Construction of pDS132::∆icsA for targeted deletion of a region of icsA gene in order to generate a live attenuated Shigella vaccine

Live attenuated Shigella vaccines have shown promise in inducing protective immune responses in human clinical trials. The aim of this study was to design and construct pDS132::∆icsA as a suicide plasmid for targeted deletion of a region of icsA gene in Shigella.

In this experimental study, species and serotypes of Shigella isolated from diarrhea samples of children at Firozabadi and Milad Hospitals of Tehran were confirmed by using serological and PCR tests. Identification primers of icsA gene were designed and then cloned to the pGEM-5zf vector and sequenced. According to icsA restriction enzyme map, 1751 bp of icsA gene was deleted by HincП restriction enzyme and the ∆icsA was constructed successfully. The pGEM∆icsA vector was digested by use of SphI and SalI enzymes and was then cloned to a suicide vector (pSD132). Precision of the process was confirmed by phenotype and genotype tests.

The Shigella dysenteriae type 1 strain was verified by serological tests and PCR. Sequence of the icsA gene in the native strain was identical to the strains submitted in the gene-bank database. Since the pDS132::∆icsA contains 1484 bp derived from icsA gene, it can be used to disrupt icsA gene as a specific suicide vector.

Application of suicide systems facilitated mutant construction in more specific and effective methods in comparison with the other primary techniques such as serial passage.