Figure 1

Construction of plasmid analysis of recombinant proteins and mouse vaccination schedule. (A) The consensus sM2 genes were cloned into pRSET A vector. (B) Purified 4sM2 protein from a prokaryotic expression system was confirmed by Coomassie Brilliant Blue staining and was detected at a molecular weight of 60 kDa. 4sM2 protein expression was verified by Western blot analysis using (C) anti 6 × His antibodies and (D) antiM2 antibodies. (E) Mice were grouped as shown in Table 2 and all groups were intramuscularly administered twice every other week. Sera were collected before the first administration and 1 week after each vaccination. Spleens were excised from three mice in each group of one set 10 days after last immunization. All mice were challenged with a lethal dose of HPAI viruses intranasally and monitored for 13 days at 2 or 24 weeks after the last immunization. At 3 and 5 dpi, lungs were excised from three mice in each group to check the virus titer. Mice from one set were sacrificed for lung histopathology at 5 dpi. Abbreviations: 4sM2, recombinant multimeric sM2 protein; NC, negative control; M, protein marker; CB, Coomassie Brilliant Blue.