WO1998017804A2 - ANTIGENE D'HELICOBACTER (η-GLUTAMYLTRANSFERASE) ET SEQUENCES CODANT CE DERNIER - Google Patents

ANTIGENE D'HELICOBACTER (η-GLUTAMYLTRANSFERASE) ET SEQUENCES CODANT CE DERNIER Download PDF

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Publication number
WO1998017804A2
WO1998017804A2 PCT/EP1997/005727 EP9705727W WO9817804A2 WO 1998017804 A2 WO1998017804 A2 WO 1998017804A2 EP 9705727 W EP9705727 W EP 9705727W WO 9817804 A2 WO9817804 A2 WO 9817804A2
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Prior art keywords
protein
pylori
sequence
polynucleotide
activity
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PCT/EP1997/005727
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English (en)
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WO1998017804A3 (fr
Inventor
Catherine Chevalier
Jean-Michel Thiberge
Agnès Labigne
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Institut Pasteur
Institut National De La Sante Et De La Recherche Medicale
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Priority to EP97945852A priority Critical patent/EP0939800A2/fr
Priority to AU51199/98A priority patent/AU5119998A/en
Publication of WO1998017804A2 publication Critical patent/WO1998017804A2/fr
Publication of WO1998017804A3 publication Critical patent/WO1998017804A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/104—Aminoacyltransferases (2.3.2)
    • C12N9/1044—Protein-glutamine gamma-glutamyltransferase (2.3.2.13), i.e. transglutaminase or factor XIII
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention concerns a polynucleotide coding
  • transpeptidase such as a gamma ( ⁇ )
  • the invention concerns more particularly the biological applications of the polynucleotide or its fragment and the corresponding encoded protein or fragment thereof, notably for the detection of H. pylori in humans or in foods or in the environment and also relates to immunogenic compositions for inducing protective antibodies against Helicobacter infection in humans or in animals such as cats or pigs.
  • H. pylori is a Gram-negative bacterium heretofore found exclusively at the mucosal surface of the antral region of the human stomach, and more particularly around the lesions and craters of gastric and duodenal ulcers. This bacterium was initially called Campylohacter pyloritis (Warren et al. (1983) Lancet 1: 1273-1275).
  • H. pylori carriers 8% present an ulcerous illness, 9% suffer from non-ulcerous dyspepsia, and 8% are asymptomatic carriers.
  • H. pylori is a microorganism that infects human gastric mucosa and is associated with active chronic gastritis. It has been shown to be an aetiological agent in gastroduodenal ulceration (Peterson, 1991), and two recent studies have reported that persons infected with H. pylori had a higher risk of developing gastric cancer (Nomura et al., 1991; Parsonnet et al . , 1991).
  • the H. pyl ori produce and release an enzyme that presents a proteolytic activity leading to the degradation of the mucus and, thus, a weakening of the natural barrier protecting the gastric mucosa.
  • GTT glutamyltranspeptidase
  • the GGT of H. pylori exhibits several features:
  • the GGT signal peptide demonstrates all the classical features of gram negative signal peptides, i.e. a charged hydrophilic N-terminal sequence (MetArgArg) and a long stretch of hydrophobic amino acids (20 AA) preceding the Ala cleavage site;
  • the C-terminal domains of the putative large (AA 311 to 401) and small (AA 501 to 615) subunits are species specific. They, therefore, represent specific antigenic targets for the H. pyl ori isolates.
  • ⁇ -Glutamyltranspeptidase catalyzes a) the transfer of
  • shock protein best expressed at 20°C and poorly expressed
  • the enzyme is located in the periplasmic space.
  • the E . col i GGT is expressed as a pro-GGT, processed later into a large and a small subunit.
  • the physiological significance of " GGT in E . col i is not yet known, however, it was recently shown that GGT was essential for the
  • the inventors have been interested in cloning the gene and identifying the protein because:
  • the invention is thus intended to supply new polynucleotides that are capable of coding for proteins
  • glutamyl residues transfer activity such as is expressed in H. pylori .
  • the invention also supplies fragments of this sequence that may be used as detection probes in H. pylori .
  • the invention aims to supply an antigen such as a protein carrying transfer catalytic activity, for
  • ⁇ -glutamyl residues transfer activity such as is
  • the purified polynucleotide is characterized in that it comprises at least a part of a coding sequence for a protein carrying
  • residues transfer activity such as is expressed in H. pylori .
