RESUMO
Objective:To grasp the distribution of fine antigenic epitope profiles of nucleoprotein (NP) and glycoprotein (GP) fragments of Crimean-Congo hemorrhagic fever virus (CCHFV) and to clarify the value of dominant antigenic epitopes in laboratory testing of Crimean-Congo hemorrhagic fever (CCHF).Methods:In a minimal synthetic short peptide consisting of 8 amino acids was segmentally expressed by CCHFV YL04057 strain using a modified bio-peptide synthesis method from 2014 to 2021 in the laboratory of Xinjiang University, College of Life Sciences. Using CCHFV polyclonal antibody or monoclonal antibody 14B7 (IgM) or CCHFV-positive sheep serum as antibodies, the minimal antigenic epitopes (BCEs) with antigenic activity on NP and GP fragments were identified by immunoblotting, and the obtained BCEs with sequence polymorphism were spatially clustered with CCHFV from different regions using the neighbor-joining method to determine the combination mode of BCEs with geographical correlation of regional distribution, to explore its application in establishing serological diagnosis. A prokaryotic expression plasmid (pET-32a), an E. coli expression plasmid (pGEX-KG) and a prokaryotic expression plasmid with an incomplete glutathione (GST188) tag (pXXGST-ST-1) were used to construct and express six dominant antigenic epitopes of different peptide lengths on NP fragments, and an indirect Enzyme-linked immunosorbent assay (ELISA) was established. CCHF sheep serum identified by immunofluorescence assay (IFA) was used as a control, and the specificity, sensitivity and overall compliance of the recombinant proteins with different peptide lengths of antigenic epitopes with IFA assay results were statistically analyzed. Results:CCHFV, NP and GP fragments had a total of 30 antigenically active BCEs, among which the core intermediate fragment NP2 (aa 170 th-305 th), which had a concentration of antigenic epitopes in the NP fragment, has 6 BCEs, and the NP1 (aa 1 st-200 th) and NP3 (aa 286 th-482 nd) at both ends have 9 BCEs; the Gc (aa 1 st-558 th) and Gn (aa 533 th-708 th) fragments of the GP fragment have 14 BCEs and a long antigenic peptide (AP) containing 15 amino acids, and the amino acid sequence homology of the NP fragment BCEs was 97.1% and that of the GP fragment BCEs was 89.1%. There was a significant difference ( P=0.0281, P<0.05). Among the 9 BCEs with sequence polymorphism in the GP fragment, 6 combined BCEs from GnEc1, GnE2, GnE4, GcE3, GcE6 and GcAP-4 (Ap) could cluster 15 CCHFV strains from different regions of the world into 5 geographical taxa, AsiaⅠ, AsiaⅡ, AficaⅠ, AficaⅡ and Europe. The constructs expressing PET-32a-NP (full length), PGEX-KG-NP2 (aa 170 th-305 th), pGEX-KG-NP2-1 (aa 235 th-275 th), PGEX-KG-NP2-1-1 (aa 237 th-256 th), pXXGST-1-NP2-1-2 (aa 250 th-265 th) and PGEX KG-NP2-1-3 (aa 260 th-276 th), six recombinant proteins CCHFV NP rabbit polyclonal antiserum (pAb) Western Blotting reaction positive, 33 sheep sera tested by IFA XHF as a reference, the sensitivity of the assay established by indirect ELISA using the recombinant proteins constructed from two fragments of NP2 and NP2-1 as antigens. The sensitivity, specificity and overall compliance were the best, with 73.4% (11/15) and 66.7% (10/15) for sensitivity, 100% (18/18) and 94.4% (17/18) for specificity, and 87.9% (29/33) and 81.8% (27/33) for overall compliance. Conclusion:CCHFV NP and GP are distributed with a high number of BCEs with antigenic immunoreactivity, among which the dominant antigenic epitopes are of high value in the laboratory serological diagnosis of CCHF.