3.1 Characteristics of Cap-3M2e VLPs
Six different recombinant Cap-3M2e proteins were expressed in E.
coli and purified using Ni2+-NTA
column. SDS-PAGE and Western blot showed that these recombinant Cap-3M2e
proteins were successfully expressed and purified (Fig. 1d). These
recombinant Cap-3M2e proteins can be recognized well with the anti-PCV2
polyclonal antibody and 14C2 monoclonal antibody (Anti-IAV M2 protein),
indicating that these recombinant proteins retained the reactogenicity
of M2e and the Cap protein (Fig. 1e). Endotoxin levels in these
recombinant proteins were less than 0.18 EU/mg.
TEM images showed that these recombinant Cap-3M2e proteins could
self-assemble into VLPs (Fig. 2a). DLS test results showed that the
diameter of these Cap-3M2e VLPs were larger than that of Cap VLPs (Fig.
2b). The zeta potential of these Cap-3M2e VLPs were lower than that of
Cap VLPs, which were caused by the strong negative charge of M2e
molecules (Fig. 2c). The particle size and surface charge of these
Cap-3M2e VLPs are consistent, indicating that the permutation of M2e of
IAV from different species does not affect the morphology and surface
charge of these VLPs (Fig. 2).
3.2 Humoral immune
effects
All groups induced consistent PCV2-specific antibodies and neutralizing
antibodies, indicated that the insertion of three copies of M2e in
different orders did not affect the immunogenicity of the Cap VLPs (Fig.
3a and 3b). Total M2e-specific antibodies in Cap-3M2e VLPs groups were
consistent, indicated the permutation of M2e did not affect the level of
total M2e-specific antibodies (Fig. 3c). However, there were significant
differences in levels of antibodies against M2e derived from human,
swine and avian IAV in sera of each group. The Cap-hsaM2e VLPs and
Cap-hasM2e VLPs groups induced higher anti-human IAV M2e antibodies than
others (Fig. 3d). Anti-human IAV M2e antibody in the Cap-hasM2e VLPs
group was 7.3 times that of the Cap-sahM2e VLPs and Cap-ashM2e VLPs
groups (Fig. 3d). The Cap-shaM2e VLPs and Cap-sahM2e VLPs groups induced
higher anti-swine IAV M2e antibodies than other groups (Fig. 3e).
Anti-swine IAV M2e antibody in the Cap-shaM2e VLPs group was 6.4 times
that of the Cap-ashM2e VLPs group (Fig. 3e). Meanwhile, anti-avian IAV
M2e antibodies in the Cap-ahsM2e VLPs and Cap-ashM2e VLPs were the
highest (Fig. 3f). The anti-avian IAV M2e antibody in Cap-ahsM2e VLPs
group was 8 times that of the Cap-sahM2e VLPs group (Fig. 3f).