  • the invention particularly concerns a polynucleotide that is capable of hybridizing with coding genes for a protein carrying transfer catalytic activity, for example,
  • the polynucleotide is characterized in that it carries the information required for the production of a protein
  • glutamyl residues transfer activity or the transfer activity of one of its fragments, which protein or fragment is capable of forming an immunologic complex with antibodies directed against, respectively, a protein
  • glutamyl residues transfer activity such as is expressed in H. pylori , or a fragment of the protein.
  • the sequence is also characterized in that it comprises at least a part of a fragment of about 2 kb corresponding to the restriction fragment containing at least four restriction sites among the following: Sau 3A, Hind III, Pstl, Sau 3A, Pstl, Hind III, Sph I, Sau 3A.
  • the enzymatic restriction map of this fragment is represented in FIG. 1.
  • a more specifically preferred sequence comprises at least a part of a fragment of about 1.8 kb ( ⁇ 5%) limited by the end nucleotides respectively situated at a distance of approximately 0.3 kb upstream of the restriction site Pstl, as represented in FIG. 1, and of approximately 0.1 kb downstream of site Sphl, as represented in FIG. 1.
  • This sequence is contained in the recombinant plasmid pILL308.
  • any fragment of the nucleotide sequence inserted in pILL308 or any genetic construct containing such a fragment or the 1.8 kb to 2 kb fragment is considered a "derivative" of pILL308 in the present invention.
  • sequences constituted by this fragment of 1.8 kb to 2 kb carry the coding and regulatory regions necessary for the expression of a protein carrying transfer catalytic
  • the invention concerns a recombinant sequence comprising one of the above-defined sequences, possibly in association with a promoter capable of controlling the transcription of a sequence and a DNA coding sequence for the termination signals of the transcription.
  • the nucleotide sequences of the invention are characterized in that they are capable of hybridizing with a probe formed from the sequence presenting:
  • nucleotides that are complementary to any one of the above-mentioned nucleotides.
  • probe 1 After amplification with the primers "oligo I” and “oligo II reverse", a 600 bp probe was obtained as probe 1. This nucleotide sequence hybridizes with pILL310 and pILL308, as shown on FIG. 3.
  • Probe 2 is obtained by amplification with the use of the oligo II and oligo I reverse primers.
  • Polynucleotides according to the invention comprise the above-defined "nucleotide chains" or at least part of the chains or the amplified sequences obtained by the use of the said nucleotide chains or primers .
  • the bases of the nucleotide sequences may be in a different order from that found in the genes, and/or that the bases may possibly be substituted when a probe elaborated from such sequences gives a characteristic and unequivocal answer with respect to the ability to recognize the presence of coding genes for the protein carrying transfer catalytic activity, for
  • the purified polynucleotide is characterized in that it is modified by deletion, addition, substitution, or inversion of one or more nucleotides so that the functional properties of the protein encoded by these modified sequences is either conserved or attenuated, or even deleted, as compared with
  • the invention also covers any sequence of nucleotides, hybridizable with that of the above-mentioned chains of nucleotides, such as that obtained by reverse enzymatic transcription of the corresponding RNA or by chemical synthesis .
  • the invention also concerns a polynucleotide that corresponds, according to the universal genetic code, to at least a part of the following amino acid sequence, which is included in the present invention:
  • the invention also concerns recombinant expression and cloning vectors, capable of transforming an appropriate host cell, the vectors containing at least a part of a polynucleotide defined above under the control of elements of regulation permitting its expression.
  • Preferred recombinant vectors include at least a part of the above-mentioned nucleotide sequence of approximately 2 kb.
  • the invention also includes the strains of the transformed microorganisms. These strains contain one of the above-defined polynucleotides or a recombinant vector such as previously defined.
  • Strain E. coli MC1061 (pILL308) deposited on October 16, 1996, under number 1-1775 at the Collection Nationale de Culture de Microorganismes (C.N.C.N.) at the Institut Pasteur in PARIS (FRANCE), carries plasmid pILL308 of approximately 2 kb comprising the previously mentioned restriction fragment.
