Sars cov 2 rbd rabbit pab

The expression of PVES/mRNA vaccine was verified by Western blot analysis. Briefly, HEK-293T cells (1 × 10⁶ cells/well) were seeded into a 6-well plate and incubated in a 5% CO₂ incubator at 37 °C for 24 h. The PVES/mRNA vaccine complexes at N/P ratio of 32 (5.0 μg mRNA/well) were transfected according to the previous transfection method. After 24 h, the cells were collected, and radio immunoprecipitation assay (RIPA) lysate and proteinase inhibitor were used to lyse the cells. The total protein of the cells was extracted, and a bicinchoninic acid assay kit (Beyotime, Shanghai, China) was used to measure the protein concentration. The same amount of protein was taken for SDS-polyacrylamide gel electrophoresis separation, transferred on to the PVDF membrane, blocked with 5% skimmed milk, detected with SARS-CoV-2 RBD rabbit PAb (1:1000) (Sino Biological, Beijing, China) and secondary antibody (Sino Biological, Beijing, China). The blots were visualized with Clarity Western ECL Substrate (Applygen, Beijing, China) on Chemiluminescence imaging system (Tanon-5200 Multi, Shanghai, China).

Indirect immunofluorescence assay was used to verify the intracellular expression and localization of RBD protein. HeLa cells (2 × 10⁴ cells/dish) were seeded into 35 mm glass-bottom culture dishes (Corning, New York, USA), and cultured for 24 h. Then cells were transfected with PVES/mRNA vaccine (N/P = 32) for 24 h. The cells were washed thrice with PBS and fixed with 4% paraformaldehyde (Beyotime, Shanghai, China) for 20 min. Then, the cells were washed and permeabilized with Triton X-100 (Beyotime, Shanghai, China) for 10 min. After washing with PBS thrice, the cells were blocked with blocking buffer (Beyotime, Shanghai, China) for 15 min. Similarly, the cells were washed thrice and incubated with SARS-CoV-2 RBD rabbit PAb (1:250) (Sino Biological, Beijing, China) overnight at 4 °C. After washing five times, the MFP488-conjugated goat anti-rabbit immunoglobulin G (IgG) antibodies (1:250) were added to the cells and incubated for 1 h at room temperature. Finally, the cells were incubated with DAPI for 10 min and observed by spinning disk confocal with Nikon Ti-E microscope(Ultraview VOX, PerkinElmer, USA).