[Gene cloning, prokaryotic expression and functional evaluation of intimin from enterohemorrhagic Escherichia coli O157:H7].
Nan Fang Yi Ke Da Xue Xue Bao
; 29(4): 707-10, 2009 Apr.
Article
en Zh
| MEDLINE
| ID: mdl-19403401
ABSTRACT
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 O157H7 (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.CONCLUSION:
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 O157H7 infection.
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Banco de datos:
MEDLINE
Asunto principal:
Adhesinas Bacterianas
/
Escherichia coli O157
/
Proteínas de Escherichia coli
/
Escherichia coli
Límite:
Animals
Idioma:
Zh
Revista:
Nan Fang Yi Ke Da Xue Xue Bao
Año:
2009
Tipo del documento:
Article
País de afiliación:
China