Generation Mutant Construct of pGEM-7zf:: Δ icsA (Catr) for Attenuation of Shigella flexneri 2a

Shigellosis causes the most contagious diseases, acute diarrheal disease in the world. The icsA gene plays a key role in pathogenesis in bacterial. The aim of this study was cloning of icsA gene and developing a mutant construct pGEM::ΔicsA(Catr) in order to induction recombination in a native shigella for generation a live attenuated vaccine candidate strain. by use of biochemical tests, monoclonal Antibody and PCR the native shigella strain was examined. The entire icsA gene specific primers were designed, the ΔicsA gene was cloned in pGEM-7zf vector and the nucleotide sequence was determined. According to the data of sequencing, digestion mapping of pGEMΔvirG was obtained, after digested of ΔicsA gene by EcoRV restriction enzyme a chloramphenicol antibiotic resistance (catr) gene Cloned in amidst of ΔicsA gene. The native shigella strain by using biochemical tests was confirmed. Sequence of the icsA gene in native strain was sequentially identical with the strains submitted in the Gene-Bank database.Structure accuracy using specific primers pGEM::ΔicsA(Catr) was confirmed. Using the technique of allelic exchange based on the incident of recombination in bacteria is one of the most effective methods to develop a disruption in the target genes. This mutant construct can be applied in development of a live attenuated Shigella flexneri2a vaccine candidate.