plasmid plex307-dpp4-puro  (Addgene inc)

Journal: Cell Biomaterials

Article Title: Engineered DPP4 decoy confers broad-spectrum inhibition of MERS-CoV infection

doi: 10.1016/j.celbio.2025.100018

Figure Lengend Snippet: Figure 1. Designed and engineered high-affinity DPP4 by direct evolution (A) 293T cells expressing each DPP4 mutant were incubated with the spike RBD of MERS-CoV labeled with superfolder GFP (sfGFP). (B) Random mutations were introduced into the DPP4 interface region (aa 264–350) by error-prone PCR amplification. The constructed DPP4 mutant plasmid library was diluted and transfected into 293T cells. Using a cell sorter, the cells incubated with RBD sfGFP were displayed for RDB binding (sfGFP) and DPP4-HA expression (Alexa 647) and the top 0.05% high-level-bound population was harvested. Extracted mRNA from the sorted cells was used for the next cycle. (C) Competitive inhibition activity was assessed in three types of high-affinity DPP4 clones. Each soluble DPP4 mutant was incubated with the RBD-sfGFP for 30 min and then the mixture was transferred to native DPP4-expressing 293T cells. Inhibition rate was calculated from the number of sfGFP-binding cells using flow cytometry. n = 3 technical replicates. (D) Neutralizing activity of Fc-DPP4 was evaluated against MERS-CoV pseudovirus. Fc-DPP4 is human IgG1 Fc fused N-terminal to DPP4 mutants. n = 4.

Article Snippet: Human DPP4 (Addgene #158451) was cloned into the KpnI-XhoI sites of pcDNA4TO (Thermo Fisher Scientific) with a C-terminal HA tag.

Techniques: Expressing, Mutagenesis, Incubation, Labeling, Construct, Plasmid Preparation, Transfection, Binding Assay, Inhibition, Activity Assay, Clone Assay, Cytometry

Journal: Cell Biomaterials

Article Title: Engineered DPP4 decoy confers broad-spectrum inhibition of MERS-CoV infection

doi: 10.1016/j.celbio.2025.100018

Figure Lengend Snippet: Figure 6. Engineered DPP4 decoy exhibited prophylactic efficacy against MERS-CoV infection (A) Schematic of the mouse experiment. 3 h prior to MERS-CoV infection with 1 3 105 plaque-forming units, hDPP4 transgenic mice were intranasally admin- istered 200 mg of the engineered DPP4 decoy. Lung tissues were harvested 1 and 3 days after infection. (B) Genomic MERS-CoV RNA and subgenomic RNA extracted from harvested lung tissues were evaluated as copies per mg of cellular transcripts. Viral titration of lung tissues was performed using the TCID50 assay. Data are expressed as mean ± SD (n = 4 per group). p values were determined using two-way ANOVA with Sida´ k’s multiple comparison test. Dashed lines indicate the detection limit. (C) HE staining and immunohistochemistry of MERS-CoV NP and CD3. Scale bars, 100 mm. (D) MERS-CoV NP- and CD3-positive cells were quantified as densities per mm2. For each individual, the density of three lobules (left, right cranial, and right middle) was quantified and averaged. Data are expressed as mean ± SD (n = 4 per group). p values were determined by unpaired t test.

Article Snippet: Human DPP4 (Addgene #158451) was cloned into the KpnI-XhoI sites of pcDNA4TO (Thermo Fisher Scientific) with a C-terminal HA tag.

Techniques: Infection, Transgenic Assay, Titration, TCID50 Assay, Comparison, Staining, Immunohistochemistry