EP1322763A2 - Listeria innocua, das genom und seine anwendungen - Google Patents

Listeria innocua, das genom und seine anwendungen

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Publication number
EP1322763A2
EP1322763A2 EP01982519A EP01982519A EP1322763A2 EP 1322763 A2 EP1322763 A2 EP 1322763A2 EP 01982519 A EP01982519 A EP 01982519A EP 01982519 A EP01982519 A EP 01982519A EP 1322763 A2 EP1322763 A2 EP 1322763A2
Authority
EP
European Patent Office
Prior art keywords
seq
monocytogenes
unknown
listeria monocytogenes
former
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP01982519A
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English (en)
French (fr)
Inventor
Fréderik KUNST
Philippe Glaser
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Centre National de la Recherche Scientifique CNRS
Institut Pasteur de Lille
Original Assignee
Centre National de la Recherche Scientifique CNRS
Institut Pasteur de Lille
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Filing date
Publication date
Application filed by Centre National de la Recherche Scientifique CNRS, Institut Pasteur de Lille filed Critical Centre National de la Recherche Scientifique CNRS
Publication of EP1322763A2 publication Critical patent/EP1322763A2/de
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the subject of the invention is a method making it possible to demonstrate the specific nucleotide sequences of the genome of a strain of bacteria of the genus Listeria, in particular of a strain of L. innocua or L. monocytogenes.
  • the subject of the present invention is also the genomic sequence and nucleotide sequences coding for Listeria innocua polypeptides, such as cell envelope polypeptides, secreted or specific, or involved in metabolism and in the replication process, as well as vectors including said sequences and cells or animals transformed by these vectors.
  • the invention also relates to the comparison of these nucleotide sequences with those coding for the polypeptides of Listeria monocytogenes, strain EGDe or L.
  • the invention also relates to methods for detecting these nucleic acids or polypeptides and to kits for diagnosing contamination by bacteria of the genus Listeria and kits for typing contaminating strains.
  • the invention also relates to a method of selecting compounds capable of modulating the bacterial infection caused by other Listeria and a method of biosynthesis or biodegradation of molecules of interest using said nucleotide sequences or said polypeptides.
  • the invention finally comprises pharmaceutical compositions, in particular vaccine compositions, for the prevention and / or treatment of bacterial infections, in particular by Listeria, in particular monocytogenes, and compositions containing antibodies directed against specific polypeptides of L.
  • Listeriosis is the most lethal food-borne infection (approximately 30% mortality).
  • Listeria monocytogenes has the unusual property of being able to cross three barriers: the intestinal barrier, the blood-brain barrier and the placental barrier. The clinical manifestations of listeriosis include meningitis, meningoencephalitis, abortion and septicemia.
  • This infection is opportunistic and mainly affects pregnant women, babies, the elderly and people who are immunosuppressed, especially people with AIDS. This disease also affects healthy individuals and is responsible for a significant number of epidemics due to contaminated food products.
  • Listeria monocytogenes is also of veterinary importance with a main risk for sheep (sheep) and cattle. Listeria monocytogenes is particularly resistant to stress or extreme conditions and it is important to look for its presence carefully not only for food safety problems but also for environmental safety issues.
  • listeriosis is very variable depending on the contaminating Listeria strain. In the extreme, some strains could be considered dangerous and others harmless (like Listeria innocua). Thus, while Listeria contaminations are very frequent, the number of cases described is low. In this perspective, the availability of a tool to identify the risk associated with contamination (depending on the genomic type of the strain and the number of bacteria per gram of food) would allow manufacturers to react based on this risk .
  • the subject of the present invention is therefore a method making it possible to demonstrate nucleotide sequences specific for the genome of a strain of bacteria of the genus Listeria, in particular specific for a strain of L. innocua or L. monocytogenes, such as the strain L monocytogenes EGDe or L. monocytogenes 4b.
  • Such a method according to the invention pe ⁇ net in particular the identification of specific sequences of:
  • L. innocua compared to L. monocytogenes, in particular compared to L. monocytogenes EGDe and / or L. monocytogenes 4b;
  • L. monocytogenes in particular L. monocytogenes EGDe or L. monocytogenes 4b, compared to L. innocua;
  • Said method according to the invention is preferably characterized in that it comprises at least the following steps: a) alignment of the nucleotide sequences of L. monocytogenes, in particular those of L. monocytogenes EGDe and / or L. monocytogenes 4b, and those of L. innocua according to the invention; and b) processing the data obtained by this alignment to isolate said specific sequences.
  • the method according to the invention is characterized in that the nucleotide sequences of L. monocytogenes, in particular those of L. monocytogenes EGDe and / or L. monocytogenes 4b are chosen from the genomic nucleotide sequences: - such as described in French patent application N ° 00 04629 filed on
  • the method according to the invention is characterized in that the nucleotide sequences specific for L. inocua or L. monocytogenes, in particular those of L. monocytogenes EGDe and / or L. monocytogenes 4b, hybridize in high stringency conditions with the sequences respectively nucleotides, or their complementary sequence, of L. inocua or L. monocytogenes, in particular those of L. monocytogenes EGDe and / or L. monocytogenes 4b.
  • the present invention relates to the nucleotide and polypeptide sequences of Listeria innocua and the comparison of the corresponding sequences with those of Listeria monocytogenes strain EGDe and / or 4b.
  • the invention relates in particular to:
  • nucleic sequences SEQ ID Nos. 3892 to 4025 specific for Listeria monocytogenes 4b compared to Listeria innocua and Listeria monocytogenes strain EGDe, their fragments of sufficient length to retain their aforesaid specificity, their complementary sequence, primers or specific probes, the peptides encoded by these nucleic acid sequences or antibodies directed against these peptides, as well as in particular their uses, for the identification of a strain of Listeria, or for the distinction between a pathogenic or non-pathogenic strain of Listeria in a biological sample, in particular using diagnostic methods or kit as below presented or known to those skilled in the art.
  • CLIP 1 1262 contained in the genomic bank prepared from the genome of this strain and deposited at the CNCM on October 2, 2000 under the number 1-2565 as well as all the non-coding regulatory genes and sequences contained in said genome.
  • the CLIP 1 1262 strain was isolated from a dairy product. This strain is kept at the National Reference Center of Listeria at the INSTITUT PASTEUR (WHO collaborating center).
  • the Listeria monocytogenes serotype 4b strain is also identified in the present application by Listeria monocytogenes 4b and interchangeably.
  • the invention also relates to new tools for typing Listeria strains. These tools could be of the DNA "chip" type or of another type.
  • the new features of these typing tools will be as follows:
  • the present invention therefore relates to a nucleotide sequence of Listeria innocua characterized in that it corresponds to a sequence chosen from SEQ ID No. 1 to SEQ ID No. 11, SEQ ID No. 2057 and SEQ ID No. 2058, in particular from SEQ ID No. 2057 and SEQ ID No. 2058.
  • the present invention also relates to a nucleotide sequence derived from Listeria innocua, characterized in that it is chosen from: a) a nucleotide sequence comprising at least 75%, 80%, 85%, 90%, 95% or 98% of identity with a sequence chosen from SEQ ID No. 1 to SEQ ID No. 11, SEQ ID No. 2057 and SEQ ID No. 2058, in particular from SEQ ID No. 2057 and SEQ ID No. 2058; b) a nucleotide sequence hybridizing under conditions of high stringency with a sequence chosen from SEQ ID No. 1 to SEQ ID No. 11, SEQ ID No. 2057 and SEQ ID No.
  • the present invention also relates to the nucleotide sequences characterized in that they come from SEQ ID No. 1 to SEQ ID No. 11, SEQ ID No. 2057 and SEQ ID No. 2058 and in what they code for a polypeptide, chosen from the sequences SEQ ID No. 12 to SEQ ID No. 689, SEQ ID No.
  • the present invention also relates more generally to the nucleotide sequences originating from SEQ ID No. 1 to SEQ ID No. 11, SEQ ID No. 2057 and SEQ ID
  • nucleotide sequences characterized in that they comprise a nucleotide sequence chosen from: a) a nucleotide sequence coding for a polypeptide, chosen from the sequences SEQ ID No. 12 to SEQ ID No. 689, SEQ ID No. 2053 to SEQ ID No. 2056 and SEQ ID No. 2059 to SEQ ID No. 2601, in particular from SEQ ID No. 2059 to SEQ ID No. 2601; b) a nucleotide sequence comprising at least 75%, 80%, 85%, 90%, 95% or 98% of identity with a nucleotide sequence coding for a polypeptide, chosen from the sequences SEQ ID No.
  • the present invention also relates to a nucleotide sequence of Listeria monocytogenes serotype 4b of sequence SEQ ID No. 1068 to SEQ ID No. 2041 and SEQ ID No. 2872 to SEQ ID No. 3891, in particular SEQ ID No. 2872 to SEQ ID No. 3891.
