Serological Characterization Of Antigenecity Of Epitopes In The Brucella Outer Membrane Protein Omp31

Brucellosis is a zoonosis caused by bacteria Brucella spp that invades both human andanimal bodies. The symptoms of the disease in human include fever, tiredness, and pains in thejoints. If not treated well in time, the disease may become chronicle, lasting long even life time.Brucellosis can cause miscarriage, abortion, and infertility in animals, posing serious threat tothe live stock industry. Therefore, the preventing of spread of this disease is becoming the mostpressing problem in our local region. The most effective way to control the spreading of thedisease in animals and humans is vaccination that requires research to identify the mosteffective antigens.Omp31is a transmembrane lipid protein whose β-sheets folded into a bucket shape with astrong signal peptide at the N end. To identify antigenic epitopes in the protein that were locatedout the membrane, hydrophilicity, flexibility, and antigenic index of all subsequences of aminoacids were analyzed using software DNASTAR; the48-74th and193-227th amino acidresidues of the protein were identified as potential epitopes. The antigenicity of the two epitopeswas tested by fusing the coding DNA sequences to another Brucella Lumathine Synthase gene（BLS）; the fusion proteins were injected into rabbits for antiserum.To construct an expression vector for the fusion protei, an epitope coding sequenceomp31_193-227was PCR amplified from the genomic DNA. Then, the coding sequence was fusedto BLS gene that was carried on an expression plasmid pET28a in the laboratory collection. Inthe resulting plasmid pET-28a-omp31_193-227-bls, the9amino acids at the N terminus of BLSwere replaced with the fragment omp31_193-227. The recombinant expression vector wastransferred into a genetically engineered E. coli strain TL129for expression of the fusionprotein after induction by IPTG. After ultrasonic disintegration, the expressed protein waspurified by Ni affinity column. The preparation of antiserum against omp31_193-227-bls is still inprogress. Meanwhile, protein BLS and fusion protein Omp31_48-74–BLS expressed and purified fromconstructed expression vectors in the laboratory were injected into rabbits to obtain antiserum.The antiserum of rabbits against Omp31_48-74–BLS showed that the antigenecity of was betterthan BLS alone as assayed by ELISA, indicating the fusion protein can be a candidate forvaccination.