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. 2014 Jun 19;9(6):e99201. doi: 10.1371/journal.pone.0099201

Figure 2. Epitope screening with HA chimeras.

Figure 2

(A) Schematic diagram of chimeric HAs. A series of chimeric HAs were generated by domain swapping between VN1194 HA (blue) and H1pdm HA. (B) Formations of chimeric HAs simulated by MOE based on the crystal structure of VN1194 HA (PDB ID: 2IBX, blue) and H1pdm HA (PDB ID: 3LZG, gray). (C) Expression of the chimeric HAs in mammalian cells. The chimeric HA expression vectors were co-transfected with pRetroX-Tet-Off Advanced into 293T cells. Cells were collected at 24 hpt and analyzed by Western blotting. Upper panel, expression patterns of the HAs; lower panel, loading control (GAPDH). Cells transfected with an empty vector (lane 1) and the expression vectors of H1pdm HA (lane 2), VN1194 HA (lane 3), H5/1 chimera (lane 4), H1/5 chimera (lane 5), and globH5 chimera (lane 6) were analyzed. (D) Interaction between the chimeric HAs and OM-b, AY-2C2 and YH-1A1mAbs. Cells described in (B) were incubated with mAbs, and the reactivity was analyzed by flow cytometry. Histograms of levels of reactivity against each mAbs in cells expressing the indicating HAs.