Camvir1

The cells were permeabilized on the glass slide with 100% cold acetone. Subsequently, the fixed cells were probed with anti-HPV16 L1 antibody CAMVIR-1 (Abcam, Cambridge, UK) and captured with anti-mouse IgG-FITC (Sigma). Immune-stained cell monolayers were thoroughly washed with PBS and covered with mounting medium with DAPI (Abcam). The immunofluorescence images were inspected under an inverted microscope at 40× magnification. Transfection efficiency was determined by the ratio of FITC (green)-positive cells to DAPI (blue)-stained cells.

Sf9 cells were infected with recombinant baculovirus for three days, harvested, and cell-lysates or purified protein were analyzed by SDS electrophoresis and Coomassie staining, or Western blotting using Camvir-1 (1:10,000 dilution; Abcam, Cambridge, UK), or antiserum to HPV16 L2 aa 11–200 (1:5,000 dilution; R. Roden, Johns Hopkins) and a goat anti-mouse IgG-HRP (horseradish peroxidase) coupled secondary antibody (Biorad) to verify the recombinant protein.

Cells were washed with PBS and total protein was harvested using RIPA lysis buffer (Beyotime, China). 20ul denatured protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- PAGE, GenScript, USA) and transferred onto polyvinylidene fluoride membranes (PVDF, Millipore, USA). After blocking with 5% non-fat milk for 1 h at room temperature, the membranes were incubated with primary antibodies (HPV16-L1-specific antibody CamVir1, Abcam, Cambridge, MA, USA, 1:3000 and anti-β-actin, Bioss, China, bs-0061R, 1:1000) at 4 °C overnight. The membranes were washed and exposed to corresponding horseradish peroxidase (HRP)-conjugated goat anti-rat (1:5000, Mengbio, China, MS002A) or goat anti-rabbit (1:1000, Beyotime, China, A0208) secondary antibodies diluted in TBST buffer for 2 h at room temperature. Finally, the protein bands were visualized with an enhanced chemiluminescence (ECL) kit (Millipore, USA).

Plant-expressed L1/L2 chimeras were quantified by capture ELISA using a modified polyvinyl alcohol (PVA)-blocking ELISA method (Studentsov et al., 2002 (link)), as described in Pineo et al. (2013 (link)). Briefly, a 96-well Nunc Maxisorp microtitre plate (Thermo Fisher, USA) was coated with 1:2,000 CamVir1 (Abcam, UK; a mouse anti HPV-16 L1 MAb) overnight at 4°C, washed and blocked with PVA buffer. Diluted plant cell extract was added to the wells and incubated for 1 h at 37°C, followed by a washing step and the addition of rabbit anti-HPV-16 polyclonal serum (1:1,000) overnight at 4°C. After washing, swine anti-rabbit horseradish peroxidase (HRP) conjugate (1:5,000; DAKO, Denmark) was added to wells, plates were incubation for 30 min at 37°C and the proteins were detected with OPD substrate (DAKO). Plates were developed in the dark, the reaction was stopped with 0.5M H₂SO₄ and the absorbance was detected at 490 nm. Total soluble protein (TSP) was determined using a Lowry protein assay (BioRad, USA) as per the manufacturer's instructions, with a bovine plasma IgG standard (BioRad, USA) to normalize the ELISA data.

Cells were lysed in RIPA buffer containing protease inhibitor cocktail (Roche) and 20 µg of total protein was separated by SDS-PAGE. Antibodies: Mouse monoclonal anti-L1 (1∶1000, CAMVIR-1, Abcam), mouse monoclonal anti-c-Myc (1∶1000, 9E10, Santa Cruz Biotechnology), mouse monoclonal anti-β-actin (1∶5000, 8H10D10, Cell Signaling Technology), rabbit polyclonal anti-IFITM1 (1∶1000, 11727-3-AP, Proteintech), rabbit polyclonal anti-IFITM3 (1∶500, H00010410-D03P, Novus Biologicals) and HRP-conjugated secondary antibodies (1∶10,000, Jackson ImmunoResearch Laboratories). Proteins were visualized using Clarity Western ECL substrate (Bio-Rad) and the ChemiDoc XRS System (Bio-Rad).

Yeast cell lysate supernatants or purified VLP samples were diluted in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer, boiled at 100 °C for 10 min, electrophoresed through 10% TGX Stain-Free SDS-PAGE gel (Bio-Rad, Hercules, CA, USA) and proteins were detected by Stain-Free technology (Bio-Rad, Hercules, CA, USA). Then gel was transferred to a PVDF membrane using a Bio-Rad semi-dry apparatus. The membrane was blocked with 5% skim milk in TBS-T and probed with the anti-L1 mAb [CamVir 1] (Abcam, Cambridge, UK) at dilution of 1:2000, and a secondary antibody directed against mouse IgG conjugated to Peroxidase. Bound antibodies were be detected using an enhanced chemiluminescence detection kit (GE Health care, Chicago, IL, USA).