  • the present invention also relates to a nucleotide sequence of Listeria monocytogenes serotype 4b characterized in that it is chosen from: a) a nucleotide sequence comprising at least 75%, 80%, 85%, 90%, 95% or 98% identity with SEQ ID No. 1068 to SEQ ID No. 2041, SEQ ID No. 2872 to SEQ ID No 3891, in particular with SEQ ID No. 2872 to SEQ ID No. 3891; b) a nucleotide sequence hybridizing under conditions of high stringency with SEQ ID No. 1068 to SEQ ID No. 2041, SEQ ID No. 2872 to SEQ ID
  • SEQ ID No. 3891 in particular with SEQ ID No. 2872 to SEQ ID No. 3891; c) a nucleotide sequence complementary to SEQ ID No. 1068 to SEQ ID No. 2041, SEQ ID No. 2872 to SEQ ID No. 3891, in particular from SEQ ID No. 2872 to SEQ ID No. 3891 or complementary to a sequence nucleotide as defined in a) or b), or an RNA nucleotide sequence corresponding to one of the sequences a) or b); d) a nucleotide sequence of a fragment representative of SEQ ID No. 1068 to SEQ ID No. 2041, SEQ ID No. 2872 to SEQ ID No. 3891, in particular from SEQ ID No.
  • the present invention also relates to the nucleotide sequences characterized in that they come from SEQ ID No. 1068 to SEQ ID No. 2041, SEQ ID No. 2872 to SEQ ID No. 3891, in particular from SEQ ID No. 2872 to SEQ ID No.
  • polypeptide 3891 and in that they code for a polypeptide, chosen from the sequences SEQ ID No. 690 to SEQ ID No. 1067, SEQ ID No. 2049 to SEQ ID No. 2052 and SEQ ID No. 2602 to SEQ ID No. 2871, in particular from SEQ ID No. 2602 to SEQ ID No. 2871.
  • the present invention also relates more generally to the nucleotide sequences originating from SEQ ID No. 1068 to 2041, SEQ ID No. 2872 to SEQ ID No. 3891, in particular from SEQ ID No. 2872 to SEQ ID No. 3891, and coding for a polypeptide of E monocytogenes, as they can be isolated from S ⁇ Q ID No. 690 to 1067, S ⁇ Q ID No. 2049 to S ⁇ Q ID No. 2052 and S ⁇ Q ID No. 2602 to S ⁇ Q ID No. 2871, in particular from S ⁇ Q ID No. 2602 to S ⁇ Q ID No. 2871.
  • nucleotide sequences characterized in that they comprise a nucleotide sequence chosen from: a) a nucleotide sequence coding for a polypeptide, chosen from the sequences SEQ ID No. 690 to SEQ ID No. 1067, SEQ ID No. 2602 to SEQ ID No. 2871, in particular from SEQ ID No. 2602 to SEQ ID No. 2871; b) a nucleotide sequence comprising at least 75%, 80%, 85%, 90%, 95% or 98% of identity with a nucleotide sequence coding for a polypeptide, chosen from the sequences SEQ ID No. 690 to SEQ ID No 1067, SEQ ID No. 2602 to SEQ ID No.
  • nucleic acid nucleic or nucleic acid sequence, polynucleotide, oligonucleotide, polynucleotide sequence, nucleotide sequence, terms which will be used interchangeably in the present description, is intended to denote a precise sequence of nucleotides, modified or not, making it possible to define a fragment or region of a nucleic acid, which may or may not contain unnatural nucleotides, and which may correspond both to double-stranded DNA, single-stranded DNA and to transcripts of said DNAs.
  • the nucleic acid sequences according to the invention also include PNA (Peptid Nucleic Acid).
  • nucleotide sequences in their natural chromosomal environment that is to say in the natural state.
  • sequences which have been isolated and / or purified that is to say that they have been taken directly or indirectly, for example by copying, their environment having been at least partially modified.
  • This also means the nucleic acids obtained by chemical synthesis.
  • percentage of identity between two nucleic acid or amino acid sequences within the meaning of the present invention is meant a percentage of identical nucleotides or amino acid residues between the two sequences to compare, obtained after the best alignment, this percentage being purely statistical and the differences between the two sequences being distributed randomly and over their entire length.
  • the term “best alignment” or “optimal alignment” is intended to denote the alignment for which the percentage of identity determined as below is the highest. Sequence comparisons between two nucleic acid or amino acid sequences are traditionally carried out by comparing these sequences after having optimally aligned them, said comparison being carried out by segment or by "comparison window” to identify and compare the regions. sequence similarity locale.
  • the optimal alignment of the sequences for the comparison can be carried out, besides manually, by means of the algorithm of local homology of Smith and Waterman (1981, Ad. App. Math. 2: 482), by means of the algorithm of local homology by Neddleman and Wunsch (1970, J. Mol. Biol. 48: 443), using the similarity search method of Pearson and Lipman (1988, Proc. Natl. Acad. Sci. USA 85: 2444 ), using computer software using these algorithms (GAP, BESTFIT, BLAST P, BLAST N, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI).
  • the BLAST program is preferably used with the BLOSUM 62 matrix.
  • the PAM or PAM250 matrices can also be used.
  • the percentage of identity between two nucleic acid or amino acid sequences is determined by comparing these two optimally aligned sequences, the nucleic acid or amino acid sequence to be compared can include additions or deletions by compared to the reference sequence for optimal alignment between these two sequences.
  • the percentage identity is calculated by determining the number of identical positions for which the nucleotide or the amino acid residue is identical in the two sequences, by dividing this number of identical positions by the total number of positions compared and by multiplying the result obtained by 100 to obtain the percentage of identity between these two sequences.
  • nucleic acid sequences having a percentage identity of at least 75%, preferably 80%, 85% or 90%, more preferably 95% or even 98%, after optimal alignment with a reference sequence is meant the nucleic acid sequences having, with respect to the reference nucleic acid sequence, certain modifications such as in particular a deletion, a truncation, an elongation, a chimeric fusion and / or a substitution, in particular punctual, and whose sequence nucleic acid present at least 75%, preferably 80%, 85%, 90%, 95% or 98%, of identity after optimal alignment with the reference nucleic sequence.
  • They are preferably sequences whose complementary sequences are capable of hybridizing specifically with the reference sequences.
  • the specific hybridization conditions or high stringency will be such that they ensure at least 75%, preferably 80%, 85%, 90%, 95% or 98% identity after optimal alignment between one of the two sequences and its complementary sequence.
  • Hybridization under conditions of high stringency means that the conditions of temperature and ionic strength are chosen in such a way that they allow hybridization to be maintained between two complementary DNA fragments.
  • high stringency conditions of the hybridization step for the purpose of defining the polynucleotide fragments described above are advantageously as follows.
  • DNA-DNA or DNA-RNA hybridization is carried out in two stages: (1) prehybridization at 42 ° C for 3 hours in phosphate buffer (20 mM, pH 7.5) containing 5 x SSC (1 x SSC corresponds to a 0.15 M NaCl + 0.015 M sodium citrate solution), 50% formamide, 7% sodium dodecyl sulfate (SDS), 10 x Denhardt's, 5% dextran sulfate and 1% salmon sperm DNA; (2) actual hybridization for 20 hours at a temperature depending on the size of the probe (ie: 42 ° C, for a probe of size> 100 nucleotides) followed by 2 washes of 20 minutes at 20 ° C in 2 x SSC + 2% SDS, 1 wash for 20 minutes at 20 ° C in 0.1 x SSC + 0.1% SDS.
  • the last washing is carried out in 0.1 x SSC + 0.1% SDS for 30 minutes at 60 ° C. for a probe of size> 100 nucleotides.
  • the high stringency hybridization conditions described above for a polynucleotide of defined size can be adapted by the skilled person for oligonucleotides of larger or smaller size, according to the teaching of Sambrook et al. (1989, Molecular cloning: a laboratory manual, 2 nd Ed. Cold Spring Harbor).
  • fragment representative of sequences according to the invention is intended to denote any nucleotide fragment having at least 15 nucleotides, preferably at least 30, 75, 150, 300 and 450 consecutive nucleotides of the sequence from which it is derived.
  • nucleic sequence coding for a biologically active fragment of a polypeptide, as defined below.
  • fragment is also meant the intergenic sequences, and in particular the nucleotide sequences carrying the regulatory signals (promoters, terminators, or even enhancers, etc.).
  • ORFs sequences ORFs for "Open Reading Frame"
  • ORFs sequences ORFs for "Open Reading Frame"
  • initiation codon and a stop codon or between two stop codons
  • polypeptides preferably at least 100 amino acids, such as for example, without limitation, the ORFs sequences which will be described later.
  • the numbering of the nucleotide sequences ORFs which will be used subsequently in the present description corresponds to the numbering of the amino acid sequences of the proteins encoded by said ORFs.
  • the representative fragments according to the invention can be obtained for example by specific amplification such as PCR or after digestion with appropriate restriction enzymes of nucleotide sequences according to the invention, this method being described in particular in the work by Sambrook et al. .. Said representative fragments can also be obtained by chemical synthesis when their size is not too large, according to methods well known to those skilled in the art.
  • sequences containing sequences of the invention we also mean the sequences which are naturally framed by sequences which have at least 75%, 80%, 85%, 90%, 95% or 98% d identity with the sequences according to the invention.
