Figure 1.
PapMV amino acid sequence and structure.
PapMV capsid protein amino acid sequence with each site of fusion and a bioinformatic secondary structure prediction (adapted from Lecours 2006).
Figure 2.
PapMV-HA11 recombinant proteins.
The three PapMV-HA11 fusions produced have characteristics similar to those of PapMV nanoparticles. (A) The sequence of the PapMV-CP-HA11 proteins produced. (B) Bacterial lysate of the culture before induction (first lane), after induction with IPTG (second lane), and after successful purification with nickel beads, third lane, of PapMV-CP-HA11-12 (lane 1–3), 187 (lane 4–6) and C (lane7–9). (C)Transmission electron microscope images of each HA11 fusion. (D) Size of VLPs recorded by dynamic light scattering (DLS).
Figure 3.
Immunoprecipitation and western blot of PapMV with or without HA11 fusion.
PapMV was immunoprecipitated with anti-PapMV mouse serum (lane 1) and with anti-HA11 monoclonal antibody (lane 2). PapMV-HA11-12, 187 and C are immunoprecipitated by anti-HA11 monoclonal antibody (lane 3–5) confirming that the HA11 peptide is at the surface.
Figure 4.
Structural changes in PapMV CP in the different recombinant nanoparticles induced by an increase in temperature.
Each of the recombinant nanoparticles (PapMV-HA11-12, PapMV-HA11-187 and PapMV-HA11-C) at a concentration of 0.1 mg/ml were heated in steps of 1°C and secondary structure changes of the protein was monitored by circular dichroism. The read-out was performed at a wave length of 208 nm. The arrows show the point of inflection for each of the nanoparticles. The black bar represents the body temperature of mice (36.9°C).
Figure 5.
Aggregation of recombinant nanoparticles upon heating.
Each of the recombinant nanoparticles at a concentration of 0.1 mg/ml were heated by steps of 5°C and DLS spectra of the samples was measured. The approximate size of the particles measured indicates the level of aggregation of the samples. The arrows show the point of inflection for each of the nanoparticles. The black bar represents the body temperature of mice (36.9°C).
Figure 6.
Stable nanoparticles are more immunogenic in animals.
Balb/C mice (5 per groups) were immunized twice with a 14-day interval with 100 µg s.c. of PapMV-HA11-12, PapMV-HA11-187 or PapMV-HA11-C, respectively. The total IgG (A) or the IgG2a (B) humoral response directed to the HA11 peptide was measured by ELISA. Also, the total IgG (C) and IgG2a (D) directed to the PapMV CP was measured by ELISA. *** P<0.0001 **P<0.01.
Table 1.
Forward and reverse oligonucleotides used to produce the seven PapMV-HA11 recombinant proteins.