Fig. 1: Construction and purification of recombinant HA proteins.
a Schematic representation of the full length HA protein of H9N2 (MsCon) virus. Precursor HA0 :1-560 amino acids (aa), HA1:19-338 aa, HA2: 339-560 aa, TM = transmembrane domain (525-547 aa) CT = cytosolic tail domain (548−560 aa). b Schematic representation of the soluble HA protein of H9N2 (MsCon) virus. The soluble H9HA was generated by removing the TM and CT domains (525-560 aa) and fusing the C-terminus of HA to 30 aa long trimerisation foldon sequence of the trimeric protein fibritin from T4 bacteriophage c His tag purification of the recombinant proteins. The expected sizes of the purified CD83 scFv antibody, rH9HA, and rH9HA-CD83 scFv are 30, 70, and 100 kDa respectively. Lane 1: control supernatant from the untransfected cells Lane 2: CD83 scFv Lane 3: rH9HA Lane 4: rH9HA-CD83 scFv. For the purification of the recombinant proteins, the harvested S2 cell culture supernatants containing recombinant protein bound to the metal ions (copper sulfate was used as an inducer of metallothionein promoter) were loaded onto uncharged “UNOsphere” resin derivatized with iminodiacetic acid functioning as a chelating ligand (Profinity™IMAC, Bio-Rad). Proteins were eluted with elution buffer containing 50 mM NaH2PO4, 300 mM NaCl and 50 mM imidazole. The purified proteins were analysed by 10% SDS-PAGE followed by Coomassie staining. All blots were derived from the same experiment and were processed in parallel.
