WO1992007074A1 - Polypeptide of a growth factor receptor family, application in the diagnosis and treatment of myeloproliferative diseases - Google Patents

Polypeptide of a growth factor receptor family, application in the diagnosis and treatment of myeloproliferative diseases Download PDF

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WO1992007074A1
WO1992007074A1 PCT/FR1990/000762 FR9000762W WO9207074A1 WO 1992007074 A1 WO1992007074 A1 WO 1992007074A1 FR 9000762 W FR9000762 W FR 9000762W WO 9207074 A1 WO9207074 A1 WO 9207074A1
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leu
ser
pro
arg
ala
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PCT/FR1990/000762
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French (fr)
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Martine Charon
Sylvie Gisselbrecht
Jean-François PENCIOLELLI
Michèle SOUYRI
Pierre Tambourin
Paule Varlet
Isabelle Vigon
Françoise Wendling
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Institut National De La Sante Et De La Recherche Medicale (Inserm)
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Priority to PCT/FR1990/000762 priority Critical patent/WO1992007074A1/en
Priority to JP2515169A priority patent/JPH06501915A/en
Priority to CA002094261A priority patent/CA2094261C/en
Priority to US08/078,311 priority patent/US5925750A/en
Priority claimed from CA002094261A external-priority patent/CA2094261C/en
Publication of WO1992007074A1 publication Critical patent/WO1992007074A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1036—Retroviridae, e.g. leukemia viruses
    • 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
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/702—Specific hybridization probes for retroviruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • a polypeptide from the growth factor receptor family Application to the diagnosis and therapy of myeloproliferative diseases.
  • Myeloproliferative diseases are diseases in which hematopoietic stem cells have an alteration in their differentiation capacity and / or an alteration in their dependence on specific growth factor.
  • the blood cells come from a small number of stem cells capable of self-renewal, which generate progenitor cells irreversibly committed to the production of one or a few hematopoietic lines. Precise control of each differentiation step is necessary to ensure a stable rate of the different specialized cells as well as to offer a precise response to stress stimuli.
  • the controls are usually the result of interactions between cells, either by contact with the hematopoietic micro-environment or by the release of specific cytokines.
  • the failure of the control systems leads either to cytopenias or to an uncontrolled cell proliferation which can affect one or more lines depending on the nature of the lesion.
  • the proliferation of several hematopoietic lines can lead to myeloproliferative disease. Such diseases can be induced by retroviruses carrying oncogenes such as the MPLV retrovirus short for the expression "myeloproliferative leukemia virus" which is a retrovirus which is defective for its replication.
  • the murine MPLV retrovirus is known to cause severe hematological disorder in adult mouse strains of most strains, characterized by dramatic proliferation and differentiation of several hematopoietic lines.
  • the most acute aspect of this disease is the suppression of in vitro dependence on growth factors of most hematopoietic progenitor cells.
  • the natural MPLV isolate has been described as a complex of two viral entities: a murine F-MuLV (Friend replication competent ecotropic murine leukemia virus) and a replication defective virus now designated by the term MPLV. Pseudotyping the defective particles with other ecotropic or amphotropic MuLVS viruses makes it possible to reproduce the initial disease.
  • Proviral MPLV DNA and F-MuLV DNA have been shown to be structurally close except in the envelope region.
  • the inventors have now identified a sequence of cell origin transduced into the rearranged gene of the MPLV envelope and which turns out to be conserved in the mammalian genome.
  • the invention therefore relates to a polypeptide capable of playing a role when it is produced by a virus of the MPLV type, in the disorders caused during myeloproliferative diseases.
  • the invention also relates to nucleotide sequences coding for this polypeptide.
  • the inventors have remarked, quite interestingly, that the protein sequence of the polypeptide of the invention has marked analogies with certain amino acid sequences of growth factor receptor.
  • the invention therefore makes it possible to identify the mechanisms of the pathology linked to infection with the MPLV retrovirus and to propose means for the detection of a pathology of the same type in humans and, where appropriate, for its treatment.
  • the invention therefore relates to a polypeptide characterized in that it corresponds to the chain of amino acids designated by SEQ ID NO1 in the list of sequences, either in that it comprises the chain SEQ ID NO 1 or a fragment of this sequence as soon as the polypeptide meets at least one of the following conditions:
  • amino acid sequence having a homology of at least 80%, preferably 88%, with the fragment represented by SEQ ID NO 2, contained in the sequence of amino acids SEQ ID NO 1.
  • the ability of a given polypeptide to behave like a growth factor receptor can be characterized by using one of the following tests.
  • the invention makes it possible to detect the potential capacity of a polyppetide of the invention to behave like a receptor and to fix a ligand. In an equivalent way, it can be investigated whether the expression of the polypeptide tested, by the MPLV virus previously modified by the nucleotide sequence coding for this polypeptide, integrated into the site usually containing the nucleotide sequence v-mpl, makes it possible to obtain the in vitro proliferation of cells transformed with MPLV containing the tested polypeptide.
  • the conditions for carrying out this test are those used for the experiments reported below, corresponding to the immortalization by MPLV, of bone marrow cells in culture.
  • MPLV MPLV at its site usually containing the sequence v-mpl
  • a nucleotide sequence of the invention previously devoid of a selected nucleotide fragment, which one seeks to determine if it is involved in the transmission of the signal.
  • MPLV thus modified can be infected, for example with bone marrow cells as described in the examples. It is then observed whether the elimination of the nucleotide fragment referred to above results in the absence of cell proliferation.
  • Another test for detecting whether a polypeptide of the invention behaves as a receptor capable of faith to bind a chosen molecule and to transmit a signal after the binding of this molecule is as follows:
  • cells which do not naturally express the polypeptide tested are transformed with the latter.
  • the cells thus transformed are placed in the presence of a selected molecule capable of behaving like a ligand and their capacity to respond to this molecule is determined, in particular by observing whether or not there is cell proliferation.
  • polypeptides meeting the definition of the invention given above are for example polypeptides comprising a chain of amino acids WSXWS, in which X is any amino acid and preferably X corresponds to arginine or serine.
  • sequence W S A W S corresponds to the fragment included in v-mpl or in the equivalent sequence in mice, and the sequence W S S W S corresponds to the h-mpl fragment of the human cell gene.
  • the peptide W S X W S may be involved in the in vivo differentiation effect of hematopoietic lines, observed during infection by an MPLV retrovirus.
  • the invention also relates to all of the polypeptides satisfying the above conditions capable of inducing or participating in cell differentiation in vitro of hematopoietic lines.
  • polypeptides according to the invention are the peptides characterized in that they comprise a chain of amino acids corresponding either to the chain identified in the list of sequences under the reference SEQ ID NO 3.
  • Preferred polypeptides corresponding to the general definition above are the polypeptides corresponding to one of the sequences identified under the following references SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7 , SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11.
  • Another polypeptide according to the invention is characterized in that it is coded by a nucleotide sequence capable of hybridizing under stringent conditions with the specific probes of MPLV and corresponding to the fragments SACI-PSTI and PSTI-PSTI of 300 base pairs each shown in Figure 1.
  • probes can be DNA or RNA.
  • polypeptide encoded by a nucleotide sequence capable of hybridizing with one of the preceding probes also relates to a polypeptide falling within the preceding definitions, characterized in that it responds to one of the amino acid sequences identified in the list of sequences, under the following references SEQ ID NO 12, SEQ ID NO 13 .
  • the invention further relates to a growth factor receptor, characterized in that it comprises the amino acid sequence identified by SEQ ID NO 1.
  • the invention also relates to a soluble form of the receptor comprising in particular the extracellular domain represented by the polypeptide sequence designated by SEQ ID NO 7 in humans or SEQ ID NO 10 in mice.
  • fusion proteins characterized in that they comprise a polypeptide of the invention in association with a determined amino acid sequence, in particular with the gp70 of MPLV, in particular in that it is the amino acid sequence SEQ ID NO 14.
  • fusion proteins can be obtained with the protein encoded by the gag gene or with virus glycoproteins, for example other than the retrovirus, in particular a VSV glycoprotein.
  • the invention also relates to a nucleotide sequence characterized in that it codes for a polypeptide described above.
  • nucleotide sequences are those which correspond to any of the nucleotide sequences SEQ ID NO15, NO16, NO17, NO18, NO19, NO20 or to a sequence complementary to these sequences or to a sequence capable of hybridizing with one of the above sequences under stringent conditions.
  • nucleotide sequences constituted by RNA which correspond to the nucleotide sequences described above.
  • the invention also relates to a recombinant cell host, characterized in that it comprises a nucleotide sequence as given above.
  • the nucleotide sequence inserted into the cell host is integrated into the genome of this host.
  • Different cellular hosts can be chosen for carrying out the invention according to the desired application. As an example for the search for molecules possibly having an affinity with the polypetides of the invention, it is possible to use cellular hosts such as bacteria, viruses, or phages, cells of insects or mammals (COS , CHO) etc.
  • a particularly preferred cellular host is the MPLV virus.
  • the invention relates to the MPLV virus containing in its genome a nucleotide sequence coding for the peptide represented by the sequence SEQ ID NO2 in the form of a fusion protein with the viral glycoprotein gp70.
  • the invention also relates to recombinant vectors characterized in that they comprise a nucleotide sequence as described above, at a site which is not essential for its replication, under the control of the regulatory sequences necessary for its expression in a determined cellular host.
  • Vectors which are particularly suitable for carrying out the invention are, for example, the vaccinia virus or the baculovirus.
  • nucleotide probes comprising at least 9 nucleotides and preferably from 15 to 30 nucleotides, capable of hybridizing with a nucleotide sequence according to the invention, under stringent conditions, this probe being optionally labeled.
  • the probes used can be specific for the nucleotide sequences of the invention or non-specific when looking for a sequence belonging to a larger group.
  • Markers suitable for producing the probes of the invention are conventional radioactive markers or even enzymatic markers or any other suitable marker.
  • the invention further relates to polyclonal or monoclonal antibodies, characterized in that they recognize one of the polypeptides according to the invention.
  • Particularly interesting antibodies are directed against the extracellular domain of the polypeptide encoded by the c-mpl gene.
  • monoclonal antibodies are obtained according to conventional methods of antibody production.
  • the method of Kolher and Milstein will be used, in particular monoclonal antibodies are prepared by cell fusion between myeloma cells and spleen cells of mice previously immunized with a polypeptide according to the invention, in accordance to the classic process.
  • the identification by the inventors of the polypeptide responsible in the MPLV virus for myeloproliferative disease allows the development of means for the in vitro detection of an abnormal expression of a polypeptide described in the preceding pages, indicative of a pathological state. .
  • the invention comprises a method for the in vitro detection of an abnormal expression of a polypeptide of the invention, characterized in that it comprises:
  • abnormal expression of the polypeptide is meant any expression of abnormal quantitative level, as well as any expression of a modified polypeptide whose functioning would be impaired.
  • Another method for the in vitro detection of the abnormal expression of the polypeptide of the invention can be carried out by the in vitro detection of the abnormal expression of a nucleotide sequence coding for a peptide according to the invention.
  • Such a method includes:
  • step c) repeating step a) so as to obtain a detectable quantity of the nucleotide sequences sought;
  • PCR are given for example in application EP 0200362 and in application EP 0229701.
  • the invention also relates to a kit for the in vitro detection of the abnormal expression of the polypeptides according to the invention, characterized in that it comprises polyclonal or monoclonal antibodies capable of reacting with said polypeptide, in the case if necessary a reagent for the detection of the antigen-antibody immunological reaction.
  • kit can be envisaged for carrying out different types of tests, notably RIA or ELISA tests.
  • the invention also relates to a method for detecting the affinity of a molecule for a polypeptide according to the invention, characterized by:
  • the invention further relates to a medicament characterized in that it comprises a polypeptide corresponding to the preceding definitions, in soluble form, in combination with an acceptable pharmaceutical vehicle.
  • a medicament can be used to remedy the abnormal production of the polypeptides of the invention in the cells of a patient.
  • the medicament according to the invention can act according to a competition reaction between the soluble form of a polypeptide present as an active ingredient in this medicament and the polypeptides abnormally present in the treated patient.
  • FIG. 1 (A) Restriction endonuclease map of clone MPLV 107 and clone F-MuLV 57. Restriction sites are given for the following enzymes: B: BamHI, C: ClaI, E : EcoRI, H: HindIII, K: Kpnl, P: PstI, Pv: PyuII, SI: Sacl, SII: SacII, X: Xbal. The black frame corresponds to the specific region of MPLV. The two probes derived from the MPLV envelope are indicated by dashes under the restriction map.
  • (B) Demonstration of the specificity of the probes derived from the MPLV envelope.
  • NIH 3T3 cells infected with the amphotropic pseudotype 4070 of MPLV are used as source of poly A + RNA (5 ⁇ g, lines A), the clone F-MuLV 57 (1 ⁇ g, lines B), and the clone 2 Mus dunni non-producer infected with MPLV (15 ⁇ q, lines C).
  • Northern blot fingerprints hybridized with the E57BS probe derived from the F-MuLV envelope obtained, labeled according to the nick-translation technique (sample 1), or with the two RNA probes derived from the MPLV envelope, SacI -PstI (sample no.
  • Lanes M represent lambda / HindIII molecular weight marker DNA.
  • the black arrows and the light arrows indicate the positions of the genomic and subgenomic RNAs of the envelope of F-MuLV and MPLV respectively.
  • Figure 2 (A) Schematic representation of the rearranged env region of MPLV.
  • the hatched box represents mpl, the light boxes represent the env sequences of F-MuLV.
  • the asterisk in the MPL domain indicates the stop codon.
  • (B) Amino acid sequence deduced from the env region of MPLV.
  • the reading frame for 284 amino acids is given.
  • Amino acids 1 to 100 correspond to sequences derived from the F-MuLV env gene.
  • the 184 specific amino acids of mpl are boxed.
  • the arrowheads indicate the junction of the two portions of the F-MuLV envelope and the junction with the specific sequence mpl, respectively.
  • the asterisks indicate the sites N-glycosylation potentials (Asn-X-Ser / Thr).
  • the underlined sequences represent the signal peptide of gp70 and the hydrophobic transmembrane domain is written in bold.
  • SacI-PstI and Pstl-PstI RNA are those described in the "experimental procedures" section.
  • FIG. 5 Establishment in vitro of cell lines infected with MPLV.
  • FIG. 6 Southern analysis of proviral integration sites in cultures infected with MPLV. The DNAs prepared 5, 21, 97 days after infection were digested with EcoRI and the fingerprints were hybridized with the v-mpl RNA probes. At day 97, L159 included myeloblasts, L173 of mast cells, L223 of megakaryocytes and erythroblasts. The arrow indicates c-mpl.
  • Figure 7 Comparison of amino acid sequences of the v-mpl extracellular domain with that of the hematopoietic cytokine receptors.
  • the extracellular domains of the murine IL-3 receptor, the murine EPO receptor, the murine IL-4 receptor, the ⁇ chain of the IL-2 receptor, the human IL- receptor 6 and human and mouse IL-7 receptors were aligned with v-mpl. Preserved amino acid residues are boxed. The consensus sequence is that which has been described by Itoh et al, 1990 (Science Vol. 247, p. 324-327).
  • the following experiments relate to the identification and characterization of the molecular rearrangements occurring at the level of the envelope of the MPLV retrovirus.
  • a cDNA library was prepared using poly A + RNA from NIH 3T3 cells productively affected with the amphotropic pseudotype of MPLV.
  • the cDNA clones comprising the complete env region of MPLV were obtained and two specific MPLV probes were prepared: these are the Sacl - PstI and PstI - PstI fragments, of 300 base pairs each (FIG. 1A).
  • the specificity of these two MPLV probes is shown in Figure 1B. They recognize the genomic (7.4 kb) and sub-genomic RNAs obtained by splicing (2.4 kb) but do not hybridize with F-MuLV or with amphotropic RNAs.
  • mice inoculated with the supernatant from cultures transfected with F-MuLV DNA alone were healthy 6 months after infection.
  • all the mice inoculated with the supernatant from the cultures transfected with the DNAs of MPLV3 and F-MuLV rapidly developed splenomegaly, hyperleukocytosis and polycythemia and died two months after inoculation.
  • the progenitor cells of the sick animals were then examined in vitro to determine what their hematopoietic growth factor needs were.
  • the rearranged env genes of the MPLV cDNAs and the genomic clone were sequenced and found to be identical.
  • the nucleotide sequence was analyzed showing that the env gene for MPLV includes sequences derived from the env gene for F-MuLV and non-viral sequences.
  • FIG. 2A two deletions appeared in the env gene of F-MuLV: the first between positions 5969 and 6505 and the second between positions 6615 and 7513 (Koch et al, 1983, J. Virol. 45, 1-9).
  • the env gene for MPLV is therefore a complex region composed between the 5 'and 3' ends of 191 base pairs of the 5 'end of the F-MuLV env gene (up to position 5969) followed by 110 pairs of base of the central region of the F-MuLV env gene (between positions 6506 and 6615) then by a non-viral region of 623 nucleotides and finally the 3 ′ part of the 15E protein of F-MuLV (from position 7513 ).
  • the MPLV env gene has an open reading frame of 284 amino acids starting from the ATG initiation codon of gp70 and ending at a TAG termination codon inside the specific sequence of MPLV (FIG. 2B) and potentially codes for an env fusion protein with a molecular weight of 31 kilodaltons.
  • This env-vmpl fusion protein comprises 64 amino acids of the NH 2 -terminal part of the gp70 of F-MuLV including the signal peptide, 36 amino acids of the central region of the env gene of F-MuLV and 184 specific amino acids of MPLV.
  • a hydrophobicity curve (Kyte et al, 1982, J. Mol Biol. 157, 105-132) of the amino acid sequence of the MPLV env gene product revealed, in addition to the 34 hydrophobic amino acids of the signal peptide. gp70, that the specific domain of MPLV contained a region of 22 uncharged amino acids extending from amino acid Ile 143 to amino acid Leu 165, which can correspond to a membrane domain.
  • the env nature protein of MPLV would thus consist of an extracellular domain of 109 amino acids with a potential glycosylation site, a transmembrane domain of 22 amino acids and an intracytoplasmic domain of
  • the leukemogenic properties of MPLV can thus be attributed to the presence of a new oncogene, v-mpl, transduced from cellular sequences. preserved in the phylogeny of mammals and transcribed in normal murine hematopoietic tissues.
  • bone marrow cells were infected in vitro with MPLV free of helper virus, obtained in a packaging cell line psi-CRE ( Danos and Mulligan, 1988, PNAS USA 85, 6460-6464).
