Fig. 1: Design and expression of HA6 protein originated from H9 subtype AIV.

a Protein interaction site selection, HA1 (red), HA2 (cyan), HA1-HA2 interaction sites (yellow). b Red represents the HA1 domain with non-interacting regions removed, exposing new amino acid sites. c After stability optimization, AlphaFold2 simulates the tertiary structure of HA6. Green represents the mutation sites optimized for stability using RosettaDesign, and yellow represents the F63D and L73D mutations. d Prokaryotic expression of HA2 protein from H9 subtype AIV: 1–3 supernatants after induction at 16, 25, and 37 °C, 4–6 pellets after induction at 16, 25, and 37 °C. e Purification of expressed HA2 protein: 1–2 supernatant and precipitate from bacterial lysis, 3–6 wash and dissolve the residue with 0, 2, 4, 8 M urea. f Prokaryotic expression of HA6 protein: 1–3 supernatants after induction at 16, 25, and 37 °C, 4–6 pellets after induction at 16, 25, and 37 °C. g. Purification of HA6 protein: 1 pellet lysate supernatant, 2 elution with 20 mM imidazole, 3–6 elution with 100 mM imidazole, 7 elution with 250 mM imidazole. h Indirect ELISA to determine the binding capacity of CR6261-SCFV to HA2 and HA6. i Indirect ELISA to determine the binding capacity of H9 subtype AIV immune serum to HA2 and HA6. j Competitive ELISA to measure the competitive ability of HA6 immune serum against CR6261-SCFV. k Competitive ELISA to evaluate the competitive ability of HA2 immune serum against CR6261-SCFV. Statistical analysis was performed using two-tailed t-tests. Error bar, mean ± SD.