US 5,470,729 A · Method of isolating restriction fragment deletions in Vibrio cholerae, and products thereof

Abstract

Methods of isolating deletion mutants of Vibrio cholerae. In one method, the deletion is predetermined by digestion with restriction endonucleases of known specificity. The deletions are inserted into the Vibrio cholerae chromosome by in vivo recombination between a plasmid carrying the desired deletion, with adjacent flanking sequences, and the Vibrio cholerae chromosome. In another method, an initial in vivo recombination event of homologous sequences from the recombinant plasmid into the chromosome provides a selectable marker at this site. A second in vivo recombination event between homologous flanking sequences results in excision of proficient genes from the chromosome with the end product being a deletion mutation. Also provided are methods for the isolation and characterization of a new Vibrio cholerae strain having a deletion in the ctx gene, as defined by Acc I, Xba I, Cla I and/or restriction endonuclease sites and further having a deletion in the gene encoding zonula occludens toxin (zot).

Claims16

Dependent claims

A culture of Vibrio cholerae comprising a strain of the Ogawa or Inaba serotype having the chromosomal DNA coding for the A subunit and B subunit of Vibrio cholerae toxin deleted from the toxin locus to confer avirulence and retaining the capacity to colonize the intestine of a host animal, and having a second DNA fragment coding for zonula occludens toxin deleted.

A method of isolating deletion mutants of Vibrio cholerae having the cholera toxin A subunit and zonula occludens toxin genes deleted, but expressing a DNA encoder the B subunit of Vibrio cholerae toxin comprising:

(a) obtaining a plasmid having a Vibrio cholerae DNA fragment which encodes cholera toxin, a DNA fragment which encodes zonula occludens toxin, and a gene for a selectable marker of foreign origin, wherein said plasmid is incapable of extrachromosomal replication in Vibrio cholerae;
(b) mating a microorganism carrying the plasmid of step (a) with a virulent strain of Vibrio cholerae containing said DNA which encodes cholera toxin and DNA which encodes zona occludens toxin;
(c) selecting for Vibrio cholerae expressing said selectable marker from the V. cholerae chromosome;
(d) screening for Vibrio cholerae produced by step (c) which are sensitive to a selective agent and no longer express the selectable marker;
(e) obtaining a second plasmid comprising V. cholerae chromosomal sequences flanking a DNA for a gene which encodes a resistance to mercury and a DNA which encoding the B subunit of Vibrio cholerae toxin, and a gene for a second selectable marker, wherein said second plasmid is incapable of extrachromosomal replication in Vibrio cholerae;
(f) mating a microorganism carrying said second plasmid with said Vibrio cholerae produced in step (d);
(g) selecting for Vibrio cholerae expressing said second selectable marker from the V. cholerae chromosome;
(h) growing the selected product of step (g) in the absence of a second selective agent;
(i) screening said Vibrio cholerae recited in step (h) for V. cholerae that have a mercury resistance gene and a DNA encoding the B subunit of V. cholerae toxin.

6 more elements

Dependencies are inferred from the claim text.

Facts

Application

07/931,943Filed 12 Aug 1992 · continuation in part

Priority

4 Mar 1983

Granted

28 Nov 1995

Term ends

Not estimatedThe source records don’t support an estimate.

Classification14 codes

A61K 39/107
Bacterial antigens: vibrio
C07K 14/28
From bacteria: from Vibrionaceae
C12N 1/205
Bacteria; Culture media therefor: bacterial isolates
C12N 15/01
Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or…:…

Family

Serial472,276Parent of this continuation-in-part · filed 4 Mar 1983

Serial472,276Parent of this continuation · filed 4 Mar 1983

Serial581,406Parent of this continuation-in-part · filed 17 Feb 1984

Serial867,633Parent of this continuation · filed 27 May 1986

Serial363,383Parent of this continuation-in-part · filed 5 Jun 1989

Serial533,315Parent of this continuation · filed 5 Jun 1990

US 4,935,364 AParent of this continuation-in-part · filed 19 Jun 1990

Serial821,872Parent of this continuation-in-part · filed 16 Jan 1992

US 5,135,862 AParent of this continuation-in-part · filed 4 Aug 1992

US 5,470,729 AThis patent

From the US filing history. Foreign family members aren’t listed.

Method of isolating restriction fragment deletions in Vibrio cholerae, and products thereof

Life of this patent

Method of isolating restriction fragment deletions in Vibrio cholerae, and products thereof

Life of this patent

Abstract

Claims16