Fig 1.
Western blot analysis of cell lysates infected by the Baculo-P1-3CD-gp41 construct.
The proteins were separated by SDS-PAGE, electrotransferred to a nitrocellulose membrane, and probed using anti-VP2 MAb (MAb979) as the primary antibody (A). Hi-5 cells used for EV71 VLP large-scale production were infected with Baculo-P1-3CD-gp41 at MOI 5, and protein was analyzed using an anti-VP1 Mab produced in-house (B). Lane1: Baculo-P1-3CD-CMV-IE infected cell lysates; Lane2: Baculo-P1-3CD-lef3 infected cell lysates; Lane3: Baculo-P1-3CD-gp41 infected cell lysates; Lane4: Baculo-P1-3CD-chitinase infected cell lysates. The cells were harvested from 3 dpi.
Table 1.
Comparison of VLP yield according to promoter type (units: mg/L).
Fig 2.
Western blot analysis of Hi-5 cell lysates infected with Baculo-P1-3CD-gp41 at MOI 5 during different times.
Hi-5 cell lysate protein was analyzed using an anti-VP2 MAb (MAb979). The cells were harvested from 1–6 dpi.
Fig 3.
TEM image of EV71 C4a-gp41 VLP produced by Baculo-P1-3CD-gp41.
The VLP preparation was purified by sucrose gradient ultracentrifugation.
Fig 4.
Humoral immune response induced by EV71 C4a VLP-gp41 in mice.
The titer of EV71 antibody in each of 5 groups of mice that received EV71 VLP (5μg), VLP (5μg) with alum, or phosphate-buffered saline (PBS) as a control according to immunization schedule as indicated by the arrows. (A) Titers of total IgG antibodies agains EV71 VLP were determined by ELISAs. (B) The titer of the neutralizing antibody against EV71 C4a was assayed by the titer of the serum in a microneutralization assay. The virus titer of EV71 C4a was 100 TCID50. (circle, wild type; square, VLP 5μg; triangle, VLP 5μg with alum). (C) Mice received EV71 VLP (5μg), VLP (5μg) with alum, or control and each titer was tested for cross-reactivity in mice against other EV71 subtypes by assaying the titer of the serum in a microneutralization assay. The virus titers of EV71 B3, B4, and C5 were each 100 TCID50, respectively (dot bar, EV71 B3 type; black bar, EV71 B4 type; white bar, EV71 C5 type; diagonal line bar, EV71 C4a). These data represent the means of two replicates and error bars indicate SD of the mean. Data statically analysis was performed by one way ANOVA, p < 0.01.