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Background: The aim of this study was to employ gene knockout technique in Brucella melitensis Rev1 to attain the attenuated mutant strain. Therefore, we deleted the perosamine synthetase encoding gene (per), one of the LPS O-chain coding genes, by homologous recombination. Materials and Methods: Homologous recombination was performed using a suicide vector containing deletion cassette, which comprised kanamycin resistance sequence (kanR) flanking with the per upstream and downstream sequences. Deletion cassette was constructed by PCR reactions using specific primers, and cloned into pBluescriptIISK(-) to construct suicide vector. The suicide vector was transformed into Brucella by electroporation. Results: The results were confirmed deletion cassette and suicide vector construction. After mutant strain selection, deletion of the per gene was confirmed by PCR using specific primers, and sequencing. Also, loss of the per gene function was confirmed by comparison of RT-PCR products from wild type and mutant strain. Conclusion: The mutant Brucella had deficiency in its LPS O-chain structure, so in addition to reduce error in diagnostic tests to discriminate between vaccinated and infected animal, it can to characterize as a candidate vaccine with later immunological tests.

Keywords: brucella melitensis Rev1, gene deletion, perosamine synthetase, suicide vector

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