  • the test was carried out in an agarose medium at a low cell concentration to avoid stimulating the formation of colonies by endogenous factors secreted by accessory cells. In repeated experiments, only a few autonomous colonies were detected.
  • these permanent cell lines are obtained from the growth of a single or a few multipotent strains infected with MPLV, in which the restriction of differentiation capacities occurs at a later stage.
  • the malignant character of the cell lines has been demonstrated by making a subcutaneous injection of 2 ⁇ 10 6 cells in syngeneic mice irradiated with a sublethal dose (5 Gy) or in nude mice. No tumor developed at the site of inoculation when cells from a culture less than 4 months old were grafted, but 6 of 10 cell lines inoculated after more than 7 months produced hematopoietic tumors of the nature of these cell lines grafted after a latency of around 30 days.
  • MPLV alone is capable of directly transforming hematopoietic progenitors engaged in differentiation and multipotentials and leads to the rapid emergence of different immortalized cell lines independent of growth factors which retain the capacity to differentiate spontaneously.
  • the env-mpl polypeptide has the general characteristics of a transmembrane protein: it contains the signal peptide of gp70 at its N-terminal part and a unique transmembrane domain, indicating that the N-terminal part of the molecule is extracellular and the part C-terminal is intra-cytoplasmic.
  • the amino acid sequence of the extracellular domain of protein v-mpl has similarities to the hematopoietic receptors of recently cloned cytokines such as the ⁇ chain of IL2R, IL3R, IL4R, IL6R, IL7R, GM-CSFR, G-CFSR, EPO-R, as well as to the receptor for prolactin.
  • this sequence contains a WSXWS motif in the extracellular domain close to the transmembrane region and does not contain a consensus sequence for proteinkinase activity in the intra-cytoplasmic domain. It has also been noted that the cytoplasmic domain of v-mpl contains many prolines (14/19, 12%) and serines (13/119, 11%) as is the case with other receptors. As a result, MPLV transduced a truncated form of a hematopoietic growth factor receptor. The expression of the endogenous c-mpl gene observed only in spleen cells, bone marrow and fetal liver confirms this hypothesis.
  • NIH 3T3 and Mus dunni cells were used.
  • the isolation of clone 2 of Mus dunni not producing MPLV was described in the publication of
  • amphotropic MPLV pseudotype was obtained by superinfecting clone 2 of Mus dunni with the amphotropic helper virus 4070 A (Chattopadhyay et al, 1981; J. Virol. 39, 777-791).
  • mice DBA / 2J, C57BL / 6J and nude mice were obtained from Iffa Credo (l'Arbesle, France) and bred under pathogen-free conditions. Animals 6 to 8 weeks old were used in all of the experiments.
  • RNA was purified according to the guanidium thiocyanate / CsCl method (Chirgwin et al, 1979; Biochemistry 18, 5294-5299) and the poly A + RNAs were selected by chromatography on an oligo dT-cellulose column.
  • RNA transfers were carried out on Hybond C-extra nitrocellulose (Amersham) as described by Thomas, 1980 (Proc. Natl. Acad. Sci. USA 77, 5201-5205).
  • the membranes were prehybridized for 5 hours at 55oC in 50% formamide, 4x SSC, 0.05 M Na / Na 2 phosphate, 1x Denhardt, 500 ⁇ g / ml yeast tRNA and 250 ⁇ g / ml sperm DNA herring. 10 7 cpm of a 32 P labeled RNA probe were added and the hybridization was carried out for 40 hours at 55 ° C. The membranes were washed twice 5 minutes at room temperature in 2x SSC - 0.1% SDS, 2 times 30 minutes at 65oC in 2X SSC - 0.1% SDS and 2 times 30 minutes at 65oC in 0.1x SSC - 0.1% SDS.
  • the DNAs were digested with appropriate restriction endonucleases according to the conditions indicated by the manufacturer and deposited on a 0.8% agarose gel. After electrophoresis, the DNAs were transferred to nitrocellulose membranes according to the method of Southern (1975; J. Mol Biol. 98, 503-518). The membranes were hybridized with 10 7 cpm of 32 p-labeled probes under conditions described for the Northern Blot.
  • the cDNA was synthesized from poly A + RNA prepared from NIH 3T3 cells in exponential growth phase, infected productively with MPLV pseudotyped by the amphotropic helper virus 4070 A (MPLV comprising the envelope of the virus 4070 A), using the Amersham cDNA synthesis kit. Blunt-ended cDNAs were ligated to a dephosphorylated pSPT18 vector digested with SmaI in the presence of T4 DNA ligase.
  • Competent LM 1035 bacteria were transformed and spread on agar dishes containing ampicillin. The colonies containing the recombinant plasmids were transferred to nitrocellulose filters.
  • the identification of the clones containing the cDNA of the envelope (env) of MPLV was carried out by in situ hybridization as described by Sambrook et al, 1989 (Cold Spring Harbor Laboratory Press), with an E57BS probe (Moreau-Gachelin et al, 1983 Biochemistry 65, 259-266), marked at 32 p by the "nick-translation" technique.
  • the high molecular weight DNA was extracted according to Souyri et al, 1983 (Proc. Natl. Acad. Sci. USA 80, 6676-6679) from clone 2 of Mus dunni non-MPLV producer and partially digested with restriction endonuclease Sau3A.
  • the DNA fragments (10 to 15 Kb) were purified by centrifugation on a sucrose gradient and ligated to BamH1 arms of the bacteriophage EMBL3 after packaging (Stratagene).
  • the recombinant phages containing the MPLV DNA were identified according to the technique of Benton et al, 1977 (Science 196, 180-182) by hybridization with RNA probes specific for MPLV.
  • the filters were prehybridized for 5 hours at 42oC in 50% formamide, 5x SSC, 5x Denhardt, 0.1% SDS, 50 mM Na / Na 2 phosphate pH 6.5, and 250 ⁇ g / ml of sperm DNA from herring (2ml per filter).
  • Hybridization with 32 Pa-labeled MPLV RNA probes was carried out for 20 hours at 42 ° C in 50% formamide, 5x SSC, 1x Denhardt, 0.1% SDS, 50 mM Na / Na 2 phosphate pH 6.5, and 250 ⁇ g / ml of herring sperm (1 ml of buffer and 2.10 6 cpm of RNA probe per filter). Filters were washed twice 10 minutes at room temperature in 2x SSC - 0.1% SDS, 30 minutes with 2x SSC - 0.1% SDS, and twice 30 minutes with 0.2x SSC - 0.1% SDS , each time at 65oC.
  • DNA sequencing DNA sequencing
  • the DNA sequence was obtained using the dideoxy chain termination method (Sanger et al, 1977; Proc. Natl. Sci. USA 74, 5463-5467) modified for the use of T7 DNA polymerase (Sequenase USB).
  • the samples were denatured for 2 minutes at 75oC and placed on a denatured acrylamide gel (6% acrylamide, 8M urea, 1x TBE).
  • Cell clones producing MPLV but free of helper virus were produced by cotransfection of psi-CRE packaging cells, with the plasmid pSV2 Neo and an approximately 10-fold excess of the plasmid pMPLV3. After selection and isolation of clones resistant to G418 (Gibco BRL), the MPLV producing clones were selected by whole cell fingerprinting according to Wendling et al, 1989 (Leukemia 3, 475-480). The fingerprints of the purified virus from the supernatants were used to select a clone producing a high titer of virus.
  • non-adherent cells were recovered and transferred to a concentration of 2.5 ⁇ 10 5 cells / ml in new vials containing a new medium. Then the cells were passed every 4 to 7 days, or more frequently, depending on the rate of cell growth that had grown.
  • the cells were seeded in a plasma coagulum culture system as described by Me Leod et al, 1978 (M.J. Murphy, ed - Springer Verlag, New York - pp 31-35). An appropriate number of cells was distributed in a volume of 0.1 ml with or without 0.25 U / ml Epo (erythropoietin) (Step 1 Human EPO, specific activity 1000 U / mg; Terry Fox Laboratory, Vancouver, Canada) . Cultures were harvested on day 2. colonies containing at least 8 benzidine-positive erythroblasts were listed as CFU-E colonies.
  • CAG GAC CAT GCT AGC TCC CAA GGC TTC TTC TAC CAC AGC AGG GCA CGG TGC TGC
  • AGC ATT ATT CAC ATC CTT GTG GAG GTG ACC ACA GCC CCG GGT ACT GTT CAC
  • GAG GAC TGG AAG GTG CTG GAG CCA TCT CTC GGT GCC CGG GGA GGG ACC CTA GAG

Abstract

Polypeptide of a growth factor receptor family, having a specific sequence and presenting all or part of the following properties: it encourages and/or is involved in the proliferation and/or differenciation of hematopoietic cell lines when obtained from the MPLV retrovirus; it is capable of acting as a hematopoietic growth factor receptor; it is recognized by antibodies directed against it. Polypeptides similar to the above. Applications in the diagnosis of the expression of said ligand polypeptide.

Description

Polypeptide de la famille d'un récepteur de facteur de croissance. Application au diagnostic et à la thérapie des maladies myéloprolifératives. A polypeptide from the growth factor receptor family. Application to the diagnosis and therapy of myeloproliferative diseases.
Les maladies myéloprolifératives sont des maladies dans lesquelles des cellules souches hématopoiétiques présentent une altération dans leur capacité de différenciation et/ou une altération dans leur dépendance vis à vis de facteur de croissance spécifique.  Myeloproliferative diseases are diseases in which hematopoietic stem cells have an alteration in their differentiation capacity and / or an alteration in their dependence on specific growth factor.
Les cellules du sang proviennent d'un petit nombre de cellules souches capables de s'auto-renouveler, qui génèrent des cellules progénitrices irréversiblement engagées vers la production d'une ou quelques lignées hématopoiétiques. Un contrôle précis de chaque étape de différenciation est nécessaire pour assurer un taux stable des différentes cellules spécialisées de même que pour offrir une réponse précise aux stimulations par le stress. Les contrôles sont habituellement le fait d'interactions entre cellules, soit par contact avec le micro-environnement hématopoiétique ou par la libération de cytokines spécifiques. La défaillance des systèmes de contrôle conduit soit à des cytopenies soit à une prolifération cellulaire incontrôlée qui peut affecter une ou plusieurs lignées selon la nature de la lésion. La prolifération de plusieurs lignées hématopoiétiques peut conduire à une maladie myéloproliférative. De telles maladies peuvent être induites par des retrovirus porteurs d'oncogènes comme le retrovirus MPLV abréviation de l'expression "myéloproliférative leukemia virus" qui est un retrovirus défectif pour sa réplication.  The blood cells come from a small number of stem cells capable of self-renewal, which generate progenitor cells irreversibly committed to the production of one or a few hematopoietic lines. Precise control of each differentiation step is necessary to ensure a stable rate of the different specialized cells as well as to offer a precise response to stress stimuli. The controls are usually the result of interactions between cells, either by contact with the hematopoietic micro-environment or by the release of specific cytokines. The failure of the control systems leads either to cytopenias or to an uncontrolled cell proliferation which can affect one or more lines depending on the nature of the lesion. The proliferation of several hematopoietic lines can lead to myeloproliferative disease. Such diseases can be induced by retroviruses carrying oncogenes such as the MPLV retrovirus short for the expression "myeloproliferative leukemia virus" which is a retrovirus which is defective for its replication.
Il est connu que le retrovirus murin MPLV provoque un désordre hématologique sévère chez des souches de souris adultes de la plupart des souches, caractérisé par une prolifération dramatique et une différenciation de plusieurs lignées hématopoiétiques. L'aspect le plus aigu de cette maladie est la suppression de la dépendance in vitro vis à vis des facteurs de croissance de la plupart des cellules progénitrices hématopoiétiques. On a décrit l'isolât MPLV naturel comme étant un complexe de deux entités virales : un virus murin F-MuLV (Friend replication compétent ecotropic murine leukemia virus) et un virus défectif pour sa replication maintenant désigné par le terme MPLV. Le pseudotypage des particules défectives avec d'autres virus MuLVS écotropes ou amphotropes permet de reproduire la maladie initiale. On a montré que l'ADN proviral de MPLV et l'ADN de F-MuLV étaient structurellement proches excepté dans la région de l'enveloppe. The murine MPLV retrovirus is known to cause severe hematological disorder in adult mouse strains of most strains, characterized by dramatic proliferation and differentiation of several hematopoietic lines. The most acute aspect of this disease is the suppression of in vitro dependence on growth factors of most hematopoietic progenitor cells. The natural MPLV isolate has been described as a complex of two viral entities: a murine F-MuLV (Friend replication competent ecotropic murine leukemia virus) and a replication defective virus now designated by the term MPLV. Pseudotyping the defective particles with other ecotropic or amphotropic MuLVS viruses makes it possible to reproduce the initial disease. Proviral MPLV DNA and F-MuLV DNA have been shown to be structurally close except in the envelope region.
Les inventeurs ont maintenant identifié une séquence d'origine cellulaire transduite dans le gène réarrangé de l'enveloppe de MPLV et qui s'avère être conservée dans le génome des mammifères.  The inventors have now identified a sequence of cell origin transduced into the rearranged gene of the MPLV envelope and which turns out to be conserved in the mammalian genome.
L'identification de cette séquence a permis de mettre en évidence son importance dans des phénomènes apparentés à une maladie myéloproliférative.  The identification of this sequence made it possible to highlight its importance in phenomena related to a myeloproliferative disease.
L'invention concerne donc un polypeptide susceptible de jouer un rôle lorsqu'il est produit par un virus de type MPLV, dans les dérèglements occasionnés lors de maladies myéloprolifératives. L'invention vise également des séquences nucléotidiques codant pour ce polypeptide.  The invention therefore relates to a polypeptide capable of playing a role when it is produced by a virus of the MPLV type, in the disorders caused during myeloproliferative diseases. The invention also relates to nucleotide sequences coding for this polypeptide.
Les inventeurs ont remarqué de façon tout à fait intéressante, que la séquence protéique du polypeptide de l'invention présente des analogies marquées avec certaines séquences d'acides aminés de récepteur de facteur de croissance. L'invention permet par conséquent d'identifier les mécanismes de la pathologie liée à l'infection par le retrovirus MPLV et de proposer des moyens pour la détection d'une pathologie de même type chez l'homme et le cas échéant pour son traitement. L'invention concerne donc un polypeptide caractérisé en ce qu'il correspond à l'enchaînement d'acides aminés désigné par SEQ ID NO1 dans la liste des séquences, soit en ce qu'il comprend l'enchaînement SEQ ID NO 1 ou un fragment de cet enchaînement dès lors que le polypeptide répond à l'une au moins des conditions suivantes : The inventors have remarked, quite interestingly, that the protein sequence of the polypeptide of the invention has marked analogies with certain amino acid sequences of growth factor receptor. The invention therefore makes it possible to identify the mechanisms of the pathology linked to infection with the MPLV retrovirus and to propose means for the detection of a pathology of the same type in humans and, where appropriate, for its treatment. The invention therefore relates to a polypeptide characterized in that it corresponds to the chain of amino acids designated by SEQ ID NO1 in the list of sequences, either in that it comprises the chain SEQ ID NO 1 or a fragment of this sequence as soon as the polypeptide meets at least one of the following conditions:
a) il est capable lorsqu'il est produit à partir du génome du retrovirus MPLV, de provoquer et/ou de favoriser in vitro ou in vivo la prolifération des lignées cellulaires hématopoiétiques,  a) it is capable, when produced from the genome of the MPLV retrovirus, of causing and / or promoting in vitro or in vivo the proliferation of hematopoietic cell lines,
b) il intervient in vitro ou in vivo, dans la différenciation cellulaire de lignées cellulaires hématopoiétiques, lorsqu'il est produit à partir du génome du retrovirus MPLV,  b) it intervenes in vitro or in vivo, in the cell differentiation of hematopoietic cell lines, when it is produced from the genome of the MPLV retrovirus,
c) il est susceptible d'intervenir in vivo dans une fonction de récepteur de facteur de croissance hématopoiétique, soit au niveau de la fixation d'un ligand, soit au niveau de la transmission d'un signal, d) il est reconnu par des anticorps dirigés contre l'enchaînement d'acides aminés représenté par la séquence SEQ ID NOl,  c) it is capable of intervening in vivo in a hematopoietic growth factor receptor function, either at the level of the binding of a ligand, or at the level of the transmission of a signal, d) it is recognized by antibodies directed against the chain of amino acids represented by the sequence SEQ ID NO1,
ou encore en ce qu'il s'agit d'une séquence d'acides aminés présentant une homologie d'au moins 80% de préférence 88% avec le fragment représenté par SEQ ID NO 2, contenu dans l'enchaînement d'acides aminés SEQ ID NO 1. or in that it is an amino acid sequence having a homology of at least 80%, preferably 88%, with the fragment represented by SEQ ID NO 2, contained in the sequence of amino acids SEQ ID NO 1.
On peut caractériser la capacité d'un polypeptide donné, de se comporter comme un récepteur de facteur de croissance, en mettant en oeuvre l'un des tests suivants.  The ability of a given polypeptide to behave like a growth factor receptor can be characterized by using one of the following tests.
L'invention permet de détecter la capacité potentielle d'un polyppetide de l'invention de se comporter comme un récepteur et de fixer un ligand. On peut en effet de façon équivalente rechercher si l'expression du polypeptide testé, par le virus MPLV préalablement modifié par la séquence nucléotidique codant pour ce polypeptide, intégrée au site contenant habituellement la séquence nucléotidique v-mpl, permet d'obtenir la prolifération in vitro de cellules transformées avec MPLV contenant le polypeptide testé. The invention makes it possible to detect the potential capacity of a polyppetide of the invention to behave like a receptor and to fix a ligand. In an equivalent way, it can be investigated whether the expression of the polypeptide tested, by the MPLV virus previously modified by the nucleotide sequence coding for this polypeptide, integrated into the site usually containing the nucleotide sequence v-mpl, makes it possible to obtain the in vitro proliferation of cells transformed with MPLV containing the tested polypeptide.
Les conditions de réalisation de ce test sont celles utilisées pour les expériences rapportées ci- après, correspondant à l'immortalisâtion par MPLV, de cellules de moelle osseuse en culture.  The conditions for carrying out this test are those used for the experiments reported below, corresponding to the immortalization by MPLV, of bone marrow cells in culture.
