Improvement of the attenuated mutant strain of Brucella melitensis Rev1 as a potential vaccine candidate

Abstract

Vaccination is the best approach to prevent brucellosis. The existing vaccines such as Rev1 are associated with disadvantages such as abortion in pregnant animals. They also induce antibodies to the O-polysaccharide which may complicate diagnosis. Therefore, it is necessary to generate an appropriate vaccine. The aim of this study is the improvement of the available mutant strain of Brucella melitensis Rev1, in which the prosamine synthetize coding sequence from the O-PS region has been replaced with kanR coding sequence, via the replacement of kanR with GFP reporter encoding sequence by using homologous recombination. The syntactic deletion cassette, containing the GFP encoding sequence flanked with the upstream and downstream sequences of kanR encoding sequence from Rev1 mutant strain, was cloned into pBluescriptIISK(-) vector by SacI and KpnI, and the suicide vector pBluescriptSK-∆kanR/gfp was electroporated into mutant strain. The constructs were confirmed by using PCR, restriction mapping and DNA sequencing. The loss of kanR function in transfected cells was confirmed by using the GFP phenotyping with flow cytometry and fluorescence microscopy, and following the phenotypic characterization of the mutant strain on selective media and the genomic DNA analysis with specific primers. This kanR-free Rev1 mutant strain lack the O-polysaccharide and have a reduced ability to enter and survive in the host cell. Therefore, it could be evaluated as a potential vaccine candidate if eliciting protective immunity.