Figure 3.

In vitro expression of consensus H7 HAs. BHK-21 cells (1 × 10⁶) were transfected with 3 μg pCH7-22, pCH7-24, pCH7-26, pCH7-28, and pVAX1 (empty vector) using Lipofectamine 3000 Transfection Reagent (Invitrogen). After incubation for 5 h, the medium was replaced with fresh medium containing 10% fetal bovine serum. At 24 h post-transfection, the cells were washed with PBS, fixed with 4% paraformaldehyde (pH 7.4) for 30 min, permeabilized with 0.2% Triton X-100 in PBS for 30 min, and then stained with Hoechst 33258 for 30 min. Indirect immunofluorescence staining was performed with monoclonal antibodies CR9114 (broad-spectrum anti-stem neutralizing antibody) or P52E03 (targeting the HA head of subtype H7) and then FITC-labeled goat anti-human IgG. Imaging was conducted under a fluorescence microscope (Nikon Eclipse TE2000).