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Background: The major aim of this study was synthesis and assay of antimicrobial activity of peptide D28 and its new analogues derivatives as dimeric peptides. Materials and Methods: Three antimicrobial peptides known as D28, Di-D28-Lys,Di-Cys-D28 including 20, 41, 42 residues were synthesized respectively. For peptide synthesis, solid phase peptide synthesis method using blocked amino acids with flourenyl methoxy carbonyl group and for peptide purification HPLC were used. Peptides compositions were confirmed by amino acid analysis and SDS-PAGE electrophoresis. Antimicrobial tests against Staphylococcus aureus and Pseudomonas aeruginosa were performed as disk and well diffusion on plate and by adding to liquid broth culture (Broth macrodilution) in different concentrations. Results: Three required peptides (D28, Di-D28-Lys, Di-Cys-D28) successfully were synthesized. All three peptides were effective against S. aureus, but Di-Cys-D28 on the contrary to two other ones, showed no antimicrobial activity against P. aeruginosa. The inhibitory activity of Di-D28-Lys against P. aeruginosa was more than that of D28 peptide. Conclusion: Improvement of antimicrobial peptides activity through dimerization depends on the methods of dimerization and the strain of bacterium. Di-D28-Lys peptide in comparison with D28 and Di-Cys-D28 showed wide range and more antimicrobial activity. Therefore, Di-D28-Lys peptide could be a suitable antibiotic candidate for future studies.

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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.

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Background: Shigella dysenteriae one of the main causes of diarrhea in humans, but there is no vaccine against it. IpaD protein is one of the most important virulence factors in pathogenic shigella. The cloning N-terminal ipaD genes with ctB genes that have a role in adjuvant and carrier as recombinant vaccine can caused enhance the mucosal immune response.

Materials and Methods: Designing primers for genes ctB and ipaD were carried out based on the construction of gene cassettes, respectively. PCR reactions were performed to amplify the fragments and amplified fragments were cloned into pGEM-Teasy vector. Both the vector cut by restriction enzymes HindIII and XhoI and ipaD gene to gene ctB finally were Fusion. The ctB-ipaD gene cassette and expression vector pET28a(+) cuted by SalI and HindIII restriction enzymes. The cloning ctB-ipaD cassette was performed in the expression vector and expression of gene cassettes.

Results: In this study, the N-terminal ipaD as vaccine candidate antigen was genetically linked to the C terminal of ctB which has a carrier and adjuvant role. Fusing ctB-ipaD in the expression vector pET28a(+) is confirmed by sequencing, PCR and digested with restriction enzymes. The recombinant proteins produced is confirmed by SDS-PAGE and Western blot.

Conclusion: According to previous and similar studies, product cassette ctB-ipaD and expression its was expected. Is hoped to protein expression of this gene cassette and the production of antibodies could be achieved the candidate vaccine against Shigella.

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Background: Shigella is the causative agent of human shigellosis and its lipopolysaccharide is detected by TLR4. TLR4 belongs to Toll-like receptors family and many immunological pathways are triggered when these receptors are stimulated. Many researches showed increasing in TLR4 expression in mesenchymal stem cells through lipopolysaccharide treatment. The main goal of this study is detecting the optimum lipopolysaccharide between shigella strains through stimulation of immune system for vaccine studies.

Materials and Methods: In this experimental study human bone marrow derived mesenchymal stem cells were treated with three distinct concentrations (0.1, 0.01, and 0.001) of shigella (S. flexneri, S. dysenteriae, S. sonnei) extract containing lipopolysaccharide. Then TLR4 expression in mRNA level was investigated by RT-PCR and Q-PCR. The cells treated with phosphate buffered saline have been considered as a control group.

Results: Expression of TLR4 was shown in all of case groups except treatment with concentration 0.001 of extracts from sonnei and dysenteriae and also control group. The variations in the expression of TLR4 was dose-dependent in all of case groups. The maximum expression of TLR4 related to treatment with extract from shigella flexneri strain and the minimum expression related to treatment with shigella sonnei extract. The use of lipopolysaccharide from E. coli as a positive control indicated that lipopolysaccharide in shigella extracts is responsible for the increased expression of TLR4.

Conclusion: The TLR4 expression level was increasesed by S. flexneri extract, so it could be recommended for increasing vaccine efficiency.