Abstract: 【Objective】To construct a prokaryotic expression system for monkeypox virus B20R protein and evaluate the immunogenicity of the fusion protein obtained from prokaryotic expression, to provide technical support for subsequent monkeypox virus detection and the development of novel treatment methods. 【Method】Referring to the monkeypox virus genome sequence published in GenBank, the DNA sequence of B20R gene was optimized according to codon preference of Escherichia coli. B20R gene was synthesized, and cloned to expression vectors pET-28a and pET-32a to construct prokaryotic expression vectors. The two constructed prokaryotic expression vectors were individually transformed into E.coli BL21 competent cells. After IPTG induced expression, both SDS-PAGE and Western blotting were performed to detect the expression of B20R fusion protein. The induced expression conditions of B20R fusion protein were optimized by the control variable method, and Ni-column affinity chromatography was used for purification. Kunming mice were immunized with the purified B20R fusion protein through intraperitoneal injection. Blood samples were collected regularly from Kunming mice, and the serum antibody titers were detected using ELISA. 【Result】The B20R gene was successfully cloned into the pET-32a expression vector resulting in the construction of prokaryotic expression vector pET-32a-B20R.After induction with IPTG, the B20R fusion protein could be expressed in the main form of insoluble inclusion bodies in E. coli BL21 competent cells. The optimal induced expression conditions for B20R fusion protein were 0.6 mmol/L IPTG induction at 37 ℃ for 12 h, and the optimal imidazole elution concentration for Ni-column affinity chromatography purification was 40 mmol/L. Immunization of Kunming mice with purified B20R fusion protein could rapidly induce strong humoral immunity in Kunming mice, and the high antibody level could still be maintained on the 42^nd day after immunization, with an antibody titer as high as 1∶409600. 【Conclusion】The constructed prokaryotic expression vector of monkeypox virus B20R protein can successfully express the B20R fusion protein in the form of inclusion bodies in ^{E. coli} BL21 cells, and the B20R fusion protein has good immunogenicity, which can rapidly induce strong humoral immunity in Kunming mice.