Journal: bioRxiv

Article Title: Analysis of the SARS-CoV-2 spike protein revealed that blocked receptor-binding domain antigenicity decreases the production of neutralizing antibodies in vivo

doi: 10.1101/2023.02.23.529497

Figure Lengend Snippet: Illustrations of the interaction between RBD/SS-RBD and ACE2 receptor and the mechanism by which RBD is undetectable with a NAb but detectable with an anti-6-Histag mAb. : Kinetics in RBD expression were detected by intracellular staining with a NAb against the S1 subunit in HEK293 cells. RBD plasmids administered at 1,6 and 1,1 μg/well were studied in parallel. The empty pUC57 vector was used as a negative control. : Kinetics of RBD expression were detected by FACS analysis with intracellular staining and anti-6 His-tag mAb. : Kinetics of SS-RBD expression were detected with intracellular staining by NAb against the S1 subunit. : The complex structure of SARS-CoV-2 RBD bound to hACE2. The core and external subdomains in the SARS-CoV-2 RBD are colored cyan and orange, respectively. Human ACE2 subdomains I and II are colored violet and green, respectively. The contacting sites are further delineated in . : A representation of the SARS-CoV-2 RBD structure. The critical contact sites of the amino acids are marked according to Wang et al . . : The signal peptide sequence (SS) consists of 19 amino acids from the beginning. A cleavage site is at amino acid 13. A partial immunogenic epitope in the SS is marked in red letters. The glycan site, “NLT,” or “N” is marked with a blue highlight. Structure-based sequence alignments of the RBD related to and . The secondary structure elements are defined based on an ESPript algorithm and labeled based on the RBD structure reported in the study. Dashed lines represent alpha strands and arrows represent beta strands. Three glycosylation sites (N) are highlighted in blue. Two antigenic epitopes, η1 and η2, are highlighted in yellow. Critical receptor binding domains consisting of essential amino acids are highlighted in green, and the number of positions of the amino acids is shown at the top.

Article Snippet: Rabbit monoclonal antibodies against 6-His-tags (12698S) were purchased from Cell Signaling Technology.

Techniques: Expressing, Staining, Plasmid Preparation, Negative Control, Sequencing, Labeling, Binding Assay

Journal: bioRxiv

Article Title: Analysis of the SARS-CoV-2 spike protein revealed that blocked receptor-binding domain antigenicity decreases the production of neutralizing antibodies in vivo

doi: 10.1101/2023.02.23.529497

Figure Lengend Snippet: Confocal microscopy imaging for the localization and expression of the full-length spike protein and fragments at 24 hrs p.t. in . A549 and HEK293 cells were grown on glass coverslips overnight; the cells were transfected with plasmid DNAs, incubated for 24 hrs, fixed, and processed for immunofluorescence staining . The full-length spike protein and fragments’ subcellular distribution was stained with an anti-6-his tag mAb and FICT secondary Ab (green), and nuclei were stained with DAPI (blue). Bar: 10 μm. : A549 and HEK293 cells were also calculated as the percentage of cells positively transfected for the full-length spike protein and fragments under immunofluorescence microscopy. The graph presents the averages from triplicate samples. The asterisks indicate significant differences, as demonstrated by an unpaired T-test (* P < 0.05; **P < 0.01; ***P < 0.001); ns, not significant. The data are presented as the mean ± SE from three samples.

Article Snippet: Rabbit monoclonal antibodies against 6-His-tags (12698S) were purchased from Cell Signaling Technology.

Techniques: Confocal Microscopy, Imaging, Expressing, Transfection, Plasmid Preparation, Incubation, Immunofluorescence, Staining, Microscopy