  • the invention also concerns an antigen that is a
  • the encoded antigen has the sequence of FIG. 4D. MFJtSFLKTIGLGVIAI ⁇ LGLLSPl ⁇ AASYPPIK ⁇
  • the invention also includes mutated strains of H. pylori containing an insert of a sequence encoding a heterologous epitope in the ggt gene. Such a mutant may be created by the methods described in WO96/14408 and WO90/11354.
  • Such H. pylori , attenuated strains can be used, for example, to induce an immune response against the heterologous epitope.
  • the immune response can be a cellular response.
  • the protein and its fragments correspond, according to the universal genetic code, to the above-defined sequences of nucleotides, in particular to at least a part of the sequence of approximately 2 kb and preferably a 1646 bp corresponding to gamma glutamyl transferase of H. pylori (GGT) .
  • the protein of the invention is also characterized in that it can be obtained through: 1) transformating host cells, bacteria, or eukaryotic cells, including yeast, such as Saccharomyces cerevisae or Pichia pastoris as operated by means of a recombinant vector as previously defined; 2) culturing, in an appropriate medium, the transformed host cells; and 3) recovering the protein from these cells or directly from the culture.
  • the invention also concerns the production of GGT or of fragments of GGT by this process .
  • the protein and its fragments which may also be obtained by chemical synthesis, advantageously have a high degree of purity and are used to form polyclonal antibodies according to standard techniques.
  • the invention also includes such polyclonal antibodies, as well as monoclonal antibodies capable of recognizing specifically the above-mentioned protein and its fragments.
  • the invention concerns a particular sequence of nucleotides located upstream of the coding region and corresponding to a peptide signal sequence. It is characterized by the following chain of amino acids :
  • the invention covers the use of such a signal peptide and its corresponding nucleotide sequence for the preparation of hybrid molecules corresponding to a foreign nucleotide sequence coding for a foreign protein or peptide and the peptide signal or its corresponding nucleotide sequence of the invention obtainable from H. pylori genome.
  • the invention also concerns the biological applications of the polynucleotide, the corresponding proteins, the monoclonal or polyclonal antibodies, and the protein GGT or its fragments.
  • the invention covers also the use of the polynucleotides, particularly the polynucleotide coding for GGT and hybridizing with the plasmid pILL308, for the induction of an immune response against H. pylori infection.
  • This elaboration comprises, in particular, denaturing the double-stranded sequences in order to obtain a mono-stranded sequence that may be used as a probe.
  • the invention therefore concerns detection probes characterized in that they comprise at least a part of an above-defined sequence of nucleotides or purified polynucleotides .
  • the invention also includes any probe which, with regard to its polynucleotide, is distinguished from the previously mentioned probe only by substitutions or alterations of nucleotides not causing modification of its hybridization properties with the genome of H. pylori .
  • the DNA fragment used as a probe contains a sufficient number of nucleotides to obtain the required specificity and the formation of a stable hybrid. It is possible to use fragments attaining several kb, but high specificity results are also obtained with shorter fragments of approximately 25 to 40 nucleotides.
  • Appropriate probes for this type of detection are advantageously marked by a radioactive element or by any other marker allowing its recognition in the hybridized state with the preparation enclosing the DNA to be studied.
  • these probes are brought into contact with a biological specimen containing the bacteria, or directly with these bacteria or other nucleic acids, in conditions permitting the hybridization of a polynucleotide of the probe with a complementary sequence contained in the tested product.
  • the hybridization method may, for example, be implemented on spots. This method involves, after denaturation of the DNA previously obtained from bacteria originating from antral biopsies, deposit of an aliquot quantity of this DNA on nitrocellulose membranes, hybridization of each membrane in usual conditions with the probe, and detection, in standard fashion, of the hybrid formed.
  • Hybridization method may also be conducted according to the Southern technique. This method involves the electrophoretic separation in agarose gel of the fragments of DNA engendered after treatment of the DNA by restriction enzymes, transfer after alkaline denaturation on appropriate membranes, and hybridization with the probe in usual conditions . It is not always necessary to proceed to the preliminary expression of the DNA; it is enough that the DNA should be rendered accessible to the probe.
  • Detection for the specific identification of the H. pylori may also be carried out by DNA amplification techniques (PCR) . These techniques are notably described in U.S. Patent Nos. 4,683,202 and 4,683,195.
  • primers of 20 to 30 nucleotides are used, preferably approximately 25 nucleotides.
