(A) Phylogenetic tree of the 18 hemagglutinin subtypes of influenza A viruses based on amino acid sequences. Group 1 and group 2 subtypes are listed in red and blue, respectively, and the HAs used in recombinant protein NMHC are listed in green. The amino acid distance scale bar denotes a distance of 0.1. (B) NMH consists of the conserved gene segments of NP, M2e, and HA2, which are connected by linker (GSAGSAG). NMHC consists of NMH and linker followed by SEC2. (C) NMHC, SEC2, and NMH purified and renatured from BL21(DE3) lysate. Lane 1: renatured NMHC; lane 2: marker; lane 3: purified SEC2; lane 4: renatured NMH. (D) The structure of the recombinant protein NMHC about homology modeling. The light blue, light green, and purple portions are NP, M2e, and HA2(H1, H3, H7) fragments, respectively, and red portion fragment is SEC2 domain. (E) Murine splenocytes proliferation induced by NMHC, NMH + SEC2, SEC2, NMH, and PBS were observed at 72 hr. (F) Proliferation index was quantified by MTS assay. Scale bar = 200 μm. Data are represented as mean ± SD (n = 3). *p<0.05, ns, not significant. Source files of the gel used for the qualitative analyses are available in Figure 1—source data 1.