Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1996-06-28
2002-11-05
Kunz, Gary L. (Department: 1647)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023100, C536S024320
Reexamination Certificate
active
06476213
ABSTRACT:
Helicobacter pylori
(also designated as
H.pylori
) is a Gram-negative bacterium which, to date, has been found exclusively on the surface of the mucosa of the stomach in man.
In common with most bacteria,
H.pylori
is sensitive to a medium which is at acid pH but, nevertheless, is able to tolerate acidity in the presence of physiological concentrations of urea (Marshall et al. (1990) Gastroenterol. 99: 697-702). By hydrolysing the urea to form carbon dioxide and ammonia, which are released into the microenvironment of the bacterium, the
H.pylori
urease enables the bacterium to survive in the acidic environment of the stomach. Recently, studies carried out on animal models have provided data suggesting that the urease is an important factor in the colonization of the gastric mucosa (Eaton et al. (1991) Infect. Immun. 59: 2470-2475). The urease is also suspected of causing injury, either directly or indirectly, to the gastric mucosa.
Currently,
Helicobacter pylori
(
H.pylori
) is recognized as being the etiological agent of antral gastrites, and appears to be one of the cofactors required for the development of ulcers. Furthermore, it appears that the development of gastric carcinomas may be associated with the presence of
H.pylori.
In order to develop novel sensitive and specific means for detecting in-vitro infections due to bacteria of the
Helicobacter pylori
species, the inventors have been taking an interest in the system for regulating the mobility of these bacteria.
With this aim in view, they have been interested in different modifications of the
H.pylori
strains, modifications which did not affect the recognition of these bacteria by sera from infected patients but which nevertheless rendered it possible to avoid obtaining reactions of the “false positive” type, in particular with bacteria of the Campylobacter family, for example
Campylobacter jejuni.
Furthermore, the inventors observed that it was possible, if need be, for the modified bacteria which were obtained to be employed in constructing immunogenic compositions or compositions used for vaccination. In this respect, the invention proposes, in particular, live attenuated bacterial strains.
In a first step, the inventors identified and isolated the gene f1bA which is involved in the regulation of the biosynthesis of the flagella of
H.pylori
and, as a consequence, in the regulation of the mobility of the bacterium. The biosynthesis of the flagella comprises synthesizing flagellins A and B and synthesizing the sheath. The f1bA gene regulates both the synthesis of flagellins A and B and the synthesis of the sheath which contains these flagellins. The inventors established that the f1bA gene was also important in that it regulated the biosynthesis of the anchoring protein of the bacterium, also termed the “hook”.
The invention therefore relates to a nucleotide sequence from the f1bA gene regulating the biosynthesis of the proteins of the
Helicobacter pylori
flagella, characterized in that it is able to hybridize, under conditions of high stringency, with a probe corresponding to a nucleotide fragment from
H. pylori
which has been amplified using two oligonucleotides having the following sequences:
OLF1bA-1: ATGCCTCGAGGTCGAAAAGCAAGATG (SEQ ID NO:1).
OLF1bA-2: GAAATCTTCATACTGGCAGCTCCAGTC (SEQ ID NO:2), or able to hybridize, under conditions of high stringency, with these oligonucleotides.
Such a sequence can be obtained by the steps of:
screening a genomic library containing the chromosomal DNA of an
H. pylori
strain with a probe corresponding to a nucleotide fragment from
H. pylori
which has been amplified using two oligonucleotides having the following sequences:
OLF1bA-1: ATGCCTCGAGGTCGAAAAGCAAGATG (SEQ ID NO:1).
OLF1bA-2: GAAATCTTCATACTGGCAGCTCCAGTC (SEQ ID NO:2), or able to hybridize, under conditions of high stringency, with these oligonucleotides,
recovering the DNA sequences which hybridize with said probe,
subcloning the DNA sequences which have been obtained in an appropriate vector of the plasmid type and selecting those modified vectors which hybridize, under conditions of high stringency, with the probe corresponding to the DNA fragment from
H. pylori
which has been amplified using oligonucleotides OLF1bA-1 and OLF1bA-2,
sequencing the DNA fragments contained in the plasmid vectors which hybridize with the abovementioned probe and determining the open reading frame contained in these fragments.