  • These primers are included in one of the above-defined sequences of are nucleotides or likely to hybridize with a part of one of the above-defined nucleotide sequences, or of their complementary sequences.
  • One of the sequences is capable of being linked to a nucleotide sequence of one of the DNA fragment strains to be amplified, this sequence being situated at an end of this fragment, for example the 5' end.
  • the other sequence is capable of being linked to a nucleotide sequence of the second DNA fragment strain to be amplified; this latter sequence being situated at the end opposite the above- mentioned end of this fragment (the 3' end in the given example) .
  • Preferred primers are included in the nucleotide sequence of restriction fragment Hind III-Sau 3A in FIG. 2.
  • fragments concerned are those situated respectively at each end of the restriction sequence Hind IV-Pstl.
  • a process of the in vitro detection of the presence of H. pylori in a biological sample includes the following steps :
  • the biological sample in question is brought into contact with a nucleotide probe according to the invention, in conditions permitting the production of an hybridization complex formed between the probe and the sequence of nucleotides; and 3) the detection of the hybridization complex.
  • the invention thus provides the tools necessary for rapid detection of H. pylori , both in si tu and in the environment, with a high degree of specificity and without having recourse to culturing.
  • Such detection tools are particularly useful in studying the natural reservoir of these bacteria, as well as the methods of transmission, circulation, and contamination. Furthermore, in the in vi tro diagnosis performed on the biopsies, the use of these probes makes it possible to gain a considerable amount of time compared to current techniques, which said techniques, moreover, require a technology which can only be applied in specialized services, i.e. bacteriological techniques or the use of electronic microscopy.
  • the invention also concerns the immunological applications of the coded protein, particularly for the elaboration of specific antiserum and of polyclonal and monoclonal antibodies.
  • the polyclonal antibodies are formed, according to standard techniques, by injecting the protein into animals obtaining the antiserums from the animals, and then obtaining the antibodies from the serums, e.g. by affinity chromatography.
  • the monoclonal antibodies are produced in the usual manner by fusing myeloma cells with the cells of animals previously immunized by means of the proteins of this invention.
  • splenic cells from female rats are used.
  • the invention concerns an in vi tro detection method for the presence of H. pylori in a biological sample in which it is likely to be contained.
  • This procedure is characterized in that it comprises:
  • the invention also concerns probes for detecting the possible presence of H. pylori in a biological sample, characterized in that they comprise at least a part of an above-defined sequence of nucleotides.
  • the base of the sequences of nucleotides under consideration may be in an order differing from that found in the genes, and/or that these bases may, if required, be substituted when a probe elaborated from such sequences gives a characteristic and unequivocal response regarding the ability to recognize the presence of coding genes for protein carrying transfer
  • the invention also covers any sequence of nucleotides hybridizable with that of the above-mentioned chains of nucleotides, such as can be obtained by reverse enzymatic transcription of the corresponding RNA or by chemical synthesis .
  • the invention also covers any probe which, with regard to its sequence of nucleotides, is distinguished from the previously-mentioned probes only by substitutions and alterations not causing any modification to its hybridization properties with the genome of H. pylori .
  • the above-mentioned probes may be used in in vi tro processes for diagnosing the presence of H. pylori in a biological sample such as described above.
  • the invention also concerns nucleotide primers of approximately 10 to approximately 40 nucleotides, these primers being included in one of the above-defined nucleotide sequences I, II, III or IV or being likely to hybridize with a part of the above-mentioned nucleotide sequences or with their complementary sequences (particularly in the above-mentioned conditions of hybridization) .
  • the invention also concerns the use of these primers in the above-mentioned in vi tro diagnosis methods, for the preliminary amplification of the quantity of nucleotides of H. pylori likely to be contained in the biological specimen according to the above-described process.
  • the above-defined protein production process may, if required, comprise a preliminary amplification step of the coding gene for the protein whose production is sought, this step being carried out with primer couples of the invention in accordance with the above-described process.
  • the invention also concerns the protein or peptide coded by the above-described nucleotide sequences.
  • the invention particularly concerns the above-described amino acid sequence of 53 kDa ⁇ 10%.
  • the molecular weight of the antigen of the invention has been measured on an electrophoresis gel (SDS PAGE) with a standard molecular weight kit commercialized by BIORAD.
  • the invention also concerns the polyclonal or monoclonal antibodies directed against all or part of the above-described protein and likely to form an immunologic complex with them.
