Gene replacement in Bordetella pertussis by transformation with linear DNA.
We replaced the wild-type TOX operon of Bordetella pertussis with in vitro mutated, detoxified alleles by electroporetic transformation using unmarked linear DNA. Uptake of DNA was selected by transient ampicillin resistance and two simultaneous recombination events resulted in gene-replacement at the natural locus with no integration of heterologous DNA. TOX alleles were stable without selection and recombinant strains secreted non-toxic, fully assembled, protective pertussis toxin (PT) analogues with kinetics similar to the parental vaccine strain under production-scale fermentation conditions. Strains generated in this way are suitable for the production of recombinant whole-cell or component whooping cough vaccines that require no chemical modification of PT.
Connaught Centre for Biotechnology Research, Willowdale, Ontario, Canada.
G R
GR
Zealey
S M
SM
Loosmore
R K
RK
Yacoob
S A
SA
Cockle
L J
LJ
Boux
L D
LD
Miller
M H
MH
Klein
D000483:Alleles; D000818:Animals; D001886:Bordetella pertussis / Q000235:genetics*; D004269:DNA, Bacterial; D004591:Electrophoresis, Polyacrylamide Gel; D051379:Mice; D009154:Mutation; D009876:Operon; D037342:Pertussis Toxin; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014169:Transformation, Bacterial; D014614:Vaccines, Synthetic / Q000008:administration & dosage / Q000235:genetics; D010566:Virulence Factors, Bordetella / Q000235:genetics* / Q000378:metabolism; D014917:Whooping Cough / Q000517:prevention & control
D004269:DNA, Bacterial; D011993:Recombinant Fusion Proteins; D014614:Vaccines, Synthetic; D010566:Virulence Factors, Bordetella; D037342:Pertussis Toxin