Gene cloning, prokaryotic expression and functional evaluation of intimin from enterohemorrhagic Escherichia coli O157:H7 / 南方医科大学学报

ABSTRACT

<p><b>OBJECTIVE</b>To obtain highly purified intimin encoded by the eae gene and study its adhesion activity.</p><p><b>METHODS</b>The eae gene was amplified from enterohemorrhagic Escherichia coli O157:H7 (EHEC) chromosome by PCR and cloned into pMD19-T vector. The eae gene was cut from pMD19-T vector and subcloned into the prokaryotic expression plasmid pET28a(+), and expressed in E.coli BL21(DE3). The recombinant protein was purified with Ni(2+)-chelating affinity chromatography followed by identification with SDS-PAGE and Western blotting. The purified intimin was detected by immunofluorescence staining to test its adhesion.</p><p><b>RESULTS</b>The 2805-bp eae gene fragment was obtained, and the recombinant expression plasmid pET28a(+)-eae was successfully expressed in E.coli BL21 (DE3). The molecular weight of the recombinant protein was 97 000. Purified recombinant intimin was recognized by rabbit anti-O157 antiserum, and bound to the surface of HEp-2 cells as revealed by immunofluorescence staining.</p><p><b>CONCLUSION</b>Highly purified and immunoreactive intimin has been successfully obtained, which can adhere to the surface of HEp-2 cells. The acquisition of recombinant intimin provides the basis for studying its interaction with the host receptors during EHEC O157:H7 infection.</p>