Fig. 1

ClyA porin carrying RBD forms RBD-PP. A Schematic diagram of the preparation of RBD-PP. B Schematic diagram of the ClyA-RBD fusion protein. Red represents the receptor-binding motif (RBM). C After SDS–PAGE, the total bacterial protein products and purified PP and RBD-PP samples were analyzed by silver staining. The red arrow indicates ClyA, and the blue arrow indicates ClyA-RBD. D Western blotting was performed using an anti-SARS-CoV-2 spike S1 antibody. E The distributions of the particle sizes of the bacterial outer membrane overexpressing ClyA-RBD (RBD-OM), the intermediate process of the surfactant being gradually inserted into the outer membrane and self-assembly, and the purified RBD-PP. In a representative TEM image of RBD-OM, the small white circles in the white box mark the nanopores on the outer membrane, bar = 100 nm. F PyMOL was utilized to visualize homology modeling results; at the top is a schematic representation of the polymeric chains formed by monomers. Red represents RBD, and green represents the linkers. In the middle is the sporadic nanopore structure observed under TEM, bar = 10 nm. At the bottom is the final stable RBD-PP and PP electron microscope image, and the bottom left corner is an enlarged image of the area in the white frame. G The top panel shows the DLS analysis of RBD-PP and PP, and the bottom panel shows the statistical analysis of particle size (n = 3). H Denatured and nondenatured samples of ClyA and ClyA-RBD were analyzed by SDS–PAGE. Arrows indicate the positions of the denatured monomer and nondenatured polymer. I The binding ability of RBD-PP with hACE2 (n = 3). J Six monoclonal antibodies that can neutralize SARS-CoV-2 were allowed to react with RBD-PP (n = 3). The half-maximal inhibitory concentrations (IC₅₀ values) of the neutralizing antibodies MM43, MM57, R004, R001, D001, and D002 were 1.41 μg/ml, 0.41 μg/ml, 0.234 μg/ml, 0.11 μg/ml, 0.646 μg/ml, and 46.76 μg/ml, respectively. Data are shown as the mean ± SD. Statistical significance was calculated via one-way ANOVA (J), two-way ANOVA (I) or Student’s t test (G) to obtain p values: ****p < 0.0001, and *p < 0.05