Designing and Construction of the Suicide Vector pDS۱۳۲-ΔkanR to Delete Kanamycin Resistance Sequence in Mutant Strain of Brucella melitensis Rev۱ 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 Rev۱ 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 pDS۱۳۲-Δkanr suicide vector to delete kanamycin resistance sequence (kanR) in a mutant strain of Brucella melitensis rev۱ to generate a candidate vaccine strain. Materials and Methods: In this study, the plasmid pDS۱۳۲ 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 pDS۱۳۲ vector to construct the suicide vector pDS۱۳۲-ΔkanR.  Results: The pDS۱۳۲-ΔkanR contains ۵۹۰bp upstream sequences and ۴۲۱bp 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 Rev۱. Conclusions: The use of suicide pDS۱۳۲-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.