lengend Snippet: Extraction and detection of the PWD MEFA A1 domain and PWD MEFA proteins. (A) SDS-PAGE Coomassie blue staining of the PWD MEFA A1 domain and PWD MEFA. (B) Western blot with anti-CT rabbit serum (1:3,000 [Sigma]; homologue to anti-LT antiserum). (C) Western blot with anti-Stx2e anti-mouse serum (1:1,500). (D) Western blot with anti-STa rabbit serum (1:2,000). (E) Western blot with anti-FedF (F18) mouse serum (1:1,500). (F) Western blot with anti-FaeG (K88) mouse serum (1:1,500). IRDye-labeled goat anti-mouse or anti-rabbit IgG (1:5,000 [LI-COR]) was used as the secondary antibody. M, protein marker (in kilodaltons [Precision Plus Protein prestained standards; Bio-Rad]). Arrows indicate the A1 domain (lane 1) and entire PWD MEFA (lane 2) proteins; total inclusion body proteins from the E. coli BL21(DE3) host strain were used as the control, indicated by “(-).”

Article Snippet: The optimized chimeric PWD MEFA A1 DNA nucleotide fragment (with substitutions of epitope-coded nucleotides) was synthesized by GenScript (Piscataway, NJ) and initially cloned in vector pUC57 and then pET28α.

Techniques: Extraction, SDS Page, Staining, Western Blot, Labeling, Marker, Control

Journal: Applied and Environmental Microbiology

Article Title: Application of a Novel Epitope- and Structure-Based Vaccinology-Assisted Fimbria-Toxin Multiepitope Fusion Antigen of Enterotoxigenic Escherichia coli for Development of Multivalent Vaccines against Porcine Postweaning Diarrhea

doi: 10.1128/AEM.00274-20

Figure Lengend Snippet: Mouse IgG antibody titers (log10) specific to ETEC fimbriae and toxins associated with PWD. Serum samples from each mouse in the group immunized with PWD MEFA protein with dmLT adjuvant (○), with PWD MWFA protein without dmLT (■), or PBS as the control (▲) were 2-fold serially diluted and incubated in ELISA plate wells coated with CT (Sigma [100 ng per well]), F18 fimbriae (100 ng per well), K88 fimbriae (100 ng per well), MBP-STb recombinant protein (100 ng per well), MPB-Stx2e recombinant protein (100 ng per well), or STa-ovalbumin conjugate (10 ng per well). The mean titer and standard deviation in each group are indicated by bars.

Article Snippet: The optimized chimeric PWD MEFA A1 DNA nucleotide fragment (with substitutions of epitope-coded nucleotides) was synthesized by GenScript (Piscataway, NJ) and initially cloned in vector pUC57 and then pET28α.

Techniques: Adjuvant, Control, Incubation, Enzyme-linked Immunosorbent Assay, Recombinant, Standard Deviation

Journal: Applied and Environmental Microbiology

Article Title: Application of a Novel Epitope- and Structure-Based Vaccinology-Assisted Fimbria-Toxin Multiepitope Fusion Antigen of Enterotoxigenic Escherichia coli for Development of Multivalent Vaccines against Porcine Postweaning Diarrhea

doi: 10.1128/AEM.00274-20

Figure Lengend Snippet: Mouse serum antibody in vitro neutralization activities against adherence of K88 fimbrial or F18 fimbrial bacteria and enterotoxicity of STa and CT toxins. (A) Serum samples from mice immunized with PWD MEFA, PWD MEFA adjuvanted with dmLT, F18 FedF recombinant protein (as the positive control), or PBS as the control in adherence inhibition against F18 fimbrial bacteria 8516 to IPEC-J2 pig intestinal cells. (B) Serum samples from mice immunized with PWD MEFA, PWD MEFA adjuvanted with dmLT, K88 FaeG recombinant protein (as the positive control), or PBS as the control in adherence inhibition against K88+ ETEC strain 3030-2 to IPEC-J2 cells. The numbers of adherent bacteria (CFU) were converted to percentages, with CFU from cells treated with the control serum at 100%. (C) Mouse serum samples from two immunized groups (PWD MEFA and PWD MEFA with dmLT adjuvant) or the control in prevention of STa toxin from stimulation of cGMP in T-84 cells to show antibody neutralization activity against STa enterotoxicity. T-84 cells incubated with 2 ng STa toxin premixed with PBS, control mouse serum, or serum from each immunized group were measured for intracellular cGMP levels (pmol/ml) by using a cGMP EIA kit (Enzo Life). (D) Mouse serum samples from the two immunized groups (PWD MEFA and PWD MEFA with dmLT adjuvant) or the control in prevention of CT (LT homologue) from stimulation of cAMP in T-84 cells to show antibody neutralization activity against CT enterotoxicity. T-84 cells incubated with 10 ng CT toxin premixed with PBS, control mouse serum, or each immunized mouse serum were measured for intracellular cAMP levels (pmol/ml) by using a cAMP EIA kit (Enzo Life). ***, P < 0.001; **, P < 0.01.

