Abstract: 【Objective】In this study, the prokaryotic expression vector of monkeypox virus(MPXV) E2L protein was constructed, and its immunogenicity was evaluated in Kunming mice after expression, purification and identification, which could provide important information for the development of monkeypox virus diagnostic reagents, vaccines, and specific therapeutic drugs. 【Method】The target gene E2L was synthesized based on the optimal codons of genome sequence of monkeypox virus available in GenBank, and cloned into the expression vector pET-28a(+) to construct the prokaryotic expression vector pET-28a-E2L and then transformed into BL21(DE3) Escherichia coli competent cells. The optimal induction expression conditions of fusion protein were explored by controlled variables, and the expression of E2L protein was identified by SDS-PAGE and Western blotting. The fusion protein was purified by affinity chromatography on a His-Bind column and the purified E2L protein was immunized in Kunming mice by intraperitoneal injection, and from the day of immunization(day 0), the serum of Kunming mice was collected every 7 d by tail amputation method, and serum antibody titer was evaluated by ELISA. 【Result】After transforming BL21(DE3) E. coli competent cells with the constructed prokaryotic expression vector pET-28a-E2L, the fusion protein E2L could be successfully expressed after IPTG induction, and the expression was mainly in the form of inclusion bodies. The optimal induced expression conditions for the fusion protein E2L were 16 h at 40 ℃ with 1.0 mmol/L IPTG, and the optimal imidazole elution concentration for purification by affinity chromatography on His-Bind columns was 100 mmol/L. The results of immunity test in Kunming mice showed that the purified fusion protein E2L induced a high level of humoral immunity in Kunming mice, and the serum of Kunming mice still maintained a high level of antibody and the titer of antibody was reached 1∶70400 on the 42^nd day of immunization, indicating that fusion protein E2L had a good immunogenicity. 【Conclusion】The monkeypox virus E2L protein can be efficiently expressed in the prokaryotic expression system, the purified fusion protein E2L can produce strong humoral immunity in Kunming mice, indicating a good immunogenicity of E2L, which can be used in the development of novel monkeypox virus detection methods and preventive vaccines.