Pour détecter si une séquence nucléotidique codant pour un polypeptide de l'invention est susceptible d'intervenir dans la transmission d'un signal, on peut modifier MPLV en son site contenant habituellement la séquence v-mpl, par une séquence nucléotidique de l'invention, préalablement dépourvue d'un fragment nucléotidique choisi, dont on cherche à déterminer s'il intervient dans la transmission du signal. MPLV ainsi modifié peut être infecté, par exemple avec des cellules de moelle osseuse selon ce qui est décrit dans les exemples. On observe alors si l'élimination du fragment nucléotidique dont il est question ci-dessus, entraine l'absence de prolifération cellulaire.  To detect whether a nucleotide sequence coding for a polypeptide of the invention is capable of intervening in the transmission of a signal, it is possible to modify MPLV at its site usually containing the sequence v-mpl, by a nucleotide sequence of the invention , previously devoid of a selected nucleotide fragment, which one seeks to determine if it is involved in the transmission of the signal. MPLV thus modified can be infected, for example with bone marrow cells as described in the examples. It is then observed whether the elimination of the nucleotide fragment referred to above results in the absence of cell proliferation.
Un autre test pour détecter si un polypeptide de l'invention se comporte comme un récepteur capable à la foias de fixer une molécule choisie et de transmettre un signal après la fixation de cette molécule est le suivant :  Another test for detecting whether a polypeptide of the invention behaves as a receptor capable of faith to bind a chosen molecule and to transmit a signal after the binding of this molecule is as follows:
des cellules n'exprimant pas naturellement le polypeptide testé sont transformées avec ce dernier. Les cellules ainsi transformées sont mises en présence d'une molécule choisie susceptible de se comporter comme un ligand et on détermine leur capacité à répondre à cette molécule, notamment en observant s'il y a ou non prolifération cellulaire.  cells which do not naturally express the polypeptide tested are transformed with the latter. The cells thus transformed are placed in the presence of a selected molecule capable of behaving like a ligand and their capacity to respond to this molecule is determined, in particular by observing whether or not there is cell proliferation.
Des polypeptides particulièrement intéressants répondant à la définition de l'invention précédemment donnée sont par exemple des polypeptides comprenant un enchaînement d'acides aminés W S X W S, dans lequel X est un acide aminé quelconque et de préférence X correspond à l'arginine ou à la serine. Particularly interesting polypeptides meeting the definition of the invention given above are for example polypeptides comprising a chain of amino acids WSXWS, in which X is any amino acid and preferably X corresponds to arginine or serine.
La séquence W S A W S correspond au fragment compris dans v-mpl ou dans la séquence équivalente chez la souris, et la séquence W S S W S correspond au fragment h-mpl du gène cellulaire humain.  The sequence W S A W S corresponds to the fragment included in v-mpl or in the equivalent sequence in mice, and the sequence W S S W S corresponds to the h-mpl fragment of the human cell gene.
Il a été remarqué de façon tout à fait intéressante, que le peptide W S X W S peut être impliqué dans l'effet de différenciation in vivo de lignées hématopoiétiques, observé lors d'une infection par un retrovirus MPLV.  It has been remarked, quite interestingly, that the peptide W S X W S may be involved in the in vivo differentiation effect of hematopoietic lines, observed during infection by an MPLV retrovirus.
L'invention vise également l'ensemble des polypeptides satisfaisant aux conditions précédentes capables d'induire ou de participer à la différenciation cellulaire in vitro de lignées hématopoiétiques.  The invention also relates to all of the polypeptides satisfying the above conditions capable of inducing or participating in cell differentiation in vitro of hematopoietic lines.
D'autres polypeptides préférés selon l'invention sont les peptides caractérisés en ce qu ' ils comprennent un enchaînement d ' acides aminés correspondant soit à l'enchaînement identifié dans la liste des séquences sous la référence SEQ ID NO 3.  Other preferred polypeptides according to the invention are the peptides characterized in that they comprise a chain of amino acids corresponding either to the chain identified in the list of sequences under the reference SEQ ID NO 3.
Des polypeptides préférés répondant à la définition générale ci-dessus sont les polypeptides correspondant à l'une des séquences identifiées sous les références suivantes SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11.  Preferred polypeptides corresponding to the general definition above are the polypeptides corresponding to one of the sequences identified under the following references SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7 , SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11.
Un autre polypeptide selon l'invention est caractérisé en ce qu'il est codé par une séquence nucléotidique capable d'hybrider dans des conditions stringentes avec les sondes spécifiques de MPLV et correspondant aux fragments SACI-PSTI et PSTI-PSTI de 300 paires de bases chacun représentés à la figure 1.  Another polypeptide according to the invention is characterized in that it is coded by a nucleotide sequence capable of hybridizing under stringent conditions with the specific probes of MPLV and corresponding to the fragments SACI-PSTI and PSTI-PSTI of 300 base pairs each shown in Figure 1.
Ces sondes peuvent être de l'ADN ou de l'ARN.  These probes can be DNA or RNA.
Entre aussi dans le cadre de l'invention un polypeptide codé par une séquence nucléotidique capable d'hybrider avec l'une des sondes précédentes. L'invention concerne aussi un polypeptide entrant dans les définitions précédentes, caractérisé en ce qu'il répond à l'un des enchaînements d'acides aminés identifiés dans la liste des séquences, sous les références suivantes SEQ ID NO 12, SEQ ID NO 13. Also within the scope of the invention is a polypeptide encoded by a nucleotide sequence capable of hybridizing with one of the preceding probes. The invention also relates to a polypeptide falling within the preceding definitions, characterized in that it responds to one of the amino acid sequences identified in the list of sequences, under the following references SEQ ID NO 12, SEQ ID NO 13 .
L'invention vise de plus un récepteur de facteur de croissance, caractérisé en ce qu'il comprend la séquence d'acides aminés identifiée par SEQ ID NO 1.  The invention further relates to a growth factor receptor, characterized in that it comprises the amino acid sequence identified by SEQ ID NO 1.
L'invention vise aussi une forme soluble du récepteur comprenant notamment le domaine extracellulaire représenté par la séquence polypeptidique désignée par SEQ ID NO 7 chez l'homme ou SEQ ID NO 10 chez la souris.  The invention also relates to a soluble form of the receptor comprising in particular the extracellular domain represented by the polypeptide sequence designated by SEQ ID NO 7 in humans or SEQ ID NO 10 in mice.
Font également partie de l'invention, des protéines de fusion, caractérisées en ce qu'elles comprennent un polypeptide de l'invention en association avec un enchaînement d'acides aminés déterminé, notamment avec la gp70 du MPLV, en particulier en ce qu'il s'agit de la séquence d'acides aminés SEQ ID NO 14.  Also part of the invention are fusion proteins, characterized in that they comprise a polypeptide of the invention in association with a determined amino acid sequence, in particular with the gp70 of MPLV, in particular in that it is the amino acid sequence SEQ ID NO 14.
D'autres protéines de fusion peuvent être obtenues avec la protéine codée par le gène gag ou avec des glycoprotéines de virus, par exemple autre que le retrovirus, notamment une glycoprotéine de VSV.  Other fusion proteins can be obtained with the protein encoded by the gag gene or with virus glycoproteins, for example other than the retrovirus, in particular a VSV glycoprotein.
L'invention concerne également une séquence nucléotidique caractérisée en ce qu'elle code pour un polypeptide décrit ci-dessus.  The invention also relates to a nucleotide sequence characterized in that it codes for a polypeptide described above.
Des séquences nucléotidiques préférées sont celles qui correspondent à l'une quelconque des séquences nucléotidiques SEQ ID NO15, NO16, NO17, NO18, NO19, NO20 ou à un enchaînement complémentaire de ces séquences ou encore à une séquence capable d'hybrider avec l'une des séquences ci-dessus dans des conditions stringentes. Entrent également dans le cadre de l'invention les séquences nucléotidiques constituées par de l'ARN et qui correspondent aux enchaînements nucléotidiques ci-dessus décrits. L'invention vise également un hôte cellulaire recombinant, caractérisé en ce qu'il comprend une séquence nucléotidique telle que donnée ci-dessus. De préférence la séquence nucléotidique insérée dans l'hôte cellulaire est intégrée dans le génome de cet hôte. Différents hôtes cellulaires peuvent être choisis pour la réalisation de l'invention selon l'application recherchée. A titre d'exemple pour la recherche de molécules présentant éventuellement une affinité avec les polypetides de l'invention, on pourra utiliser des hôtes cellulaires tels que des bactéries, des virus, ou des phages, des cellules d'insectes ou de mammifères (COS, CHO) etc.. Preferred nucleotide sequences are those which correspond to any of the nucleotide sequences SEQ ID NO15, NO16, NO17, NO18, NO19, NO20 or to a sequence complementary to these sequences or to a sequence capable of hybridizing with one of the above sequences under stringent conditions. Also within the scope of the invention are the nucleotide sequences constituted by RNA and which correspond to the nucleotide sequences described above. The invention also relates to a recombinant cell host, characterized in that it comprises a nucleotide sequence as given above. Preferably, the nucleotide sequence inserted into the cell host is integrated into the genome of this host. Different cellular hosts can be chosen for carrying out the invention according to the desired application. As an example for the search for molecules possibly having an affinity with the polypetides of the invention, it is possible to use cellular hosts such as bacteria, viruses, or phages, cells of insects or mammals (COS , CHO) etc.
Un hôte cellulaire particulièrement préféré est le virus MPLV. En particulier l'invention concerne le virus MPLV contenant dans son génome une séquence nucléotidique codant pour le peptide représenté par la séquence SEQ ID NO2 sous forme d'une protéine fusion avec la glycoprotéine virale gp70.  A particularly preferred cellular host is the MPLV virus. In particular, the invention relates to the MPLV virus containing in its genome a nucleotide sequence coding for the peptide represented by the sequence SEQ ID NO2 in the form of a fusion protein with the viral glycoprotein gp70.
L'invention concerne également des vecteurs recombinants caractérisés en ce qu'ils comprennent une séquence nucléotidique telle que décrite précédemment, en un site non essentiel pour sa replication, sous le contrôle des séquences régulatrices nécessaires à son expression dans un hôte cellulaire déterminé.  The invention also relates to recombinant vectors characterized in that they comprise a nucleotide sequence as described above, at a site which is not essential for its replication, under the control of the regulatory sequences necessary for its expression in a determined cellular host.
Des vecteurs particulièrement adaptés pour la réalisation de l'invention sont par exemple le virus vaccine ou le baculovirus.  Vectors which are particularly suitable for carrying out the invention are, for example, the vaccinia virus or the baculovirus.
Font également partie de l'invention des sondes nucléotidiques comprenant au moins 9 nucléotides et de préférence de 15 à 30 nucléotides, capables d'hybrider avec une séquence nucléotidique selon l'invention, dans des conditions stringentes, cette sonde étant le cas échéant marquée.  Also part of the invention are nucleotide probes comprising at least 9 nucleotides and preferably from 15 to 30 nucleotides, capable of hybridizing with a nucleotide sequence according to the invention, under stringent conditions, this probe being optionally labeled.
Les sondes mises en oeuvre peuvent être spécifiques des séquences nucléotidiques de l'invention ou non spécifiques lorsqu'on recherche une séquence appartenant à un groupe plus large. The probes used can be specific for the nucleotide sequences of the invention or non-specific when looking for a sequence belonging to a larger group.
Des marqueurs adaptés à la réalisation des sondes de l'invention sont des marqueurs radioactifs classiques ou encore des marqueurs enzymatiques ou tout autre marqueur adapté.  Markers suitable for producing the probes of the invention are conventional radioactive markers or even enzymatic markers or any other suitable marker.
L'invention vise par ailleurs des anticorps polyclonaux ou monoclonaux, caractérisés en ce qu'ils reconnaissent un des polypeptides selon l'invention.  The invention further relates to polyclonal or monoclonal antibodies, characterized in that they recognize one of the polypeptides according to the invention.
Des anticorps particulièrement intéressants sont dirigés contre le domaine extra-cellulaire du polypeptide codé par le gène c-mpl  Particularly interesting antibodies are directed against the extracellular domain of the polypeptide encoded by the c-mpl gene.
Ces anticorps sont obtenus selon les méthodes classiques de production d'anticorps. En particulier pour la préparation des anticorps monoclonaux on aura recours à la méthode de Kolher et Milstein en particulier on prépare des anticorps monoclonaux par fusion cellulaire entre des cellules de myélome et des cellules spléniques de souris préalablement immunisées avec un polypeptide selon l'invention, conformément au procédé classique.  These antibodies are obtained according to conventional methods of antibody production. In particular for the preparation of monoclonal antibodies, the method of Kolher and Milstein will be used, in particular monoclonal antibodies are prepared by cell fusion between myeloma cells and spleen cells of mice previously immunized with a polypeptide according to the invention, in accordance to the classic process.
L'identification par les inventeurs du polypeptide responsable dans le virus MPLV de la maladie myéloproliférative permet la mise au point de moyens pour la détection in vitro d'une expression anormale d'un polypeptide décrit dans les pages précédentes, significative d'un état pathologique.  The identification by the inventors of the polypeptide responsible in the MPLV virus for myeloproliferative disease allows the development of means for the in vitro detection of an abnormal expression of a polypeptide described in the preceding pages, indicative of a pathological state. .
Dans ce but l'invention comprend un procédé pour la détection in vitro d'une expression anormale d'un polypeptide de l'invention, caractérisé en ce qu'il comprend :  For this purpose, the invention comprises a method for the in vitro detection of an abnormal expression of a polypeptide of the invention, characterized in that it comprises:
- la mise en contact d'un échantillon biologique susceptible de contenir le polypeptide recherché, avec des anticorps tels que définis précédemment,  - bringing a biological sample capable of containing the desired polypeptide into contact with antibodies as defined above,
- la détection d'une réaction immunologique du type antigène-anticorps. Par "expression anormale" du polypeptide, on entend toute expression de niveau quantitatif anormal, de même que toute expression d'un polypeptide modifié dont le fonctionnement serait altéré. - the detection of an immunological reaction of the antigen-antibody type. By "abnormal expression" of the polypeptide is meant any expression of abnormal quantitative level, as well as any expression of a modified polypeptide whose functioning would be impaired.
Un autre procédé pour la détection in vitro de l'expression anormale du polypeptide de l'invention peut être réalisé par la détection in vitro de l'expression anormale d'une séquence nucléotidique codant pour un peptide selon l'invention. Un tel procédé comprend :  Another method for the in vitro detection of the abnormal expression of the polypeptide of the invention can be carried out by the in vitro detection of the abnormal expression of a nucleotide sequence coding for a peptide according to the invention. Such a method includes:
a) la mise en contact d'un échantillon biologique susceptible de contenir la séquence nucléotidique recherchée, utilisée comme matrice, avec une amorce nucléotidique capable de s'hybrider avec la séquence nucléotidique recherchée, en présence des 4 différents nucléosides triphosphates, et d'un agent de polymérisation, dans des conditions d'hybridation telles que pour chaque séquence nucléotidique ayant hybride avec une amorce, un produit d ' elongation de chaque amorce, complémentaire de la matrice est synthétisé;  a) bringing a biological sample capable of containing the desired nucleotide sequence, used as template, into contact with a nucleotide primer capable of hybridizing with the desired nucleotide sequence, in the presence of the 4 different nucleoside triphosphates, and of a polymerization agent, under hybridization conditions such that for each nucleotide sequence having hybridized with a primer, an extension product of each primer, complementary to the template is synthesized;
b) la séparation de la matrice et du produit d'elongation obtenu, ce dernier pouvant alors également se comporter comme une matrice;  b) separation of the matrix and of the elongation product obtained, the latter then also being able to behave like a matrix;
c) la répétition de l'étape a) de façon à obtenir une quantité détectable des séquences nucléotidiques recherchées;  c) repeating step a) so as to obtain a detectable quantity of the nucleotide sequences sought;
d) la détection du produit d'amplification des séquences nucléotidiques.  d) detection of the amplification product of the nucleotide sequences.
Des détails pour la mise en oeuvre de la technique Details for the implementation of the technique
PCR sont donnés par exemple dans la demande EP 0200362 et dans la demande EP 0229701. PCR are given for example in application EP 0200362 and in application EP 0229701.
L'invention vise également un kit pour la détection in vitro de l'expression anormale des polypeptides selon l'invention, caractérisé en ce qu'il comprend des anticorps polyclonaux ou monoclonaux susceptibles de réagir avec le dit polypeptide, le cas échéant un réactif pour la détection de la réaction immunologique antigène-anticorps. The invention also relates to a kit for the in vitro detection of the abnormal expression of the polypeptides according to the invention, characterized in that it comprises polyclonal or monoclonal antibodies capable of reacting with said polypeptide, in the case if necessary a reagent for the detection of the antigen-antibody immunological reaction.
Un tel kit peut être envisagé pour la réalisation de différents types de tests notat-ment des tests RIA ou ELISA.  Such a kit can be envisaged for carrying out different types of tests, notably RIA or ELISA tests.
L'invention a trait également à un procédé pour la détection de l'affinité d'une molécule pour un polypeptide selon l'invention, caractérisé par :  The invention also relates to a method for detecting the affinity of a molecule for a polypeptide according to the invention, characterized by:
- la mise en contact de la molécule testée avec un hôte cellulaire préalablement modifié par une séquence nucléotidique selon l'invention, dans des conditions permettant l'expression de cette séquence, de façon à obtenir un polypeptide selon l'invention comportant au moins un site susceptible d'interagir avec la molécule testée, exposé à la surface de l'hôte cellulaire,  - bringing the tested molecule into contact with a cellular host previously modified with a nucleotide sequence according to the invention, under conditions allowing the expression of this sequence, so as to obtain a polypeptide according to the invention comprising at least one site capable of interacting with the molecule tested, exposed on the surface of the cell host,
- la détection de la formation d'un complexe entre la molécule testée et le polypeptide.  - detecting the formation of a complex between the molecule tested and the polypeptide.
L'invention concerne par ailleurs un médicament caractérisé en ce qu'il comprend un polypeptide répondant aux définitions précédentes, sous forme soluble, en combinaison avec un véhicule pharmaceutique acceptable. Un tel médicament peut être mis en oeuvre pour remédier à la production anormale des polypeptides de l'invention dans les cellules d'un malade.  The invention further relates to a medicament characterized in that it comprises a polypeptide corresponding to the preceding definitions, in soluble form, in combination with an acceptable pharmaceutical vehicle. Such a medicament can be used to remedy the abnormal production of the polypeptides of the invention in the cells of a patient.
Le médicament selon l'invention peut agir selon une réaction de compétition entre la forme soluble d'un polypeptide présent à titre de principe actif dans ce médicament et les polypeptides présents de façon anormale chez le patient traité.  The medicament according to the invention can act according to a competition reaction between the soluble form of a polypeptide present as an active ingredient in this medicament and the polypeptides abnormally present in the treated patient.
D'autres caractéristiques et avantages de l'invention apparaissent dans les figures et dans les exemples qui suivent.  Other characteristics and advantages of the invention appear in the figures and in the examples which follow.
