2.1 Expression and purification of recombinant proteins

Triple M2e peptides derived from human (hM2e), swine (sM2e) and avian (aM2e) IAV were combined with the C-terminus of Cap protein in different arrangements using Gly-Gly-Gly-Gly linker. (Fig. 1a-1c). These sequences were inserted into the pET28a vector by BamHI and HindIII digestion enzymes, and transformed into E. coli BL21 (DE3). Then these transformed cells were induced expression at 20°C for 15 h by isopropyl-β-d-thiogalactoside (IPTG) (0.2 mM). These recombinant proteins were purified by using Ni-NTA His·Bind Resin and identified by SDS-PAGE and Western blot. The concentrations of these purified proteins were determined with a BCA protein assay kit (Solarbio, Beijing, China). The endotoxin concentrations were measured by ToxinSensor Single Tests Kit (GenScript, Piscataway, NJ, USA). The VLPs pattern was drawn from the PDB accession number 3R0R.