Journal: Frontiers in Microbiology
Article Title: Induction of Avian β-Defensin 2 Is Possibly Mediated by the p38 MAPK Signal Pathway in Chicken Embryo Fibroblasts After Newcastle Disease Virus Infection
Figure Lengend Snippet: Recombinant expression of AvBD2 protein and antiviral activity of AvBD2 against NDV. (A) 12% SDS–PAGE analysis of His-tagged recombinant AvBD2 protein expressed in E . coli BL21 (DE3) cells. Lane 1: Supernatant; lane 2: Inclusion body; lane 3: Total protein of Rosetta containing AvBD2 without IPTG induction; lanes 4–6: Total protein of Rosetta containing AvBD2 at 2, 4, 6 h after induction with IPTG, respectively; lane 7: Purified recombinant AvBD2 protein; lane M: Protein marker. (B) 12% SDS–PAGE analysis of His-tagged AvBD2 protein expressed in Expi293 cells. Lane 1: purified AvBD2 with a His-tag on the C-terminal; lane 2: purified vector with a His-tag on the C-terminal; lane M: protein molecular weight marker. (C) Protection of CEFs from NDV infection by using the CCK-8 assay. Red curve , NDV (1 MOI) and AvBD2 protein (3.75–120 ng/μl) were mixed for 1.5 h at 37°C. Cells were washed and then incubated with the NDV-AvBD2 protein mixture for another 1.5 h at 37°C. Green curve , NDV (1 MOI) and equal volume of PB were mixed and then incubated with CEFs for 1.5 h at 37°C. Cells were washed and incubated with AvBD2 protein and equal volume of DMEM without serum for another 1.5 h. Purple curve , AvBD2 protein (3.75–120 ng/μl) and equal volume of DMEM without serum were mixed to incubate with CEFs for 1.5 h at 37°C. Cells were washed and then the mixtures of NDV (1 MOI) and equal volume of PB were added and incubated with CEFs for another 1.5 h at 37°C. Then, the cells were washed, and 2% DMEM were added and incubated for 48 h at 37°C. Then CCK-8 solution (5 mg/ml, 10 μl/well) was added to the plate. The absorbance at 450 nm was measured using a microplate reader after the incubation at 37°C for 1.5 h. The vector was used as control for each group. Cells without virus-protein mixture were also used as a negative control. Cells only infected with the virus served as a positive control. The experiment was performed in triplicate.
Article Snippet: In brief, CEFs in different multiplicity of infection (MOI) (0.01, 0.1, 1, 10, and 100) at different time points (24, 36, and 48 h post-infection, hpi) was added to the CCK-8 solution (5 mg/ml, 10 μl/well) before incubation at 37°C for 1 h. Absorbance was then measured at a wavelength of 450 nm by using a microplate reader (model 680; Bio-Rad Laboratories, Hercules, CA, United States).
Techniques: Recombinant, Expressing, Activity Assay, SDS Page, Purification, Marker, Plasmid Preparation, Molecular Weight, Infection, CCK-8 Assay, Incubation, Negative Control, Positive Control