Engineered and construction of pDS132:: virG as suicide vector for targeted gene deletion of virG from Shigella flexneri 2a in order to generation a live attenuated Shigella vaccine

Background & Objective: Shigella are Gram negative bacteria capable of inducing their entry into non-phagocytic cells via secretion of various effector proteins called invasion plasmid antigens (Ipas). The most important of them is VirG protein. Live attenuated Shigella vaccines have indicated promise in inducing protective immune responses in human clinical trials. In current situation, constructions of Shigella vaccine candidate strains based on classical allelic exchange systems are considered. The aim of this research was engineered and constructed pDS132:: virG as a suicide plasmid for targeted deletion regions of virG gene by using allele exchange method in Shigella flexneri 2a. Method & Materials: In this applied study, species and serotype of shigella was confirmed by using serological and Polymerase Chain Reaction (PCR) tests. Detection primers of virG gene were designed and cloned to pGEM-5zf vector and finally, sequencing was done. According to virG restriction enzyme map, 1751 bp of virG gene was removed by using of HincП restriction enzyme and the virG was successfully constructed. The pGEM virG vector was digested by use of SphI and SalI enzymes and then cloned to pSD132 as suicide vector. Precision of process were verified through phenotype and genotype experiment. Results: The Shigella flexneri type 2a strain was verified by serological and PCR tests. Sequence of the virG gene in native strain was sequentially identical with the strains submitted in the Gene-Bank database. Since the pDS132:: virG contains 1484 bp which derived from virG gene, therefore, it can be utilized for interference in virG gene as specific suicide vector in shigella flexneri 2a. Conclusion: Application of suicide systems facilitated mutant construction in more specific and effective method in comparison with the other early techniques such as serial passage.