Figure 1.
Prediction and mapping strategy of the epitopes in the central region of CCHFV-NP.
(a) Epitope prediction for amino acid residues 237–305 of the NP sequence of the YL04057 strain using DNAStar Protean software. The secondary structure, flexibility plot, hydrophilicity, surface probability, and antigenicity index for NP237−305 were taken into consideration. (b) Epitope predictions of the NP237−305 fragment of the YL04057 strain based on various principles. (c) Schematic of the BCE mapping strategy with six 16–22mer overlapping biosynthetic peptides spanning the NP237−305 fragment. The blue band represents the full length nucleoprotein. The red band represents the immunodominant fragments of YL04057 NP.
Figure 2.
Prokaryotic expression and immunoblotting analysis of Y1–Y6 fused proteins.
(a) SDS-PAGE analysis of expressed pXXGST-1 (CK) and Y1-Y6 peptides fused with a GST (Y1, Y3 and Y6) or GST188 tag (Y2, Y4 and Y5). (b) Western blotting of fusion proteins Y1–Y6 using the rabbit polyclonal antibody against CCHFV-NP. (c) Western blotting of fusion proteins Y1–Y6 using the mouse IgM-type monoclonal antibody 14B7 against CCHFV. The arrows represent expressed target peptides in SDS-PAGE and the corresponding positive antigenic-peptides in Western Blotting analysis.
Figure 3.
SDS-PAGE identification and Western blotting analysis of the minimal epitopes on NP237−305 using pAb.
(a) Thirteen 8mer peptides (P1–P13) corresponding to the Y1 protein. (b) Nine 8mer peptides (P14–P22) corresponding to the Y2 protein. (c) Ten 8mer peptides (P23–P32) corresponding to the Y3 protein. (d) Nine 8mer peptides (P33–P41) corresponding to the Y4 protein. The arrows stand for 8mer peptides which display a positive antigen-antibody reaction in Western Blotting analysis.
Figure 4.
The synthetic 8mer peptide sequences derived from a span of the immunodominant peptides Y1, Y2, Y3, and Y4 respectively.
The yellow and magenta highlighting represents the common sequences among peptides which react with pAb or mAb using Western blotting analysis.
Figure 5.
Minimal epitope identification on CCHFV-NP using mAb 14B7.
(a) A reactivity profile of the 8mer peptides P14–P22 corresponding to Y2 using Western blotting analysis. (b) Sequences of the 8mer peptides and their positions in NP. Magenta highlighting indicates the common sequence identified as the minimal BCE (E5) on NP using 14B7. The arrows represent 8mer peptides which display positive antigen-antibody reactions using Western Blotting analysis.
Figure 6.
Western blot of five 8-mer peptides containing identified BCEs with or without one residue variation performed using positive sheep sera with a confirmed history of CCHFV infection.
Five randomly selected 8-mer peptides containing identified BCEs (a) or BCEs with one residue variation (b) expressed as GST188 fusion protein in E. coli. A serum sample of healthy sheep with no history of CCHFV infection was used as a negative control. CK was a GST188 protein tag. The arrows represent 8mer peptides displaying positive antigen-antibody reactions based upon Western Blotting analysis.
Figure 7.
Amino acid sequence comparison of the NP170−305 fragment from the YL04057 strain (ACM78470. 1) and other CCHFV strains using the ClustalW program.
The GenBank codes and sources are shown at left. The five minimal epitopes E1, E2a, E2b, E3, and E4 recognized by pAb are highlighted in yellow, and the variable amino acids within the minimal epitopes are highlighted in red. Dots (.) indicate identical amino acids within the ten strains.
Figure 8.
Location and three-dimensional structure of the epitopes identified using pAb and mAb 14B7 on 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 the five minimal epitopes E1–E4. (b) E1–E4 sites on the CCHFV-NP stalk domain. (c) Surface properties of CCHFV-NP. The molecular surfaces of E1 (red), E2a (blue), E2b (magenta), E3 (orange) and E4 (yellow) are shown. (d) The structural representations show the location and spatial conformation of epitope E5 (tetrapeptide “NGYL”) identified by Amb 14B7. Residues N258, G259, Y260, and L261 are shown in different colors. The figures were generated using the PyMOL molecular graphics system.