  • modified nucleotide sequence any nucleotide sequence obtained by mutagenesis according to techniques well known to those skilled in the art, and comprising modifications with respect to the normal sequences, for example mutations in the regulatory and / or promoter sequences of the expression of the polypeptide, in particular leading to a modification of the level of expression or of the activity of said polypeptide.
  • modified nucleotide sequence is also meant any nucleotide sequence coding for a modified polypeptide as defined below.
  • the representative fragments according to the invention can also be probes or primers, which can be used in methods of detection, identification, assay or amplification of nucleic sequences.
  • a probe or primer is defined, within the meaning of the invention, as being a fragment of single-stranded nucleic acids or a denatured double-stranded fragment comprising for example from 12 bases to a few kb, in particular from 15 to a few hundred bases, preferably from 15 to 50 or 100 bases, and having a specificity of hybridization under determined conditions to form a hybridization complex with a target nucleic acid.
  • the probes and primers according to the invention can be labeled directly or indirectly with a radioactive or non-radioactive compound by methods well known to those skilled in the art, in order to obtain a detectable and / or quantifiable signal (patent FR 78 10975 and bDNA of Chiron EP 225 807 and EP 510 085).
  • the unlabeled polynucleotide sequences according to the invention can be used directly as a probe or primer.
  • sequences are generally marked to obtain sequences which can be used for numerous applications.
  • the labeling of the primers or probes according to the invention is carried out with radioactive elements or with non-radioactive molecules.
  • the non-radioactive entities are selected from ligands such as biotin, avidin, streptavidin, dioxygenin, haptens, dyes, luminescent agents such as radioluminescent, chemoluminescent, bioluminescent, fluorescent, phosphorescent agents.
  • the polynucleotides according to the invention can thus be used as a primer and / or probe in methods using in particular the PCR technique (polymerase chain reaction) (Rolfs et al., 1991, Berlin: Springer-Verlag).
  • This technique requires the choice of pairs of oligonucleotide primers framing the fragment which must be amplified.
  • the amplified fragments can be identified, for example after agarose or polyacrylamide gel electrophoresis, or after a chromatographic technique such as gel filtration or ion exchange chromatography, and then sequenced.
  • the specificity of the amplification can be controlled by using the nucleotide sequences of polynucleotides of the invention as template, plasmids containing these sequences or even the amplification products derived therefrom.
  • the amplified nucleotide fragments can be used as reagents in hybridization reactions in order to demonstrate the presence, in a biological sample, of a target nucleic acid of sequence complementary to that of said amplified nucleotide fragments.
  • the invention also relates to the nucleic acids capable of being obtained by amplification using primers according to the invention.
  • Other techniques for amplifying the target nucleic acid can advantageously be used as an alternative to PCR (PCR-like) using pairs of primers of nucleotide sequences according to the invention.
  • PCR-like is meant to denote all the methods implementing direct or indirect reproductions of the nucleic acid sequences, or in which the labeling systems have been amplified, these techniques are of course known. In general, it is the amplification of DNA by a polymerase; when the original sample is an RNA, a reverse transcription should be carried out beforehand.
  • the target polynucleotide to be detected is an mRNA
  • an enzyme of reverse transcriptase type in order to obtain a cDNA from the mRNA contained in the biological sample.
  • the cDNA obtained will then serve as a target for the primers or probes used in the amplification or detection method according to the invention.
  • the probe hybridization technique can be performed in various ways (Matthews et al., 1988, Anal. Biochem., 169, 1-25).
  • the most general method consists in immobilizing the nucleic acid extracted from cells of different tissues or cells in culture on a support (such as nitrocellulose, nylon, polystyrene) and incubating, under well defined conditions, the target nucleic acid immobilized with the probe. After hybridization, the excess probe is eliminated and the hybrid molecules formed are detected by the appropriate method (measurement of radioactivity, fluorescence or enzymatic activity linked to the probe).
  • a support such as nitrocellulose, nylon, polystyrene
  • the latter can be used as capture probes.
  • a probe called a “capture probe”
  • a probe is immobilized on a support and is used to capture by specific hybridization the target nucleic acid obtained from the biological sample to be tested and the target nucleic acid is then detected.
  • a second probe called a “detection probe”, marked by an easily detectable element.
  • the antisense oligonucleotides that is to say those whose structure ensures, by hybridization with the target sequence, an inhibition of the expression of the corresponding product. Mention should also be made of sense oligonucleotides which, by interaction with proteins involved in the regulation of the expression of the corresponding product, will induce either an inhibition or an activation of this expression.
  • the probes or primers according to the invention are immobilized on a support, covalently or non-covalently.
  • the support can be a DNA chip or a high or medium density filter, also objects of the present invention (patents WO 97/29212, WO 98/27317, WO 97/10365 and WO 92/10588).
  • DNA chip or high density filter is intended to denote a support on which DNA sequences are fixed, each of which can be identified by its geographic location. These chips or filters differ mainly in their size, the material of the support, and possibly the number of DNA sequences attached to them.
  • the probes or primers according to the first invention can be fixed on solid supports, in particular DNA chips, by various manufacturing methods.
  • a synthesis can be carried out in situ by photochemical addressing or by ink jet.
  • Other techniques consist in carrying out an ex situ synthesis and in fixing the probes on the support of the DNA chip by mechanical, electronic or inkjet addressing. These different methods are well known to those skilled in the art.
  • a nucleotide sequence (probe or primer) according to the invention therefore allows the detection and / or amplification of specific nucleic sequences.
  • the detection of these said sequences is facilitated when the probe is fixed to a DNA chip, or to a high density filter.
  • DNA chips or high density filters makes it possible to determine the expression of genes in an organism having a genomic sequence close to L. monocytogenes or innocua and the typing of the strain in question.
  • the genomic sequence of L. innocua and the partial sequences of L. monocytogenes 4b serve as a basis for the construction of these DNA chips or filter.
  • the preparation of these filters or chips consists in synthesizing oligonucleotides, corresponding to the 5 ′ and 3 ′ ends of the genes or to more internal fragments to amplify fragments of a suitable size, for example between approximately 300 and 800 bases.
  • oligonucleotides are chosen using the genomic sequence and its annotations disclosed by the present invention.
  • the pairing temperature of these oligonucleotides at the corresponding places on the DNA should be approximately the same for each oligonucleotide. This makes it possible to prepare DNA fragments corresponding to each gene by the use of appropriate PCR conditions in a highly automated environment.
  • the amplified fragments are then immobilized on filters or supports in glass, silicon or synthetic polymers and these media are used for hybridization.
  • filters and / or chips and of the corresponding annotated genomic sequence makes it possible to study the expression of large sets, or even of all of the genes in the microorganisms associated with Listeria innocua and L. monocytogenes 4b, by preparing the complementary DNAs, and by hybridizing them to the DNA or to the oligonucleotides immobilized on the filters or the chips.
  • the filters and / or the chips make it possible to study the variability of the strains or of the species, by preparing the DNA of these organisms and by hybridizing them to the DNA or to the oligonucleotides immobilized on the filters or the chips.
  • the nucleotide sequences according to the invention can be used in DNA chips to carry out the analysis of mutations. This analysis is based on the constitution of chips capable of analyzing each base of a nucleotide sequence according to the invention. In particular, it will be possible to implement micro-sequencing techniques on a DNA chip.
  • the mutations are detected by extension of immobilized primers hybridizing to the matrix of the sequences analyzed, just in position adjacent to that of the mutated nucleotide sought.
  • a single-stranded matrix, RNA or DNA, of the sequences to be analyzed will advantageously be prepared according to conventional methods, from products amplified according to PCR type techniques.
  • the single-stranded DNA or RNA matrices thus obtained are then deposited on the DNA chip, under conditions allowing their specific hybridization to the immobilized primers.
  • a thermostable polymerase for example Tth or Taq DNA polymerase, specifically extends the 3 'end of the immobilized primer with a labeled nucleotide analog complementary to the nucleotide at the variable site position; for example, thermal cycling is carried out in the presence of fluorescent dideoxyribonucleotides.
  • the experimental conditions will be adapted in particular to the chips used, to the immobilized primers, to the polymerases used, and to the chosen labeling system.
  • microsequencing compared to techniques based on probe hybridization, is that it makes it possible to identify all the variable nucleotides with optimal discrimination under homogeneous reaction conditions; used on DNA chips, it allows optimal resolution and specificity for routine and industrial detection of mutations in multiplex.
  • the use of high density filters and / or chips thus makes it possible to obtain new knowledge on the regulation of genes in organisms of industrial importance, and in particular the listeria propagated under various conditions. It also allows rapid identification of the differences between the genomes of the strains used in multiple industrial applications.
  • a DNA chip or filter can be an extremely useful tool for the determination, detection and / or identification of a microorganism.
  • the DNA chips according to the invention are also preferred, which also contain at least one nucleotide sequence of a microorganism other than Listeria monocytogenes 4b or Listeria innocua, immobilized on the support of said chip.
  • the microorganism chosen is from bacteria of the genus Listeria (hereinafter designated as bacteria associated with L. monocytogenes), or variants of Listeria monocytogenes EGD-e.
