Figure 1.
Selection and comparison of transfected cell clones expressing E2 protein using ELISA.
E2 protein expressing titers were detected and compared between 60 G418-resistant and IFA-positive cell clones (A). Among the 60 cell clones, 12 cell clones with a relative high titer of E2 were selected and further compared using ELISA (B). The no. 12 cell clone, which had the highest ELISA titer value was selected for further characterization (as shown by the arrow).
Figure 2.
Immunofluorescence and flow cytometry analysis of BCSFV-E2 cell passages.
The tenth (F10), twentieth (F20), and thirtieth (F30) generations of BCSFV cells were analyzed by indirect immunofluorescence and flow cytometry. Both assays show a high percentage of cells expressing CSFV E2.
Figure 3.
SDS-PAGE analysis of mE2 in culture and purified medium.
M, Molecular marker; 1, supernatant of BCSFV-E2; 2 and 3, Immunoaffinity-purified mE2; –, non-reduced; +, reduced with β-mercaptoethanol.
Figure 4.
Western blot analysis of mE2 in culture supernatants.
BHK-21 cells and BCSFV-E2 cells culture supernatants were separated in the absence (+) or presence (+) of β-mercaptoethanol or treated with peptide: N-glycosidase F (PNGase F) followed by Western blot analysis with 12C4 monoclonal antibody.
Figure 5.
Production of mE2 by BCSFV-E2 cells.
(A) Confluent BCSFV-E2 cells were incubated in maintenance medium for six days. ELISA was used to quantify the titers of mE2 antigen accumulated in the supernatant every day (24 h). (B) The culture medium of confluent BCSFV-E2 cells was harvested every 4–6 days and replaced with fresh medium, and the mE2 antigen titers in the harvested culture supernatants were determined by ELISA. (C) The culture supernatants of different passages of cell line were determined by ELISA.
Figure 6.
ELISA antibody development after vaccination and challenge infection.
Pigs were vaccinated with mE2 antigen with four kinds of adjuvants. The pigs were given a booster vaccination four weeks after the first vaccination. The pigs were then challenged with a virulent CSFV strain at 32 weeks post-vaccination. Pigs inoculated with PBS were used as controls. The antibody titers are presented as blocking rates (A) and the reciprocal of the highest dilution of serum for which a blocking rate of ≥40% was obtained (B).
Figure 7.
Rectal temperatures post-challenge.
Vaccinated and control pigs were challenged with the virulent CSFV Shimen strain. After challenge, rectal temperatures of each pig were measured daily for 14 days.
Table 1.
Neutralizing antibody response following vaccination and challenge infection.
Table 2.
Clinical outcomes of pigs following viral challenge.