Fine mapping of the antigenic epitopes of the Gc protein of Guertu virus.

Guertu virus (GTV), a newly discovered member of the genus Banyangvirus in the family Phenuiviridae, poses a potential health threat to humans and animals. The viral glycoprotein (GP) binds to host cell receptors to induce a neutralizing immune response in the host. Therefore, identification of the B-cell epitopes (BCEs) in the immunodominant region of the GTV Gc protein is important for the elucidation of the virus-host cell interactions and the development of GTV epitope assays and vaccines. In this study, an improved overlapping biosynthetic peptide method and rabbit anti-GTV Gc polyclonal antibodies were used for fine mapping of the minimal motifs of linear BCEs of the GTV Gc protein. Thirteen BCE motifs were identified from eleven positive 16mer-peptides, namely EGc1 (19KVCATTGRA27), EGc2 (58KKINLKCKK66), EGc3 (68SSYYVPDA75), EGc4 (75ARSRCTSVRR84), EGc5 (79CTSVRRCRWA88), EGc6 (90DCQSGCPS97), EGc7 (96PSHFTSNS103), EGc8 (115AGLGFSG121), EGc9 (148ENPHGVI154), EGc10 (179KVFHPMS185), EGc11 (230QAGMGVVG237), EGc12 (303RSHDSQGKIS312), and EGc13 (430DIPRFV435). Of these, 7 could be recognized by GTV IgG-positive sheep sera. Three-dimensional structural analysis revealed that all 13 BCEs were present on the surface of the Gc protein. Sequence alignment of the 13 BCEs against homologous proteins from 10 closely related strains of severe fever with thrombocytopenia syndrome virus from different geographical regions revealed that the amino acid sequences of EGc4, EGc5, EGc8, EGc11, and EGc12 were highly conserved, with 100% similarity. The remaining 8 epitopes (EGc1, EGc2, EGc3, EGc6, EGc7, EGc9, EGc10, and EGc13) showed high sequence similarity in the range of 71.43%-87.50%. These 13 BCEs of the GTV Gc protein provide a molecular foundation for future studies of the immunological properties of GTV glycoproteins and the development of GTV multi-epitope assays and vaccines.

[Prokaryotic expression and purification of nucleoprotein of Guertu virus and its establishment of ELISA detection method].

Objective: To obtain purified protein antigen of guertu virus (GTV) nucleoprotein (NP) and establish a rapid and accurate enzyme-linked immunosorbent assay (ELISA) method for detection of GTV antibody. Methods: Codon optimized GTV NP encoding genes were synthesized, cloned into the pet32a (+) vector, and recombinant expression plasmids were constructed and transformed into BL21 (DE3). Recombinant protein (rNP) obtained from the optimized expression were purified over a Ni column and identified by SDS-PAGE and Western blot. The purified protein was used as the antigen to optimize the reaction conditions, and an indirect ELISA assay for GTV IgG antibody was developed and optimized, which was evaluated and initially applied. Results: The prokaryotic expression plasmid pet32a-NP was successfully constructed, the recombinant protein was highly expressed in E. coli in the form of inclusion bodies, the size was about 44 kD, and the results of Western blot indicated that the recombinant protein had good antigenicity with GTV positive serum. The optimized ELISA (GTV-rNP-iELISA) established in this study showed strong specificity, high sensitivity, and the coefficient of variation within and between batches is less than 10%, and has good repeatability; the detection results are consistent with the IFA detection results. Using the established ELISA method to detect 162 sheep sera from some regions of Xinjiang in 2017-2019, the total positive rate of antibodies was 39.8%. Conclusions: The GTV NP antibody detection ELISA method has good sensitivity, reproducibility, and specificity and has the potential to be a powerful tool for the diagnosis and serological investigation of GTV infection.

Immunogenicity of chimeric peptide gene of Xinjiang hemorrhagic fever virus glycoprotein / 国际生物医学工程杂志

Objective:To investigate the effects of different chimerism strategies and different immune ways on the two antigen-dominant regions of Xinjiang hemorrhagic fever virus (XHFV) glycoprotein.Methods:The 5' end was added or not added with interleukin-2 (IL-2) signal peptide and the general-purpose auxiliary T cell epitopes as different design strategies. GcⅠ and GcⅡ and the epitopes previously identified on GcⅠ (Gc 233-248, Gc 241-256 and Gc 281-296) were fused and constructed into the eukaryotic expression vector pVAX1 and the prokaryotic expression vector pET-28a. The recombinant prokaryotic plasmid transformed into E.coli BL21 was induced and purified, and the recombinant eukaryotes were extracted by indirect immunofluorescent assay. BALB/c mice were immunized by protein immunity, gene immunity, and DNA prime-protein boost immunity. The IgG antibody level was measured by ELISA. The immune effect was evaluated by the proliferation of T-lymphocytes and the content of cytokines in the spleen. Results:The results of double enzyme digestion and sequencing showed that eight recombinant plasmids were successfully constructed, and the recombinant eukaryotes were successfully expressed in vitro by fluorescence microscopy. After three times of immunization, the IgG level and the proliferation of T-lymphocytes in the spleen of mice in the experimental group were significantly higher than those in the control group ( P<0.01). The mass concentration test results of Th2 cytokines IL-4 and Th1 cytokines interferon-gamma (IFN-γ) revealed that the response of the DNA prime-protein boost immunity was biased to Th1. Conclusions:The multi-epitope chimeric vaccine of XHFV glycoprotein was successfully constructed, and the target antigen could be expressed effectively in vivo. The immune groups stimulated stronger humoral and cellular immune responses compared with the control group. Among them, the immune effect of pVAX1-ST(GcⅠe+GcⅡ) combined with recombinant protein r(GcⅠe+GcⅡ) was the best, and it is expected to be a new candidate vaccine for XHFV.