Gene cloning, prokaryotic expression and functional evaluation of intimin from enterohemorrhagic Escherichia coli O157:H7.
- Author:
Li-juan PENG
1
;
Yong ZHOU
;
Yu YANG
;
Chang-ye HUI
;
Wei ZHAO
;
Cheng-song WAN
Author Information
- Publication Type:Journal Article
- MeSH: Adhesins, Bacterial; biosynthesis; genetics; isolation & purification; metabolism; Animals; Blotting, Western; Cell Adhesion; Cell Line; Cloning, Molecular; Escherichia coli; genetics; Escherichia coli O157; Escherichia coli Proteins; biosynthesis; genetics; isolation & purification; metabolism; Gene Expression; Plasmids; genetics
- From: Journal of Southern Medical University 2009;29(4):707-710
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo obtain highly purified intimin encoded by the eae gene and study its adhesion activity.
METHODSThe 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.
RESULTSThe 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.
CONCLUSIONHighly 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.