Designing and Construction of the Suicide Vector pDS132-ΔkanR to Delete Kanamycin Resistance Sequence in Mutant Strain of Brucella melitensis Rev1 Mutant Strain in order to Generate a Candidate Vaccine Strain

Background and Aim: Brucellosis is a common disease between humans and animals. Vaccination is the best approach to prevent brucellosis. Existing vaccines such as Rev1 are associated with disadvantages such as abortion in pregnant animals. Therefore, it is necessary to generate an appropriate vaccine that is produced by molecular methods. The aim of this study was designing and construction of pDS132-Δkanr suicide vector to delete kanamycin resistance sequence (kanR) in a mutant strain of Brucella melitensis rev1 to generate a candidate vaccine strain. Materials and Methods: In this study, the plasmid pDS132 was used as a suicide vector to delete target gene. In order to construct the deletion cassette, two homologous fragments were separately designed and constructed by PCR, and tandemly cloned into the pBluescriptSK(-) vector using appropriate restriction enzymes. Then, the deletion cassette was digested from the recombinant vector by terminal restriction enzymes, and sub cloned into the pDS132 vector to construct the suicide vector pDS132-ΔkanR.  Results: The pDS132-ΔkanR contains 590bp upstream sequences and 421bp downstream sequences on the deletion cassette fragment; it can be used as specific suicide vector to disrupt the kanR sequence in genome of mutant strain of Brucella melitensis Rev1. Conclusions: The use of suicide pDS132-ΔkanR system facilitated mutant construction which is a more specific and effective system in comparison with the other positive marker-dependent suicide systems and primary techniques such as serial passages