Figure 1 : (A) Carte de restriction par les endonucléases de restriction du clone MPLV 107 et du clone F-MuLV 57. Les sites de restriction sont donnés pour les enzymes suivantes : B :BamHI, C :ClaI, E :EcoRI, H :HindIII, K :Kpnl, P :PstI, Pv : PyuII, SI : Sacl, SII :SacII, X : Xbal. Le cadre noir correspond à la région spécifique de MPLV. Les deux sondes dérivées de l'enveloppe de MPLV sont indiquées par des traits sous la carte de restriction. Figure 1: (A) Restriction endonuclease map of clone MPLV 107 and clone F-MuLV 57. Restriction sites are given for the following enzymes: B: BamHI, C: ClaI, E : EcoRI, H: HindIII, K: Kpnl, P: PstI, Pv: PyuII, SI: Sacl, SII: SacII, X: Xbal. The black frame corresponds to the specific region of MPLV. The two probes derived from the MPLV envelope are indicated by dashes under the restriction map.
(B) Démonstration de la spécificité des sondes dérivées de l'enveloppe de MPLV. Les cellules NIH 3T3 infectées avec le pseudotype amphotrope 4070 de MPLV sont utilisées comme source d'ARN poly A+ (5 μg, lignes A), le clone F-MuLV 57 (1 μg, lignes B), et le clone 2 Mus dunni non producteur infecté par MPLV (15 μq , lignes C). Les empreintes Northern blot ont hybride avec la sonde E57BS dérivée de l'enveloppe de F-MuLV obtenue, marquée selon la technique de nick-translation (échantillon nº 1), ou avec les deux sondes ARN dérivées de l'enveloppe de MPLV, SacI-PstI (échantillon n° 2) et Pstl-Pstl (échantillon nº 3). Les lignes M représentent l'ADN lambda/HindIII marqueur de poids moléculaire. Les flèches noires et les flèches claires indiquent les positions des ARN génomiques et sousgénomiques de l'enveloppe de F-MuLV et MPLV respectivement. (B) Demonstration of the specificity of the probes derived from the MPLV envelope. NIH 3T3 cells infected with the amphotropic pseudotype 4070 of MPLV are used as source of poly A + RNA (5 μg, lines A), the clone F-MuLV 57 (1 μg, lines B), and the clone 2 Mus dunni non-producer infected with MPLV (15 μq, lines C). Northern blot fingerprints hybridized with the E57BS probe derived from the F-MuLV envelope obtained, labeled according to the nick-translation technique (sample 1), or with the two RNA probes derived from the MPLV envelope, SacI -PstI (sample no. 2) and Pstl-Pstl (sample no. 3). Lanes M represent lambda / HindIII molecular weight marker DNA. The black arrows and the light arrows indicate the positions of the genomic and subgenomic RNAs of the envelope of F-MuLV and MPLV respectively.
Figure 2 : (A) Représentation schématique de la région env réarrangée de MPLV. Le cadre hachuré représente mpl, les cadres clairs représentent les séquences env de F-MuLV. L'astérisque dans le domaine MPL indique le codon stop. Figure 2: (A) Schematic representation of the rearranged env region of MPLV. The hatched box represents mpl, the light boxes represent the env sequences of F-MuLV. The asterisk in the MPL domain indicates the stop codon.
(B) Séquence d'acides aminés déduite de la région env de MPLV. Le cadre de lecture de 284 acides aminés est donné. Des acides aminés 1 à 100 correspondent aux séquences dérivées du gène env de F- MuLV. Les 184 acides aminés spécifiques de mpl sont encadrés. Les têtes de flèches indiquent la jonction des deux portions de l'enveloppe de F-MuLV et la jonction avec la séquence spécifique mpl, respectivement. Les astérisques indiquent les sites potentiels de N-glycosylation (Asn-X-Ser/Thr). Les séquences soulignées représentent le peptide signal de la gp70 et le domaine transmembranaire hydrophobe est écrit en caractère gras. (B) Amino acid sequence deduced from the env region of MPLV. The reading frame for 284 amino acids is given. Amino acids 1 to 100 correspond to sequences derived from the F-MuLV env gene. The 184 specific amino acids of mpl are boxed. The arrowheads indicate the junction of the two portions of the F-MuLV envelope and the junction with the specific sequence mpl, respectively. The asterisks indicate the sites N-glycosylation potentials (Asn-X-Ser / Thr). The underlined sequences represent the signal peptide of gp70 and the hydrophobic transmembrane domain is written in bold.
Figure 3 : Zoo blot Figure 3: Zoo blot
10 μg d'ADN de haut poids moléculaire, digéré par ECORI, de souris ICFW (ligne 1) et de Mus spretus (ligne 2), de rat (ligne 3), de vison (ligne 4), de vache (ligne 5), de chien (ligne 6) et d'humain (ligne 7) ont été hybrides avec les sondes ARN SacI-PstI et Pstl-PstI dans des conditions d'hybridation en forte stringence et lavés selon les protocoles détaillés dans la partie "procédures expérimentales". Figure 4 : Expression de c-mpl dans différents organes de souris.  10 μg of high molecular weight DNA, digested by ECORI, ICFW mice (line 1) and Mus spretus (line 2), rat (line 3), mink (line 4), cow (line 5) , dog (lane 6) and human (lane 7) were hybridized with the SacI-PstI and Pstl-PstI RNA probes under high stringency hybridization conditions and washed according to the protocols detailed in the "experimental procedures" section. ". Figure 4: Expression of c-mpl in different mouse organs.
Les empreintes Northern blot d'ARN poly A+ (5 μg) de cerveau de souris (ligne 1), de foie (ligne 2), de glande salivaire (ligne 3), de rate (ligne 4), de rein (ligne 5), de testicule (ligne 6), de thymus (ligne 7) ou de foie foetal (ligne 8) ont hybride avec les sondesNorthern blot imprints of poly A + RNA (5 μg) of mouse brain (line 1), liver (line 2), salivary gland (line 3), spleen (line 4), kidney (line 5 ), testis (line 6), thymus (line 7) or fetal liver (line 8) hybridized with probes
ARN SacI-PstI et Pstl-PstI. Les conditions d'hybridation et de lavage sont celles décrites dans la partie "procédures expérimentales". SacI-PstI and Pstl-PstI RNA. The hybridization and washing conditions are those described in the "experimental procedures" section.
Figure 5 : Etablissement in vitro des lignées cellulaires infectées par MPLV. Figure 5: Establishment in vitro of cell lines infected with MPLV.
Les cellules de moelle osseuse de souris C57BL/6 normales ont été infectées in vitro avec MPLV exempt de virus auxiliaire. Les cercles représentent les valeurs moyennes ± déviation standard des cellules non adhérentes pour cinq cultures infectées (cercles noirs) ou cinq cultures contrôle (cercles blancs). Les transferts des populations cellulaires non adhérentes sont indiqués en pointillés. Figure 6 : Analyse Southern des sites d'intégration proviraux dans les cultures infectées par MPLV. Les ADN préparés 5, 21, 97 jours après l'infection ont été digérés avec EcoRI et les empreintes ont été hybridées avec les sondes ARN de v-mpl. Au jour 97, L159 comportait des myéloblastes, L173 des mastocytes, L223 des mégacaryocytes et des erythroblastes. La flèche indique c-mpl. The bone marrow cells of normal C57BL / 6 mice were infected in vitro with MPLV free of helper virus. The circles represent the mean values ± standard deviation of the non-adherent cells for five infected cultures (black circles) or five control cultures (white circles). Transfers from non-adherent cell populations are shown in dotted lines. Figure 6: Southern analysis of proviral integration sites in cultures infected with MPLV. The DNAs prepared 5, 21, 97 days after infection were digested with EcoRI and the fingerprints were hybridized with the v-mpl RNA probes. At day 97, L159 included myeloblasts, L173 of mast cells, L223 of megakaryocytes and erythroblasts. The arrow indicates c-mpl.
Figure 7 : Comparaison des séquences d'acides aminés du domaine extracellulaire de v-mpl avec celui des récepteurs des cytokines hématopoiétiques. Figure 7: Comparison of amino acid sequences of the v-mpl extracellular domain with that of the hematopoietic cytokine receptors.
Les domaines extracellulaires du récepteur de IL-3 murin, du récepteur murin de l'EPO, du récepteur murin de l'IL-4, de la chaîne β du récepteur de l'IL-2, du récepteur humain de l'IL-6 et des récepteurs humains et murins de l'IL-7 ont été alignés avec v-mpl. Les résidus d'acides aminés conservés sont encadrés. La séquence consensus est celle qui a été décrite par Itoh et al, 1990 (Science Vol. 247, p. 324-327). The extracellular domains of the murine IL-3 receptor, the murine EPO receptor, the murine IL-4 receptor, the β chain of the IL-2 receptor, the human IL- receptor 6 and human and mouse IL-7 receptors were aligned with v-mpl. Preserved amino acid residues are boxed. The consensus sequence is that which has been described by Itoh et al, 1990 (Science Vol. 247, p. 324-327).
EXEMPLES EXAMPLES
Clonage moléculaire du provirus MPLV Molecular cloning of the MPLV provirus
Les expériences suivantes se rapportent à l'identification et à la caractérisation des réarrangements moléculaires intervenus au niveau de l'enveloppe du retrovirus MPLV.  The following experiments relate to the identification and characterization of the molecular rearrangements occurring at the level of the envelope of the MPLV retrovirus.
Pour caractériser la région réarrangée du gène env, une banque d'ADNc a été préparée en utilisant les ARN poly A+ des cellules NIH 3T3 affectées de façon productive avec le pseudotype amphotrope de MPLV. Les clones d'ADNc comportant la région env complète de MPLV ont été obtenus et deux sondes spécifiques de MPLV ont été préparées : il s'agit des fragments Sacl - PstI et PstI - PstI, de 300 paires de bases chacun (figure 1A). La spécificité de ces deux sondes de MPLV est représentée à la figure 1B. Elles reconnaissent les ARN génomiques (7,4 kb) et sous-génomiques obtenus par épissage (2,4 kb) mais n'hybrident pas avec F-MuLV ou avec des ARN amphotropes. To characterize the rearranged region of the env gene, a cDNA library was prepared using poly A + RNA from NIH 3T3 cells productively affected with the amphotropic pseudotype of MPLV. The cDNA clones comprising the complete env region of MPLV were obtained and two specific MPLV probes were prepared: these are the Sacl - PstI and PstI - PstI fragments, of 300 base pairs each (FIG. 1A). The specificity of these two MPLV probes is shown in Figure 1B. They recognize the genomic (7.4 kb) and sub-genomic RNAs obtained by splicing (2.4 kb) but do not hybridize with F-MuLV or with amphotropic RNAs.
Pour cloner un provirus MPLV biologiquement actif, une banque génomique a été préparée à partir de clones de cellules Mus Dunni non productrices, contenant une unique copie du provirus MPLV (Penciolelli et al, 1987). Parmi les 1,5 x 106 phages recombinants criblés avec les deux sondes spécifiques de MPLV, un clone unique a été obtenu (MPLV 107). Des analyses de restriction ont montré que ce clone contient le génome complet de MPLV à l'exception de la partie LTR 3' (figure 1A). To clone a biologically active MPLV provirus, a genomic library was prepared from clones of non-producing Mus Dunni cells, containing a single copy of the MPLV provirus (Penciolelli et al, 1987). Among the 1.5 × 10 6 recombinant phages screened with the two MPLV specific probes, a single clone was obtained (MPLV 107). Restriction analyzes have shown that this clone contains the complete MPLV genome with the exception of the 3 ′ LTR part (FIG. 1A).
Pour démontrer que cette entité moléculaire était responsable des caractéristiques de la maladie de myéloprolifération aiguë causée par MPLV, un provirus complet a été construit par ligation de MPLV 107 au clone F-MuLV 57 3' LTR. La construction résultante (MPLV3) a été cotransfectée avec l'ADN du virus auxiliaire F-MuLV dans les cellules NIH 3T3 selon un ratio molaire de 10/1 (MPLV3/F-MuLV). Après quelques passages cellulaires, le surnageant viral a été injecté par voie intraveineuse à des souris jeunes adultes DBA/2 connues pour leur résistance à l'érythroleucémie précoce induite par F-MULV (Ruscetti et al, 1981, J. Exp. Med. 154, 907-920). To demonstrate that this molecular entity was responsible for the characteristics of the acute myeloproliferative disease caused by MPLV, a complete provirus was constructed by ligation of MPLV 107 to the clone F-MuLV 57 3 'LTR. The resulting construct (MPLV3) was cotransfected with the DNA of the helper virus F-MuLV in NIH 3T3 cells at a molar ratio of 10/1 (MPLV3 / F-MuLV). After some cell passages, the viral supernatant was injected intravenously into young adult DBA / 2 mice known for their resistance to early erythroleukemia induced by F-MULV (Ruscetti et al, 1981, J. Exp. Med. 154, 907 -920).
Les animaux inoculés avec le surnageant des cultures transfectées avec l'ADN de F-MuLV seul étaient sains 6 mois après l'infection. Au contraire toutes les souris inoculées avec le surnageant des cultures transfectées avec les ADN de MPLV3 et F-MuLV ont rapidement développé une splénomégalie, une hyperleucocytose et une polycythémie et sont mortes deux mois après l'inoculation. Les cellules progénitrices des animaux malades ont été ensuite examinées in vitro pour déterminer quels étaient leurs besoins en facteur de croissance hématopoiétique. 100% des cellules progénitrices tardives des globules rouges (colonie formant des unités érythroïdes CFU-E) ont formé des colonies d' érythrocytes hémoglobinisés sans l'addition d'érythropoïétine (EPO), alors que 62% des cellules formant les colonies CFU-C dans la rate et 30% des CFU-C dans la moelle osseuse ont proliféré et se sont différenciées sans l'addition exogène de facteurs stimulant les colonies. Elles ont conduit à des colonies matures de granulocytes, de monocytes, de mégacaryocytes, d'érythrocytes, et à des colonies multipotentes contenant différentes lignées cellulaires.  Animals inoculated with the supernatant from cultures transfected with F-MuLV DNA alone were healthy 6 months after infection. On the contrary, all the mice inoculated with the supernatant from the cultures transfected with the DNAs of MPLV3 and F-MuLV rapidly developed splenomegaly, hyperleukocytosis and polycythemia and died two months after inoculation. The progenitor cells of the sick animals were then examined in vitro to determine what their hematopoietic growth factor needs were. 100% of the late progenitor cells of red blood cells (colony forming CFU-E erythroid units) formed colonies of hemoglobinized erythrocytes without the addition of erythropoietin (EPO), while 62% of cells forming CFU-C colonies in the spleen and 30% of CFU-C in the bone marrow proliferated and differentiated without the exogenous addition of colony stimulating factors. They have led to mature colonies of granulocytes, monocytes, megakaryocytes, erythrocytes, and to multipotent colonies containing different cell lines.
Ainsi le clone MPLV3 était biologiquement actif et ses propriétés ne pouvaient être distinguées de celles de l'isolat MPLV d'origine. Séquence et structure de la région env de MPLV Thus the clone MPLV3 was biologically active and its properties could not be distinguished from those of the original MPLV isolate. Sequence and structure of the MPLV env region
Les gènes env réarrangés des ADNc de MPLV et du clone génomique ont été séquences et se sont avérés identiques. La séquence nucléotidique a été analysée montrant que le gène env de MPLV comprend des séquences dérivées du gène env de F-MuLV et des séquences non virales. Comme l'illustre la figure 2A deux délétions sont apparues dans le gène env de F-MuLV : la première entre les positions 5969 et 6505 et la seconde entre les positions 6615 et 7513 (Koch et al, 1983, J. Virol. 45, 1-9). Le gène env de MPLV est donc une région complexe composée entre les extrémités 5' et 3' de 191 paires de base de l'extrémité 5' du gène env de F- MuLV (jusqu'à la position 5969) suivie par 110 paires de base de la région centrale du gène env de F-MuLV (entre les positions 6506 et 6615) puis par une région non virale de 623 nucléotides et finalement la partie 3' de la protéine 15E de F-MuLV (à partir de la position 7513).  The rearranged env genes of the MPLV cDNAs and the genomic clone were sequenced and found to be identical. The nucleotide sequence was analyzed showing that the env gene for MPLV includes sequences derived from the env gene for F-MuLV and non-viral sequences. As illustrated in FIG. 2A, two deletions appeared in the env gene of F-MuLV: the first between positions 5969 and 6505 and the second between positions 6615 and 7513 (Koch et al, 1983, J. Virol. 45, 1-9). The env gene for MPLV is therefore a complex region composed between the 5 'and 3' ends of 191 base pairs of the 5 'end of the F-MuLV env gene (up to position 5969) followed by 110 pairs of base of the central region of the F-MuLV env gene (between positions 6506 and 6615) then by a non-viral region of 623 nucleotides and finally the 3 ′ part of the 15E protein of F-MuLV (from position 7513 ).
Le gène env de MPLV a un cadre de lecture ouvert de 284 acides aminés à partir du codon d'initiation ATG de la gp70 et se terminant à un codon de terminaison TAG à l'intérieur de la séquence spécifique de MPLV (figure 2B) et code potentiellement pour une protéine de fusion env d'un poids moléculaire de 31 kilodaltons. Cette protéine de fusion env-vmpl comprend 64 acides aminés de la partie NH2-terminale de la gp70 de F-MuLV incluant le peptide signal, 36 acides aminés de la région centrale du gène env de F-MuLV et 184 acides aminés spécifiques de MPLV. The MPLV env gene has an open reading frame of 284 amino acids starting from the ATG initiation codon of gp70 and ending at a TAG termination codon inside the specific sequence of MPLV (FIG. 2B) and potentially codes for an env fusion protein with a molecular weight of 31 kilodaltons. This env-vmpl fusion protein comprises 64 amino acids of the NH 2 -terminal part of the gp70 of F-MuLV including the signal peptide, 36 amino acids of the central region of the env gene of F-MuLV and 184 specific amino acids of MPLV.
