Method of producing thy a-strains of vibrio cholerae, such...

C - Chemistry – Metallurgy – 12 – N

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C12N 15/74 (2006.01) C07K 14/245 (2006.01) C07K 14/28 (2006.01) C12N 1/20 (2006.01) C12N 1/21 (2006.01) C12N 9/10 (2006.01) C12N 15/31 (2006.01) C12N 15/54 (2006.01) A61K 39/00 (2006.01) A61K 39/106 (2006.01)

Patent

CA 2329255

A method of producing a thy A-strain of vibrio cholerae comprising the step of site-directed mutagenesis in the V. cholerae chromosome at the locus of the thy A gene SEQ ID NO: 1 of FIG. 1, is described. Particularly, a .DELTA. thy A strain of of Vibrio cholerae lacking the functionality of the thy A is disclosed. This strain may comprise one or several episomal autonomously replicating DNA elements, such as plasmids, having an optionally foreign, e.g. E. coli, functional thy A gene that enables the strain to grow in the absence of thymine in the growth medium, and optionally having a structural gene encoding a homologous or heterologous protein. Further, proteins encoded by a structural thy A gene and the 5'-flanking region are described as SEQ ID NO: 4 of FIG. 4 and SEQ ID NO: 5 of FIG. 5, respectively. Additionally, a vaccine comprising a Vibrio cholerae L. thy A strain of the invention or a thy A-strain of Vibrio cholerae produced by the method of the invention is disclosed.

L'invention concerne une méthode de production d'une souche thy A<-> de Vibrio cholerae comprenant l'étape de mutagénèse ciblée dans le chromosome V. cholerae au locus du gène thy A SEQ ID NO:1 de la FIG.1. L'invention concerne notamment une souche DELTA thy A de Vibrio cholerae ne présentant pas la fonctionnalité de thy A. Cette souche peut comprendre un ou plusieurs éléments d'ADN épisomique à réplication autonome, tels que des plasmides, ayant un gène thy A fonctionnel facultativement étranger, par exemple E. coli, permettant à la souche de se développer en l'absence de thymine dans le milieu de croissance, et ayant facultativement un gène structurel codant une protéine homologue ou hétérologue. En outre, l'invention concerne des protéines codées par un gène thy A structurel et la région adjacente 5' définie par SEQ ID NO:4 de FIG.4 et SEQ ID NO:5 de FIG.5, respectivement. De plus, un vaccin comprenant une souche DELTA thy A de Vibrio cholerae de l'invention ou une souche thy A<-> de Vibrio cholerae, produit selon la méthode de l'invention, est également décrit.

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