Abstract
E. coli O157:H7, one of the major EHEC serotypes, is capable of developing bloody diarrhea, hemorrhagic colitis (HC), and fatal hemolytic uremic syndrome (HUS) and is accompanied by high annual economic loss worldwide. Due to the increased risk of HC and HUS development following antibiotic therapy, the prevention of infections caused by this pathogen is considered to be one of the most effective ways of avoiding the consequences of this infection. The main aim of the present study was to design, express, and purify a novel chimeric protein to develope human vaccine candidate against E. coli O157:H7 containing loop 2–4 of E. coli O157:H7, outer membrane protein A (OmpA), and B subunit of E. coli heat labile enterotoxin (LTB) which are connected by a flexible peptide linker. Several online databases and bioinformatics software were utilized to choose the peptide linker among 537 analyzed linkers, design the chimeric protein, and optimize the codon of the relative gene encoding this protein. Subsequently, the recombinant gene encoding OmpA-LTB was synthesized and cloned into pET-24a (+) expression vector and transferred to E. coli BL21(DE3) cells. The expression of OmpA-LTB chimeric protein was then carried out by induction of cultured E. coli Bl21 (DE3) cells with 1mM isopropyl-β-D-thiogalactopyranoside (IPTG). The purification of OmpA-LTB was then performed by nickel affinity chromatography. Expression and purification were analyzed by sodium dodecyl sulphate poly acrylamide gel electrophoresis. Moreover, the identity of the expressed protein was analyzed by western blotting. SDS-PAGE and western immunoblotting confirmed the successful expression of a 27 KDa recombinant protein after 24 hours at 37°C post-IPTG induction. OmpA-LTB was then successfully purified, using nickel affinity chromatography under denaturing conditions. The yield of purification was 12 mg per liter of culture media. Ultimately, we constructed the successful design and efficient expression and purification of OmpA-LTB divalent under the above-mentioned conditions.
Highlights
The Enterohemorrhagic Escherichia coli, EHEC, which is a well-known foodborne and waterborne pathogen, evolves from nonpathogenic E. coli by acquiring a number of virulence factors
The extensive antibody response to outer membrane protein A (OmpA) of E. coli O157:H7 and other Enterobacteriacea species has been ascertained by many studies [19,20, 34,35,36]
It is obvious this multifunctional outer membrane protein is highly conserved among Enterobacteriaceae
Summary
The Enterohemorrhagic Escherichia coli, EHEC, which is a well-known foodborne and waterborne pathogen, evolves from nonpathogenic E. coli by acquiring a number of virulence factors. According to CDC reports, O157:H7 is one of the four major foodborne pathogens causing diarrhea [7] This pathogen is mainly transmitted to human population through the consumption of contaminated food products, including uncooked meat, unpasteurized dairy products, and vegetables contaminated by cattle feces. Since this serotype is a part of normal flora of cattle intestinal tract, ’the infected livestock shed these bacteria in high levels in their faces. The first step in the pathogenicity of E. coli O157:H7 is adherence and colonization of the bacteria in the human intestine Certain adhesion factors such as outer membrane protein A, or OmpA, and intimin play significant roles in colonization of this bacteria and initiation of pathogenesis. Fact, the unique and specific structure of four-surface hydrophilic loops exposed to the N-terminal of OmpA enables this protein to induce host-innate and adoptive immune responses [21,22,23]
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