Une courbe d'hydrophobicité (Kyte et al, 1982, J. Mol Biol. 157, 105-132) de la séquence d'acides aminés du produit du gène env de MPLV a révélé en plus des 34 acides aminés hydrophobes du peptide signal de la gp70, que le domaine spécifique de MPLV contenait une région de 22 acides aminés non chargés s'étendant de l'acide aminé Ile 143 à l'acide aminé Leu 165, qui peut correspondre à un domaine membranaire. La protéine env nature de MPLV serait ainsi constituée d'un domaine extracellulaire de 109 acides aminés avec un site potentiel de glycosylation, un domaine transmembranaire de 22 acides aminés et un domaine intracytoplasmique deA hydrophobicity curve (Kyte et al, 1982, J. Mol Biol. 157, 105-132) of the amino acid sequence of the MPLV env gene product revealed, in addition to the 34 hydrophobic amino acids of the signal peptide. gp70, that the specific domain of MPLV contained a region of 22 uncharged amino acids extending from amino acid Ile 143 to amino acid Leu 165, which can correspond to a membrane domain. The env nature protein of MPLV would thus consist of an extracellular domain of 109 amino acids with a potential glycosylation site, a transmembrane domain of 22 amino acids and an intracytoplasmic domain of
119 acides aminés sans séquence pour une activité kinase (Hanks et al, 1988, Science Vol. 241, p. 42-52). 119 amino acids without sequence for kinase activity (Hanks et al, 1988, Science Vol. 241, p. 42-52).
Une recherche parmi les données EMBL (nucléotides et protéines) a indiqué que la séquence spécifique de MPLV, désignée par v-mpl ne correspondait pas à un gène identifié jusqu'à présent.  A search of the EMBL (nucleotides and proteins) data indicated that the specific sequence of MPLV, designated by v-mpl, did not correspond to a gene identified so far.
Région env de MPLV contenant une séquence cellulaire unique fortement conservée chez les mammifères MPLV env region containing a unique highly conserved cell sequence in mammals
La présence d'un locus c-mpl éventuel chez la souris, le rat, le vison, la vache, le chien et l'homme a été recherchée. L'hybridation de l'ADN digéré par EcoRI avec les deux sondes ARN de v-mpl a permis de détecter des bandes nettes dans des conditions d'hybridation stringentes, indiquant la présence d'une contrepartie cellulaire (c-mpl) à la séquence v-mpl dans les 6 espèces testées (figure 3) .  The presence of a possible c-mpl locus in mice, rats, mink, cows, dogs and humans was investigated. Hybridization of the DNA digested by EcoRI with the two v-mpl RNA probes made it possible to detect clear bands under stringent hybridization conditions, indicating the presence of a cellular counterpart (c-mpl) to the sequence v-mpl in the 6 species tested (Figure 3).
On a ensuite recherché l'expression de c-mpl dans différents tissus de souris. Des empreintes Northern blots d'ARN poly A+ préparées à partir de foie foetal et à partir de différents organes de souris adultes ont hybride avec des sondes ARN v-mpl. Comme le montre la figure 4 une seule bande d'ARNm de 3,0 kb a pu être détectée dans la rate adulte (ligne 4) et dans le foie foetal (ligne 8). Un transcript similaire était également présent dans la moelle osseuse. Au contraire, aucun transcript n'a été détecté dans le cerveau, le foie, les glandes salivaires, le rein, les testicules ou le thymus de souris adultes. We then looked for the expression of c-mpl in different mouse tissues. Northern blot fingerprints of poly A + RNA prepared from fetal liver and from different organs of adult mice hybridized with v-mpl RNA probes. As shown in FIG. 4, a single 3.0 kb mRNA band could be detected in the adult spleen (line 4) and in the fetal liver (line 8). A similar transcript was also found in the bone marrow. On the contrary, no transcript was detected in the brain, the liver, the salivary glands, the kidney, the testes or the thymus of adult mice.
Les propriétés leucémogènes de MPLV peuvent être ainsi attribuées à la présence d'un nouvel oncogène, v-mpl, transduit à partir de séquences cellulaires conservé dans la phylogénie des mammifères et transcrit dans des tissus hématopoiétiques normaux murins. The leukemogenic properties of MPLV can thus be attributed to the presence of a new oncogene, v-mpl, transduced from cellular sequences. preserved in the phylogeny of mammals and transcribed in normal murine hematopoietic tissues.
MPLV transforme des progéniteurs hematopoietigues in vitro MPLV transforms hematopoietigues progenitors in vitro
Pour déterminer si MPLV pouvait directement transformer des cellules hématopoiétiques et pour analyser la nature des cellules cibles du virus, des cellules de moelle osseuse ont été infectées in vitro avec MPLV exempt de virus auxiliaire, obtenu dans une lignée cellulaire d'empaquetage psi-CRE (Danos et Mulligan, 1988, P.N.A.S. USA 85, 6460-6464). Le test a été réalisé dans un milieu d'agarose à une faible concentration cellulaire pour éviter de stimuler la formation de colonies par des facteurs endogènes sécrétés par des cellules accessoires. Dans des expériences répétées, seules quelques colonies autonomes ont été détectées. Cependant lorsque l'infection a été réalisée avec des cellules de moelle osseuse enrichies, en progéniteurs immatures mis en division, par un prétraitement des souris avec le 5- fluorouracil (5-FU) (Hodgson et Bradley, 1979, Nature Vol. 281, p. 381-382) des colonies se sont développées spontanément en nombre significatif. Environ la moitié étaient des colonies d'une seule lignée telles que des colonies de mégacaryocytes ou de granulocytes ou des colonies érythroïdes. Les autres colonies étaient des colonies mixtes dont environ 20% présentaient trois ou plus de trois lignées de différenciation. Des expériences de repiquage de colonies contenant une ou deux lignées de différenciation n'ont pas conduit à la production de colonies secondaires indiquant que ces colonies résultaient de progéniteurs irréversiblement engagés dans la différenciation. Au contraire plus de 65% des colonies contenant plusieurs lignées de différenciation (12/18) ont produit un nombre variable de colonies secondaires (de 7 à 286) exprimant une ou deux lignées de différenciation mais également des colonies macroscopiques dans lesquelles au moins trois lignées étaient présentes. Certaines de ces colonies (3/18) ont produit des colonies mixtes tertiaires. Ceci indique que MPLV est capable de promouvoir la prolifération et la différenciation terminale à la fois de cellules souches multipotentes et des cellules progénitrices déjà engagées dans la différenciation. To determine whether MPLV could directly transform hematopoietic cells and to analyze the nature of the target cells of the virus, bone marrow cells were infected in vitro with MPLV free of helper virus, obtained in a packaging cell line psi-CRE ( Danos and Mulligan, 1988, PNAS USA 85, 6460-6464). The test was carried out in an agarose medium at a low cell concentration to avoid stimulating the formation of colonies by endogenous factors secreted by accessory cells. In repeated experiments, only a few autonomous colonies were detected. However, when the infection was carried out with cells of bone marrow enriched, in immature progenitors divided, by a pretreatment of the mice with 5-fluorouracil (5-FU) (Hodgson and Bradley, 1979, Nature Vol. 281, p. 381-382) colonies developed spontaneously in significant numbers. About half were single line colonies such as megakaryocyte or granulocyte colonies or erythroid colonies. The other colonies were mixed colonies, of which about 20% had three or more than three differentiation lines. Colonization experiments containing one or two differentiation lines did not lead to the production of secondary colonies indicating that these colonies were the result of irreversibly differentiated progenitors. On the contrary, more than 65% of the colonies containing several differentiation lines (12/18) produced a variable number of secondary colonies (from 7 to 286) expressing one or two differentiation lines but also macroscopic colonies in which at least three lines were present. Some of these colonies (3/18) produced mixed tertiary colonies. This indicates that MPLV is capable of promoting the proliferation and terminal differentiation of both multipotent stem cells and progenitor cells already engaged in differentiation.
MPLV immortalise des cellules de moelle osseuse en culture et induit leur différenciation MPLV immortalizes bone marrow cells in culture and induces their differentiation
Lorsque des cellules de moelle osseuse infectées par MPLV et provenant soit de souris normales soit de souris prétraitées avec 5-FU ont été mises en culture, une augmentation du pourcentage de 10 à 20 fois de la fréquence de cellules progénitrices a été observée en même temps qu'une augmentation dans le pourcentage de colonies indépendantes de facteurs de croissance. L'évolution de telles cultures est montrée à la figure 5. Alors que dans les cultures non infectées les cellules ont une faible croissance, les cultures infectées par MPLV contiennent des cellules non adhérentes se divisant rapidement qui peuvent être transférées dans de nouveaux flacons dépourvus de cellules stromales nourricières adhérentes. Les cellules ont continué à proliférer en formant des lignées cellulaires permanentes poussant en suspension et comportant des érythroblastes en phase terminale de différenciation, des mégacaryocytes et des polymorphonucléaires en association avec des cellules blastiques immatures. Lorsque ces cellules sont cultivées en milieu semi-solide, différents types de colonies autonomes contenant des cellules matures se sont développées.  When MPLV infected bone marrow cells from either normal mice or mice pretreated with 5-FU were cultured, a 10-20 fold percentage increase in the frequency of progenitor cells was observed at the same time than an increase in the percentage of colonies independent of growth factors. The evolution of such cultures is shown in Figure 5. While in uninfected cultures the cells have low growth, cultures infected with MPLV contain non-adherent rapidly dividing cells which can be transferred to new vials lacking Adhering nourishing stromal cells. The cells continued to proliferate by forming permanent cell lines growing in suspension and comprising terminally differentiated erythroblasts, megakaryocytes and polymorphonuclear cells in association with immature blast cells. When these cells are grown in semi-solid medium, different types of autonomous colonies containing mature cells have developed.
Quatre à six semaines après l'infection la majorité des lignées cellulaires évoluaient vers un phénotype plus restreint qui restait apparemment stable pendant des mois de culture continue. Un examen morphologique des cellules en suspension et des colonies obtenues dans le milieu semi-solide a montré que parmi les 24 lignées, une lignée restait multipotente, cinq contenaient des mégacaryocytes matures et des érythroblastes hémoglobinisés, cinq étaient composés de cellules mégacaryocytiques , cinq étaient des mastocytes, quatre des cellules myelomonocytiques, deux contenaient des cellules érythroblastiques en phase de différenciation et deux correspondaient à des cellules blastiques immatures. Des expériences ont eu lieu pour déterminer si ces cultures étaient de type polyclonal ou monoclonal. Il en résulte que ces lignées cellulaires permanentes sont obtenues à partir de la croissance d'une seule ou de quelques souches multipotentes infectées par MPLV, dans lesquelles la restriction des capacités de différenciation se produit à un stade ultérieur. Le caractère malin des lignées cellulaires a été démontré en faisant une injection sous-cutanée de 2 x 106 cellules chez des souris syngéniques irradiées avec une dose subléthale (5 Gy) ou chez des souris nude. Aucune tumeur ne s'est développée au site de l'inoculation lorsque les cellules provenant d'une culture ayant moins de 4 mois ont été greffées mais 6 des 10 lignées cellulaires inoculées après plus de 7 mois ont produit des tumeurs hématopoiétiques de la nature de ces lignées cellulaires greffées après une latence d'environ 30 jours. Four to six weeks after infection, the majority of cell lines evolved into a more restricted phenotype which remained apparently stable. for months of continuous cultivation. A morphological examination of the cells in suspension and of the colonies obtained in the semi-solid medium showed that among the 24 lines, one line remained multipotent, five contained mature megakaryocytes and hemoglobinized erythroblasts, five were composed of megakaryocytic cells, five were mast cells, four of the myelomonocytic cells, two contained differentiated erythroblastic cells and two corresponded to immature blast cells. Experiments were carried out to determine whether these cultures were of the polyclonal or monoclonal type. As a result, these permanent cell lines are obtained from the growth of a single or a few multipotent strains infected with MPLV, in which the restriction of differentiation capacities occurs at a later stage. The malignant character of the cell lines has been demonstrated by making a subcutaneous injection of 2 × 10 6 cells in syngeneic mice irradiated with a sublethal dose (5 Gy) or in nude mice. No tumor developed at the site of inoculation when cells from a culture less than 4 months old were grafted, but 6 of 10 cell lines inoculated after more than 7 months produced hematopoietic tumors of the nature of these cell lines grafted after a latency of around 30 days.
Pour rechercher si la croissance autonome des cellules résultait de la production d'un facteur de croissance des milieux conditionnés concentrés 10 fois de différentes lignées cellulaires ont été testés sur la lignée cellulaire indicatrice FDC-P1 (Dexter et al, 1980, J. Exp. Med. 152, 1036-1047). Aucune incorporation de 3H thymidine n'a pu être détectée. La croissance continue n'était donc pas soutenue par la sécrétion d'IL3 ou de GM-CSF. De plus des analyses Northern blot n'ont pas révélé l'ARNm de l'IL3, du GM- CSF, du G-CSF ou de l'EPO dans douze lignées cellulaires examinées excepté pour une lignée cellulaire qui exprimait l'ARNm GM-CSF. To test whether the autonomous growth of cells resulted from the production of a growth factor of conditioned media concentrated 10 times from different cell lines were tested on the indicator cell line FDC-P1 (Dexter et al, 1980, J. Exp. Med. 152, 1036-1047). No incorporation of 3 H thymidine could be detected. Continued growth was therefore not supported by the secretion of IL3 or GM-CSF. In addition, Northern blot analyzes did not reveal the mRNA of IL3, GM-CSF, G-CSF or EPO in twelve cell lines examined except for a cell line which expressed the GM- mRNA CSF.
Ainsi ces observations indiquent que MPLV seul est capable de transformer directement des progéniteurs hématopoiétiques engagés dans la différenciation et multipotentiels et conduit à l'émergence rapide de différentes lignées cellulaires immortalisées indépendantes de facteurs de croissance qui conservent la capacité de se différencier spontanément.  Thus, these observations indicate that MPLV alone is capable of directly transforming hematopoietic progenitors engaged in differentiation and multipotentials and leads to the rapid emergence of different immortalized cell lines independent of growth factors which retain the capacity to differentiate spontaneously.
Les expériences décrites précédemment ont montré que la pathogénicité de MPLV n'était pas due à des modifications majeures dans sa partie LTR. L'analyse de la séquence de la région env de MPLV a montré que le gène env de MPLV ne contenait pas de séquences apparentées à des séquences de virus MCF mais qu'une séquence de 1,5 kb de l'enveloppe de F-MuLV était délétée et remplacée par une nouvelle séquence non virale de 0,7 kb qui ne présentait pas d'homologie avec des gènes connus. Cette nouvelle séquence, v-mpl est d'origine cellulaire et est conservée parmi les mammifères y compris l'homme. Le proto-oncogène c-mpl est transcrit sous forme d'un ARNm de 3,0 kb dans la rate de souris adulte et dans la moelle osseuse et dans le foie foetal. La localisation chromosomique c-mpl est le chromosome 4 de la souris et le chromosome humain 1-p34.  The experiments described above have shown that the pathogenicity of MPLV was not due to major modifications in its LTR part. Analysis of the sequence of the MPLV env region showed that the MPLV env gene did not contain sequences related to sequences of MCF virus but that a 1.5 kb sequence of the envelope of F-MuLV was deleted and replaced with a new 0.7 kb non-viral sequence that did not show homology with known genes. This new sequence, v-mpl is of cellular origin and is conserved in mammals including man. The proto-oncogene c-mpl is transcribed as a 3.0 kb mRNA in the spleen of adult mice and in bone marrow and in the fetal liver. The chromosomal location c-mpl is the mouse chromosome 4 and the human chromosome 1-p34.
Le polypeptide env-mpl présente les caractéristiques générales d'une protéine transmembranaire : il contient le peptide signal de la gp70 à sa partie N-terminale et un domaine transmembranaire unique, indiquant que la partie N- terminale de la molécule est extracellulaire et la partie C-terminale est intra-cytoplasmique. La séquence d'acides aminés du domaine extracellulaire de la protéine v-mpl présente des similitudes avec les récepteurs hématopoiétiques des cytokines récemment clones comme la chaîne β de IL2R, IL3R, IL4R, IL6R, IL7R, GM-CSFR, G-CFSR, EPO-R, de même qu'avec le récepteur de la prolactine. Ainsi cette séquence contient un motif W S X W S dans le domaine extracellulaire proche de la région transmembranaire et ne contient pas de séquence consensus pour une activité protéinekinase dans le domaine intra-cytoplasmique. On a remarqué également que le domaine cytoplasmique de v-mpl contient beaucoup de prolines (14/19, 12%) et de serines (13/119, 11%) comme c'est le cas d'autres récepteurs. Il en résulte que MPLV a transduit une forme tronquée d'un récepteur de facteur de croissance hématopoiétique. L'expression du gène c-mpl endogène observé uniquement dans les cellules spléniques, la moelle osseuse et le foie foetal confirme cette hypothèse. The env-mpl polypeptide has the general characteristics of a transmembrane protein: it contains the signal peptide of gp70 at its N-terminal part and a unique transmembrane domain, indicating that the N-terminal part of the molecule is extracellular and the part C-terminal is intra-cytoplasmic. The amino acid sequence of the extracellular domain of protein v-mpl has similarities to the hematopoietic receptors of recently cloned cytokines such as the β chain of IL2R, IL3R, IL4R, IL6R, IL7R, GM-CSFR, G-CFSR, EPO-R, as well as to the receptor for prolactin. Thus, this sequence contains a WSXWS motif in the extracellular domain close to the transmembrane region and does not contain a consensus sequence for proteinkinase activity in the intra-cytoplasmic domain. It has also been noted that the cytoplasmic domain of v-mpl contains many prolines (14/19, 12%) and serines (13/119, 11%) as is the case with other receptors. As a result, MPLV transduced a truncated form of a hematopoietic growth factor receptor. The expression of the endogenous c-mpl gene observed only in spleen cells, bone marrow and fetal liver confirms this hypothesis.
PROCEDURES EXPERIMENTALES EXPERIMENTAL PROCEDURES
Cellules, virus et souris Cells, viruses and mice
Des cellules NIH 3T3 et Mus dunni ont été utilisées. L'isolement du clone 2 de Mus dunni non producteur de MPLV a été décrit dans la publication de NIH 3T3 and Mus dunni cells were used. The isolation of clone 2 of Mus dunni not producing MPLV was described in the publication of
Penciolelli et al, 1987 (J. Virol. 61, 579-583). Le pseudotype MPLV amphotrope a été obtenu en surinfectant le clone 2 de Mus dunni avec le virus auxiliaire amphotrope 4070 A (Chattopadhyay et al, 1981; J. Virol. 39, 777-791). Penciolelli et al, 1987 (J. Virol. 61, 579-583). The amphotropic MPLV pseudotype was obtained by superinfecting clone 2 of Mus dunni with the amphotropic helper virus 4070 A (Chattopadhyay et al, 1981; J. Virol. 39, 777-791).
