English

E A Aly Marwa,A Amin Magdy,M Essam Tamer,S Hanora Amro

doi:10.5897/ajb2015.15163

Abstract

Enterohemorrhagic Escherichia coli are important human food-borne pathogens. Recently, Shiga toxin-producing E. coli (STEC) causes life-threatening hemolytic-uremic syndrome (HUS). In this study, Stx2B gene, a subunit of Shiga toxin, was amplified via polymerase chain reaction (PCR) from the chromosomal DNA of clinical fecal sample using appropriate primers. The PCR product was cloned to commercially available plasmid pH6HTN His6HaloTag® T7 containing two purification tags, namely, six histadine tag and Halo tag. The integrity of the constructed plasmid was confirmed using restriction enzyme mapping and sequencing. Then, Stx2B protein expressed after induction with isopropyl β-D-1-thiogalactopyranoside (IPTG) in E. coli JM109 (DE3) under the control of the T7 promotor. The two step purification trains were used to purify native Stx2B. First step purification was Ni-immobilized metal ion affinity chromatography (IMAC) column, followed by second step using HaloLink resin. The native Stx2B was obtained after column cleavage of halo-tag using HaloTEV protease. Maximum protein expression of Stx2B economically was obtained using 1 mM IPTG for 4 h at 37°C. Protein identity was confirmed by a band at ~11.4 kDa using 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and StxB2 yield was 450 µg ml-1 confirmed by Bradford assay. Recombinant Stx2B protein was produced in highly pure yield using HaloTag technology. Key words: Escherichia coli O157:H7, StxB gene, expression, HaloTag technology, purification.

Highlights

Enterohemorrhagic Escherichia coli (EHEC) strains are important human food-borne pathogens (Kaper et al, 2004)
The magnitude of the social and economic impacts caused by EHEC infections is high, no licensed vaccine or effective therapy is presently available for human use
The digestion of double restriction the pH6HTN His6HaloTag® T7 vector and Stx2 insert with Fast Digest XbaI/NotI restriction enzymes resulting in the linearization of vector producing a band size ~3974 bp (Figure 1A; Lane2), while the polymerase chain reaction (PCR) product of stx2B gene was observed at ~270 bp, (Figure 1B; Lane 1)

Summary

Introduction

Enterohemorrhagic Escherichia coli (EHEC) strains are important human food-borne pathogens (Kaper et al, 2004). The clinical manifestations of EHEC infections range from watery diarrhea or hemorrhagic colitis (HC), to the most severe outcome, the life-threatening hemolytic-uremic syndrome (HUS) (Nataro and Kaper, 1998). The magnitude of the social and economic impacts caused by EHEC infections is high, no licensed vaccine or effective therapy is presently available for human use. The immune-prophylactic potential of the Stx1B subunit has been proven in the publication from different laboratory such as Boyd et al (1991) and Acheson et al (1996). The immune-modulatory potential of recombinant Shiga toxin B subunit (rStxB) protein in BALB/c mice was evaluated. Animal protection with recombinant StxB was conferred through both humoral and cellular immune responses (Gu et al, 2011). The large scale production of stx in E. coli for toxoid vaccine antigen was achieved by Hideyuki et al (2013)

Methods

Results

Discussion

Conclusion