Article Snippet: The optimized chimeric PWD MEFA A1 DNA nucleotide fragment (with substitutions of epitope-coded nucleotides) was synthesized by GenScript (Piscataway, NJ) and initially cloned in vector pUC57 and then pET28α.

Techniques: In Vitro, Neutralization, Bacteria, Recombinant, Positive Control, Control, Inhibition, Adjuvant, Activity Assay, Incubation

Journal: Applied and Environmental Microbiology

Article Title: Application of a Novel Epitope- and Structure-Based Vaccinology-Assisted Fimbria-Toxin Multiepitope Fusion Antigen of Enterotoxigenic Escherichia coli for Development of Multivalent Vaccines against Porcine Postweaning Diarrhea

doi: 10.1128/AEM.00274-20

Figure Lengend Snippet: Mouse serum antibody in vitro neutralization activity against STb and Stx2e toxin cytotoxicity from a Vero cell assay. Vero cells incubated with 300 μl filtrates of STb+ strain 8020 or 100 μl filtrates of Stx2e+ strain 9168 showed cytotoxicity. (Top row) Normal Vero cells, cells treated with 300 μl 8020 filtrates, 300 μl 8020 filtrates premixed with 150 μl control mouse serum (1:4.7; in a total volume of 700 μl), 300 μl 8020 filtrates premixed with 150 μl (1:4.7), 25 μl (1:28), or 10 μl (1:70) mouse serum of the group immunized with PWD MEFA with or without dmLT adjuvant. (Bottom row) Normal Vero cells, cells treated with 100 μl 9168 filtrates, 100 μl 9168 filtrates premixed with 18.8 μl control mouse serum (1:53 in a total volume of 1,000 μl), 100 μl 9168 filtrates premixed with 18.8 μl (1:53), 6.3 μl (1:159), or 3 μl (1:333) mouse serum of the group immunized with PWD MEFA with or without dmLT adjuvant.

Article Snippet: The optimized chimeric PWD MEFA A1 DNA nucleotide fragment (with substitutions of epitope-coded nucleotides) was synthesized by GenScript (Piscataway, NJ) and initially cloned in vector pUC57 and then pET28α.

Techniques: In Vitro, Neutralization, Activity Assay, Incubation, Control, Adjuvant

Journal: Applied and Environmental Microbiology

Article Title: Application of a Novel Epitope- and Structure-Based Vaccinology-Assisted Fimbria-Toxin Multiepitope Fusion Antigen of Enterotoxigenic Escherichia coli for Development of Multivalent Vaccines against Porcine Postweaning Diarrhea

doi: 10.1128/AEM.00274-20

Figure Lengend Snippet: E. coli bacterial strains and plasmids used in the study

Article Snippet: The optimized chimeric PWD MEFA A1 DNA nucleotide fragment (with substitutions of epitope-coded nucleotides) was synthesized by GenScript (Piscataway, NJ) and initially cloned in vector pUC57 and then pET28α.

Techniques: Plasmid Preparation, Synthesized

Journal: Applied and Environmental Microbiology

Article Title: Application of a Novel Epitope- and Structure-Based Vaccinology-Assisted Fimbria-Toxin Multiepitope Fusion Antigen of Enterotoxigenic Escherichia coli for Development of Multivalent Vaccines against Porcine Postweaning Diarrhea

doi: 10.1128/AEM.00274-20

Figure Lengend Snippet: PCR primers used in the study to generate the A1 domain of PWD MEFA and PWD MEFA genes

Article Snippet: The optimized chimeric PWD MEFA A1 DNA nucleotide fragment (with substitutions of epitope-coded nucleotides) was synthesized by GenScript (Piscataway, NJ) and initially cloned in vector pUC57 and then pET28α.

Techniques: Sequencing, Amplification