Fig. 3

Analysis of the HA2 protein linear epitopes using WB and IFA. a Schematic representation of the HA truncated fragments used for epitope mapping (left). The corresponding results of WB were shown in right and the reactive fragments were containing ⁴³³NAELLVL⁴³⁹ (or ⁹⁵NAELLVL¹⁰¹, HA2 numbering) sequence in full length of HA. b The minimal epitope mapping of 3C12 using WB. The 433-439aa sequences was cloned into pGEX-4 T-1 vector and expressed in E. coli BL21 then subjected to incubation with mAb 3C12. c Reaction of the mAb with the minimal epitope were evaluated by IFA. The three epitopes were cloned into pCAGGS vector and transfected into 293 T cells, respectively. d The location of the epitope recognized by 3C12 in the HA molecule was determined by PyMOL using the PDB 1jsd (H9N2 HA). The corresponding positions were colored and pointed out with lines. Since the Alanine residue of the position 96 (HA2) did not exist on the surface, we could not map it. All the residues were represented by HA2 numbering. e The structure of HA molecule showed by cartoon using the PDB 1jsd. HA with helicx A (red), C (yellow), and D (blue) and B loop (orange) in one monomer colored distinctly