Construction of a diphtheria toxin A fragment-C180 peptide fusion protein which elicits a neutralizing antibody response against diphtheria toxin and pertussis toxin. Infect Immun 1992 Dec;60(12):5071-7
Date
12/01/1992Pubmed ID
1452339Pubmed Central ID
PMC258279DOI
10.1128/iai.60.12.5071-5077.1992Scopus ID
2-s2.0-0026459612 (requires institutional sign-in at Scopus site) 25 CitationsAbstract
A genetically engineered gene fusion was constructed which encoded a nontoxic derivative of the A fragment of diphtheria toxin joined to the C180 peptide of the S1 subunit of pertussis toxin. The product of this gene fusion, termed the DTA-C180 protein, was purified from the periplasm of Escherichia coli to approximately 80% purity. The DTA-C180 protein possessed an apparent molecular weight of 43,000 by reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The DTA-C180 protein was cleaved into two tryptic peptides, which migrated with apparent molecular weights of approximately 22,000. One tryptic peptide reacted with diphtheria antitoxin, while the other tryptic peptide reacted with anti-C180 peptide immunoglobulin G. The DTA-C180 protein did not inhibit protein synthesis or stimulate clustering morphology in Chinese hamster ovary cells. The DTA-C180 protein elicited an immune response, in guinea pigs, against both the DTA and C180 peptide components of the fusion protein, with alum being a more efficient adjuvant than Freund's adjuvant for eliciting neutralization titers. Neutralization titers elicited by DTA-C180 protein were weaker than those elicited by diphtheria toxoid and pertussis toxin 9K/129G, a genetically engineered double mutant of pertussis toxin. Three doses of DTA-C180 protein yielded a neutralization titer of 1/750 against pertussis toxin in Chinese hamster ovary cells and a neutralization titer of 1/50 against diphtheria toxin in Vero cells. This is the first report of a protein derived from a recombinant S1 subunit that elicits a neutralizing titer against pertussis toxin.
Author List
Barbieri JT, Armellini D, Molkentin J, Rappuoli RAuthor
Joseph T. Barbieri PhD Professor in the Microbiology and Immunology department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
Adenosine Diphosphate RiboseAnimals
Antibodies, Bacterial
CHO Cells
Cricetinae
Diphtheria Toxin
Guinea Pigs
Neutralization Tests
Peptide Fragments
Pertussis Toxin
Recombinant Fusion Proteins
Trypsin
Virulence Factors, Bordetella