Plasmid Transfection and Capsid Protein Expression

Plasmids pVAX-C and pVAX were prepared using the EndoFree Plasmid Kit (Tiangen, Beijing, China) and were transfected into COS7 cells using the TransIntro^TM EL Transfection Reagent (TransGen Biotech, Beijing, China) when the cells were growing at around 80% confluent in six-well plates. The expression of the capsid protein in the cells post 48 h transfection was confirmed by indirect immunofluorescence as described previously [21 (link)]. Briefly, transfected cells were washed with phosphate-buffered-saline (PH 7.2) (PBS), then fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton X-100 in PBS and blocked with 5% BSA in PBS (BSA-PBS). After that, the cells were incubated with rabbit anti-capsid protein polyclonal antibody (prepared by our lab) as the primary antibody diluted 1:100 in BSA-PBS for 2 h, followed by incubation with the Alexa Fluor 488-conjugated goat anti-rabbit IgG (Thermo Fisher, Lafayette, CO, USA) as the secondary antibody was diluted 1:2000 in BSA-PBS for 2 h at room temperature. The cell nuclei were counterstained with 4′, 6-diamidino-2-phenylindole (DAPI) for 10 min at room temperature. The cells were examined by fluorescence microscopy (Nikon, Tokyo, Japan).