  • the invention concerns the antibodies, especially monoclonal antibodies, obtained by means of a peptide sequence of at least approximately 10 to 15 amino acids of the sequence that comprises amino acids 550 to 615 and/or amino acids 311 to 401 of FIG. 4.
  • Preferred monoclonal antibodies of the invention are directed against all or part of the sequence limited by the amino acids situated at positions 501 to 615 and/or 311 to 401 of the sequence of FIG. 4.
  • the invention also relates to the use of the above- mentioned antibodies to implement the in vi tro methods and the in vi tro kits as described above for diagnosing the infection of an individual by H. pylori .
  • the invention also concerns immunogenic compositions comprising all or part of the above-described protein encoded by the polynucleotide corresponding to the nucleotide sequence inserted in plasmid pILL308, and in particular all or part of a polypeptide sequence limited by the amino acids situated at positions 501 to 615 and/or 311 to 401 of the sequence depicted in FIG. 4 in association with a pharmaceutically acceptable vehicle.
  • One of the advantages of the immunogenic composition according to the invention characterized in that it contains all or part of the GGT in association with a mixture of antigens of H. pylori as urease A or B (URE A or URE B) and/or HspA is to obtain a complementary target.
  • GGT it is located differently from URE B and HspA (WO 95/714093) .
  • Such immunogenic compositions may be used as vaccine compositions in preventing the infection of an individual by H. pylori, and can be administered by any route, such as oral, intravenous, intramuscular, in combination with an adjuvant or not, and in a liposomal vesicle or not.
  • the invention is adapted to the animal to be treated.
  • the urease will be prepared according to the teachings of PCT/WO95/14093.
  • the composition can contain the antigen of the invention associated with a urease and/or HSPA specific to the Helicobacter strains specific to the animal to be infected or treated.
  • the invention also concerns pharmaceutical composition
  • pharmaceutical composition comprising one (or several) antibodies such as described above, and in particular, antibodies likely to form an immunologic complex with all or part of the peptide sequence limited by the amino acids situated at positions 501 to 615 and/or 311 to 401 of the sequence of FIG. 4 in association with a pharmaceutically acceptable vehicle.
  • compositions may be used in the treatment of pathologies linked to the infection of an individual by H. pylori , notably gastritis, and gastric and duodenal ulcers.
  • the term "primer” means the sequences I, II, III or IV described herein, and the term “probe” means probe 1 or 2 described herein or any sequence capable of hybridizing under stringent conditions with the polynucleotide according to the invention.
  • protein is to be understood in the context of the invention as a chain of amino acids, whatever its length, and includes the term “peptide.”
  • fragment means any amino acid sequence shorter by at least one amino acid than the parent sequence and comprising a length of amino acids, preferably at least 6 residues, that are consecutive in the parent sequence.
  • Figure 1 is a restriction map of plasmid pILL308.
  • Figure 2 contains primer sequences I, II, III, and IV of the present invention.
  • Figure 3 contains the restriction maps of regions of the plasmids pILL310 and pILL308 hybridizing to probes 1 and 2.
  • Figures 4A and 4B compare the GGT amino acid sequence of H. pylori with the corresponding sequences from other organisms .
  • Figure 4C contains the open phase nucleotide sequence of the first 583 amino acids of H. pylori GGT.
  • Figure 4D contains the sequence of the first 583 amino acids of H. pylori GGT.
  • Figure 5 is an immunoblot of various extracts of H. pylori 85P using rabbit antisera against human transferase.
  • Figure 6 is a restriction map of regions of plasmids pILL310, pILL308, and pILL309 hybridizing with probe 1 or probe 2.
  • Figure 7 is a restriction map of plasmids pILL309 and pILL311 illustrating the 714 bp-internal deletion in the ggt open reading frame of plasmid pILL311.
  • H. pylori isolates encode a GGT that catalyzes the transfer of gamma glutamyl residues from glutathione to amino acids, and might therefore participate to the intracellular translocation of amino acids.
  • This enzymatic activity accounts for one of the key biochemical assays that leads to the identification of H. pylori .
  • a polyclonal rabbit antiserum raised against a mammalian transferase a 53 kDa band was revealed by Western blot in the cytoplasmic fraction of strain 85P described in J. Bacteriol. 173:1920-1931 (1991).
