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Human cytomegalovirus (HCMV), a member of the human herpesvirus family, is a common opportunistic virus causing severe ailments and deaths in people with immature or compromised immune systems. UL23 is a virion protein found in the tegument and is expressed in the cytoplasm in HCMV infected cells. However, UL23 is dispensable for viral replication in cultured cells and little is currently known about its function. In order to further study of UL23, polyclonal antibody of UL23 was prepared. UL23 gene fragment was cloned from HCMV Towne by PCR and ligated into pET28a (+). The recombinant plasmid pET28a (+)-UL23 was transformed into E.coli BL21(DE3) to induce expression of the target protein. Then we efficiently purified the recombinant protein affinity chromatography under unique denaturation conditions. Recombinant UL23 protein was used as immunogen to inoculate New Zealand white rabbits and the sera was collected after the fourth immunization. UL23 Polyclonal antibody was purified from antisera using CNBr-activated Sepharose 4 beads. Our UL23 Polyclonal antibody showed specific reaction with UL23 in ELISA, Western-blot and immunofluorescence. More importantly, UL23 Polyclonal antibody could specifically recognize UL23 protein in HCMV infected cells, which laid a foundation for further study of HCMV UL23.