Des souris DBA/2J, C57BL/6J et des souris nude ont été obtenues de Iffa Credo (l'Arbesle, France) et élevées dans des conditions exemptes d'agents pathogènes. Des animaux âgés de 6 à 8 semaines ont été utilisés dans toutes les expériences.  DBA / 2J, C57BL / 6J and nude mice were obtained from Iffa Credo (l'Arbesle, France) and bred under pathogen-free conditions. Animals 6 to 8 weeks old were used in all of the experiments.
Préparation d'ARN et analyse Northern Blot RNA preparation and Northern Blot analysis
L'ARN total a été purifié selon la méthode au guanidium thiocyanate/CsCl (Chirgwin et al, 1979; Biochemistry 18, 5294-5299) et les ARN poly A+ ont été sélectionnés par chromatographie sur colonne oligo dT- cellulose. The total RNA was purified according to the guanidium thiocyanate / CsCl method (Chirgwin et al, 1979; Biochemistry 18, 5294-5299) and the poly A + RNAs were selected by chromatography on an oligo dT-cellulose column.
Pour l'analyse Northern Blot, 5 μg d'ARN poly A+ ont été dénaturés dans un tampon de glyoxal selon Me Master et Carmichael, 1977 (Proc. Natl. Acad. Sci. USA 74, 4835-4838). L'électrophorèse a eu lieu dans des gels d'agarose 1,1% dans 10 mM de tampon de phosphate de Na/Na2. Les transferts d'ARN ont été réalisés sur nitrocellulose Hybond C-extra (Amersham) tel que décrit par Thomas, 1980 (Proc. Natl. Acad. Sci. USA 77, 5201-5205). Les membranes ont été préhybridées pendant 5 heures à 55ºC dans 50% de formamide, 4x SSC, 0,05 M Na/Na2 phosphate, 1x Denhardt, 500 μg/ml ARNt de levure et 250 μg/ml d'ADN de sperme de hareng. 107 cpm d'une sonde ARN marquée au 32P ont été ajoutés et l'hybridation a été réalisée pendant 40 heures à 55ºC. Les membranes ont été lavées deux fois 5 minutes à la température ambiante dans 2x SSC - 0,1% SDS, 2 fois 30 minutes à 65ºC dans 2X SSC - 0,1% SDS et 2 fois 30 minutes à 65ºC dans 0,1x SSC - 0,1% SDS. For the Northern Blot analysis, 5 μg of poly A + RNA were denatured in a glyoxal buffer according to Me Master and Carmichael, 1977 (Proc. Natl. Acad. Sci. USA 74, 4835-4838). The electrophoresis took place in 1.1% agarose gels in 10 mM Na / Na 2 phosphate buffer. The RNA transfers were carried out on Hybond C-extra nitrocellulose (Amersham) as described by Thomas, 1980 (Proc. Natl. Acad. Sci. USA 77, 5201-5205). The membranes were prehybridized for 5 hours at 55ºC in 50% formamide, 4x SSC, 0.05 M Na / Na 2 phosphate, 1x Denhardt, 500 μg / ml yeast tRNA and 250 μg / ml sperm DNA herring. 10 7 cpm of a 32 P labeled RNA probe were added and the hybridization was carried out for 40 hours at 55 ° C. The membranes were washed twice 5 minutes at room temperature in 2x SSC - 0.1% SDS, 2 times 30 minutes at 65ºC in 2X SSC - 0.1% SDS and 2 times 30 minutes at 65ºC in 0.1x SSC - 0.1% SDS.
Analyse Southern Blot Southern blot analysis
Les ADN ont été digérés avec des endonucléases de restriction appropriées selon les conditions indiquées par le fabricant et déposés sur un gel d'agarose à 0,8%. Après l'électrophorèse, les ADN ont été transférés sur des membranes de nitrocellulose selon la méthode de Southern (1975; J. Mol Biol. 98, 503-518). Les membranes ont été hybridées avec 107 cpm de sondes marquées au 32p dans des conditions décrites pour le Northern Blot. The DNAs were digested with appropriate restriction endonucleases according to the conditions indicated by the manufacturer and deposited on a 0.8% agarose gel. After electrophoresis, the DNAs were transferred to nitrocellulose membranes according to the method of Southern (1975; J. Mol Biol. 98, 503-518). The membranes were hybridized with 10 7 cpm of 32 p-labeled probes under conditions described for the Northern Blot.
Construction de la banque de l'ADNc de l'enveloppe de MPLV Construction of the MPLV envelope cDNA library
L'ADNc a été synthétisé à partir de l'ARN poly A+ préparé à partir de cellules NIH 3T3 en phase exponentielle de croissance, infectées de façon productive avec MPLV pseudotypé par le virus amphotrope auxiliaire 4070 A (MPLV comportant l'enveloppe du virus 4070 A), en utilisant le kit de synthèse d'ADNc de Amersham. Des ADNc à extrémité franche ont été ligués à un vecteur pSPT18 déphosphorylé digéré avec Smal en présence d'ADN T4 ligase. The cDNA was synthesized from poly A + RNA prepared from NIH 3T3 cells in exponential growth phase, infected productively with MPLV pseudotyped by the amphotropic helper virus 4070 A (MPLV comprising the envelope of the virus 4070 A), using the Amersham cDNA synthesis kit. Blunt-ended cDNAs were ligated to a dephosphorylated pSPT18 vector digested with SmaI in the presence of T4 DNA ligase.
Des bactéries LM 1035 compétentes ont été transformées et étalées sur des boites d'agar contenant de l'ampicilline. Les colonies contenant les plasmides recombinants ont été transférées sur des filtres de nitrocellulose. L'identification des clones contenant l'ADNc de l'enveloppe (env) de MPLV a été réalisée par hybridation in situ telle que décrite par Sambrook et al, 1989 (Cold Spring Harbor Laboratory Press), avec une sonde E57BS (Moreau-Gachelin et al, 1983 Biochimie 65, 259-266), marquée au 32p par la technique de "nick-translation". Competent LM 1035 bacteria were transformed and spread on agar dishes containing ampicillin. The colonies containing the recombinant plasmids were transferred to nitrocellulose filters. The identification of the clones containing the cDNA of the envelope (env) of MPLV was carried out by in situ hybridization as described by Sambrook et al, 1989 (Cold Spring Harbor Laboratory Press), with an E57BS probe (Moreau-Gachelin et al, 1983 Biochemistry 65, 259-266), marked at 32 p by the "nick-translation" technique.
Construction de la banque génomique de MPLV Construction of the MPLV genomic library
L'ADN de haut poids moléculaire a été extrait selon Souyri et al, 1983 (Proc. Natl. Acad. Sci. USA 80, 6676-6679) à partir du clone 2 de Mus dunni non producteur de MPLV et partiellement digéré avec l'endonucléase de restriction Sau3A. Les fragments d'ADN (10 à 15 Kb) ont été purifiés par centrifugation sur gradient de sucrose et ligués à des bras BamH1 du bactériophage EMBL3 après encapsidation (Stratagene). Après encapsulation in vitro (Gigapack, Stratagene), les phages recombinants contenant l'ADN de MPLV ont été identifiés selon la technique de Benton et al, 1977 (Science 196, 180-182) par hybridation avec des sondes ARN spécifiques de MPLV. Les filtres ont été préhybridés 5 heures à 42ºC dans 50% formamide, 5x SSC, 5x Denhardt, 0,1% SDS, 50 mM de phosphate Na/Na2 pH 6,5, et 250 μg/ml d'ADN de sperme de hareng (2ml par filtre). L'hybridation avec des sondes ARN de MPLV marqué au 32p a été réalisée pendant 20 heures à 42ºC dans 50% formamide, 5x SSC, 1x Denhardt, 0,1% SDS, 50 mM de phosphate Na/Na2 pH 6,5, et 250 μg/ml de sperme de hareng (1ml de tampon et 2.106 cpm de sonde ARN par filtre). Les filtres ont été lavés deux fois 10 minutes à température ambiante dans 2x SSC - 0,1% SDS, 30 minutes avec 2x SSC - 0,1% SDS, et 2 fois 30 minutes avec 0,2x SSC - 0,1% SDS, chaque fois à 65ºC. Séguençage de l'ADN The high molecular weight DNA was extracted according to Souyri et al, 1983 (Proc. Natl. Acad. Sci. USA 80, 6676-6679) from clone 2 of Mus dunni non-MPLV producer and partially digested with restriction endonuclease Sau3A. The DNA fragments (10 to 15 Kb) were purified by centrifugation on a sucrose gradient and ligated to BamH1 arms of the bacteriophage EMBL3 after packaging (Stratagene). After in vitro encapsulation (Gigapack, Stratagene), the recombinant phages containing the MPLV DNA were identified according to the technique of Benton et al, 1977 (Science 196, 180-182) by hybridization with RNA probes specific for MPLV. The filters were prehybridized for 5 hours at 42ºC in 50% formamide, 5x SSC, 5x Denhardt, 0.1% SDS, 50 mM Na / Na 2 phosphate pH 6.5, and 250 μg / ml of sperm DNA from herring (2ml per filter). Hybridization with 32 Pa-labeled MPLV RNA probes was carried out for 20 hours at 42 ° C in 50% formamide, 5x SSC, 1x Denhardt, 0.1% SDS, 50 mM Na / Na 2 phosphate pH 6.5, and 250 μg / ml of herring sperm (1 ml of buffer and 2.10 6 cpm of RNA probe per filter). Filters were washed twice 10 minutes at room temperature in 2x SSC - 0.1% SDS, 30 minutes with 2x SSC - 0.1% SDS, and twice 30 minutes with 0.2x SSC - 0.1% SDS , each time at 65ºC. DNA sequencing
La séquence d'ADN a été obtenue en utilisant la méthode dideoxy de terminaison de chaîne (Sanger et al, 1977; Proc. Natl. Sci. USA 74, 5463-5467) modifiée pour l'utilisation de l'ADN T7 polymerase (Sequenase USB). Les échantillons ont été dénaturés 2 minutes à 75ºC et placés sur un gel d'acrylamide dénaturé (6% d'acrylamide, 8M urée, 1x TBE). The DNA sequence was obtained using the dideoxy chain termination method (Sanger et al, 1977; Proc. Natl. Sci. USA 74, 5463-5467) modified for the use of T7 DNA polymerase (Sequenase USB). The samples were denatured for 2 minutes at 75ºC and placed on a denatured acrylamide gel (6% acrylamide, 8M urea, 1x TBE).
L'analyse de séquences, la comparaison des séquences nucléotidiques et protéiques entre mpl et les gènes inclus dans la banque EMBL ont été faites en utilisant les programmes FASTP (Lipman et al, 1985; Science 227, 1435-1441) et PC-GENE (Intelligenetics Inc. et Genofit SA).  Sequence analysis, comparison of nucleotide and protein sequences between mpl and the genes included in the EMBL library were carried out using the FASTP (Lipman et al, 1985; Science 227, 1435-1441) and PC-GENE ( Intelligenetics Inc. and Genofit SA).
Infection in vitro de cellules hématopoiétiques et établissement de lignées cellulaires In vitro infection of hematopoietic cells and establishment of cell lines
Des clones cellulaires producteurs de MPLV mais dépourvus de virus auxiliaire ont été réalisés par cotransfection de cellules d'empaquetage psi-CRE, avec le plasmide pSV2 Neo et un excès d'environ 10 fois du plasmide pMPLV3. Après sélection et isolement de clones résistants au G418 (Gibco BRL), les clones producteurs de MPLV ont été sélectionnés par empreinte des cellules entières selon Wendling et al, 1989 (Leukemia 3, 475-480). Les empreintes du virus purifié à partir des surnageants ont été utilisées pour sélectionner un clone produisant un titre élevé de virus. 5 millions de cellules de moelle osseuse normale ou 1,5x106 cellules de souris adultes mâles C57BL/6 prétraitées avec le 5-fluorouracil (150 mg/kg de poids corporel, 4 jours avant) ont été suspendues dans un 1ml de surnageant infectieux. L'incubation a été effectuée pendant 2 heures à 37ºC dans une atmosphère contenant 5% de CO2 dans l'air. Les cellules ont ensuite été placées dans un milieu semi-solide ou cultivées à une concentration de 2,5x106 cellules/ml dans des flacons de culture de 25 cm2 contenant 8 ml de milieu Dulbecco modifié selon Iscove (IMDM) complétés avec 20% de sérum de veau foetal inactivé par la chaleur (Flow Laboratories). Après 10 à 12 jours des cellules non adhérentes ont été récupérées et transférées à une concentration 2,5x105 cellules/ml dans de nouveaux flacons contenant un nouveau milieu. Ensuite les cellules ont été passées tous les 4 à 7 jours, ou plus fréquemment, selon le taux de croissance cellulaire ayant poussé. Cell clones producing MPLV but free of helper virus were produced by cotransfection of psi-CRE packaging cells, with the plasmid pSV2 Neo and an approximately 10-fold excess of the plasmid pMPLV3. After selection and isolation of clones resistant to G418 (Gibco BRL), the MPLV producing clones were selected by whole cell fingerprinting according to Wendling et al, 1989 (Leukemia 3, 475-480). The fingerprints of the purified virus from the supernatants were used to select a clone producing a high titer of virus. 5 million cells of normal bone marrow or 1.5 × 10 6 cells of adult male C57BL / 6 mice pretreated with 5-fluorouracil (150 mg / kg of body weight, 4 days before) were suspended in a 1 ml of infectious supernatant. The incubation was carried out for 2 hours at 37ºC in an atmosphere containing 5% CO 2 in the air. The cells were then placed in a semi-solid medium or cultured at a concentration of 2.5 × 10 6 cells / ml in 25 cm 2 culture flasks containing 8 ml of Dulbecco medium modified according to Iscove (IMDM) supplemented with 20%. heat-inactivated fetal calf serum (Flow Laboratories). After 10 to 12 days non-adherent cells were recovered and transferred to a concentration of 2.5 × 10 5 cells / ml in new vials containing a new medium. Then the cells were passed every 4 to 7 days, or more frequently, depending on the rate of cell growth that had grown.
Tests sur les cellules proqénitrices Progenitor cell tests
Pour les colonies CFU-E, les cellules ont été ensemencées dans un système de culture à coagulum de plasma tel que décrit par Me Leod et al, 1978 (M.J. Murphy, ed - Springer Verlag, New York - pp 31-35). Un nombre approprié de cellules a été réparti dans un volume de 0,1 ml avec ou sans 0,25 U/ml Epo (érythropoïétine) (Etape 1 EPO humaine, activité spécifique 1000 U/mg; Terry Fox Laboratoire, Vancouver, Canada). Les cultures ont été récoltées au jour 2. les colonies contenant au moins 8 érythroblastes positifs avec la benzidine ont été répertoriés en tant que colonies CFU-E.  For the CFU-E colonies, the cells were seeded in a plasma coagulum culture system as described by Me Leod et al, 1978 (M.J. Murphy, ed - Springer Verlag, New York - pp 31-35). An appropriate number of cells was distributed in a volume of 0.1 ml with or without 0.25 U / ml Epo (erythropoietin) (Step 1 Human EPO, specific activity 1000 U / mg; Terry Fox Laboratory, Vancouver, Canada) . Cultures were harvested on day 2. colonies containing at least 8 benzidine-positive erythroblasts were listed as CFU-E colonies.
