[Gene cloning, prokaryotic expression and functional evaluation of intimin from enterohemorrhagic Escherichia coli O157:H7].
OBJECTIVE
To obtain highly purified intimin encoded by the eae gene and study its adhesion activity.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Peng
Li-juan
LJ
Department of Medical Microbiology, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou 510515, China. pljuan221@163.com
Zhou
Yong
Y
Yang
Yu
Y
Hui
Chang-ye
CY
Zhao
Wei
W
Wan
Cheng-song
CS
D018829:Adhesins, Bacterial / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D002448:Cell Adhesion; D002460:Cell Line; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D019453:Escherichia coli O157; D029968:Escherichia coli Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D015870:Gene Expression; D010957:Plasmids / Q000235:genetics
D018829:Adhesins, Bacterial; D029968:Escherichia coli Proteins; C073819:eaeA protein, E coli