Extracellular PagC-HlyAs fusion protein for the generation and identification of Salmonella-specific antibodies.
The outer membrane protein, PagC, of Salmonella typhimurium was converted into a secreted protein by linking the 61-amino-acid long, C-terminal signal sequence of the E. coli hemolysin protein (HlyAs) to the mature PagC peptide. This PagC-HlyAs fusion protein was expressed and efficiently secreted into the culture supernatant by E. coli upon complementation with the hemolysin secretion proteins HlyB and HlyD. Polyclonal antibodies raised against this fusion protein not only recognized PagC in the membrane fraction of all salmonellae by Western blotting, but also reacted with proteins of smaller size in other gram-negative bacteria tested. A monoclonal antibody against the PagC-HlyAs fusion protein recognized only PagC in membrane fractions. The antibody-binding domain was determined using synthetic peptides derived from specific PagC domains. Sera from Salmonella-infected human patients and from a rabbit infected with S. typhimurium did not react with PagC in immunoblots, suggesting that PagC may not be recognized as a major antigen by the humoral immune system.
Mollenkopf
H J
HJ
Theodor-Boveri-Institut für Biowissenschaften (Biozentrum), Lehrstuhl für Mikorbiologie, Universität Würzburg, Germany.
Gentschev
I
I
Bubert
A
A
Schubert
P
P
Goebel
W
W
D000595:Amino Acid Sequence; D000818:Animals; D000907:Antibodies, Bacterial / Q000097:blood / Q000276:immunology*; D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D006090:Gram-Negative Bacteria / Q000276:immunology; D006460:Hemolysin Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D006801:Humans; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000276:immunology; D012475:Salmonella / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D012480:Salmonella Infections / Q000097:blood; D012486:Salmonella typhimurium / Q000235:genetics / Q000276:immunology; D013045:Species Specificity
D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D006460:Hemolysin Proteins; C036507:Hlya protein, E coli; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; C066766:pagC protein, Salmonella typhimurium