Pour les colonies CFU-C les tests ont été réalisés dans 0,5 ml d'agarose (Seaplaque agarose, FMC) dans des cultures sur plaques Linbro (CT-CV 96) selon la méthode Me Leod et al 1978 précitée. Pour les cultures contrôles, la formation de colonies a été stimulée au maximum par l'addition de 5% (vol/vol) d'un milieu conditionné préparé à partir de cellules de rate stimulées avec un agent mitogène (PWMSCM) et 1 U/ml Epo. Les cultures ont été incubées dans une atmosphère saturée d'humidité contenant 5% de CO2. Après 7 jours d'incubation , les cultures ont été retirées des puits, placées sur des lames et fixées dans un tampon phosphate contenant 5% de glutaraldehyde (pH7), colorées avec soit la benzidine, la myeloperoxydase ou l'acétyleholinesterase puis avec l'hématoxyline pour déterminer la composition cellulaire de chaque colonie. SEQUENCE ID Nº1 For the CFU-C colonies, the tests were carried out in 0.5 ml of agarose (Seaplaque agarose, FMC) in cultures on Linbro plates (CT-CV 96) according to the Me Leod et al 1978 method mentioned above. For the control cultures, the formation of colonies was stimulated to the maximum by the addition of 5% (vol / vol) of a conditioned medium prepared from spleen cells stimulated with a mitogenic agent (PWMSCM) and 1 U / ml Epo. The cultures were incubated in an atmosphere saturated with humidity containing 5% CO 2 . After 7 days of incubation, the cultures were removed from the wells, placed on slides and fixed in a phosphate buffer containing 5% of glutaraldehyde (pH7), stained with either benzidine, myeloperoxidase or acetyleholinesterase then with hematoxylin to determine the cell composition of each colony. SEQUENCE ID Nº1
5 10 15 | | |5 10 15 | | |
1 Met Pro Ser Trp Ala I Leu Phe Met Val Thr Ser Cys Leu Leu Leu1 Met Pro Ser Trp Ala I Leu Phe Met Val Thr Ser Cys Leu Leu Leu
16 Ala Pro Gln Asn Leu Ala Gln Val Ser Ser Gln Asp Val Ser Leu16 Ala Pro Gln Asn Leu Ala Gln Val Ser Ser Gln Asp Val Ser Leu
31 Leu Ala Ser Asp Ser Glu Pro Leu Lys Cys Phe Ser Arg Thr Phe31 Leu Ala Ser Asp Ser Glu Pro Leu Lys Cys Phe Ser Arg Thr Phe
46 Glu Asp Leu Thr Cys Phe Trp Asp Glu Glu Glu Ala Ala Pro Ser46 Glu Asp Leu Thr Cys Phe Trp Asp Glu Glu Glu Ala Ala Pro Ser
61 Gly Thr Tyr Gln Leu Leu Tyr Ala Tyr Pro Arg Glu Lys Pro Arg61 Gly Thr Tyr Gln Leu Leu Tyr Ala Tyr Pro Arg Glu Lys Pro Arg
76 Ala Cys Pro Leu Ser Ser Gln Ser Met Pro His Phe Gly Thr Arg76 Ala Cys Pro Leu Ser Ser Gln Ser Met Pro His Phe Gly Thr Arg
91 Tyr Val Cys Gln Phe Pro Asp Gln Glu Glu Val Arg Leu Phe Phe91 Tyr Val Cys Gln Phe Pro Asp Gln Glu Glu Val Arg Leu Phe Phe
106 Pro Leu His Leu Trp Val Lys Asn Val Phe Leu Asn Gln Thr Arg106 Pro Leu His Leu Trp Val Lys Asn Val Phe Leu Asn Gln Thr Arg
121 Thr Gln Arg Val Leu Phe Val Asp Ser Val Gly Leu Pro Ala Pro121 Thr Gln Arg Val Leu Phe Val Asp Ser Val Gly Leu Pro Ala Pro
136 Pro Ser Ile Ile Lys Ala Met Gly Gly Ser Gln Pro Gly Glu Leu136 Pro Ser Ile Ile Lys Ala Met Gly Gly Ser Gln Pro Gly Glu Leu
151 Gln Ile Ser Trp Glu Glu Pro Ala Pro Glu Ile Ser Asp Phe Leu151 Gln Ile Ser Trp Glu Glu Pro Ala Pro Glu Ile Ser Asp Phe Leu
166 Arg Tyr Glu Leu Arg Tyr Gly Pro Arg Asp Pro Lys Asn Ser Thr166 Arg Tyr Glu Leu Arg Tyr Gly Pro Arg Asp Pro Lys Asn Ser Thr
181 Gly Pro Thr Val Ile Gln Leu Ile Ala Thr Glu Thr Cys Cys Pro181 Gly Pro Thr Val Ile Gln Leu Ile Ala Thr Glu Thr Cys Cys Pro
196 Ala Leu Gln Arg Pro His Ser Ala Ser Ala Leu Asp Gln Ser Pro196 Ala Leu Gln Arg Pro His Ser Ala Ser Ala Leu Asp Gln Ser Pro
211 Cys Ala Gln Pro Thr Met Pro Trp Gln Asp Gly Pro Lys Gln Thr211 Cys Ala Gln Pro Thr Met Pro Trp Gln Asp Gly Pro Lys Gln Thr
226 Ser Pro Ser Arg Glu Ala Ser Ala Leu Thr Ala Glu Gly Gly Ser226 Ser Pro Ser Arg Glu Ala Ser Ala Leu Thr Ala Glu Gly Gly Ser
241 Cys Leu Ile Ser Gly Leu Gln Pro Gly Asn Ser Tyr Trp Leu Gln241 Cys Leu Ile Ser Gly Leu Gln Pro Gly Asn Ser Tyr Trp Leu Gln
256 Leu Arg Ser Glu Pro Asp Gly Ile Ser Leu Gly Gly Ser Trp Gly256 Leu Arg Ser Glu Pro Asp Gly Ile Ser Leu Gly Gly Ser Trp Gly
271 Ser Trp Ser Leu Pro Val Thr Val Asp Leu Pro Gly Asp Ala Val271 Ser Trp Ser Leu Pro Val Thr Val Asp Leu Pro Gly Asp Ala Val
236 Ala Leu Gly Leu Gln Cys Phe Thr Leu Asp Leu Lys Asn Val Thr236 Ala Leu Gly Leu Gln Cys Phe Thr Leu Asp Leu Lys Asn Val Thr
301 Cys Gln Trp Gln Gln Gln Asp His Ala Ser Ser Gln Gly Phe Phe301 Cys Gln Trp Gln Gln Gln Asp His Ala Ser Ser Gln Gly Phe Phe
316 Tyr His Ser Arg Ala Arg Cys Cys Pro Arg Asp Arg Tyr Pro Ile316 Tyr His Ser Arg Ala Arg Cys Cys Pro Arg Asp Arg Tyr Pro Ile
331 Trp Glu Asn Cys Glu Glu Glu Glu Lys Thr Asn Pro Gly Leu Gln331 Trp Glu Asn Cys Glu Glu Glu Glu Lys Thr Asn Pro Gly Leu Gln
346 Thr Pro Gln Phe Ser Arg Cys His Phe Lys Ser Arg Asn Asp Ser346 Thr Pro Gln Phe Ser Arg Cys His Phe Lys Ser Arg Asn Asp Ser
361 Ile Ile His Ile Leu Val Glu Val Thr Thr Ala Pro Gly Thr Val361 Ile Ile His Ile Leu Val Glu Val Thr Thr Ala Pro Gly Thr Val
376 His Ser Tyr Leu Gly Ser Pro Phe Trp Ile His Gln Ala Val Arg376 His Ser Tyr Leu Gly Ser Pro Phe Trp Ile His Gln Ala Val Arg
391 Leu Pro Thr Pro Asn Leu His Trp Arg Glu Ile Ser Ser Gly His391 Leu Pro Thr Pro Asn Leu His Trp Arg Glu Ile Ser Ser Gly His
406 Leu Glu Leu Glu Trp Gln His Pro Ser Ser Trp Ala Ala Gln Glu406 Leu Glu Leu Glu Trp Gln His Pro Ser Ser Trp Ala Ala Gln Glu
421 Thr Cys Tyr Gln Leu Arg Tyr Thr Gly Glu Gly His Gln Asp Trp421 Thr Cys Tyr Gln Leu Arg Tyr Thr Gly Glu Gly His Gln Asp Trp
436 Lys Val Leu Glu Pro Pro Leu Gly Ala Arg Gly Gly Thr Leu Glu436 Lys Val Leu Glu Pro Pro Leu Gly Ala Arg Gly Gly Thr Leu Glu
451 Leu Arg Pro Arg Ser Arg Tyr Arg Leu Gln Leu Arg Ala Arg Leu451 Leu Arg Pro Arg Ser Arg Tyr Arg Leu Gln Leu Arg Ala Arg Leu
466 Asn Gly Pro Thr Tyr Gln Gly Pro Trp Ser Ser Trp Ser Asp Pro466 Asn Gly Pro Thr Tyr Gln Gly Pro Trp Ser Ser Trp Ser Asp Pro
481 Thr Arg Val Glu Thr Ala Thr Glu Thr Ala Trp Ile Ser Leu Val481 Thr Arg Val Glu Thr Ala Thr Glu Thr Ala Trp Ile Ser Leu Val
496 Thr Ala Leu His Leu Val Leu Gly Leu Ser Ala Val Leu Gly Leu496 Thr Ala Leu His Leu Val Leu Gly Leu Ser Ala Val Leu Gly Leu
511 Leu Leu Leu Arg Trp Gln Phe Pro Ala His Tyr Arg Arg Leu Arg511 Leu Leu Leu Arg Trp Gln Phe Pro Ala His Tyr Arg Arg Leu Arg
526 His Ala Leu Trp Pro Ser Leu Pro Asp Leu His Arg Val Leu Gly526 His Ala Leu Trp Pro Ser Leu Pro Asp Leu His Arg Val Leu Gly
541 Gln Tyr Leu Arg Asp Thr Ala Ala Leu Ser Pro Pro Lys Ala Thr541 Gln Tyr Leu Arg Asp Thr Ala Ala Leu Ser Pro Pro Lys Ala Thr
556 Val Ser Asp Thr Cys Glu Glu Val Glu Pro Ser Leu Leu Glu Ile556 Val Ser Asp Thr Cys Glu Glu Val Glu Pro Ser Leu Leu Glu Ile
571 Leu Pro Lys Ser Ser Glu Arg Thr Pro Leu Pro Leu cys Ser Ser 586 Gln Ala Gln Met Asp Tyr Arg Arg Leu Gln Pro Ser Cys Leu Gly571 Leu Pro Lys Ser Ser Glu Arg Thr Pro Leu Pro Leu cys Ser Ser 586 Gln Ala Gln Met Asp Tyr Arg Arg Leu Gln Pro Ser Cys Leu Gly
601 Thr Met Pro Leu Ser Val Cys Pro Pro Met Ala Glu Ser Gly Ser601 Thr Met Pro Leu Ser Val Cys Pro Pro Met Ala Glu Ser Gly Ser
616 Cys Cys Thr Thr His Ile Ala Asn His Ser Tyr Leu Pro Leu Ser616 Cys Cys Thr Thr His Ile Ala Asn His Ser Tyr Leu Pro Leu Ser
631 Tyr Trp Gln Gln Pro 631 Tyr Trp Gln Gln Pro
Nombre de résidus : 635  Number of residues: 635
Poids moléculaire : 71244  Molecular Weight: 71244
------------------------------------------- -------------------------------------------
Composition acides aminésAmino acid composition
======================= ========================
41 Ala 22 Cys 19 His 8 Met 39 Thr 41 Ala 22 Cys 19 His 8 Met 39 Thr
39 Arg 41 Gln 17 Ile 18 Phe 21 Trp39 Arg 41 Gln 17 Ile 18 Phe 21 Trp
12 Asn 38 Glu 78 Leu 60 Pro 19 Tyr12 Asn 38 Glu 78 Leu 60 Pro 19 Tyr
23 Asp 36 Gly 12 Lys 64 Ser 28 Val SEQUENCE ID Nº2 23 Asp 36 Gly 12 Lys 64 Ser 28 Val SEQUENCE ID Nº2
5 10 15 | | |5 10 15 | | |
1 Leu Glu Leu Arg Pro Arg Ser Arg Tyr Arg Leu Gln Leu Arg Ala1 Leu Glu Leu Arg Pro Arg Ser Arg Tyr Arg Leu Gln Leu Arg Ala
16 Arg Leu Asn Gly Pro Thr Tyr Gln Gly Pro Trp Ser Ser Trp Ser16 Arg Leu Asn Gly Pro Thr Tyr Gln Gly Pro Trp Ser Ser Trp Ser
31 Asp Pro Thr Arg Val Glu Thr Ala Thr Glu Thr Ala Trp Ile Ser31 Asp Pro Thr Arg Val Glu Thr Ala Thr Glu Thr Ala Trp Ile Ser
46 Leu Val Thr Ala Leu His Leu Val Leu Gly Leu Ser Ala Val Leu46 Leu Val Thr Ala Leu His Leu Val Leu Gly Leu Ser Ala Val Leu
61 Gly Leu Leu Leu Leu Arg Trp Gln Phe Pro Ala His Tyr Arg Arg61 Gly Leu Leu Leu Leu Arg Trp Gln Phe Pro Ala His Tyr Arg Arg
76 Leu Arg His Ala Leu Trp Pro Ser Leu Pro Asp Leu His Arg Val76 Leu Arg His Ala Leu Trp Pro Ser Leu Pro Asp Leu His Arg Val
91 Leu Gly Gln Tyr Leu Arg Asp Thr Ala Ala Leu Ser Pro Pro Lys91 Leu Gly Gln Tyr Leu Arg Asp Thr Ala Ala Leu Ser Pro Pro Lys
106 Ala Thr Val Ser Asp Thr Cys Glu Glu Val Glu Pro Ser Leu Leu106 Ala Thr Val Ser Asp Thr Cys Glu Glu Val Glu Pro Ser Leu Leu
121 Glu Ile Leu Pro Lys Ser Ser Glu Arg Thr Pro Leu Pro Leu Cys121 Glu Ile Leu Pro Lys Ser Ser Glu Arg Thr Pro Leu Pro Leu Cys
136 Ser Ser Gln Ala Gln Met Asp Tyr Arg Arg Leu Gln Pro Ser Cys136 Ser Ser Gln Ala Gln Met Asp Tyr Arg Arg Leu Gln Pro Ser Cys
151 Leu Gly Thr Met Pro Leu Ser Val Cys Pro Pro Met Ala Glu Ser151 Leu Gly Thr Met Pro Leu Ser Val Cys Pro Pro Met Ala Glu Ser
166 Gly Ser Cys Cys Thr Thr His Ile Ala Asn His Ser Tyr Leu Pro166 Gly Ser Cys Cys Thr Thr His Ile Ala Asn His Ser Tyr Leu Pro
181 Leu Ser Tyr Trp Gln Gln Pro 181 Leu Ser Tyr Trp Gln Gln Pro
Nombre de résidus : 187  Number of residues: 187
Poids moléculaire : 21112 SEQUENCE ID Nº3 Molecular Weight: 21112 SEQUENCE ID Nº3
5 10 15 | | |5 10 15 | | |
1 Ile Leu Leu Leu Ser Tyr Ala Ala Asn ArgI Arg Gly Leu Pro Ser1 Ile Leu Leu Leu Ser Tyr Ala Ala Asn ArgI Arg Gly Leu Pro Ser
16 Trp Leu Leu Gly Pro Trp Ser Phe Pro Val Thr Val Asp Leu Pro16 Trp Leu Leu Gly Pro Trp Ser Phe Pro Val Thr Val Asp Leu Pro
31 Gly Glu Ala Val Ile Ile Gly Leu Gln Cys Phe Thr Leu Asp Leu31 Gly Glu Ala Val Ile Ile Gly Leu Gln Cys Phe Thr Leu Asp Leu
46 Lys Met Val Thr Cys Gln Trp Gln Gln Gln Asp Arg Thr Ser Ser46 Lys Met Val Thr Cys Gln Trp Gln Gln Gln Asp Arg Thr Ser Ser
61 Gln Gly Phe Phe Arg Kis Ser Arg Thr Arg Cys Cys Pro Thr Asp61 Gln Gly Phe Phe Arg Kis Ser Arg Thr Arg Cys Cys Pro Thr Asp
76 Arg Asp Pro Thr Trp Glu Lys Cys Glu Glu Glu Glu Pro Arg Pro76 Arg Asp Pro Thr Trp Glu Lys Cys Glu Glu Glu Glu Pro Arg Pro
91 Gly Ser Gln Pro Ala Leu Val Ser Arg Cys His Phe Lys Ser Arg 06 Asn Asp Ser Val Ile His Ile Leu Val Glu Val Thr Thr Ala Gln21 Gly Ala Val His Ser Tyr Leu Gly Ser Pro Phe Trp Ile His Gln 36 Ala Val Leu Leu Pro Thr Pro Ser Leu His Trp Arg Glu Val Ser51 Ser Gly Arg Leu Glu Leu Glu Trp Gln His Gln Ser Ser Trp Ala 66 Ala Gln Glu Thr Cys Tyr Gln Leu Arg Tyr Thr Gly Glu Gly Arg 31 Glu Asp Trp Lys Val Leu Glu Pro Ser Leu Gly Ala Arg Gly Gly 96 Thr Leu Glu Leu Arg Pro Arg Ala Arg Tyr Ser Leu Gln Leu Arg 11 Ala Arg Leu Asn Gly Pro Thr Tyr Gln Gly Pro Trp Ser Ala Trp26 Ser Pro Pro Ala Arg Val Ser Thr Gly Ser Glu Thr Ala Trp Ile 41 Thr Leu Val Thr Ala Leu Leu Leu Val Leu Ser Leu Ser Ala Leu56 Leu Gly Leu Leu Leu Leu Lys Trp Gln Phe Pro Ala His Tyr Arg 71 Arg Leu Arg His Ala Leu Trp Pro Ser Leu Pro Asp Leu His Arg 36 Val Leu Gly Gln Tyr Leu Arg Asp Thr Ala Ala Leu Ser Pro Ser 01 Lys Ala Thr Val Thr Asp Ser Cys Glu Glu Val Glu Pro Ser Leu 16 Leu Glu Ile Leu Pro Lys Ser Ser Glu Ser Thr Pro Leu Pro Leu 31 Cys Pro Ser Gln Pro Gln Met Asp Tyr Arg Gly Leu Gln Pro Cys 46 Leu Arg Thr Met Pro Leu Ser Val Cys Pro Pro Met Ala Glu Thr 61 Gly Ser Cys Cys Thr Thr His Ile Ala Asn His Ser Tyr Leu Pro 76 Leu Ser Tyr Trp Gln Gln Pro 91 Gly Ser Gln Pro Ala Leu Val Ser Arg Cys His Phe Lys Ser Arg 06 Asn Asp Ser Val Ile His Ile Leu Val Glu Val Thr Thr Ala Gln21 Gly Ala Val His Ser Tyr Leu Gly Ser Pro Phe Trp Ile His Gln 36 Ala Val Leu Leu Pro Thr Pro Ser Leu His Trp Arg Glu Val Ser51 Ser Gly Arg Leu Glu Leu Glu Trp Gln His Gln Ser Ser Trp Ala 66 Ala Gln Glu Thr Cys Tyr Gln Leu Arg Tyr Thr Thr Gly Glu Gly Arg 31 Glu Asp Trp Lys Val Leu Glu Pro Ser Leu Gly Ala Arg Gly Gly 96 Thr Leu Glu Leu Arg Pro Arg Ala Arg Tyr Ser Leu Gln Leu Arg 11 Ala Arg Leu Asn Gly Pro Thr Tyr Gln Gly Pro Trp Ser Ala Trp26 Ser Pro Pro Ala Arg Val Ser Thr Gly Ser Glu Thr Ala Trp Ile 41 Thr Leu Val Thr Ala Leu Leu Leu Val Leu Ser Leu Ser Ala Leu56 Leu Gly Leu Leu Leu Leu Lys Trp Gln Phe Pro Ala His Tyr Arg 71 Arg Leu Arg His Ala Leu Trp Pro Ser Leu Pro Asp Leu His Arg 36 Val Leu Gly Gln Tyr Leu Arg Asp Thr Ala Ala Leu Ser Pro Ser 01 Lys Ala Thr Val Thr Asp Ser Cys Glu Glu Val Glu Pro Ser Leu 16 Leu Glu Ile Leu Pro Lys Ser Ser Glu Ser Thr Pro Leu Pro Leu 31 Cys Pro Ser Gln Pro Gln Met Asp Tyr Arg Gly Leu Gln Pro Cys 46 Leu Arg Thr Met Pro Leu Ser Val Cys Pro Pro Met Ala Glu Thr 61 Gly Ser Cys Cys Cys Thr Thr His Ile Ala Asn His Ser Tyr Leu Pro 76 Leu Ser Tyr Trp Gln Gln Pro
