Journal: Infection and Immunity

Article Title: Recombinant outer membrane vesicles delivering eukaryotic expression plasmid of cytokines act as enhanced adjuvants against Helicobacter pylori infection in mice

doi: 10.1128/iai.00313-23

Figure Lengend Snippet: Construction and characterization of recombinant OMVs. Construction of recombinant cytokine OMVs and their pattern diagram for immunization as adjuvant (A). HEK-293T and GES-1 cells were divided into four groups each: 100 µg of OMVs delivering cytokine IFN-γ (EGFP) was added to the first group, 100 µg of OMVs delivering cytokine IL-17A (mCherry) was added to the second, 50 µg each of OMVs delivering cytokine IFN-γ (EGFP) or IL-17A (mCherry) was added to the third, and 100 µg of OMVs delivering empty plasmid followed by incubation in a cell culture incubator. qPCR was used to detect the amount of plasmids encapsulated in the OMVs, and DNase treatment can eliminate interference from plasmids adhering to the surface of OMVs. The group without DNase treatment serves as the control group (B). Eukaryotic expression plasmids are highly expressed in both HEK-293T and GES-1 cells as observed by laser confocal microscopy. Bars: 40 µm (C). The expression of IL-17A and IFN-γ in cells by RT-qPCR (D) or quantitative ELISA (E). The X-axis represents the cytokine expression levels from HEK-293T or GES-1 cells produced by the OMVs delivering the eukaryotic expression plasmid of cytokine IFN-γ (EGFP), IL-17A (mCherry) or both.

Article Snippet: The protein concentrations of the obtained OMVs and OMPs were quantified using a Bicinchoninic Acid (BCA) Assay Kit (Thermo Fisher, Rockford, IL, USA) according to the manufacturer’s instructions. . Construction of recombinant plasmids and electroporation To construct the recombinant expression plasmids of pIRES-IL-17A/mCherry and pIRES-IFN-γ/EGFP, the IL-17A and IFN-γ genes were amplified from mouse spleen cells by RT-PCR and cloned into the plasmid pIRES (BD Biosciences Clontech, Palo Alto, CA, USA) and digested using suitable restriction enzymes to produce the plasmids pIRES-IL-17A or pIRES-IFN-γ.

Techniques: Recombinant, Adjuvant, Plasmid Preparation, Incubation, Cell Culture, Expressing, Confocal Microscopy, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Produced

Journal: Infection and Immunity

Article Title: Recombinant outer membrane vesicles delivering eukaryotic expression plasmid of cytokines act as enhanced adjuvants against Helicobacter pylori infection in mice

doi: 10.1128/iai.00313-23

Figure Lengend Snippet: Detection of IL-17A and IFN-γ levels during the immunization process. The expression levels of IL-17A (A and C) and IFN-γ (B and D) in gastric mucosa of mice were measured by quantitative PCR at the eighth week of immunization and the second week after H. pylori challenge. A total of 18 mice were sacrificed and gastric mucosa was taken for detection, of which 9 mice in each group were repeated twice. The data were combined for statistical analysis and expressed as mean ± SD per group. All statistical differences were comparisons between two groups and assessed using one-way ANOVA followed by Bonferroni’s multiple-pair comparison. P < 0.05 and P < 0.01 represent the differences between the related groups.

Article Snippet: The protein concentrations of the obtained OMVs and OMPs were quantified using a Bicinchoninic Acid (BCA) Assay Kit (Thermo Fisher, Rockford, IL, USA) according to the manufacturer’s instructions. . Construction of recombinant plasmids and electroporation To construct the recombinant expression plasmids of pIRES-IL-17A/mCherry and pIRES-IFN-γ/EGFP, the IL-17A and IFN-γ genes were amplified from mouse spleen cells by RT-PCR and cloned into the plasmid pIRES (BD Biosciences Clontech, Palo Alto, CA, USA) and digested using suitable restriction enzymes to produce the plasmids pIRES-IL-17A or pIRES-IFN-γ.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Comparison

Journal: Infection and Immunity

Article Title: Recombinant outer membrane vesicles delivering eukaryotic expression plasmid of cytokines act as enhanced adjuvants against Helicobacter pylori infection in mice

doi: 10.1128/iai.00313-23

Figure Lengend Snippet: Recombinant OMVs as adjuvants induce a stronger Th1/Th17 balanced immune response. MLN cells and splenocytes of mice were isolated after 8 weeks of immunization, and the expression levels of cytokines IL-17 and IFN-γ in different groups were assessed by quantitative ELISA after immunization with UreB (A and C) or WCV (B and D) as vaccine antigens combined with recombinant OMVs or CT as adjuvants. A total of 18 mice were sacrificed, and gastric mucosa was taken for detection (nine mice in each group, in duplicate). The data were combined for statistical analysis and expressed as mean ± SD per group. All statistical differences were comparisons between two groups and assessed using one-way ANOVA followed by Bonferroni’s multiple-pair comparison. The significant differences between the clusters of groups shown in the figure represent the significant differences between the groups in the cluster compared to the other cluster. P < 0.05 and P < 0.01 represent the differences between the related groups.

Article Snippet: The protein concentrations of the obtained OMVs and OMPs were quantified using a Bicinchoninic Acid (BCA) Assay Kit (Thermo Fisher, Rockford, IL, USA) according to the manufacturer’s instructions. . Construction of recombinant plasmids and electroporation To construct the recombinant expression plasmids of pIRES-IL-17A/mCherry and pIRES-IFN-γ/EGFP, the IL-17A and IFN-γ genes were amplified from mouse spleen cells by RT-PCR and cloned into the plasmid pIRES (BD Biosciences Clontech, Palo Alto, CA, USA) and digested using suitable restriction enzymes to produce the plasmids pIRES-IL-17A or pIRES-IFN-γ.

Techniques: Recombinant, Isolation, Expressing, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Infection and Immunity

Article Title: Recombinant outer membrane vesicles delivering eukaryotic expression plasmid of cytokines act as enhanced adjuvants against Helicobacter pylori infection in mice

doi: 10.1128/iai.00313-23

Figure Lengend Snippet: Vaccine formulation strategy for immunization using recombinant H. pylori OMVs as adjuvants a

Article Snippet: The protein concentrations of the obtained OMVs and OMPs were quantified using a Bicinchoninic Acid (BCA) Assay Kit (Thermo Fisher, Rockford, IL, USA) according to the manufacturer’s instructions. . Construction of recombinant plasmids and electroporation To construct the recombinant expression plasmids of pIRES-IL-17A/mCherry and pIRES-IFN-γ/EGFP, the IL-17A and IFN-γ genes were amplified from mouse spleen cells by RT-PCR and cloned into the plasmid pIRES (BD Biosciences Clontech, Palo Alto, CA, USA) and digested using suitable restriction enzymes to produce the plasmids pIRES-IL-17A or pIRES-IFN-γ.

Techniques: Formulation, Recombinant