  • a DNA chip or a filter according to the invention is a very useful element in certain kits or necessary for the detection and / or identification of microorganisms, in particular bacteria belonging to the species Listeria monocytogenes or the associated microorganisms , also objects of the invention.
  • DNA chips or filters according to the invention containing probes or primers specific for Listeria innocua or monocytogenes, are very advantageous elements of kits or necessary for the detection and / or quantification of the expression of genes Listeria innocua or monocytogenes (or associated microorganisms).
  • the control of gene expression is a critical point for optimizing the growth and yield of a strain, either by allowing the expression of one or more new genes, or by modifying the expression of genes already present in the cell.
  • the present invention provides all the naturally active sequences in L. innocua allowing the expression of genes. It thus allows the determination of all the sequences expressed in L. innocua. It also provides a tool for identifying genes whose expression follows a given pattern. To achieve this, the DNA of all or part of the genes of L. innocua and monocytogenes can be amplified using primers according to the invention, then fixed to a support such as for example glass or nylon or a DNA chip, in order to build a tool to monitor the expression profile of these genes.
  • This tool consisting of this support containing the coding sequences, serves as a hybridization matrix for a mixture of labeled molecules reflecting the messenger RNAs expressed in the cell (in particular the labeled probes according to the invention).
  • each control sequence present upstream of the segments serving as probes and to monitor their activity using an appropriate means such as a reporter gene (luciferase, ⁇ -galactosidase, GFP).
  • a reporter gene luciferase, ⁇ -galactosidase, GFP
  • the invention also relates to the polypeptides encoded by a nucleotide sequence according to the invention, preferably, by a fragment representative of the preceding sequences and corresponding to an ORF sequence.
  • the Listeria innocua polypeptides encoded by the sequences SEQ ID No. 12 to SEQ ID No. 689, SEQ ID Nos. 2042 and 2043, SEQ ID Nos. 2047 and 2048, SEQ ID Nos. 2053 to 2056 and SEQ ID Nos. 2059 to 2601 in particular by SEQ ID Nos. 2059 to 2601, or those of Listeria monocytogenes EGDe, characterized in that they are chosen from the polypeptides coded by the sequences SEQ ID No.
  • SEQ ID No. 1067 SEQ ID No. 2049 to SEQ ID No. 2052 and SEQ ID Nos. 2602 to 2871, notably among SEQ ID Nos. 2602 to 2871, or those of Listeria monocytogenes 4b, characterized in that they are chosen from the polypeptides coded by the sequences SEQ ID No. 3892 to SEQ ID No. 4025, are subject of the invention.
  • the invention also includes the polypeptides characterized in that they comprise a polypeptide chosen from: a) a polypeptide according to the invention; b) a polypeptide having at least 80%, preferably 85%, 90%, 95% and 98% identity with a polypeptide according to the invention; c) a fragment of at least 5 amino acids of a polypeptide according to the invention, or as defined in b); d) a biologically active fragment of a polypeptide according to the invention, or as defined in b) or c); and e) a polypeptide according to the invention, or as defined in b), c) or d) modified.
  • the nucleotide sequences coding for the polypeptides described above are also subject of the invention.
  • polypeptides includes any amino acid sequence used to generate an antibody response.
  • polypeptide includes any amino acid sequence used to generate an antibody response. It should be understood that the invention does not relate to polypeptides in natural form, that is to say that they are not taken in their natural environment. On the other hand, it relates to those which could have been isolated or obtained by purification from natural sources, or else obtained by genetic recombination, or by chemical synthesis, and which they can then comprise non-natural amino acids as will be described more far.
  • polypeptide having a certain percentage of identity with another which will also be designated by homologous polypeptide, is intended to denote the polypeptides having, with respect to the natural polypeptides, certain modifications, in particular a deletion, addition or substitution of at least an amino acid, truncation, elongation, chimeric solution and / or mutation, or polypeptides with post-translational modifications.
  • homologous polypeptides those whose amino acid sequence have at least 80%, preferably 85%, 90%, 95% and 98% of homology with the amino acid sequences of the polypeptides according to the invention are preferred. .
  • equivalent amino acids is intended here to denote any amino acid capable of being substituted for one of the amino acids of the basic structure without, however, essentially modifying the biological activities of the corresponding peptides as defined by after.
  • Leucine can thus be replaced by valine or isoleucine, aspartic acid by glutamine acid, glutamine by asparagine, arginine by lysine, etc., the reverse substitutions being naturally possible in the same conditions.
  • homologous polypeptides also correspond to the polypeptides encoded by the homologous or identical nucleotide sequences, as defined above and thus include, in the present definition, polypeptides which are mutated or correspond to inter or intra species variations, which may exist in Listeria, and which correspond in particular to truncations, substitutions, deletions and / or additions, of at least one amino acid residue.
  • the percentage of identity between two polypeptides is calculated in the same way as between two nucleic acid sequences.
  • the percentage of identity between two polypeptides is calculated after optimal alignment of these two sequences, over a window of maximum homology.
  • the same algorithms can be used as for the nucleic acid sequences.
  • biologically active fragment of a polypeptide according to the invention is intended to denote in particular a fragment of polypeptide, as defined below, having at least one of the biological characteristics of the polypeptides according to the invention, in particular in that it is able to exercise in general even partial activity, such as for example:
  • polypeptide fragment according to the invention is intended to denote a polypeptide comprising at least 5 amino acids, preferably 10, 15, 25, 50, 100 and 150 amino acids.
  • Polypeptide fragments can also be prepared by chemical synthesis, from hosts transformed by an expression vector according to the invention which contain a nucleic acid allowing the expression of said fragment, and placed under the control of regulatory elements and / or appropriate expression.
  • modified polypeptide of a polypeptide according to the invention is intended to denote a polypeptide obtained by genetic recombination or by chemical synthesis as described below, which exhibits at least one modification with respect to the normal sequence. These modifications can be carried in particular on amino acids necessary for the specificity or the efficiency of the activity, or at the origin of the structural conformation, of the charge, or of the hydrophobicity of the polypeptide according to the invention. It is thus possible to create polypeptides of equivalent, increased or decreased activity, or of equivalent specificity, narrower or wider.
  • modified polypeptides mention should be made of the polypeptides in which up to five amino acids can be modified, truncated at the N or C-terminus, or else deleted, or added.
  • Chemical synthesis also has the advantage of being able to use unnatural amino acids or non-peptide bonds. Thus, it may be advantageous to use unnatural amino acids, for example in D form, or analogs of amino acids, in particular suffering forms.
  • the present invention provides the nucleotide sequence of the Listeria innocua genome and the partial sequence of Listeria monocytogenes serotype 4b, as well as certain polypeptide sequences.
  • the invention relates to a nucleotide sequence according to the invention, characterized in that it codes for a polypeptide of Listeria innocua or monocytogenes 4b or one of its fragments involved in the biosynthesis of amino acids.
  • the invention relates to a nucleotide sequence according to the invention, characterized in that it codes for a polypeptide of Listeria innocua or monocytogenes 4b or one of its fragments involved in the biosynthesis of cofactors, prosthetic groups and transporters .
  • the invention relates to a nucleotide sequence according to the invention, characterized in that it codes for a cell envelope polypeptide or present on the surface of Listeria innocua or monocytogenes 4b or for one of its fragments.
  • the invention relates to a nucleotide sequence according to the invention, characterized in that it codes for a polypeptide of Listeria innocua or monocytogenes 4b or one of its fragments involved in the cellular machinery.
  • the invention relates to a nucleotide sequence according to the invention, characterized in that it codes for a polypeptide of Listeria innocua or monocytogenes 4b or one of its fragments involved in the central intermediate metabolism.
  • the invention relates to a nucleotide sequence according to the invention, characterized in that it codes for a polypeptide of Listeria innocua or monocytogenes 4b or one of its fragments involved in energy metabolism.
  • the invention relates to a nucleotide sequence according to the invention, characterized in that it codes for a polypeptide of Listeria innocua or monocytogenes 4b or one of its fragments involved in the metabolism of fatty acids and phospholipids.
  • the invention relates to a nucleotide sequence according to the invention, characterized in that it codes for a polypeptide of Listeria innocua or monocytogenes 4b or one of its fragments involved in the metabolism of nucleotides, purines, pyrimidines or nucleosides.
  • the invention relates to a nucleotide sequence according to the invention, characterized in that it codes for a polypeptide of Listeria innocua or monocytogenes 4b or one of its fragments involved in the regulatory functions.
  • the invention relates to a nucleotide sequence according to the invention, characterized in that it codes for a Listeria innocua or monocytogenes 4b polypeptide or one of its fragments involved in the replication process.
  • the invention relates to a nucleotide sequence according to the invention, characterized in that it codes for a Listeria innocua or monocytogenes 4b polypeptide or one of its fragments involved in the transcription process.
  • the invention relates to a nucleotide sequence according to the invention, characterized in that it codes for a Listeria innocua or monocytogenes 4b polypeptide or one of its fragments involved in the translation process.
