Objective The study aims to investigate the immunological functions of the nucleocapsid (N) protein of the novel coronavirus Omicron (BA.1, BA.2) and evaluate the differences among different N proteins of mutant strains in immunogenicity. Methods By aligning sequences, the mutation sites of the Omicron (BA.1, BA.2) N protein relative to prototype strain of the novel coronavirus (Wuhan-Hu-1) were determined. The pET-28a-N-Wuhan-Hu-1 plasmid was used as template to construct pET-28a-BA.1/BA.2-N through single point mutation or homologous recombination. The three kinds of N protein were expressed in prokaryotic system, purified through Ni-NTA affinity chromatography, and then immunized into mice. The titer and reactivity of the polyclonal antibody, as well as the expression level of IL-1β and IFN-γ in mouse spleen cells, were detected using indirect ELISA and Western blot assay. Results The constructed prokaryotic expression plasmids were successfully used to express the Wuhan-Hu-1 N, BA.1 N, and BA.2 N proteins in E.coli BL21(DE3) at 37 DegreesCelsius for 4 hours. The indirect ELISA test showed that the titers of polyclonal antibody prepared by three N proteins were all 1:51 200. All three N proteins can increase the expression of IFN-γ and IL-1β cytokines, but the effect of Omicron N protein in activing two cytokines was more obvious than that of Wuhan-Hu-1 N protein. Conclusion The study obtained three new coronavirus N proteins and polyclonal antibodies, and confirmed that mutations in the amino acid sites of the N protein can affect its immunogenicity. This provides a basis for developing rapid diagnostic methods targeting N protein of different novel coronavirus variants.
Animals ; Mice ; SARS-CoV-2/genetics* ; Coronavirus Nucleocapsid Proteins/immunology* ; Nucleocapsid Proteins/isolation & purification* ; COVID-19/immunology* ; Antibodies, Viral/immunology* ; Mice, Inbred BALB C ; Interferon-gamma/metabolism* ; Interleukin-1beta/metabolism* ; Female ; Escherichia coli/metabolism* ; Mutation ; Humans

Objective To express the monkeypox virus (MPXV) A23R protein in Escherichia coli and purify by Ni-NTA affinity column, and to prepare mouse antiserum against MPXV A23R. Methods The recombinant plasmid pET-28a-MPXV-A23R was constructed and transformed into Escherichia coli BL21 to induce the expression of A23R protein. After optimizing the conditions of expression, A23R protein was highly expressed. Recombinant A23R protein was purified by Ni-NTA affinity column and identified by Western blot analysis. The purified protein was used to immunize mice for preparing the A23R polyclonal antibody, and the antibody titer was detected by ELISA. Results The expression of A23R recombinant protein reached the peak under the induced conditions of 0.6 mmol/L isopropyl-β-D-thiogalactoside (IPTG), 37 DegreesCelsius and 20 hours. The purity of the protein was about 96.07% and was identified by Western blot analysis. The mice were immunized with recombinant protein, and the titer of antibody reached 1:102 400 at the 6th week after immunization. Conclusion MPXV A23R is expressed highly and purified with a high purity and its antiserum from mouse is obtained with a high titre.
Animals ; Mice ; Monkeypox virus ; Antibodies ; Enzyme-Linked Immunosorbent Assay ; Blotting, Western ; Recombinant Proteins ; Escherichia coli/genetics*

Based on previous report that the Chinese herb Ligustrum lucidum (LL) extract directly inhibited hepatitis C virus (HCV) replicase (NS5B) activity, the active components of LL extract to inhibit HCV NS5B activity and their inhibition mode were investigated in this study. LL extract was separated using ethyl acetate and thin layer chromatography (TLC). The inhibitory activity of separated fractions on HCV NS5B was analyzed by the inhibitory assay of NS5B activity. The results showed that only fractions 1 and 2 inhibited NS5B activity, and fraction 2 possessed higher inhibitory activity than fraction 1. HPLC analysis combined with inhibitory assays indicated that ursolic acid and oleanolic acid are the active components within fractions 1 and 2 to inhibit NS5B activity, separately. Moreover, oleanolic acid possessed higher inhibitory activity than ursolic acid. Further inhibition mode analysis found that both oleanolic acid and ursolic acid suppressed NS5B activity as noncompetitive inhibitors. The Ki values of ursolic acid and oleanolic acid were about 4.7 microg x mL(-1) (10 micromol x kg(-1)) and 2.5 microg x mL(-1) (5.5 micromol x kg(-1)), respectively. Taken together, these results demonstrated that oleanolic acid and ursolic acid suppressed NS5B activity as noncompetitive inhibitors, implying that the two natural products have potential value for HCV therapy.