[PubMed] [Google Scholar] 26. mice contain high levels of antibodies (IgG) against recombinant chimeric protein. Conclusion: The recombinant chimeric protein could be a potential antigen candidate for the development of a subunit vaccine against is facultative intracellular pathogens that infect humans and many domestic animals such as cows, sheep, and goats. Infection causes abortion and infertility in the animals and undulant fever in humans (brucellosis) and is endemic in many developing countries. Brucellosis is a zoonotic infection, leading to clinically infectious diseases and economic consequences (1, 2). The efforts of disease eradication and infection prevention have been made through the use of vaccines and health guidelines (3, 4). The control of brucellosis is attempted by vaccine administration using strain 19, Rev1, and RB-51 vaccines. Despite the efficacy of vaccination, these vaccines have some disadvantages, such as the ability to cause disease in humans and abortion in pregnant animals, and difficulty in the diagnostic validation of infection stages in vaccinated animals (5C7). Recombinant subunit vaccines have predetermined compositions with suitable homogeneity; they can be controlled to ensure good production and are completely inert. Because of the problems derived from the utilization of attenuated and killed vaccines in humans and animals, similar to other infectious diseases vaccines, development of a beneficial subunit vaccine against brucellosis is desirable. However, the success of subunit vaccines to stimulate the immune response depends on the optimization of the antigen and adjuvant (s) and selection of the delivery system (8). Intracellular and cell surface components have recently been considered as protective antigens, but only few antigenic components W-2429 have suitable immunogenic activity, for example, lumazine synthase, BLS (Cytoplasm); ribosomal protein L7/L12 (Cytoplasm); sugar-binding 39-kDa protein, p39 (periplasm); Bp26 periplasmic immunogenic protein, Bp26 (periplasm); molecular W-2429 chaperone, DnaK (cytoplasm); outer membrane protein, Omp16,19,25,31 (outer membrane); Cu/Zn superoxide dismutase, SodC (periplasm); SurA Peptidyl-prolyl cis-trans isomerase, SurA (periplasm) and Trigger factor, Tig/TF (cytoplasm). Despite the immunogenicity of these antigens, the desirable protection against bacteria could be improved using a multiple subunit vaccine. Omp31, TF, and Bp26, have been characterized as potential immunogenic and protective antigens and have been previously studied in NKSF2 whole and portion form to determine their protective immunogenicity (9, 10). In this study, we developed a new structural model containing three putative antigenic determinants of antigens, in the murine model. MATERIALS AND METHODS According to previous researches (11C19), we chose three antigenic determinants of TF, 485 amino W-2429 acids, Bp26, 25 amino acids (87C111) and Omp31, 27 amino acids (48C74), fused together by EAAAK rigid linkers to avoid the construction changes in final composition; also these rigid linker maintain the conformation of protein by lowest changes in structure. The segment arrangement of chimera was determined by changing the three antigenic determinants to construct the best structure experiments and optimization. After design and prediction, the chimeric gene was synthesized and subsequently cloned into pET-28a (+) to construct pET-chimeric protein (pET-CP) plasmids (Biomatik, Ontario, Canada). Gene expression and purification of recombinant protein. The pET-CP was transformed into BL21 (DE3) strain (Novagen, Merck KGaA, Germany). Preparation of competent (BL21) and transformation of it was performed using calcium chloride and heat shock method respectively. The transformed clones were inoculated into 5 ml Luria Bertani (LB) medium (Merck, Germany), containing 50g/ml kanamycin (Sigma-Aldrich, Germany) and overnight growth at 37 C. The culture was used to inoculate 1000 ml LB medium-kanamycin. The incubation was continued with agitation (300 rpm) to 0.5 OD value at 600 nm; Isopropyl–D-1-Thiogalactopyranoside (IPTG) (Sigma-Aldrich, Germany) was added (final concentration of 1mM) to induce the gene expression at 37 C for duration of 4 hours; the concentration of IPTG was optimized by adding various amounts of IPTG to determine the best concentration (IPTG gradient). The culture was harvested by centrifugation at (10000g, 10 min, 4 C), then resuspended in lyses buffer (8 M.