Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Patent
1993-10-21
1998-01-06
Ketter, James S.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
4352523, 4353201, C12P 2102, C12N 120, C12N 1574
Patent
active
057053618
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to a plasmid for bacterial expression of pertussis toxin, useful in vaccine preparation.
2. Brief Description of Related Art
Increasing concern regarding the side-effects associated with immunization using whole cell preparations of B. pertussis has led to a decreasing acceptability of vaccines against whooping cough. This has in turn resulted in an increase in the incidence of the disease. It is imperative that an effective, nonreactogenic vaccine of high acceptability be developed. Several potentially protective antigens are candidates for inclusion in a purified multi-component vaccine, including toxins, such as pertussis toxin (PT) and adenylate cyclase, and also several cell surface or secreted antigens like filamentous haemagglutinin and serotype-specific fimbriae (Robinson, A., et al. 1985. Pertussis vaccine: present status and future prospects. Vaccine 3:11-22; and Robinson. A., and L. A. E. Ashworth. 1988. Acellular and defined-component vaccines against pertussis, p. 399-417. In A. C. Wardlaw, and R. Parton (ed.). Pathogenesis and Immunity in Pertussis. John Wiley and Sons, Chichester). However, Bordetella pertussis is a nutritionally fastidious microorganism. Purification of antigens obtained directly from B. pertussis is hampered by antigenic variation controlled by the bvg-positive regulatory locus (Gross, R., and R. Rappuoli. 1988. Positive regulation of pertussis toxin expression. Proc. Natl. Acad. Sci. 85:3913-3917; Knapp, S., and J. J. Mekalanos. 1988. Two trans-acting regulatory genes (vir and mod) control antigenic modulation in Bordetella pertussis. J. Bacterial. 170:5059-5066; and Weiss, A. A., and S. Falkow. 1984. Genetic analysis of phase change in Bordetella pertussis. Infect. Immun. 43:263-269), poor bacterial growth rates, low yields, and the presence of reactogenic contaminants. To overcome those problems, attempts have been made to obtain expression of recombinant antigens in E. coli using strong transcriptional and translational signals. The five genes encoding PT have been expressed independently of one another in E. coli under the control of the lambda P.sub.L promoter (Burnette, W. N., et al. 1988. Direct expression of Bordetella pertussis toxin subunits to high levels in Escherichia coli. Biotechnol. 6:699-706). The serotype 2 fimbrial sub-unit has also been expressed with the aid of the lambda P.sub.L and P.sub.R promoters (Walker, M. J., et al. 1990. engineering upstream transcriptional and translational signals of Bordetella pertussis serotype 2 fimbrial subunit protein for efficient expression in Escherichia coli: in vitro auto-assembly of the expressed product into filamentous structures. Mol. Microbiol. 4.39-47). In both cases the recombinant proteins failed to assemble into products that were immunologically identical with the native antigens. Pertussis holotoxin can be produced in recombinant B. parapertussis and B. bronchiseptica strains under the control of the original B. pertussis promoter. However, the level of expression detected was less than that for wild-type B. pertussis and the expression was also subject to modulation and bvg-controlled phase variation. Recombinant broad-host-range plasmids carrying the PT operon were also extremely unstable, being subject to both plasmid loss and gene deletions (Locht, C., et al. 1986. Molecular cloning of pertussis toxin genes. Nucl. Acids Res. 14:3251-3261).
The invention described in the following is based on the above-mentioned prior art and on the problem of making pertussis toxin a central component of new-generation vaccines.
SUMMARY OF THE INVENTION
Thus, one embodiment of the invention relates to a hybrid transposon that can be produced by inserting a pertussis toxin operon (PT operon) between the inverted repeats of a transposon.
That hybrid transposon is accordingly promoterless. For the purpose of expression, it is to be under the control of a native promoter after chromosomal integration.
The hybrid transposon according to the i
REFERENCES:
patent: 4883761 (1989-11-01), Keith et al.
Walker et al., Infection and Immunity, vol. 59, Nov. 1991, pp. 4238-4248.
Herrero et al., V. Bacteriol., vol. 172, Nov. 1990, pp. 6557-6567.
Bloom, Rev. Infect. Diseases, vol. 11, Supplement 2, 1989, pp. 5460-5466.
Knapp et al., vol. 170, J. Bacteriol., 1988, pp. 5059-5066.
Timmis Kenneth
Walker Mark
Gesellschaft fur Biotechnologische Forschung mbH (GBF)
Ketter James S.
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