  • the invention relates to a nucleotide sequence according to the invention, characterized in that it codes for a polypeptide of Listeria innocua or monocytogenes 4b or one of its fragments involved in the protein transport and binding process .
  • the invention relates to a nucleotide sequence according to the invention, characterized in that it codes for a Listeria innocua or monocytogenes 4b polypeptide or one of its fragments involved in the adaptation to atypical conditions.
  • the invention relates to a nucleotide sequence according to the invention, characterized in that it codes for a polypeptide of Listeria innocua or monocytogenes 4b or one of its fragments in the sensitivity to drugs and the like.
  • the invention relates to a nucleotide sequence according to the invention, characterized in that it codes for a polypeptide of Listeria innocua or monocytogenes 4b or one of its fragments involved in the functions relating to transposons.
  • the invention relates to a nucleotide sequence according to the invention, characterized in that it codes for a specific polypeptide of Listeria innocua or monocytogenes 4b or one of its fragments.
  • the invention relates to a polypeptide according to the invention, characterized in that it is a polypeptide of Listeria innocua or monocytogenes 4b or one of its fragments involved in biosynthesis amino acids.
  • the invention relates to a polypeptide according to the invention, characterized in that it is a Listeria polypeptide innocua or monocytogenes 4b or one of its fragments involved in the biosynthesis of cofactors, prosthetic groups and transporters.
  • the invention relates to a polypeptide according to the invention, characterized in that it is a cell envelope or surface polypeptide of Listeria innocua or monocytogenes 4b or a of its fragments.
  • the invention relates to a polypeptide according to the invention, characterized in that it is a polypeptide of Listeria innocua or monocytogenes 4b or one of its fragments involved in the machinery cellular.
  • the invention relates to a polypeptide according to the invention, characterized in that it is a polypeptide of Listeria innocua or monocytogenes 4b or one of its fragments involved in metabolism central intermediary. In another aspect, preferably, the invention relates to a polypeptide according to the invention, characterized in that it is a polypeptide of Listeria innocua or monocytogenes 4b or one of its fragments involved in metabolism energy.
  • the invention relates to a polypeptide according to the invention, characterized in that it is a polypeptide of Listeria innocua or monocytogenes 4b or one of its fragments involved in metabolism fatty acids and phospholipids.
  • the invention relates to a polypeptide according to the invention, characterized in that it is a polypeptide of Listeria innocua or monocytogenes 4b or one of its fragments involved in metabolism nucleotides, purines, pyrimidines or nucleosides.
  • the invention relates to a polypeptide according to the invention, characterized in that it is a polypeptide of Listeria innocua or monocytogenes 4b or one of its fragments involved in the functions of regulation.
  • the invention relates to a polypeptide according to the invention, characterized in that it is a polypeptide of Listeria innocua or monocytogenes 4b or one of its fragments involved in the process replication. In another aspect, preferably, the invention relates to a polypeptide according to the invention, characterized in that it is a polypeptide of Listeria innocua or monocytogenes 4b or one of its fragments involved in the process of transcription. In another aspect, preferably, the invention relates to a polypeptide according to the invention, characterized in that it is a polypeptide of Listeria innocua or monocytogenes 4b or one of its fragments involved in the process translation.
  • the invention relates to a polypeptide according to the invention, characterized in that it is a polypeptide of Listeria innocua or monocytogenes 4b or one of its fragments involved in the process protein transport and binding.
  • the invention relates to a polypeptide according to the invention, characterized in that it is a polypeptide of Listeria innocua or monocytogenes 4b or one of its fragments involved in the adaptation to atypical conditions.
  • the invention relates to a polypeptide according to the invention, characterized in that it is a polypeptide of Listeria innocua or monocytogenes 4b or one of its fragments in sensitivity to drugs and the like.
  • the invention relates to a polypeptide according to the invention, characterized in that it is a polypeptide of Listeria innocua or monocytogenes 4b or one of its fragments involved in the functions relating to transposons.
  • the invention relates to a polypeptide according to the invention, characterized in that it is a specific polypeptide of Listeria innocua or monocytogenes 4b or one of its fragments.
  • the present invention also relates to the nucleotide and / or polypeptide sequences according to the invention, characterized in that said sequences are recorded on a recording medium whose shape and nature facilitate the reading, analysis and / or exploitation of said sequence (s).
  • These supports can also contain other information extracted from the present invention, in particular analogies with already known sequences, and / or information concerning the nucleotide sequences and / or polypeptides of other microorganisms in order to facilitate the comparative analysis and the exploitation of the results obtained.
  • these recording media particular preference is given to media readable by a computer, such as magnetic, optical, electrical or hybrid media, in particular computer floppy disks, CD-ROMs, computer servers. Such recording media are also subject of the invention.
  • the recording media according to the invention are very useful for the choice of primers or nucleotide probes for the determination of genes in Listeria innocua or monocytogenes 4b or strains close to this organism.
  • these supports for the study of the genetic polymorphism of strains close to Listeria irmocua or monocytogenes 4b, in particular by the determination of the regions of collinearity, is very useful insofar as these supports provide not only the sequence nucleotide of the genome of Listeria innocua or monocytogenes 4b, but also the genomic organization in said sequence.
  • the uses of recording media according to the invention are also objects of the invention.
  • sequence comparison software such as the Blast software, or the software of the GCG kit, described above.
  • the invention also relates to the cloning and / or expression vectors, which contain a nucleotide sequence according to the invention.
  • the vectors according to the invention preferably comprise elements which allow the expression and / or the secretion of the nucleotide sequences in a determined host cell.
  • the vector must then include a promoter, translation initiation and termination signals, as well as suitable regions for transcription regulation. It must be able to be maintained stably in the host cell and may possibly have specific signals which specify the secretion of the translated protein. These various elements are chosen and optimized by a person skilled in the art according to the cell host used. To this end, the nucleotide sequences according to the invention can be inserted into vectors with autonomous replication within the chosen host, or be integrative vectors of the chosen host.
  • Such vectors are prepared by methods commonly used by those skilled in the art, and the resulting clones can be introduced into an appropriate host by standard methods, such as lipofection, electroporation, heat shock, or chemical methods .
  • the vectors according to the invention are for example vectors of plasmid or viral origin. They are useful for transforming host cells in order to clone or express the nucleotide sequences according to the invention.
  • the invention also includes host cells transformed with a vector according to the invention.
  • the cell host can be chosen from prokaryotic or eukaryotic systems, for example bacterial cells but also yeast cells or animal cells, in particular mammalian cells. You can also use insect cells or plant cells.
  • the preferred host cells according to the invention are in particular prokaryotic cells, preferably bacteria belonging to the genus Listeria, to the species Listeria innocua or monocytogenes 4b, or the microorganisms associated with the species Listeria innocua or monocytogenes 4b.
  • the invention also relates to plants and animals, except humans, which comprise a transformed cell according to the invention.
  • the cells transformed according to the invention can be used in processes for the preparation of recombinant polypeptides according to the invention.
  • the methods for preparing a polypeptide according to the invention in recombinant form characterized in that they use a vector and / or a cell transformed with a vector according to the invention are themselves included in the present invention.
  • a cell transformed with a vector according to the invention is cultivated under conditions which allow the expression of said polypeptide and said recombinant peptide is recovered.
  • the cell host can be chosen from prokaryotic or eukaryotic systems.
  • a vector according to the invention carrying such a sequence can therefore be advantageously used for the production of recombinant proteins, intended to be secreted. Indeed, the purification of these recombinant proteins of interest will be facilitated by the fact that they are present in the supernatant of the cell culture rather than inside the host cells.
  • the polypeptides according to the invention can also be prepared by chemical synthesis. Such a preparation process is also an object of the invention.
  • a person skilled in the art knows the chemical synthesis processes, for example the techniques implementing solid phases (see in particular Steward et al., 1984, Solid phase peptides synthesis, Pierce Chem. Company, Rockford, 11 1, 2nd ed. , (1984)) or techniques using partial solid phases, by condensation of fragments or by synthesis in conventional solution.
  • the polypeptides obtained by chemical synthesis and which may contain corresponding unnatural amino acids are also included in the invention.
  • the invention further relates to hybrid polypeptides having at least one polypeptide or a fragment thereof according to the invention, and a sequence of a polypeptide capable of inducing an immune response in humans or animals.
  • the antigenic determinant is such that it is capable of inducing a humoral and / or cellular response.
  • Such a determinant may comprise a polypeptide or one of its fragments according to the invention in glycosylated form used with a view to obtaining immunogenic compositions capable of inducing the synthesis of antibodies directed against multiple epitopes.
  • Said polypeptides or their glycosylated fragments also form part of the invention.
  • hybrid molecules can consist in part of a molecule carrying polypeptides or their fragments according to the invention, associated with a possibly immunogenic part, in particular an epitope of diphtheria toxin, tetanus toxin, a surface antigen of the virus.
  • hepatitis B (patent FR 79 2181 1), the VP1 antigen of the poliomyelitis virus or any other toxin or viral or bacterial antigen.
  • the methods of synthesis of the hybrid molecules include the methods used in genetic engineering to construct hybrid nucleotide sequences coding for the polypeptide sequences sought.