Advantageously, these DNA fragments will be used to reconstitute the coding sequence of the f1bA gene, corresponding to an open reading frame comprising approximately 2196 nucleotides.
The genomic library containing the chromosomal DNA of
H.pylori
can be obtained from any
H.pylori
strain. A cosmid library may also be prepared from the chromosomal DNA of
H.pylori.
An example of a strain which can be used for constructing this library is the strain N6, which was deposited in the NCIMB on Jun. 26, 1992 under No. NCIMB40512.
The two oligonucleotide primers which are used for preparing the probe which is intended for hybridizing the sought-after DNA which is present in the
H.pylori
DNA library are selected from the conserved regions of the various proteins of the LcrD/F1bF family.
The two oligonucleotide primers, OLF1bA-1 and OLF1bA-2, enabled a fragment to be amplified which was usable as a probe and which was of 130 base pairs, having the following sequence:
(SEQ ID NO:3)
ATG CCA GGG AAG CAA ATG GCG ATT GAT GCG GAT TTA
AAT TCA GGG CTT ATT GAT GAT AAG GAA GCT AAA AAA
CGG CGC GCC GCT CTA AGC CAA GAA GCG GAT TTT TAT
GGT GCG ATG GAT GGC GCG TCT AAA TTT.
The conditions of high stringency referred to above are the following: the hybridization is carried out at 42° C. in the presence of 50% formamide in a 2×SSC buffer containing 0.1% SDS (1×SSC corresponds to 0.15 M NaCl plus 15 mM sodium citrate—pH 7.0). The washings are carried out at 68° C., for example twice during a period of one hour, using 2×SSC plus 0.1% SDS.
A nucleotide sequence which is particularly interesting in accordance with the invention is the sequence of the f1bA gene corresponding to the sequence of nucleotides depicted in
FIG. 2
(SEQ ID NO:6), or to a nucleotide sequence which hybridizes, under conditions of high stringency, with the abovementioned sequence.
According to another embodiment of the invention, the nucleotide sequence which is the subject-matter of the present application is characterized in that it encodes a protein having the amino acid sequence (SEQ ID NO:7) depicted in
FIG. 2
or an amino acid sequence possessing the same regulatory properties, with regard to the biosynthesis of the flagellar proteins of
H.pylori
, as the abovementioned sequence.
The invention also relates to a nucleotide sequence which corresponds to the previous definitions and which is modified by deletion, substitution or insertion of bases or of a fragment of a nucleotide sequence, such that:
either the f1bA gene is no longer expressed in a host cell,
or the expression of the f1bA gene in a host cell does not enable the A and B flagellins or the sheath which contains them to be biosynthesized and, if this is the case, does not enable the
H.pylori
anchoring protein or the hook, to be synthesized.
The modification to which the nucleotide sequence of the invention is subjected should be such that it is irreversible and, in particular, that it remains irreversible when this sequence is recombined with the f1bA gene which is present in a bacterium which is transformed with a nucleotide sequence which is modified in this manner. This recombination is, for example, of the “double crossing over” type. Preferably, the modification of the nucleotide sequence should not involve any substantial modification—after replacement, by this modified sequence, of the corresponding fragment of the normal f1bA gene in a given
H.pylori
strain—of the functions of the neighbouring genes.
Also included within the scope of the invention are nucleotide sequences which constitute a fragment of the f1bA gene meeting the above crit
Labigne Agnes
Suerbaum Sebastian
Finnegan Henderson Farabow Garrett & Dunner L.L.P.
Gucker Stephen
Institut Pasteur
Kunz Gary L.
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