BHK-21 cells were seeded into a 6-well plate and cultured at 37 °C with 5% CO
2. A cell monolayer at 80% confluence was transfected with either H, optiH, H-IRES-N, optiH-IRES-optiN, optiH-IRES-IFN, or an empty plasmid vector, pIRES, using
Lipofectamine 2000 (Thermo Fisher, Carlsbad, CA 92010, USA) according to the manufacturer’s instructions. At 48 h after transfection, the cells were fixed in 1 mL of 4% formaldehyde for 30 min at 37 °C and permeabilized in 1 mL of 0.5% Triton X-100 for 10 min. After three washes with phosphate-buffered saline (PBS), samples were blocked with goat serum for 30 min at 37 °C. The samples were washed and blocked after each subsequent antibody exposure to anti-CDV H protein monoclonal antibody (1C42H11 monoclonal antibody, VMRD, Pullman, WA, 99163, USA), anti-CDV N protein monoclonal antibody (CDV-NP monoclonal antibody, VMRD, USA), and anti-mink IFN-γ monoclonal antibody [19 (
link)], as appropriate for the corresponding plasmid vectors, whereas
goat anti-mouse IgG H-L (FITC) (Abcam, Cambridge 02139, UK) was used as the secondary antibody. Images were obtained using a fluorescence microscope (
EVOS FL, Thermo, USA). Five fields of vision for each image were randomly selected, and the mean number of fluorescent cells was calculated and compared to evaluate protein expression levels.
Zhao J., Sun Y., Sui P., Pan H., Shi Y., Chen J., Zhang H., Wang X., Tao R., Liu M., Sun D, & Zheng J. (2023). DNA Vaccine Co-Expressing Hemagglutinin and IFN-γ Provides Partial Protection to Ferrets against Lethal Challenge with Canine Distemper Virus. Viruses, 15(9), 1873.