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. 2019 Apr 25;10:860. doi: 10.3389/fimmu.2019.00860

Figure 2.

Figure 2

Design and characterization of the pscCtrl or pscDEC plasmids encoding the eGn and mCherry protein fusions with scFvs. (A) Schematic representation of pscCtrl and pscDEC fused with eGn or mCherry sequences. The pscCtrl (gray background) and pscDEC (white background) constructions include the signal peptide from VH of murine IgG (SP), the scFv (VH and VL) sequences, and the codon-optimized eGn or the mCherry sequences. The VH and VL sequences derived from the rat anti-DEC205 NLDC-145 mAb (scDEC) and from a control rat mAb (scCtrl) are connected together with a (G4S)4 linker and the scFv and antigen sequences are connected with a (G4S)3 linker. In (B) purified scCtrl-mCherry and scDEC-mCherry proteins collected from supernatants of transfected HEK293 cells were separated by SDS-PAGE under reducing conditions and mCherry was revealed with an anti-mCherry rabbit IgG followed by a HRP-GAR IgG. The predicted sizes of the expressed chimeric proteins are 57.6 and 57.5 kDa for the scDEC-mCherry and scCtrl-mCherry, respectively. In (C) HEK293 cells were transfected with pcDNA4 (control), peGn, pscCtrl-eGn, and pscDEC-eGn. The cell lysates were separated by SDS-PAGE under reducing conditions and eGn was revealed with an anti-RVFV HMAF followed by a HRP-GAM IgG. The predicted sizes of the recombinant proteins are 48.8 kDa for eGn, 78.8 kDa for the scCtrl-eGn, and 79 kDa for the scDEC-eGn. In (D) CHO cells expressing the murine DEC205 receptor (CHOmDEC205) or CHOneo (control) were labeled with 10 μg/ml of A647-anti-DEC205 (gray) and compared to an A647-isotype control (dash line). In (E) CHOmDEC205 were labeled with 50 μg/ml of scCtrl-mCherry and scDEC-mCherry proteins from purified supernatants. The staining with scDEC-mCherry is depicted (gray) and compared to scCtrl-mCherry (dash line). In (F) CHOmDEC205 were labeled with the 20–40 times concentrated supernatants from the same number of transfected cells with peGn, pscCtrl-eGn, and pscDEC-eGn and eGn was revealed with the RVFV HMAF (1:1,000) followed with an A488-DAM IgG. The staining is depicted (gray) compared to a control supernatant (pcDNA4 empty vector, dash line). No signal was obtained with CHOneo cells (not shown).