  • hybrid nucleotide sequences coding for a hybrid polypeptide as well as the hybrid polypeptides according to the invention characterized in that they are Recombinant polypeptides obtained by the expression of said hybrid nucleotide sequences, also form part of the invention.
  • the invention also includes the vectors characterized in that they contain one of said hybrid nucleotide sequences.
  • Host cells transformed by said vectors, transgenic animals comprising one of said transformed cells as well as methods for preparing recombinant polypeptides using said vectors, said transformed cells and / or said transgenic animals also form part of the invention.
  • the coupling between a polypeptide according to the invention and an immunogenic polypeptide can be carried out chemically, or biologically.
  • one or more binding element (s), in particular amino acids to facilitate the coupling reactions between the polypeptide according to the invention, and the immunostimulatory polypeptide
  • the covalent coupling of the immunostimulatory antigen can be produced at the N or C-terminal end of the polypeptide according to the invention.
  • the bifunctional reagents allowing this coupling are determined as a function of the end chosen to achieve this coupling, and the coupling techniques are well known to those skilled in the art.
  • the conjugates resulting from a coupling of peptides can also be prepared by genetic recombination.
  • the hybrid (conjugated) peptide can in fact be produced by recombinant DNA techniques, by insertion or addition to the DNA sequence coding for the polypeptide according to the invention, of a sequence coding for the peptide (s) ) antigen (s), immunogen (s) or hapten (s). These techniques for preparing hybrid peptides by genetic recombination are well known to those skilled in the art (see for example Makrides, 1996, Microbiological Reviews 60, 512-538).
  • said immune polypeptide is chosen from the group of peptides containing toxoids, in particular the diphtheria toxoid or the tetanus toxoid, proteins derived from Streptococcus (such as the protein for binding to human seralbumin), OMPA membrane proteins and complexes. proteins from external membranes, vesicles from external membranes or thermal shock proteins.
  • hybrid polypeptides according to the invention are very useful for obtaining monoclonal or polyclonal antibodies capable of specifically recognizing the polypeptides according to the invention. Indeed, a hybrid polypeptide according to the invention allows the potentiation of the immune response, against the polypeptide according to the invention coupled to the immunogenic molecule. Such monoclonal or polyclonal antibodies, their fragments, or chimeric antibodies, recognizing the polypeptides according to the invention, are also objects of the invention.
  • the specific monoclonal antibodies can be obtained according to the conventional method of hybridoma culture described by Kohler and Milstein (1975, Nature 256, 495).
  • the antibodies according to the invention are for example chimeric antibodies, humanized antibodies, Fab fragments, or F (ab '). They can also be in the form of immunoconjugates or labeled antibodies in order to obtain a detectable and / or quantifiable signal.
  • the antibodies according to the invention can be used in a method for the detection and / or identification of bacteria belonging to the species Listeria innocua or monocytogenes 4b or to an associated microorganism in a biological sample, characterized in that that it comprises the following stages: a) bringing the biological sample into contact with an antibody according to the invention; b) highlighting of the antigen-antibody complex possibly formed.
  • the antibodies according to the present invention can also be used in order to detect an expression of a gene of Listeria innocua or monocytogenes 4b or of associated microorganisms.
  • the presence of the expression product of a gene recognized by an antibody specific for said expression product can be detected by the presence of an antigen-antibody complex formed after contacting the strain of Listeria innocua or monocytogenes 4b or of the microorganism associated with an antibody according to the invention.
  • the bacterial strain used may have been "prepared", that is to say centrifuged, lysed, placed in a reagent suitable for constituting the medium suitable for the immunological reaction.
  • a method of detecting expression in the gene, corresponding to a Western blot which can be carried out after an electrophoresis on polyacrylamide gel of a lysate of the bacterial strain, is preferred, in the presence or in the absence of reducing conditions (SDS-PAGE).
  • the present invention also comprises the kits or kits necessary for the implementation of a method as described (for detecting the expression of a gene for
  • Listeria innocua or monocytogenes 4b or an associated microorganism or for the detection and / or identification of bacteria belonging to the species Listeria innocua or monocytogenes 4b or an associated microorganism), comprising the following elements: a ) a polyclonal or monoclonal antibody according to the invention; b) optionally, the reagents for constituting the medium suitable for the immunological reaction; c) optionally, the reagents allowing the detection of the antigen-antibody complexes produced by the immunological reaction.
  • polypeptides and antibodies according to the invention can advantageously be immobilized on a support, in particular a protein chip.
  • a protein chip is an object of the invention, and may also contain at least one polypeptide from a microorganism other than Listeria innocua or monocytogenes 4b or an antibody directed against a compound of a microorganism other than Listeria innocua or monocytogenes 4b.
  • the protein chips or high density filters containing proteins according to the invention can be constructed in the same way as the DNA chips according to the invention.
  • the latter method is preferable, when it is desired to attach proteins of large size to the support, these proteins being advantageously prepared by genetic engineering.
  • the protein chips according to the invention can advantageously be used in kits or necessary for the detection and / or identification of bacteria associated with the species Listeria innocua or monocytogenes 4b or with a microorganism, or more generally in kits or kits for the detection and / or identification of microorganisms.
  • the polypeptides according to the invention are fixed on the DNA chips, the presence of antibodies is sought in the samples tested, the fixing of an antibody according to the invention on the support of the protein chip allowing the identification of the protein of which said antibody is specific.
  • an antibody according to the invention is fixed on the support of the protein chip, and the presence of the corresponding antigen, specific for Listeria innocua or monocytogenes 4b or an associated microorganism, is detected.
  • a protein chip described above can be used for the detection of gene products, to establish an expression profile of said genes, in addition to a DNA chip according to the invention.
  • the protein chips according to the invention are also extremely useful for proteomics experiments, which studies the interactions between the different proteins of a given microorganism.
  • proteomics experiments which studies the interactions between the different proteins of a given microorganism.
  • peptides representative of the various proteins of an organism are fixed on a support. Then, said support is brought into contact with labeled proteins, and after an optional rinsing step, interactions between said labeled proteins and the peptides fixed on the protein chip are detected.
  • protein chips comprising a polypeptide sequence according to the invention or an antibody according to the invention are subject of the invention, as well as the kits or kits containing them.
  • the present invention also covers a method for detecting and / or identifying bacteria belonging to the species Listeria innocua or monocytogenes 4b or to an associated microorganism in a biological sample, which implements a nucleotide sequence according to the invention .
  • the te ⁇ ne biological sample relates in the present invention to the samples taken from a living organism (in particular blood, tissues, organs or others taken from a mammal) or a sample containing biological material, that is, DNA or RNA.
  • a biological sample also includes food compositions containing bacteria (for example cheeses, dairy products), but also food compositions containing yeasts (beers, breads) or others.
  • the third biological sample also relates to the bacteria isolated from these samples or food compositions.
  • the detection and / or identification process using the nucleotide sequences according to the invention can be of various nature.
  • a method comprising the following steps: a) optionally, isolation of the DNA from the biological sample to be analyzed, or obtaining a cDNA from the RNA of the biological sample; b) specific amplification of the DNA of bacteria belonging to the species Listeria innocua or monocytogenes 4b or to a microorganism associated with the aid of at least one primer according to the invention; c) highlighting of the amplification products. This process is based on specific amplification of DNA, in particular by an amplification chain reaction.
  • a method is also preferred comprising the following steps: a) bringing a nucleotide probe according to the invention into contact with a biological sample, the nucleic acid contained in the biological sample having, if necessary, previously been made accessible to hybridization, under conditions allowing hybridization of the probe to the nucleic acid of a bacterium belonging to the species Listeria innocua or monocytogenes 4b or to an associated microorganism; b) demonstration of the hybrid possibly formed between the nucleotide probe and the DNA of the biological sample.
  • Such a method should not be limited to the detection of the presence of the DNA contained in the biological sample to be tested, it can also be implemented to detect the RNA contained in said sample. This process includes in particular the Southern and Northern blot.
  • Another preferred method according to the invention comprises the following steps: a) bringing a nucleotide probe immobilized on a support according to the invention into contact with a biological sample, the nucleic acid of the sample, having, where appropriate , been previously made accessible for hybridization, under conditions allowing hybridization of the probe to the nucleic acid of a bacterium belonging to the species Listeria innocua or monocytogenes 4b or to an associated microorganism; b) bringing the hybrid formed into contact between the nucleotide probe immobilized on a support and the nucleic acid contained in the biological sample, where appropriate after elimination of the DNA from the biological sample which has not hybridized with the probe, with a labeled nucleotide probe according to the invention; c) highlighting of the new hybrid formed in step b).
  • This method is advantageously used with a DNA chip according to the invention, the desired nucleic acid hybridizing with a probe present on the surface of said chip, and being detected by the use of a labeled probe.
  • This process is advantageously implemented by combining a prior step of amplification of DNA or complementary DNA optionally obtained by reverse transcription, using primers according to the invention.
  • kits or kits for the detection and / or identification of bacteria belonging to the species Listeria innocua or monocytogenes 4b or to an associated microorganism characterized in that it comprises the following elements : a) a nucleotide probe according to the invention; b) optionally, the reagents necessary for carrying out a hybridization reaction; c) optionally, at least one primer according to the invention as well as the reagents necessary for a DNA amplification reaction.
