Fig 1: (a) One percent agarose gel to show PCR products corresponding to the H5HA gene of 1695 bp: lane 1, positive control (plasmid DNA with the gene H5rD); lanes 2–4, PCR product from 3 samples from transformed D. salina culture; lane 5, negative control (DNA from 1 culture sample of untransformed D. salina); MP, molecular weight marker (GeneRuler 1 kb DNA Ladder SM0311, Thermo Fisher, Waltham, MA, USA). (b) Coomassie blue-stained 12% SDS-PAGE gel showing the expression of a putative protein in transformed D. salina cultures: lane 1, TSP of WT D. salina cultures; lane 2, TSP of transformed D. salina cultures (adjusted to 70 µg); lane MP, molecular weight marker (PageRuler™ Plus Prestained Protein Ladder, 10 to 250 kDa 26619, Thermo Fisher, Waltham, MA, USA); lanes 3–6, BSA at total concentration of 0.1, 0.5, 1, and 1.5 µg, respectively. (c) Western blot of the H5rD protein with monoclonal antibodies: lane 1, H5rD protein detected with anti–avian influenza A hemagglutinin antibody (Abcam AB135382, Cambridge, MA, USA); lane 2, H5 standard protein as positive control (Abcam AB190125, Cambridge, MA, USA) molecular weight ~64 kDa without post-translational modification; lane 3, TSP sample from D. salina WT; MP, molecular weight marker (PageRuler™ Plus Prestained Protein Ladder, 10 to 250 kDa 26619, Thermo Fisher, Waltham, MA, USA). (d) Western blot detection of H5rD protein with polyclonal antibodies: lane 1, H5rD protein from TSP detected with a chicken serum (IgY anti-H5); lane 2, viral proteins from low pathogenic virus A/Chicken/México/232/94/CPA; lane 3, TSP sample from D. salina WT; lane MP, molecular weight marker (PageRuler™ Plus Prestained Protein Ladder, 10 to 250 kDa 26619, Thermo Fisher, Waltham, MA, USA); protein bands identified in lane 2 correspond to (I) HA, (II) NA / NP, (III) HA1, (IV) M1 and (V) HA2. (e) HA assay of ultrafiltered H5rD protein incubated with 1% suspension of chicken erythrocytes: row 1–3, Microalgae-produced hemagglutinin; row 4, BSA negative control serially diluted, initial concentration of 10 µg/mL; row 5, allantoic fluid from chicken embryos infected with low pathogenic virus A/Chicken/México/232/94/CPA as a positive control.