Fig. 1

Generation and verification of recombinant BCG strain CW-rBCG::RBD. (a) Schematic representation of the coding sequence within the recombinant pMV261 plasmid, encompassing the 19-KD antigen (coding gene Rv3763) promoter and signal peptide, linker sequence, and the wild-type SARS-CoV-2 RBD sequence. Illustration depicting the engineering process of the RBD antigen expressed by CW-rBCG::RBD, wherein the signal peptide of 19-KD antigen undergoes cleavage by Sec/signal peptidase II at the preferred restriction site LSG, forming a mature signal peptide with the mature sequence CSSNKSTTG. The mature signal peptide, together with the RBD protein, constitutes the fusion protein expressed by CW-rBCG::RBD. (b) Western blot analysis of CW-rBCG::RBD strain to assess the presence of the RBD antigen in subcellular fractions. The RBD antigen band was observed at approximately 35–40 kDa. A negative control was established using the parent BCG Danish strain harboring an empty pMV261 vector. The subcellular constituents of the CW-rBCG::RBD are organized within the identical lanes as those observed in the control group situated on the right-hand side. Subcellular fractions were analyzed in lanes corresponding to the cell membrane and other components: lane 2 represents the Triton X-114 soluble detergent phase extracted from the precipitate; lane 3 represents the Triton X-114 aqueous phase extracted from the precipitate; lane 4 represents the Triton X-114 detergent and amphoteric phases extracted from the supernatant of cell lysate. (c) Quantification of RBD antigen content in the cell wall and membrane of CW-rBCG::RBD per 10⁷ cfu. Integrated density values of the bands were determined via western blot experiments and quantified against RBD-His protein standards using ImageJ software. Details regarding the calculation of CW-rBCG::RBD or BCG quantities used for subcellular component separation, along with corresponding volume ratios, are provided in the Methods, Supplementary Data, and Supplementary Fig. S1b–e. The RBD antigen content in the samples was calculated by estimating the final volume ratios of different subcellular components relative to the total bacterial solution volume during the separation process (Supplementary Fig. S1). (d) Flow cytometry confirmed surface expression of RBD on CW-rBCG::RBD. Shown is one representative result from three independent experiments (Supplementary Fig. S1g). Green histogram: stained with anti-RBD antibody and FITC-conjugated secondary antibody. Yellow histogram: shown background fluorescence, anti-RBD antibody only (control).