FIGURE 1.

CuMV_TT‐VLPs constitute an efficient platform for genetically fusing the receptor‐binding motif (RBM). A, Schematic depiction of E. coli cells containing pETDuet‐1‐derived plasmid, allowing the coexpression of CuMV_TT‐RBM fusion and unmodified CMV_TT genes and production of mosaic VLPs termed (mCuMV_TT‐RBM), B, SDS‐PAGE analysis of the production and sucrose gradient purification of mCuMV_TT‐RBM. M—protein size marker (Thermo Scientific), 0 – total proteins in E.coli C2566 cells before IPTG induction; T—total proteins in E.coli C2566 cells after IPTG induction and 18h cultivation at 20℃; S—soluble proteins in E.coli C2566 cells after induction and 18 h cultivation; P—insoluble proteins in E.coli C2566 cells after induction and cultivation; 60 – 0 – sucrose gradient fractions after separation of cell lysate in Beckman SW32 rotor; mCuMV_TT‐RBM VLP containing sucrose fractions are labeled with red circle; C, native agarose gel analysis of the sucrose gradient fractions after sucrose gradient purification of mCuMV_TT‐RBM. M—DNA size marker (Thermo Scientific), 60–0– sucrose gradient fractions after separation of the cell lysate in Beckman SW32 rotor; mCuMV_TT‐RBM VLP containing sucrose fractions are labeled with red circle; D, SDS‐PAGE analysis of purified mCuMV_TT‐RBM VLPs. M—protein size marker (Thermo Scientific), asterisk (blue) refers to unmodified CuMV_TT monomer and asterisk (green) refers to genetically modified CuMV_TT‐RBM E, Electron microscopy analysis of purified mCuMV_TT‐RBM VLPs; F, DLS analysis of purified mCuMV_TT‐RBM VLPs. G, ACE2 binding to mCuMV_TT‐RBD vaccine candidate, CuMV_TT, and RBD alone were used as controls. Plates coated with 1µg/ml of ACE2. Binding was revealed using anti‐CuMV mAb. One representative of 2 similar experiments is shown