The Brucella melitensis 16M genome was examined for proteins in excess of 100 amino acids and for immunogenicity-associated genes. One subset of 32 annotated genes or open reading frames was identified, and each of these were cloned into the eukaryotic vector pcDNA3.1. Purified recombinant plasmids were used to intramuscularly (i.m.) immunize BALB/c mice. After challenge with B. melitensis 16M strain, two protective antigens were found: the periplasmic protein, bp26, and the chaperone protein, trigger factor (TF). Protective efficacy was confirmed with DNA vaccines for these two B. melitensis proteins and, when combined, protection against wild-type challenge was significantly enhanced. Both proteins were found to be immunogenic since elevated serum immunoglobulin G (IgG) antibodies without a specific IgG subclass bias were induced subsequent to i.m. DNA immunization. Antigen-restimulation assays revealed that bp26 and TF stimulated gamma interferon and only bp26 induced interleukin-4 (IL-4), IL-5, and IL-6 cytokines as measured by cytokine enzyme-linked immunospot assay. These collective results suggest that both bp26 and TF are excellent candidates for use in future vaccination studies against brucellosis.Brucella spp., facultative intracellular pathogens, are the etiological agents of brucellosis, a disease that affects livestock and humans (9). The attenuated strains such as Brucella melitensis Rev1 and B. abortus S19 and RB51 are used to control brucellosis in domesticated animals. However, these are less than ideal because of their limited efficacy and potential to cause disease in humans. Moreover, both B. abortus S19 and B. melitensis Rev1 strains induce antibodies to their lipopolysaccharide (LPS), making it difficult to differentiate vaccinated animals from those naturally infected (3,17,20). Recently, Brucella spp. have also been recognized as a bioterror threat by the Centers for Disease Control (16). Therefore, a subunit vaccine that is protective against B. melitensis is desirable.DNA vaccines offer a promising approach because they can stimulate both cellular and humoral immunity (13, 26). Furthermore, DNA vaccines have many advantages over traditional protein-based vaccines, including ease of development, induction of long-lived immunity, and minimal preparation costs. With regard to effectiveness, previous studies have already shown that DNA vaccination with sodC (22), lumazine synthase gene (27), and P39 (2) can elicit partial protection against Brucella challenge. Furthermore, in contrast to live attenuated vaccines, there are no concerns of induced disease, and the DNA vaccines are stable.With the completion of sequencing the Brucella genome, identification of novel protective antigens is feasible. In the present study, we applied a search strategy to screen the B. melitensis 16M genome for potential immunogenic antigens. By cloning these potential antigen candidates into the pcDNA3.1 vector and testing their efficacy in BALB/c mice, two protective antigens were identified.