[Optimization of expression, extraction and purification of the N-terminal region of ipaD gene in Shigella dysenteriae by proteomics analysis]

Shigella dysenteriae is one of the most important pathogens which in spite of many attempts vaccine preparation, extended researches are still in the way, Transport and surface expression of the invasion plasmid antigens [IpaD proteins] have essential role in the pathogenicity of Shigella spp. IpaD has been one of the most important proteins for Shigella vaccine candidate. Studies have shown that N - terminal region of this protein has a key role in the pathogen city and invasion. This study was done to evaluate the optimization of N-terminal region of Ipad in order to increase the production of recombinant protein. In this experimental labortary study, desired region of IpaD cloned in vector pET-28a [+]. For confirming cloning procedure, standard tests were performed. The effect of IPTG concentration, temperature and induction times on the level of protein expression were evaluated by SDS-PAGE, qualitatively. The gels were evaluated with 2-D gel analysis software [Melanie 7]. The recombinant protein was extracted by Urea and eventually purificated with affinity chromatography column. SDS-PAGE analysis showed that approximately the same amount of recombinant protein is expressed at different times, but software analysis proved that the optimized condition for the expression of recombinant protein was in the final concentration of 0.7 mM of IPTG, 37°C and 3 hours induction. According to the results every protein has its own expression after the homogenization process, and the temperature and the cells induction time length are more effective in the amount of protein production

[Molecular cloning and expression of Bacillus anthracis Lethal Factor domain 1 gene in Escherichia coli]

Anthrax is a common disease in human and livestock caused by Bacillus anthracis. Bacillus anthracis has two strong immunogen proteins: Protective antigen [PA] and lethal factor domain I [LFD1] that has been always considered as a candidate vaccine against Bacillus anthracis. The aim of this study was to express the lethal factor domain I in Escherichia coli. In this laboratory experimental study, the gene of LFD1 was detected and amplified from pXO1 plasmid by PCR. The gene was cloned with Bam H I and Xho I restriction site in cloning vector [pGEM-T easy], after isolation was sub cloned to expression vector pET28a[+]. This vector was transformed to E. coli-BL21 [DE3] to express LFD1 gene. The expression of LFD1 gene was induced by IPTG, and LFD1 protein was produced. The cloned LFD1 gene in pET28a[+] vector was confirmed by sequencing, PCR and enzymatic analysis. The expressed recombinant protein was confirmed by SDS-PAGE and Western blotting. According to immunogenicity of LFD1 protein, the produced recombinant protein can be used separately or in combination by adjuvants and delivers to design a vaccine against anthrax