Figure legends:
Figure. 1. Preparation and proteolysis analysis of CaSM, CaSM-Seq8, and Seq8 proteins. A) SDS-PAGE analysis of two dilutions (0.5× and 0.1×) of the purified recombinant proteins; B) Determination of the target proteins purity by the analysis of the SDS-PAGE bands using Image J; C) SDS-PAGE gel showing the thermal stability of the purified proteins at 37℃ and 4℃, followed by the grayscale quantification of the target bands (D); E) SDS-PAGE gel showing the proteolytic action of pepsin (Pep) trypsin (Try) on CaSM, CaSM-Seq8, and Seq8; followed by a grayscale analysis of the target bands (NC: negative control).
Figure. 2. Morphological analysis of target proteins. A, B, C, and D) Electron micrographs (top) and the radius/frequency analysis (bottom) of CaSM, CaSM-Seq8, Seq8, and native HEV virions, respectively. E) The self-assembly patterns of CaSM, CaSM-Seq8 and HEV capsid protein (PDB ID: 3HAG) as predicted by the GalaxyWeb server. F) The dimerization of Seq8 as predicted by the GalaxyWeb server.
Figure. 3. Computational analysis of the antigenicity of the target proteins. A) Protrusion index (PI) of the Seq8 residues in both CaSM-Seq8 and Seq8 alone; B) Comparison of the fluctuation of PI of the three G-H loops between Seq8 and CaSM-Seq8; C) Column bar representation of the area under the PI curve of the three G-H loops in Seq8 and CaSM-Seq8 proteins.
Figure. 4. Experimental evaluation of the antigenicity of the target proteins. A, B, C, and D) Comparison of the immunoreactivity of CaSM, CaSM-Seq8, Seq8 with FMDV-specific antibodies using indirect ELISA. The anti-FMDV antibodies used in the experiment were: A) O/Mya/98+O/HN/CHA/09-induced antibodies in pigs; B) O/JMS/00+O/GX/09-7-induced antibodies in pigs; C) purified anti-FMDV/O pAbs; D), negative sera collected from FMDV-free pigs; E) Western blotting analysis with the corresponding grayscale values. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05, and ns: p>0.05.
Figure. 5. A) Immunogenicity analysis by monitoring the induced FMDV-specific antibody titers in the mice immunized with CaSM-Seq8, Seq8, and the FMDV inactivated vaccine; **p<0.01, *p<0.05, ns: p>0.05. *p<0.05, and ns: p>0.05.
B) Structure of the HEV capsid protein (S domain in blue, M domain in purple, and P domain in green) and its self-assembly into T=1 VLP (PDB ID: 2ZTN). C) Structure of the FMDV capsid protein (PDB ID: 1FOD) with the VP1 G-H loop shown in green (red box) and a schematic of the design of the recombinant antigen Seq8 containing three different VP1 G-H loops. D) Predicted structure of the constructed CaSM-Seq8.