Fig 1.
Schematic of epitope mapping strategy.
The green band indicates the full length of the NP protein, the blue bands indicate the NP1 and NP3 segments and the red band represents the NP2 segment (a). Schematic of epitope mapping strategy involving 48 overlapping 16mer peptides spanning NP1 (b) and NP3 (c).
Fig 2.
Prokaryotic expression and immunoblotting analysis of truncated NP segments.
(a) SDS-PAGE analysis of expressed pET-32a-NP1, expressed pET-32a-NP3, the pGEX-KG vector (expressing GST-tagged protein as the negative control) and pGEX-KG-NP2. (b) Western blotting of NP1, NP2 and NP3 using rabbit pAbs against r-NP. The arrows represent the three expressed target segments on the gel and the reactive segments in the western blotting analysis.
Fig 3.
SDS-PAGE and western blotting analysis of GST188 fusion expressed 16mer peptides derived from CCHFV-NP1 and -NP3.
(a, c) SDS-PAGE analysis of 48 GST188 fusion expressed 16mer peptides. (b, d) Western blot analysis of 48 GST188 fusion expressed 16mer peptides using pAbs. CK, negative control (GST188 protein expressed by pXXGST-3 vector). The arrows indicate the 16mer peptides with a positive antigen-antibody reaction in the western blotting analysis.
Fig 4.
SDS-PAGE (upper layer) and western blotting analysis (lower layer) of the minimal epitopes on NP1 and NP3 using pAbs.
(a) Eight 8mer peptides (P49 to P56) in 16mer peptide P23. (b) Eight 8mer peptides (P57 to P64) in 16mer peptide P33. (c) Seven 8mer peptides (P65 to P71) in 16mer peptide P34. (d) Eight 8mer peptides (P72 to P79) in 16mer peptide P38. (e) Seven 8mer peptides (P80 to P86) in 16mer peptide P39. (f) Seven 8mer peptides (P87 to P93) in 16mer peptide P45. (g) Seven 8mer peptides (P94 to P100) in 16mer peptide P47. (h) Six 8mer peptides (P101 to P106) in 16mer peptide P48. The arrows indicate the peptides with a positive antigen-antibody reaction in the western blotting analysis. CK, negative control (GST188 protein expressed by pXXGST-3); CP, positive control (16mer peptide P38 identified as positive by pAbs).
Fig 5.
Synthetic 8mer peptide sequences derived from a span of the immunodominant peptides.
The yellow-brown highlighting represents the common sequences among immunodominant peptides that react with pAbs according to western blotting analysis.
Fig 6.
Western blotting of nine 8mer peptides containing identified epitopes performed using positive sera from sheep with a confirmed history of CCHFV infection.
(a) A serum sample from healthy sheep with no history of CCHFV infection was used as a negative control. (b) A positive serum sample from a sheep with a confirmed history of CCHFV infection. The arrows represent 8mer peptides exhibiting positive antigen-antibody reactions based on western blotting analysis. CK, negative control (GST188 protein expressed by pXXGST-3).
Fig 7.
Positioning of the minimal motifs of the mapped epitopes on the predicted 3D structure of CCHFV-NP.
(a) The ribbon diagram shows the overall secondary structure of CCHFV-NP from strain YL04057 (PDB code: 3U3I). The motifs within the frame indicate that the eight minimal epitopes are located on the CCHFV-NP head domain. (b) Surface properties of CCHFV-NP. The molecular surfaces of the eight minimal epitopes are shown in different colors (E1a, red; E2, magenta; E3, lemon; E4, cyan; E5a, wheat; E5a-E5b, olive; E5b, blue; E6, warm pink; E7, slate). The figures were generated using the PyMOL™ molecular graphics system.
Fig 8.
Sequence alignment of the NP1 and NP3 segments from the YL04057 strain (ACM78470.1) and other homologous CCHFV-NP proteins.
(a) The aa sequence of the NP1 segment; (b) The aa sequence of the NP3 segment. The GenBank codes and sources are shown on the left. The nine minimal epitopes E1a, E1b, E2, E3, E4, E5a, E5b, E6 and E7 recognized by pAbs are highlighted, and the variable aa residues within the minimal motif epitopes are highlighted in red. Dots (.) indicate identical aa residues within all 12 strains.