  • the protein was purified by a series of chromatography and the immuno-reactive fractions were pooled at each step.
  • the partially purified 53-kDa protein was submitted to enzymatic endoproteolysis and the N-amino acid sequences of two of the peptides thus generated was determined.
  • ⁇ -GT Glutamyltransferase
  • oligonucleotides were synthesized and used to amplify the H. pylori chromosomal DNA. A unique combination of 2 of the oligonucleotides gave a PCR product that was then used as a probe to identify a recombinant cosmid within the genomic library, and subclones of the cosmid.
  • the identified gene encodes a 555 amino-acid protein that showed high similarity with the E. coli and human GGTs and resembles the cephalosporin acylase from Pseudomonas and Bacillus with respect to their molecular organization and amino acid sequence. Whether the GGT in H. pylori consists of two polypeptide chains (a and b chains, 365 and 190 kDa, respectively) that result from the maturation of the 555 kDa precursor protein remains to be determined.
  • a 0.7 kb fragment internal to the ggt gene was replaced by a kanamycin cassette and the recombinant plasmid was used to generate a knocked out mutant of H. pylori .
  • This mutant was viable despite the fact that no residual gamma-glutamyltranspeptidase activity was detectable .
  • the invention also covers the production of recombinant large and small subunits of the GGT.
  • Example 1 Detection and partial purification of the
  • antibiotics Polymyxin, Vancomycin, Trimetroprime,
  • Buffer A 25 mM NaCl in Tris 1M
  • Buffer B 0.5 M NaCl in Tris 1M
  • VGLALSSHPLATEIGQK II GFYQGQVAELIEK
  • VH12 One recombinant cosmid (VH12) was identified, purified, and partially restricted with restriction enzyme
  • nucleotide sequence of the subclones allowed the inventors to identify an open reading frame, 1646 bp in length, encoding a polypeptide partly homologous to the ggt gene of E . coli .
  • Plasmid pIL311 (a pUcl8 derivative plasmid) in which a 714 bp-internal detection has been introduced within the ggt open reading frame, was electroporated into H. pylori strain N6.
  • the y-glutamyltransferase activity of the N6 kanamycin derivative transformants as well as that of the original isolate (N6) has been determined spectrophotometrically by measuring the p-nitroaniline released from L-y-glytamyl-3- carboxy-4-nitroanilide used together with glycylglycine . No residual y-glytamyltransferase activity was detectable in the N6 isogenic mutants.
  • the synthesis of y-glutamyltranspeptidase is not essential for H. pylori in vi tro growth.
  • the role of the GGT for survival and multiplication in vivo remains to be determined.

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Abstract

Cette invention concerne un polynucléotide purifié d'hélicobacter qui se caractérise par le fait qu'il comprend dans sa structure une séquence de codage destinée à une protéine chargée d'une activité catalytique de transfert, par exemple une activité de transfert des restes de η-glutamyl ou pour ses fragments; ou bien qu'il est capable d'être hybridé avec la séquence nucléotidique d'environ 2Kb qui est introduite dans le plasmide pILL308.
