فهرست مطالب hesaraki m

This study aims to detect the ipaD plasmid in Shigella dysenteriae type 1 and evaluate the invasive ability of a live, attenuated Shigella dysenteriae type 1 generated from deletion of the ipaD plasmid. The attenuated strain was tested in guinea pigs. We performed biochemical and antibody tests in addition to PCR analysis, to identify the strain and serotype of an isolated Shigella strain. The plasmid version of the ipaD gene was recognized. We generated a live, attenuated Shigella serotype by deletion of the plasmid version of the ipaD gene, which was confirmed by the above mentioned tests. Absence of the ipaD gene was assessed in a PCR reaction and compared to the wild type strain. Various dilutions of cultured bacteria were prepared and colony numbers counted. We prepared specific dilution which was appropriate for conducting the keratoconjunctive test. We used 6 guinea pigs that were divided into two groups, control (n=3) and experimental (n=3). C Invasion capability of produced strain was evaluated by keratoconjunctive test in guinea pigs.The produced live attenuated Shigella dysenteriae type 1 was confirmed through serological tests and PCR. Absence of ipaD was confirmed. Results of keratoconjunctive test has shown that this strain lost its ability to invade host cells. The plasmid version of ipaD gene plays a critical role in Shigella invasiveness. Deletion of mentioned gene reduces the invasion ability of these bacteria. Therefore, Live attenuated ΔipaD shigella dysenteriae can be an appropriate candidate to produce vaccine against shigellosis.

Shigellosis is caused by Shigella species. Considering the high frequency of morbidity and mortality reports and antibiotics resistance, designing and producing a vaccine against this disease is one of the goals of World Health Organization. Invasion Plasmid Antigen especially IpaC and IpaB are the major Shigella virulence agents and are also vaccine candidates. The aim of this study was to express ipaC gene which is of the virulence factors in order to investigate its immunogenicity. In this study, ipa C gene was obtained from NCBI gene bank and primers were designed. After genome extraction from Shigella dysenteriae, it was used as template for PCR amplification. The amplified ipa C gene by PCR was cloned into pTZ57R and sub-cloned into the expression vector pET-28a(+). E. coli BL21(DE3)plysS was transformed by recombinant vector pET-28a(+)/ ipa C and expression of the recombinant ipa C was investigated by IPTG induction and SDS-PAGE electrophoresis. Cloning was confirmed by PCR, enzyme digestion and sequencing. In addition, the recombinant protein was expressed by IPTG induction. Protein expression was confirmed by Ni–NTA column, Western-Blotting analysis and SDS-PAGE electrophoresis. Cloning, sub-cloning and expression of ipaC gene are confirmed in the present study. Therefore, this recombinant protein can be used for production of a recombinant vaccine against Shigella dysenteriae in future studies, after immunogenicity assay.