Prokaryotic Expression,Purification and Preliminary Immunogenicity Analyses of the A29L-A35R-M1R Fusion Protein of the Monkeypox Virus
The monkeypox virus(MPXV)is a poxvirus that can infect humans and animals.The MPXV poses a significant threat to public-health security.A safe and efficacious vaccine is needed to prevent and control the spread of the MPXV.The amino-acid sequences of A29L,A35R and M1R of the MPXV were selected to design fusion proteins using a flexible peptide(GGGGS)linkage.The whole gene was synthesized after codon optimization according to Escherichia coli codon bias,inserted into the pET-28a vector,and then we constructed the pET28a-A29L-A35R-M1R recombinant plasmid.Expression was induced by transformation into E.coli BL21(DE3)receptor cells,characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotting,and the optimal induction conditions were determined.On this basis,the fusion protein rA29L-A35R-M1R was purified by Ni-NTA affinity chromatography and immunized to mice.The immunogenicity of fusion proteins was evaluated by measuring the levels of anti-MPXV A29L,A35R and M1R-specific immunoglobulin(Ig)G and cytokines(interferon(IFN)-γ,interleukin(IL)-2 and(IL)-4)by enzyme-linked immunosorbent assays.The recombinant plasmid pET-28a-A29L-A35R-M1R was expressed in E.coli BL21(DE3)receptor cells.The optimal conditions for expression were use of IPTG(0.25 mmol/L)at 37℃ for 5 h.After immunizing mice thrice with the purified fusion proteins rA29L-A35R-M1R,mice could produce anti-A29L,A35R and M1R-specific IgG.The level of anti-rA29L-A35R-M1R IgG was the highest.The fusion protein rA29L-A35R-M1R could stimulate the body to produce high levels of IFN-γ,IL-2 and IL-4,and the differences in levels compared with those in the control group were all highly significant(P＜0.01).These data indicate that the fusion protein rA29L-A35R-M1R had good immunogenicity.Our results provide a theoretical basis for the development of novel vaccines and therapeutic biological agents for the MPXV.
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