  • kits or kits for the detection and / or identification of bacteria belonging to the species Listeria innocua or monocytogenes 4b or to an associated microorganism characterized in that it comprises the elements following: a) a nucleotide probe, called capture probe, according to the invention; b) an oligonucleotide probe, called the revelation probe, according to the invention; c) optionally, at least one primer according to the invention as well as the reagents necessary for a DNA amplification reaction.
  • kits or kits for the detection and / or identification of bacteria belonging to the species Listeria innocua or monocytogenes 4b or to an associated microorganism characterized in that it comprises the following elements: a) at least a primer according to the invention; b) optionally, the reagents necessary to carry out a DNA amplification reaction; c) optionally, a component making it possible to verify the sequence of the amplified fragment, more particularly an oligonucleotide probe according to the invention, are also objects of the present invention.
  • said primers and / or probes and / or polypeptides and / or antibodies according to the present invention used in the methods and / or kits or necessary according to the present invention are chosen from primers and / or probes and / or polypeptides and / or antibodies specific for the species Listeria innocua or monocytogenes 4b.
  • these elements are chosen from the nucleotide sequences coding for a secreted protein, among secreted polypeptides, or among antibodies directed against secreted polypeptides of Listeria innocua or monocytogenes 4b.
  • the present invention also relates to strains of Listeria innocua or monocytogenes 4b and / or associated microorganisms containing one or more mutation (s) in a nucleotide sequence according to the invention, in particular an ORF sequence, or their regulatory elements. (in particular promoters).
  • the strains of Listeria innocua or monocytogenes 4b are preferred, having one or more mutation (s) in the nucleotide sequences coding for polypeptides involved in the cellular machinery, in particular secretion, central intermediate metabolism, metabolism. energy, the processes of amino acid synthesis, transcription and translation, synthesis of polypeptides.
  • Said mutations can lead to inactivation of the gene, or in particular when they are located in the regulatory elements of said gene, to overexpression of the latter.
  • the invention further relates to the use of a nucleotide sequence according to the invention, a polypeptide according to the invention, an antibody according to the invention, a cell according to the invention, and / or d '' an animal transformed according to the invention, for the selection of organic or inorganic compound capable of modulating, regulating, inducing or inhibiting the expression of genes, and / or modifying the cellular replication of eukaryotic or prokaryotic cells or capable of inducing, inhibiting or aggravating the pathologies linked to infection with Listeria innocua or monocytogenes 4b or one of its associated microorganisms.
  • the invention also includes a method of selecting compounds capable of binding to a polypeptide or a fragment thereof according to the invention, capable of binding to a nucleotide sequence according to the invention, or capable of recognizing an antibody according to claim , and / or capable of modulating, regulating, inducing or inhibiting gene expression, and / or modifying the growth or cellular replication of eukaryotic or prokaryotic cells, or capable of inducing, inhibiting or aggravate in an animal or human organism the pathologies linked to an infection by Listeria, for example by L.
  • monocytogenes 4b or one of its associated microorganisms, characterized in that it comprises the following stages: a) bringing said compound into contact with said polypeptide, said nucleotide sequence, with a cell transformed according to the invention and / or administering said compound to an animal transformed according to the invention; b) determining the capacity of said compound to bind with said polypeptide or said nucleotide sequence, or to modulate, regulate, induce or inhibit the expression of genes, or to modulate cell growth or replication, or induce, inhibit or aggravate in said transformed animal the pathologies linked to an infection with Listeria, for example L. monocytogenes 4b or one of its associated microorganisms.
  • the cells and / or animals transformed according to the invention can advantageously serve as a model and be used in methods for studying, identifying and / or selecting compounds capable of being responsible for pathologies induced or aggravated by Listeria monocytogenes, or susceptible to prevent and / or treat these pathologies.
  • the transformed host cells in particular bacteria of the Listeria family, the transformation of which by a vector according to the invention can, for example, increase or inhibit its infectious power, or modulate the pathologies usually induced or aggravated by the infection, may be used to infect animals whose pathologies will be monitored.
  • These unprocessed animals, infected for example with transformed Listeria bacteria could serve as a study model.
  • the animals transformed according to the invention may be used in methods of selecting compounds capable of preventing and / or treating diseases due to Listeria. Said methods using said transformed cells and / or transformed animals form part of the invention.
  • the compounds which can be selected can be organic compounds such as polypeptides or carbohydrates or any other organic or inorganic compounds already known, or new organic compounds produced from molecular modeling techniques and obtained by chemical or biochemical synthesis. , these techniques being known to those skilled in the art.
  • Said selected compounds may be used to modulate the growth and / or cell replication of Listeria innocua or monocytogenes 4b or any other associated microorganism and thus to control infection by these microorganisms.
  • Said compounds according to the invention may also be used to modulate the growth and / or cell replication of all eukaryotic or prokaryotic cells, in particular tumor cells and infectious microorganisms, for which said compounds will prove to be active, the methods making it possible to determine said modulations being well known to those skilled in the art.
  • compound capable of modulating the growth of a microorganism is intended to denote any compound which makes it possible to intervene, modify, limit and / or reduce the development, growth, rate of proliferation and / or viability of said microorganism. .
  • This modulation can be carried out for example by an agent capable of binding to a protein and thus of inhibiting or potentiating its biological activity, or capable of binding to a membrane protein of the external surface of a microorganism and of block the penetration of said microorganism into the host cell or promote the action of the immune system of the infected organism directed against said microorganism.
  • This modulation can also be carried out by an agent capable of binding to a nucleotide sequence of a DNA or RNA of a microorganism and of blocking for example the expression of a polypeptide whose biological or structural activity is necessary to the growth or reproduction of said microorganism.
  • associated microorganism is intended to denote any microorganism whose gene expression can be modulated, regulated, induced or inhibited, or whose cell growth or replication can also be modulated by a compound of l 'invention.
  • associated microorganism in the present invention is also intended to denote any microorganism comprising nucleotide sequences or polypeptides according to the invention. These microorganisms may in certain cases contain polypeptides or nucleotide sequences identical or homologous to those of the invention and may also be detected and / or identified by the methods or kit for detection and / or identification according to the invention and also serve as a target for the compounds of the invention.
  • microorganism is also intended to denote any Listeria monocytogenes microorganism of any serotype.
  • the invention relates to compounds capable of being selected by a selection method according to the invention.
  • the invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a compound chosen from the following compounds: a) a nucleotide sequence according to the invention; b) a polypeptide according to the invention; c) a vector according to the invention; d) an antibody according to the invention; and e) a compound capable of being selected by a selection method according to the invention, optionally in combination with a pharmaceutically acceptable vehicle.
  • the term effective quantity is intended to denote a sufficient quantity of the said compound or antibody, or of the polypeptide of the invention, making it possible to modulate the growth of Listeria innocua or monocytogenes 4b or of an associated microorganism.
  • the invention also relates to a pharmaceutical composition according to the invention for the prevention or treatment of an infection by a bacterium belonging to the genus Listeria or by an associated microorganism.
  • the invention further relates to an immunogenic and / or vaccine composition, characterized in that it comprises one or more polypeptides according to the invention and / or one or more hybrid polypeptides according to the invention.
  • the invention also includes the use of a transformed cell according to the invention, for the preparation of a vaccine composition.
  • the invention also relates to a vaccine composition, characterized in that it contains a nucleotide sequence according to the invention, a vector according to the invention and / or a transformed cell according to the invention.
  • the invention also relates to the vaccine compositions according to the invention, for the prevention or treatment of an infection by a bacterium belonging to the genus Listeria or by an associated microorganism.
  • the immunogenic and / or vaccine compositions according to the invention intended for the prevention and / or treatment of infection by Listeria or by an associated microorganism will be chosen from the immunogenic and / or vaccine compositions comprising a polypeptide or one of its fragments corresponding to a protein, or one of its fragments, of the Listeria cell envelope.
  • the vaccine compositions comprising nucleotide sequences will preferably also comprise nucleotide sequences coding for a polypeptide or one of its fragments corresponding to a protein, or one of its fragments, of the cell envelope of Listeria.
  • polypeptides of the invention or their fragments entering into the immunogenic compositions according to the invention can be selected by techniques known to those skilled in the art, for example on the capacity of said polypeptides to stimulate the T cells, which results, for example, in their proliferation or the secretion of interleukins, and which results in the production of antibodies directed against said polypeptides.
  • mice In mice, in which a weight dose of the vaccine composition comparable to the dose used in humans is administered, the antibody reaction is tested by sampling the serum followed by a study of the formation of a complex between the antibodies present in the serum and the antigen of the vaccine composition, according to the usual techniques.
  • said vaccine compositions will preferably be in association with a pharmaceutically acceptable vehicle and, where appropriate, with one or more appropriate immunity adjuvants.
