EFFICACY OF LOCALLY PREPARED WHOLE-CELL AND ACELLULAR V AC'CINES FOR THE CONTROL OF WHOOPING COUGH DISEASE IN EGYPTIAN ENVIRONMENT
MAGDY NABIL OSMAN EZZAT;
Abstract
Bordetella pertussis strains (134), and (509) were grown on Bordet-Gengou plates supplemented with 20% sheep blood. Selected colonies from each strain were used to start a 140 L culture in a fully monitored fermenter as described by the Duth (RIV manual).
PT was purified from B. pertussis strains (134) phase I
culture supernatants (BS) as previously described. The supernatant was chromatographed on hydroxyl apatite, and fetuin columns to get the PT enriched fraction.
In SDS- PAGE, the concentrated purified toxin preparations revealed the presence of the 5 subunits ofPT in the proper ratio, in addition to minor contaminating proteins. The estimated toxin content is more than 90%.
Pertussis toxin enriched fractions were also purified on fetuin Sepharose columns loaded with the B. pertussis supernatant before and after adsorption on phenyl Sepharose, and by affinity chromatography using Sepharose blue gel. However the toxin yield is low, eventually the procedure can not be considered practical for vaccine preparation.
In alternative procedures, concentrated B. pertussis culture supernatant was used for preparation of the toxin enriched fractions, by gel filtration using Sephacryl S-300.
The B. pertussis supernatant was concentrated by ultrafiltration, diafiltration, clarified using membrane filters
0.221!, and lyophilized , the concentrated supernatants of
134, and 509 are unstable at 4°C.
Two fractions, a soluble fraction, and insoluble fraction were derived from the concentrated supernatants by centrifugation at 9000xg. Whereas highly aggregated proteins were obtained on reconstitution of the lyophilized supernatants.
PT was purified from B. pertussis strains (134) phase I
culture supernatants (BS) as previously described. The supernatant was chromatographed on hydroxyl apatite, and fetuin columns to get the PT enriched fraction.
In SDS- PAGE, the concentrated purified toxin preparations revealed the presence of the 5 subunits ofPT in the proper ratio, in addition to minor contaminating proteins. The estimated toxin content is more than 90%.
Pertussis toxin enriched fractions were also purified on fetuin Sepharose columns loaded with the B. pertussis supernatant before and after adsorption on phenyl Sepharose, and by affinity chromatography using Sepharose blue gel. However the toxin yield is low, eventually the procedure can not be considered practical for vaccine preparation.
In alternative procedures, concentrated B. pertussis culture supernatant was used for preparation of the toxin enriched fractions, by gel filtration using Sephacryl S-300.
The B. pertussis supernatant was concentrated by ultrafiltration, diafiltration, clarified using membrane filters
0.221!, and lyophilized , the concentrated supernatants of
134, and 509 are unstable at 4°C.
Two fractions, a soluble fraction, and insoluble fraction were derived from the concentrated supernatants by centrifugation at 9000xg. Whereas highly aggregated proteins were obtained on reconstitution of the lyophilized supernatants.
Other data
| Title | EFFICACY OF LOCALLY PREPARED WHOLE-CELL AND ACELLULAR V AC'CINES FOR THE CONTROL OF WHOOPING COUGH DISEASE IN EGYPTIAN ENVIRONMENT | Other Titles | كفاءة اللقاحات الخلوية واللاخلوية المحضرة محليا لمقاومة مرض السعال الديكي في البيئة المصرية | Authors | MAGDY NABIL OSMAN EZZAT | Issue Date | 1999 |
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