---

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

---

Background: Shigellosis is a major global issue of human health. To date, no effective vaccine has been found against Shigella. One of the major virulent factors in Shigella dysenteriae type 1 is Shigella enterotoxin or STx. STxB has immunogenic, adjuvant, or carrier properties. Vaccine candidate antigens can be coupled with this adjuvant for production of an appropriate vaccine. IpaD has a key role in invasion, virulence, and infection by Shigella.

Materials and Methods: In this study, the gene sequences of STXB and ipaD were obtained from gene bank and corresponding genes were prepared as synthetic construct and then transferred to E. coli BL21DE3. By PCR amplification and enzymatic digestion, protein expression levels were assessed. Its protein expression was confirmed by Western blot technique. After extraction by affinity chromatography, the recombinant protein was injected four times to guinea pigs. The pigs were, then, challenged by Shigella felexneri 2a and active toxin of E. coli O157:H7.

Results: The results showed that groups of guinea pigs challenged with 28×LD₅₀ of  toxin  completely survived. Furthermore, guinea pigs were challenged by inducing Shigella felexneri 2a in their eyes. The results showed that the control pigs got cataracts, whereas the immune pigs were in health.

Conclusion: The findings of this study suggest that this recombinant protein is a good candidate for production of a recombinant vaccine against Shigella family.