PCT/EP1997/005727 1996-10-17 1997-10-17 ANTIGENE D'HELICOBACTER (η-GLUTAMYLTRANSFERASE) ET SEQUENCES CODANT CE DERNIER WO1998017804A2 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP97945852A EP0939800A2 (fr) 1996-10-17 1997-10-17 ANTIGENE D'HELICOBACTER (gamma-GLUTAMYLTRANSFERASE) ET SEQUENCES CODANT CE DERNIER
AU51199/98A AU5119998A (en) 1996-10-17 1997-10-17 A (helicobacter) antigen (gamma-glutamyltransferase) and sequences encoding the same

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US2866496P 1996-10-17 1996-10-17
US60/028,664 1996-10-17

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Cited By (10)

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WO2000001825A1 (fr) * 1998-07-01 2000-01-13 Institut Pasteur ANTIGENE D'HELICOBACTER (η-GLUTAMYLTRANSPEPTIDASE) ET SEQUENCES CODANT POUR CET ANTIGENE
WO2000049044A1 (fr) * 1999-02-19 2000-08-24 Astrazeneca Ab Expression de polypeptides helicobacter dans pichia pastoris
WO2001080904A2 (fr) * 2000-04-22 2001-11-01 Fischer, Peter Moyen permettant de diagnostiquer et de traiter des carcinomes
WO2001087944A1 (fr) * 2000-04-29 2001-11-22 Shanghai Biowindow Gene Development Inc. Proteine 10, polypeptide humain caracterise en ce qu'il contient la sequence de la gamma glutamyl transpeptidase, et polynucleotide codant ce polypeptide
WO2003102025A1 (fr) * 2002-05-30 2003-12-11 Japan Science And Technology Agency Proteine induisant la mort cellulaire d'helicobacter pylori
US6762051B2 (en) * 1998-06-30 2004-07-13 Institut Pasteur Methods of inhibiting helicobacter pylori
WO2008046650A1 (fr) 2006-10-19 2008-04-24 Markus Gerhard Nouveau procédé pour traiter les infections par h. pylori
US7968324B2 (en) * 2004-08-13 2011-06-28 Barry J Marshall Helicobacter system and uses thereof
US8029777B2 (en) 2004-08-13 2011-10-04 Marshall Barry J Helicobacter system and uses thereof
AU2013273605B2 (en) * 2006-10-19 2016-04-14 Hepyvaxx Gmbh Novel method for treating H.pylori infections

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US6762051B2 (en) * 1998-06-30 2004-07-13 Institut Pasteur Methods of inhibiting helicobacter pylori
WO2000001825A1 (fr) * 1998-07-01 2000-01-13 Institut Pasteur ANTIGENE D'HELICOBACTER (η-GLUTAMYLTRANSPEPTIDASE) ET SEQUENCES CODANT POUR CET ANTIGENE
WO2000049044A1 (fr) * 1999-02-19 2000-08-24 Astrazeneca Ab Expression de polypeptides helicobacter dans pichia pastoris
WO2001080904A2 (fr) * 2000-04-22 2001-11-01 Fischer, Peter Moyen permettant de diagnostiquer et de traiter des carcinomes
WO2001080904A3 (fr) * 2000-04-22 2002-04-04 Fischer Peter Moyen permettant de diagnostiquer et de traiter des carcinomes
WO2001087944A1 (fr) * 2000-04-29 2001-11-22 Shanghai Biowindow Gene Development Inc. Proteine 10, polypeptide humain caracterise en ce qu'il contient la sequence de la gamma glutamyl transpeptidase, et polynucleotide codant ce polypeptide
WO2003102025A1 (fr) * 2002-05-30 2003-12-11 Japan Science And Technology Agency Proteine induisant la mort cellulaire d'helicobacter pylori
US7968324B2 (en) * 2004-08-13 2011-06-28 Barry J Marshall Helicobacter system and uses thereof
US8029777B2 (en) 2004-08-13 2011-10-04 Marshall Barry J Helicobacter system and uses thereof
US8298806B2 (en) 2004-08-13 2012-10-30 Ondek Pty. Ltd. Helicobacter system and uses thereof
US8298527B2 (en) 2004-08-13 2012-10-30 Ondek Pty. Ltd. Helicobacter system and uses thereof
US8420374B2 (en) 2004-08-13 2013-04-16 Ondek Pty. Ltd. Helicobacter system and uses thereof
JP2010506575A (ja) * 2006-10-19 2010-03-04 ゲルハルト，マルクス ピロリ菌感染を治療するための新規の方法
WO2008046650A1 (fr) 2006-10-19 2008-04-24 Markus Gerhard Nouveau procédé pour traiter les infections par h. pylori
US9090676B2 (en) * 2006-10-19 2015-07-28 Imevax Gmbh Method for treating Helicobacter pylori infections
EP2902038A1 (fr) 2006-10-19 2015-08-05 ImevaX GmbH Nouveau procédé de traitement des infections H. pylori
AU2013273605B2 (en) * 2006-10-19 2016-04-14 Hepyvaxx Gmbh Novel method for treating H.pylori infections
CN101594880B (zh) * 2006-10-19 2016-08-10 伊梅瓦克斯有限公司 治疗幽门螺旋菌感染的新方法
CN105879019A (zh) * 2006-10-19 2016-08-24 伊梅瓦克斯有限公司 治疗幽门螺旋菌感染的新方法
US9821048B2 (en) 2006-10-19 2017-11-21 Imevax Gmbh Method for treating Helicobacter pylori infections

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