Production and immunogenicity of recombinant ferric enterobactin protein (FepA).
OBJECTIVE
Ferric siderophore complexes are produced by most bacteria to acquire iron, a vital element. These complexes are transported across the outer membrane by receptor proteins commonly known as FepA (ferric enterobactin protein). In this study we attempted to evaluate the immunogenicity of the membrane protein FepA, aiming at inhibition of iron uptake to protect invasion of the host by the bacterium.


METHODS
The genomic fepA gene was amplified from Escherichia coli O157:H7. The PCR product was ligated into pET28a and was then expressed in E. coli BL21(DE3). The recombinant protein purified by nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography was injected into BALB/C mice to induce immunity. Antibody titer was determined by ELISA. Mouse groups were challenged with various doses of E. coli O157:H7, Shigella flexneri, Klebsiella pneumoniae, and Salmonella typhi to study immune response.


RESULTS
An 85-kDa recombinant protein was expressed and purified. Immunogenicity of the recombinant protein was determined by injecting BALB/C mice. The antibody produced therein could efficiently recognize and bind ferric enterobactin binding protein. Immunized mice challenged with higher doses of selected bacteria survived.


CONCLUSIONS
Significant recognition by the antibody of ferric enterobactin binding protein may lead to its application in the restriction of Enterobacteriaceae propagation.
Baghal
Seyed Mehdi Larrie
SM
Department of Biology, Shahed University, Tehran, Iran.
Gargari
Seyed Latif Mousavi
SL
Rasooli
Iraj
I
D000818:Animals; D000907:Antibodies, Bacterial / Q000096:biosynthesis; D001425:Bacterial Outer Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology*; D001483:Base Sequence; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology*; D004269:DNA, Bacterial / Q000235:genetics; D004758:Enterobactin / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D019453:Escherichia coli O157 / Q000235:genetics / Q000276:immunology / Q000378:metabolism / Q000472:pathogenicity; D029968:Escherichia coli Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology*; D005798:Genes, Bacterial; D007114:Immunization; D007501:Iron / Q000378:metabolism; D007711:Klebsiella pneumoniae / Q000276:immunology / Q000472:pathogenicity; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D011956:Receptors, Cell Surface / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D012485:Salmonella typhi / Q000276:immunology / Q000472:pathogenicity; D012763:Shigella flexneri / Q000276:immunology / Q000472:pathogenicity
D000907:Antibodies, Bacterial; D001425:Bacterial Outer Membrane Proteins; D002352:Carrier Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; C029489:enterobactin receptor; D004758:Enterobactin; D007501:Iron