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Cloning and molecular characterization of omp31 gene of the Indian isolate of Brucella melitensis

T. T. Habtamu1, R. Rathore2, K. Dhama3* and K Karthik4

1Mekelle University, College of Veterinary Medicine, Mekelle, Ethiopia. P.O. Box: 231; 2,4Division of Bacteriology and Mycology, 3Division of Pathology, Indian Veterinary Research Institute, Izatnagar, Bareilly (U.P.)-243122, India


Brucellosis, caused by members of the genus Brucella, is an important re-emerging bacterial zoonosis and a significant cause of reproductive losses in animals. The genes encoding for the Brucella major omp31 has been extensively studied in reference and vaccine strains to determine their role in protection against infection. In this study, native major omps from field isolate of Brucella melitensis and B. abortus were extracted using N-lauryl sarcosinate. From original 2 gm (wet weight) of B. abortus and 1 gm (wet weight) of B. melitensis cells, about 5 mg (0.25%) and 2 mg (0.20%) of omp suspended in 1 ml of Tris HCl was recovered, respectively. Analysis of sonicated cells and sarcosyl extracted omps of B. abortus and B. melitensis revealed the presence of native omp31 in field isolate of B. melitensis by SDS-PAGE and Western blot, however, the omp31 gene was not present in B. abortus strain. With the exception of this, there was no any other significant difference in banding pattern and immunoreactivity between the two species both before and after sarcosyl extraction. The western blot analysis performed on native omp further confirmed that the omp31 protein is one of the major immunodominant proteins in B. melitensis. Confirmation of the absence of omp31 gene in B. abortus was also made by PCR amplification. The B. melitensis omp31 gene was PCR synthesized based on its ORF sequence and directly cloned to an entry vector. DNA sequence analysis revealed an open reading frame of 240 codons, and as compared to the consensus sequence of Brucella omp31 genes, six nucleotides have been replaced in the field isolate. The predicted sequence of omp31 showed a remarkable degree of similarity (97%) to the reported omp31 sequences of Brucella species and very less significant identity with omps from other bacteria like Yersinia enterocolitica ompH, E. coli O157 ompW, and Pseudomonas aeruginosa ompM. Being the major omp in B. melitensis and B. ovis strains, omp31 might have a particular usefulness for vaccination against sheep and goat brucellosis.

Keywords: Brucella melitensis, cloning, omp31, sequencing, sheep, vaccine
To cite this article: Habtamu TT, R Rathore, K Dhama and K Karthik, 2013. Cloning and molecular characteriza- tion of omp31 gene of the Indian isolate of Brucella melitensis. Res. Opin. Anim. Vet. Sci., 3(8), 235-243.

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