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Expression system for the b subunit of cholera toxin

An expression system, a technology for cholera toxin, applied in the fields of peptides, chemical instruments and methods, fusion polypeptides, etc., to achieve the effect of cheap production process and simplified cost

Inactive Publication Date: 2007-01-24
SBL VACCIN AB
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Finally, DNA encoding antibiotic resistance may also be transferred to susceptible bacteria in individuals using the product, spreading undesired antibiotic resistance among them

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  • Expression system for the b subunit of cholera toxin
  • Expression system for the b subunit of cholera toxin
  • Expression system for the b subunit of cholera toxin

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Experimental program
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Embodiment I

[0190] A. Raw materials

[0191] The source of the ctxB gene is Vibrio cholerae serotype O1 strain 395 (Ogawa) [2]. The eltB signal sequence was obtained from plasmid pMMB68[3]. The linkage of the eltB and ctxB genes is described in [4].

[0192] The origin of replication was ColE1 obtained from pBlueScript KS (Stratagene).

[0193] The tac promoter used in pMT-ctxBthyA-2 was from plasmid pKK223-3 (Pharmacia).

[0194] The DNA sequence used for PCR amplification of the E. coli thyA gene was E. coli SY327 [5].

[0195] Kan used in the inactivation of the chromosomal Vibrio cholerae thyA locus R The resistance gene block was obtained from pUC4K (Pharmacia).

[0196] The suicide vectors used for site-directed mutagenesis of the V. cholerae thyA locus were pNQ705 [6], and pDM4 as described in [7].

[0197] The V. cholerae thyA gene was sequenced by SBL Vaccin AB and published in EMBL / Genebank under Accession No. AJ006514.

[0198] A.1 Construction of a typical biotype of Vi...

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Abstract

The present invention provides an expression system for obtaining improved cholera toxin subunit B (CTB) production, wherein the expression system comprises a Vibrio cholerae host cell lacking thyA gene function; and an expression vector comprising a functional thyA gene and a CTB gene , the CTB gene does not substantially contain the flanking sequences immediately adjacent to the 5' and 3' ends of the CTB gene in the native genome of the host cell as the source of the CTB gene. The present invention also provides methods for producing CTB, and isolated nucleic acid constructs for use as expression vectors in expression systems.

Description

[0001] The present invention relates to expression systems for the production of cholera toxin subunit B (CTB), methods of producing CTB and isolated nucleic acid constructs for use as expression vectors in the expression systems. Background of the invention [0002] The nontoxic cholera toxin subunit B (CTB) is a potent oral immunization agent that, in a wide range of experiments, has been shown to confer protection against diarrhea caused by cholera and enterotoxigenic Escherichia coli (Sanchez and Holmgren 1989 PNAS 86:481-485). This therefore makes CTB an important component of oral cholera vaccines, along with inactivated whole V. cholerae cells. Furthermore, CTB has recently attracted much attention as an immunogenic carrier for various other peptide or carbohydrate antigens and as an immunomodulator for down-regulation of immune responses. These findings raise the need to increase the yield of large-scale production of CTB, in part to facilitate the development of vacc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/67C12N15/68C12N15/74C07K14/28
CPCC07K2319/00C07K14/28C12N15/74
Inventor N·卡林M·勒本斯
Owner SBL VACCIN AB
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