Expression of Shigella flexneri ipaB gene in tobacco

Shigellosis is a leading cause of diarrhea in many developing countries and although the disease can be controlled and managed with antibiotics, the constant emergence of resistant species requiring ever newer antibacterial drugs make development of an effective vaccine necessary. The bacteria are highly contagious and since immunity to Shigella is serotypespecific a multi-serotype vaccine is required for adequate protection. Proteins encoded by Shigella invasion plasmid, which are part of the Type Three Secretion System [TTSS] of this bacteria, are good candidate as vaccine targets since they are both immunogenic and conserved between different Shigella species. The advent of molecular farming, which is a low cost system, has opened up new venues for production of recombinant proteins. In view of the difficulties encountered in expressing IpaB in Escherichia coli [E. coli], the feasibility of the expression of this protein in tobacco has been investigated. The ipaB gene was cloned in place of the Hygromycin gene in pCambia1304 containing GFP as a reporter gene. The vector was then transferred into competent Agrobacterium tumefaciens [A. tumefaciens] strain LBA4404 which was used for agro-infiltration of Nicotiana tobaccum [N. tobaccum] leaves. Transformation was confirmed by expression of GFP. The gene was also cloned in pBAD/geneIII A and transformed E. coli host containing the construct was induced using different amounts of L-arabinose as inducer. Expression of IpaB gene by both hosts was determined by Western blotting using anti-IpaB monoclonal antibody. The data obtained showed that IpaB was expressed in plant leaves but expression in E. coli was not detectable. This study showed that N. tobaccum is capable of expressing this protein without its specific chaperon and in levels detectable by Western blotting

[ investigation of molecular epidemiology of Shigella soneii isolated from clinical cases in Tehran using RAPD-PCR method]

Bloody diarrhea [Shigellosis] is caused by different species of Shigella and is often seen in children befor than under 15 years old must be aded. less than 15 years of age. This disease is extremely contagious, epidemic and endemic in communities with low level hygiene and in majority of cases is accompanied with hemolytic uremia syndrome and decreased children's growth. As the rate of infection by Shigella soneii among different ranges of age is considered as an indicator of hygiene level, this study was designed to detect the rate of infection by Shigella soneii among different ranges of ages in Tehran by Random Amplified Polymorphic DNA [RAPD-PCR] between 2002-2006. In this study totally 60 isolates of Shigella soneii taken from 36 [60%] boys and 24 [40%] girls were studied. All isolates were primary confirmed as Shigella species by biochemical [Motility, MR, Citrate, H[2]S, Indole, Lysin decarboxylase, Ornitin decarboxylase, ONPG] and serologic tests; then all isolates were finally confirmed as Shigella soneii by Random Amplified Polymorphic DNA [RAPD-PCR] test. Among all 60 patients, the highest rate of infection with Shigella soneii belonged to 1-2 year-old group [36/7%]. Furthermore, the lowest rate of infection belonged to group with more than 9 years of age [1/6%]. This study showed that RAPD PCR method had a relative good discrimination power, and was a good method for typing of Shigella isolates in molecular epidemiological studies according to its high discrimination power, typing ability, reproducibility, low cost, rapidity and easy of use