Monkeypox virus (MPXV) is a DNA virus belonging to the Orthopoxvirus genus of the Poxviridae family. It causes symptoms similar to Smallpox virus and is a zoonotic virus with widespread prevalence. Antigen detection is a fast and effective detection method. The MPXV A29 protein not only plays an important role in the virus lifecycle but also serves as a promising target for developing specific antibodies, which have significant potential for application in the diagnosis of MPXV. The coding sequences of the MPXV A29 protein, Cowpox virus (CPXV) 163 protein homolog and Vaccinia virus (VACV) A27 protein homolog were chemically synthesized, and all three recombinant proteins were expressed in