  • This type of vaccination is carried out with a particular plasmid derived from an E. coli plasmid which does not replicate in vivo and which codes only for the vaccinating protein. Animals have been immunized by simply injecting naked plasmid DNA into the muscle. This technique leads to the expression of the vaccine protein in situ and to an immune response of cell type (CTL) and of humoral type (antibody). This double induction of the immune response is one of the main advantages of the vaccination technique with naked DNA.
  • CTL cell type
  • antibody humoral type
  • compositions comprising nucleotide sequences or vectors into which said sequences are inserted, are in particular described in international application No. WO 90/1 1092 and also in international application No. WO 95/1 1307.
  • the nucleotide sequence constituting the vaccine composition according to the invention can be injected into the host after being coupled to compounds which promote the penetration of this polynucleotide inside the cell or its transport to the cell nucleus.
  • the resulting conjugates can be encapsulated in polymer microparticles, as described in international application No. WO 94/27238 (Medisorb Technologies International).
  • the nucleotide sequence preferably DNA
  • the nucleotide sequence is complexed with DEAE-dextran, with nuclear proteins, with lipids or encapsulated in liposomes or even introduced under the form of a gel facilitating its transfection into cells.
  • the polynucleotide or the vector according to the invention can also be in suspension in a buffer solution or be associated with liposomes.
  • such a vaccine will be prepared according to the technique described by Tacson et al. or Huygen et al. in 1996 or according to the technique described by Davis et al. in international application No. WO 95/1 1307.
  • Such a vaccine can also be prepared in the form of a composition containing a vector according to the invention, placed under the control of regulatory elements allowing its expression in humans or animals. It is possible, for example, to use, as a vector for the in vivo expression of the polypeptide antigen of interest, the plasmid pcDNA3 or the plasmid pcDNAl / neo, both marketed by Invitrogen (R & D Systems, Abingdon, United Kingdom). United). Such a vaccine will advantageously comprise, in addition to the recombinant vector, a saline solution, for example a sodium chloride solution.
  • a saline solution for example a sodium chloride solution.
  • pharmaceutically acceptable vehicle is intended to denote a compound or a combination of compounds entering into a pharmaceutical or vaccine composition which does not cause side reactions and which allows for example the facilitation of the administration of the active compound, the increase in its duration of life and / or its efficiency in the organism, increasing its solubility in solution or improving its conservation.
  • pharmaceutically acceptable vehicles are well known and will be adapted by those skilled in the art depending on the nature and the mode of administration of the active compound chosen.
  • these can include suitable immunity adjuvants which are known to those skilled in the art, such as, for example, aluminum hydroxide, a representative of the family of muramyl peptides. as one of the peptide derivatives of N-acetyl-muramyl, a bacterial lysate, or even the incomplete adjuvant of Freund.
  • these compounds will be administered by the systemic route, in particular by the intravenous route, by the intramuscular, intradermal or subcutaneous route, or by the oral route. More preferably, the vaccine composition comprising polypeptides according to the invention will be administered several times, over a period of time, by the intradermal or subcutaneous route.
  • dosages and dosage forms can be determined according to the criteria generally taken into account in establishing a treatment adapted to a patient such as, for example, the patient's age or body weight, the severity of his general condition, tolerance to treatment and side effects observed.
  • the invention comprises the use of a composition according to the invention, for the treatment or prevention of diseases induced or aggravated by the presence of Listeria.
  • the present invention also relates to a genomic DNA library of a bacterium of the genus Listeria, preferably, Listeria innocua or monocytogenes, preferably the strain 4b.
  • the genomic DNA banks described in the present invention cover the genome of Listeria innocua and Listeria monocytogenes 4b respectively.
  • these regions could not be cloned into said library, due to lethality problems in Escherichia coli, these regions can easily be amplified and identified by a person skilled in the art, using oligonucleotides specific for the sequences of the sequences. ends of the different clones which form the contigs.
  • the present invention also relates to methods for the isolation of a polynucleotide of interest present in a strain of Listeria and absent in another strain, which uses at least one DNA library based for example on a plasmid pcDNA2.1 containing the Listeria genome.
  • the method according to the invention for the isolation of a polynucleotide of interest can comprise the following steps: a) isolating at least one polynucleotide contained in a clone of the DNA library of Listeria origin, b) isolating: at least one genomic polynucleotide or cDNA of a listeria, said listeria belonging to a strain different from the strain used for the construction of the DNA library of step a) or, alternatively,
  • step a) at least one polynucleotide contained in a clone of a DNA library prepared from the genome of a Listeria which is different from the Listeria used for the construction of the DNA library of step a); c) hybridizing the polynucleotide of step a) to the polynucleotide of step b); d) selecting the polynucleotides of step a) which have not formed a hybridization complex with the polynucleotides of step b); e) characterize the selected polynucleotide.
  • the polynucleotide of step a) can be prepared by digestion of at least one recombinant clone with an appropriate restriction enzyme, and optionally, the amplification of the resulting polynucleotide insert.
  • the method of the invention allows a person skilled in the art to carry out comparative genomic studies between the different strains or species of the genus
  • Listeria for example between pathogenic strains and their non-pathogenic equivalents.
  • the chromosomal DNA of the strains studied was prepared by a conventional method including treatment with proteinase K and phenol extraction (Jacquet, C, et al., Monellanase K and phenol extraction (Jacquet, C, et al., Monellanase K and phenol extraction (Jacquet, C, et al., Monellanase K and phenol extraction (Jacquet, C,
  • the plasmids were prepared by a semi-automatic preparation method developed in the GMP laboratory (Genomics of Pathogenic Microorganisms of the Institut Pasteur) based on the alkaline lysis method (Birnboim, H. C , Methods Enzymol., 100: 243-255, 1983).
  • the chromosomal inserts were sequenced from their two ends using the T7 and universal primer following the recommendations of the supplier (PE-biosystems). The sequences were determined using automatic sequencers of type 377 and 3700 (PE-Biosystem).
  • the sequences were assembled using the software package developed at the University of Washington, Phred, Phrap and Consed (Ewing, B., et al., Génome Res., 8: 186-194, 1998; Gordon, D ., et al., Genome Res., 8: 195-202, 1998).
  • the finishing of the sequence was carried out using the GMPTB software package (Frangeul, L., et al., Microbiology, 145: 2625-2634, 1999).
  • the finishing step corresponds to the resequencing of the regions where the sequence is insecure and the sequencing of the regions located between the contigs. It was carried out either by sequencing PCR products or by walking on the clones of the bank.
  • the sequences of the oligonucleotides were defined using the consed and Primo software (Gordon, D., et al., 1998; Li, P., et al., Genomics, 40: 476-485, 1997).
  • the identification of the coding phases was carried out using the GMPTB software package. This program combines the results of different methods: (i) identifying open reading phases and sorting them according to their size, (ii) analyzing the probability of being coding using Genemark software (Lukashin, AV, et al., Nucleic Acids Res., 15: 1 107-1 1 15, 1998), (iii) identification of the start of translation (initiation codon and ribosome binding sequence), (iv ) similarity of protein sequence deduced with the protein sequences contained in the sequence banks using the BLASTP software.
  • the chromosomal regions of the strain L. monocytogenes of serotype 4b which are absent from the strains L. monocytogenes EGDe and L. innocua were identified using the Crossmatch / Phrap Package (Phil Green, University of Washington, Seattle, unpublished). This software makes it possible to assemble nucleotide sequences (Phrap) by masking all the sequences or parts of sequence similar to one or more reference sequences (Crossmatch).
  • the reference sequences used were: the complete genome sequence of L. monocytogenes EGD, the complete genome sequence of L. innocua and the sequence of its plasmid.
  • SEQ ID Nos. 1 - 1 1 nucleotide sequences of 10 Contigs and 1 plasmid from the Listeria innocua assembly.
  • SEQ ID Nos. 12 - 689 nucleotide sequences of specific proteins of L. innocua (absent from Z. monocytogenes-EGD).
  • SEQ ID Nos. 690 - 1067 nucleotide sequences of the specific proteins of L. monocytogenes-EGD (absent from L. innocua).
  • SEQ ID Nos. 1068 - 2041 nucleotide sequences of 974 contigs originating from the assembly of Listeria monocytogenes-4b (1,231,537 bases).
  • SEQ ID Nos. 2059-2601 nucleotide sequences of 543 genes specific for Listeria innocua Clipl 1262; with the SEQ ID identifier in the first column, the gene name in the second column, the IPF number in the third column (“Institut Pasteur” identifier number allowing the sequence to be correlated with the sequences in Table V) and in the last column the corresponding annotation.
  • SEQ ID Nos. 2602 - 2871 nucleotide sequences of the 270 genes specific for Listeria monocytogenes EGDe; with the SEQ ID identifier in the first column, the gene name in the second column, the IPF number in the third column (“Institut Pasteur” identifier number allowing the sequence to be correlated with the sequences in Table V) and in the last column the corresponding annotation.

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US20060257894A1 (en) * 2003-09-16 2006-11-16 Michel Doumith Molecular typing of listeria monocytogenes, hybridization supports and kits for said molecular typing
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US9422529B2 (en) 2010-12-22 2016-08-23 The Board Of Regents Of The University Of Texas System Alphavirus compositions and methods of use
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