Publication: Construction of Bordetella pertussis strains with enhanced production of genetically-inactivated Pertussis Toxin and Pertactin by unmarked allelic exchange
7
3
Issued Date
2012
Resource Type
Language
eng
Rights
Mahidol University
Rights Holder(s)
BioMed Central
Bibliographic Citation
BMC Microbiology. Vol. 12, (2012), 61
Suggested Citation
Wasin Buasri, Attawut Impoolsup, Chuenchit Boonchird, Anocha Luengchaichawange, Pannipa Prompiboon, Petre, Jean, Watanalai Panbangred Construction of Bordetella pertussis strains with enhanced production of genetically-inactivated Pertussis Toxin and Pertactin by unmarked allelic exchange. BMC Microbiology. Vol. 12, (2012), 61. doi:10.1186/1471-2180-12-61 Retrieved from: https://repository.li.mahidol.ac.th/handle/123456789/2726
Research Projects
Organizational Units
Authors
Journal Issue
Thesis
Title
Construction of Bordetella pertussis strains with enhanced production of genetically-inactivated Pertussis Toxin and Pertactin by unmarked allelic exchange
Other Contributor(s)
Abstract
Background: Acellular Pertussis vaccines against whooping cough caused by Bordetella pertussis present a muchimproved
safety profile compared to the original vaccine of killed whole cells. The principal antigen of acellular Pertussis
vaccine, Pertussis Toxin (PT), must be chemically inactivated to obtain the corresponding toxoid (PTd). This process,
however, results in extensive denaturation of the antigen. The development of acellular Pertussis vaccines containing
PTd or recombinant PT (rPT) with inactivated S1, Filamentous Hemagglutinin (FHA), and Pertactin (PRN) has shown that
the yield of PRN was limiting, whereas FHA was overproduced. To improve antigen yields and process economics, we
have constructed strains of Bordetella pertussis that produce enhanced levels of both rPT and PRN.
Results: Three recombinant strains of Bordetella pertussis were obtained by homologous recombination using an allelic
exchange vector, pSS4245. In the first construct, the segment encoding PT subunit S1 was replaced by two mutations
(R9K and E129G) that removed PT toxicity and Bp-WWC strain was obtained. In the second construct, a second copy of
the whole cluster of PT structural genes containing the above mutations was inserted elsewhere into the chromosome
of Bp-WWC and the Bp-WWD strain was obtained. This strain generated increased amounts of rPT (3.77 ± 0.53 μg/mL)
compared to Bp-WWC (2.61 ± 0.16 μg/mL) and wild type strain (2.2 μg/mL). In the third construct, a second copy of the
prn gene was inserted into the chromosome of Bp-WWD to obtain Bp-WWE. Strain Bp-WWE produced PRN at 4.18 ±
1.02 μg/mL in the cell extract which was about two-fold higher than Bp-WWC (2.48 ± 0.10 μg/mL) and Bp-WWD (2.31 ±
0.17 μg/mL). Purified PTd from Bp-WWD at 0.8-1.6 μg/well did not show any toxicity against Chinese hamster ovary
(CHO) cell whereas purified PT from WT demonstrated a cell clustering endpoint at 2.6 pg/well.
Conclusions: We have constructed Bordetella pertussis strains expressing increased amounts of the antigens, rPT or
rPT and PRN. Expression of the third antigen, FHA was unchanged (always in excess). These strains will be useful
for the manufacture of affordable acellular Pertussis vaccines.