Nombre de résidus : 382 Number of residues: 382
Poids moléculaire : 43069 SEQUENCE ID Nº4 Molecular Weight: 43069 SEQUENCE ID Nº4
5 10 15 | | |5 10 15 | | |
1 Leu Glu Leu Arg Pro Arg Ala Arg Tyr Ser Leu Gln Leu Arg Ala1 Leu Glu Leu Arg Pro Arg Ala Arg Tyr Ser Leu Gln Leu Arg Ala
16 Arg Leu Asn Gly Pro Thr Tyr Gln Gly Pro Trp Ser Ala Trp Ser16 Arg Leu Asn Gly Pro Thr Tyr Gln Gly Pro Trp Ser Ala Trp Ser
31 Pro Pro Ala Arg Val Ser Thr Gly Ser Glu Thr Ala Trp Ile Thr31 Pro Pro Ala Arg Val Ser Thr Gly Ser Glu Thr Ala Trp Ile Thr
46 Leu Val Thr Ala Leu Leu Leu Val Leu Ser Leu Ser Ala Leu Leu46 Leu Val Thr Ala Leu Leu Leu Val Leu Ser Leu Ser Ala Leu Leu
61 Gly Leu Leu Leu Leu Lys Trp Gln Phe Pro Ala His Tyr Arg Arg61 Gly Leu Leu Leu Leu Lys Trp Gln Phe Pro Ala His Tyr Arg Arg
76 Leu Arg His Ala Leu Trp Pro Ser Leu Pro Asp Leu His Arg Val76 Leu Arg His Ala Leu Trp Pro Ser Leu Pro Asp Leu His Arg Val
91 Leu Gly Gln Tyr Leu Arg Asp Thr Ala Ala Leu Ser Pro Ser Lys91 Leu Gly Gln Tyr Leu Arg Asp Thr Ala Ala Leu Ser Pro Ser Lys
106 Ala Thr Val Thr Asp Ser Cys Glu Glu Val Glu Pro Ser Leu Leu106 Ala Thr Val Thr Asp Ser Cys Glu Glu Val Glu Pro Ser Leu Leu
121 Glu Ile Leu Pro Lys Ser Ser Glu Ser Thr Pro Leu Pro Leu Cys121 Glu Ile Leu Pro Lys Ser Ser Glu Ser Thr Pro Leu Pro Leu Cys
136 Pro Ser Gln Pro Gln Met Asp Tyr Arg Gly Leu Gln Pro Cys Leu136 Pro Ser Gln Pro Gln Met Asp Tyr Arg Gly Leu Gln Pro Cys Leu
151 Arg Thr Met Pro Leu Ser Val Cys Pro Pro Met Ala Glu Thr Gly151 Arg Thr Met Pro Leu Ser Val Cys Pro Pro Met Ala Glu Thr Gly
166 Ser Cys Cys Thr Thr His Ile Ala Asn His Ser Tyr Leu Pro Leu166 Ser Cys Cys Thr Thr His Ile Ala Asn His Ser Tyr Leu Pro Leu
181 Ser Tyr Trp Gln 181 Ser Tyr Trp Gln
Nombre de résidus : 184 Number of residues: 184
Poids moléculaire : 20558 Molecular Weight: 20558
SEQUENCE ID N° 5 SEQUENCE ID N ° 5
Trp Gln Phe Pro Ala His Tyr Arg Arg Leu Arg His Ala Leu Trp Pro Ser Leu Pro Asp Leu His Arg Val Leu Gly Gln Tyr Leu Arg Asp Thr Ala Ala Leu Ser Pro Trp Gln Phe Pro Ala His Tyr Arg Arg Leu Arg His Ala Leu Trp Pro Ser Leu Pro Asp Leu His Arg Val Leu Gly Gln Tyr Leu Arg Asp Thr Ala Ala Leu Ser Pro
SEQUENCE ID Nº6 SEQUENCE ID Nº6
Leu Val Thr Ala Leu His Leu Val Leu Gly Leu Ser Ala Val Leu Gly Leu Leu Leu Leu Leu Val Thr Ala Leu His Leu Val Leu Gly Leu Ser Ala Val Leu Gly Leu Leu Leu Leu Leu
SEQUENCE ID Nº7 SEQUENCE ID Nº7
5 10 155 10 15
I I I I I I
1 Met Pro Ser Trp Ala Leu Phe Met Val Thr Ser Cys Leu Leu Leu 1 Met Pro Ser Trp Ala Leu Phe Met Val Thr Ser Cys Leu Leu Leu
16 Ala Pro Gln Asn Leu Ala Gln Val Ser Ser Gln Asp Val Ser Leu16 Ala Pro Gln Asn Leu Ala Gln Val Ser Ser Gln Asp Val Ser Leu
31 Leu Ala Ser Asp Ser Glu Pro Leu Lys Cys Phe Ser Arg Thr Phe31 Leu Ala Ser Asp Ser Glu Pro Leu Lys Cys Phe Ser Arg Thr Phe
46 Glu Asp Leu Thr Cys Phe Trp Asp Glu Glu Glu Ala Ala Pro Ser46 Glu Asp Leu Thr Cys Phe Trp Asp Glu Glu Glu Ala Ala Pro Ser
61 Gly Thr Tyr Gln Leu Leu Tyr Ala Tyr Pro Arg Glu Lys Pro Arg61 Gly Thr Tyr Gln Leu Leu Tyr Ala Tyr Pro Arg Glu Lys Pro Arg
76 Ala Cys Pro Leu Ser Ser Gln Ser Met Pro His Phe Gly Thr Arg76 Ala Cys Pro Leu Ser Ser Gln Ser Met Pro His Phe Gly Thr Arg
SI Tyr Val Cys Gln Phe Pro Asp Gln Glu Glu Val Arg Leu Phe PheSI Tyr Val Cys Gln Phe Pro Asp Gln Glu Glu Val Arg Leu Phe Phe
106 Pro Leu His Leu Trp Val Lys Asn Val Phe Leu Asn Gln Thr Arg106 Pro Leu His Leu Trp Val Lys Asn Val Phe Leu Asn Gln Thr Arg
121 Thr Gln Arg Val Leu Phe Val Asp Ser Val Gly Leu Pro Ala Pro121 Thr Gln Arg Val Leu Phe Val Asp Ser Val Gly Leu Pro Ala Pro
136 Pro Ser Ile Ile Lys Ala Met Gly Gly Ser Gln Pro Gly Glu Leu136 Pro Ser Ile Ile Lys Ala Met Gly Gly Ser Gln Pro Gly Glu Leu
151 Gln Ile Ser Trp Glu Glu Pro Ala Pro Glu Ile Ser Asp Phe Leu151 Gln Ile Ser Trp Glu Glu Pro Ala Pro Glu Ile Ser Asp Phe Leu
166 Arg Tyr Glu Leu Arg Tyr Gly Pro Arg Asp Pro Lys Asn Ser Thr166 Arg Tyr Glu Leu Arg Tyr Gly Pro Arg Asp Pro Lys Asn Ser Thr
1S1 Gly Pro Thr Val Ile Gln Leu Ile Ala Thr Glu Thr Cys Cys Pro1S1 Gly Pro Thr Val Ile Gln Leu Ile Ala Thr Glu Thr Cys Cys Pro
196 Ala Leu Gln Arg Pro His Ser Ala Ser Ala Leu Asp Gln Ser Pro196 Ala Leu Gln Arg Pro His Ser Ala Ser Ala Leu Asp Gln Ser Pro
211 Cys Ala Gln Pro Thr Met Pro Trp Gln Asp Gly Pro Lys Gln Thr211 Cys Ala Gln Pro Thr Met Pro Trp Gln Asp Gly Pro Lys Gln Thr
226 Ser Pro Ser Arg Glu Ala Ser Ala Leu Thr Ala Glu Gly Gly Ser226 Ser Pro Ser Arg Glu Ala Ser Ala Leu Thr Ala Glu Gly Gly Ser
241 Cys Leu Ile Ser Gly Leu Gln Pro Gly Asn Ser Tyr Trp Leu Gln241 Cys Leu Ile Ser Gly Leu Gln Pro Gly Asn Ser Tyr Trp Leu Gln
256 Leu Arg Ser Glu Pro Asp Gly Ile Ser Leu Gly Gly Ser Trp Gly256 Leu Arg Ser Glu Pro Asp Gly Ile Ser Leu Gly Gly Ser Trp Gly
271 Ser Trp Ser Leu Pro Val Thr Val Asp Leu Pro Gly Asp Ala Val271 Ser Trp Ser Leu Pro Val Thr Val Asp Leu Pro Gly Asp Ala Val
236 Ala Leu Gly Leu Gln Cys Phe Thr Leu Asp Leu Lys Asn Val Thr236 Ala Leu Gly Leu Gln Cys Phe Thr Leu Asp Leu Lys Asn Val Thr
301 Cys Gln Trp Gln Gln Gln Asp His Ala Ser Ser Gln Gly Phe Phe301 Cys Gln Trp Gln Gln Gln Asp His Ala Ser Ser Gln Gly Phe Phe
316 Tyr His Ser Arg Ala Arg Cys Cys Pro Arg Asp Arg Tyr Pro Ile316 Tyr His Ser Arg Ala Arg Cys Cys Pro Arg Asp Arg Tyr Pro Ile
331 Trp Glu Asn Cys Glu Glu Glu Glu Lys Thr Asn Pro Gly Leu Gln331 Trp Glu Asn Cys Glu Glu Glu Glu Lys Thr Asn Pro Gly Leu Gln
346 Thr Pro Gln Phe Ser Arg Cys His Phe Lys Ser Arg Asn Asp Ser346 Thr Pro Gln Phe Ser Arg Cys His Phe Lys Ser Arg Asn Asp Ser
361 Ile Ile His Ile Leu Val Glu Val Thr Thr Ala Pro Gly Thr Val 376 His Ser Tyr Leu Gly Ser Pro Phe Trp Ile His Gln Ala Val Arg 391 Leu Pro Thr Pro Asn Leu His Trp Arg Glu Ile Ser Ser Gly His361 Ile Ile His Ile Leu Val Glu Val Thr Thr Ala Pro Gly Thr Val 376 His Ser Tyr Leu Gly Ser Pro Phe Trp Ile His Gln Ala Val Arg 391 Leu Pro Thr Pro Asn Leu His Trp Arg Glu Ile Ser Ser Gly His
406 Leu Glu Leu Glu Trp Gln His Pro Ser Ser Trp Ala Ala Gln Glu 421 Thr Cys Tyr Gln Leu Arg Tyr Thr Gly Glu Gly His Gln Asp Trp 436 Lys Val Leu Glu Pro Pro Leu Gly Ala Arg Gly Gly Thr Leu Glu 451 Leu Arg Pro Arg Ser Arg Tyr Arg Leu Gln Leu Arg Ala Arg Leu 466 Asn Gly Pro Thr Tyr Gln Gly Pro Trp Ser Ser Trp Ser Asp Pro 481 Thr Arg Val Glu Thr Ala Thr Glu Thr Ala Trp Ile Ser 406 Leu Glu Leu Glu Trp Gln His Pro Ser Ser Trp Ala Ala Gln Glu 421 Thr Cys Tyr Gln Leu Arg Tyr Thr Gly Glu Gly His Gln Asp Trp 436 Lys Val Leu Glu Pro Pro Leu Gly Ala Arg Gly Gly Thr Leu Glu 451 Leu Arg Pro Arg Ser Arg Tyr Arg Leu Gln Leu Arg Ala Arg Leu 466 Asn Gly Pro Thr Tyr Gln Gly Pro Trp Ser Ser Trp Ser Asp Pro 481 Thr Arg Val Glu Thr Ala Thr Glu Thr Ala Trp Ile Ser
Nombre de résidus : 493 Number of residues: 493
Poids moléculaire : 55436 SEQUENCE ID Nº8 Molecular Weight: 55436 SEQUENCE ID Nº8
5 10 15 | | |5 10 15 | | |
1 Arg Trp Gln Phe Pro I Ala His Tyr Arg ArgI Leu Arg His Ala LeuI1 Arg Trp Gln Phe Pro I Ala His Tyr Arg ArgI Leu Arg His Ala LeuI
16 Trp Pro Ser Leu Pro Asp Leu His Arg Val Leu Gly Gln Tyr Leu16 Trp Pro Ser Leu Pro Asp Leu His Arg Val Leu Gly Gln Tyr Leu
31 Arg Asp Thr Ala Ala Leu Ser Pro Pro Lys Ala Thr Val Ser Asp31 Arg Asp Thr Ala Ala Leu Ser Pro Pro Lys Ala Thr Val Ser Asp
46 Thr Cys Glu Glu Val Glu Pro Ser Leu Leu Glu Ile Leu Pro Lys46 Thr Cys Glu Glu Val Glu Pro Ser Leu Leu Glu Ile Leu Pro Lys
61 Ser Ser Glu Arg Thr Pro Leu Pro Leu Cys Ser Ser Gln Ala Gln61 Ser Ser Glu Arg Thr Pro Leu Pro Leu Cys Ser Ser Gln Ala Gln
76 Met Asp Tyr Arg Arg Leu Gln Pro Ser Cys Leu Gly Thr Met Pro76 Met Asp Tyr Arg Arg Leu Gln Pro Ser Cys Leu Gly Thr Met Pro
91 Leu Ser Val Cys Pro Pro Met Ala Glu Ser Gly Ser Cys Cys Thr91 Leu Ser Val Cys Pro Pro Met Ala Glu Ser Gly Ser Cys Cys Thr
106 Thr His Ile Ala Asn His Ser Tyr Leu Pro Leu Ser Tyr Trp Gln106 Thr His Ile Ala Asn His Ser Tyr Leu Pro Leu Ser Tyr Trp Gln
121 Gln Pro 121 Gln Pro
Nombre de résidus : 122 Number of residues: 122
Poids moléculaire : 13816 Molecular Weight: 13816
SEQUENCE ID Nº9 SEQUENCE ID Nº9
Leu Val Thr Ala Leu Leu Leu Val Leu Ser Leu Ser Ala Leu Leu Gly Leu Leu Leu Leu Leu Val Thr Ala Leu Leu Leu Val Leu Ser Leu Ser Ala Leu Leu Gly Leu Leu Leu Leu
SEQUENCE ID Nº10 SEQUENCE ID Nº10
5 10 15 | | |5 10 15 | | |
1 Leu Glu Leu Arg Pro I Arg Ala Arg Tyr Ser Leu Gln Leu Arg Ala1 Leu Glu Leu Arg Pro I Arg Ala Arg Tyr Ser Leu Gln Leu Arg Ala
16 Arg Leu Asn Gly Pro Thr Tyr Gln Gly Pro Trp Ser Ala Trp Ser16 Arg Leu Asn Gly Pro Thr Tyr Gln Gly Pro Trp Ser Ala Trp Ser
31 Pro Pro Ala Arg Val Ser Thr Gly Ser Glu Thr Ala Trp Ile Thr31 Pro Pro Ala Arg Val Ser Thr Gly Ser Glu Thr Ala Trp Ile Thr
Nombre de résidus : 45 Number of residues: 45
Poids moléculaire : 5099 Molecular Weight: 5099
SEQUENCE ID Nº11 SEQUENCE ID Nº11
5 10 155 10 15
I I I I I I
1 Lys Trp Gln Phe Pro Ala His Tyr Arg Arg Leu Arg His Ala Leu 1 Lys Trp Gln Phe Pro Ala His Tyr Arg Arg Leu Arg His Ala Leu
16 Trp Pro Ser Leu Pro Asp Leu His Arg Val Leu Gly Gln Tyr Leu16 Trp Pro Ser Leu Pro Asp Leu His Arg Val Leu Gly Gln Tyr Leu
31 Arg Asp Thr Ala Ala Leu Ser Pro Ser Lys Ala Thr Val Thr Asp31 Arg Asp Thr Ala Ala Leu Ser Pro Ser Lys Ala Thr Val Thr Asp
46 Ser Cys Glu Glu Val Glu Pro Ser Leu Leu Glu Ile Leu Pro Lys46 Ser Cys Glu Glu Val Glu Pro Ser Leu Leu Glu Ile Leu Pro Lys
61 Ser Ser Glu Ser Thr Pro Leu Pro Leu Cys Pro Ser Gln Pro Gln61 Ser Ser Glu Ser Thr Pro Leu Pro Leu Cys Pro Ser Gln Pro Gln
76 Met Asp Tyr Arg Gly Leu Gln Pro Cys Leu Arg Thr Met Pro Leu76 Met Asp Tyr Arg Gly Leu Gln Pro Cys Leu Arg Thr Met Pro Leu
91 Ser Val Cys Pro Pro Met Ala Glu Thr Gly Ser Cys Cys Thr Thr91 Ser Val Cys Pro Pro Met Ala Glu Thr Gly Ser Cys Cys Thr Thr
106 His Ile Ala Asn His Ser Tyr Leu Pro Leu Ser Tyr Trp Gln106 His Ile Ala Asn His Ser Tyr Leu Pro Leu Ser Tyr Trp Gln
Nombre de résidus : 119 Number of residues: 119
Poids moléculaire : 13446 Molecular Weight: 13446
SEQUENCE ID Nº12 SEQUENCE ID Nº12
5 10 155 10 15
I I I I I I
1 Met Pro Ser Trp Ala Leu Phe Met Val Thr Ser Cys Leu Leu Leu 16 Ala Pro Gln Asn Leu Ala Gln Val Ser Ser Gln Asp Val Ser Leu 31 Leu Ala Ser Asp Ser Glu Pro Leu Lys Cys Phe Ser Arg Thr Phe 46 Glu Asp Leu Thr Cys Phe Trp Asp Glu Glu Glu Ala Ala Pro Ser 61 Gly Thr Tyr Gln Leu Leu Tyr Ala Tyr Pro Arg Glu Lys Pro Arg 76 Ala Cys Pro Leu Ser Ser Gln Ser Met Pro His Phe Gly Thr Arg 91 Tyr Val Cys Gln Phe Pro Asp Gln Glu Glu Val Arg Leu Phe Phe106 Pro Leu His Leu Trp Val Lys Asn Val Phe Leu Asn Gln Thr Arg121 Thr Gln Arg Val Leu Phe Val Asp Ser Val Gly Leu Pro Ala Pro136 Fro Ser Ile Ile Lys Ala Met Gly Gly Ser Gln Pro Gly Glu Leu151 Gln Ile Ser Trp Glu Glu Pro Ala Pro Glu Ile Ser Asp Phe Leu166 Arg Tyr Glu Leu Arg Tyr Gly Pro Arg Asp Pro Lys Asn Ser Thr181 Gly Pro Thr Val Ile Gln Leu Ile Ala Thr Glu Thr Cys Cys Pro196 Ala Leu Gln Arg Pro His Ser Ala Ser Ala Leu Asp Gln Ser Pro211 Cys Ala Gln Pro Thr Met Pro Trp Gln Asp Gly Pro Lys Gln Thr226 Ser Pro Ser Arg Glu Ala Ser Ala Leu Thr Ala Glu Gly Gly Ser 241 Cys Leu Ile Ser Gly Leu Gln Pro Gly Asn Ser Tyr Trp Leu Gln256 Leu Arg Ser Glu Pro Asp Gly Ile Ser Leu Gly Gly Ser Trp Gly271 Ser Trp Ser Leu Pro Val Thr Val Asp Leu Pro Gly Asp Ala Val286 Ala Leu Gly Leu Gln Cys Phe Thr Leu Asp Leu Lys Asn Val Thr 301 Cys Gln Trp Gln Gln Gin Asp His Ala Ser Ser Gln Gly Phe Phe 316 Tyr His Ser Arg Ala Arg Cys Cys Pro Arg Asp Arg Tyr Pro Ile 331 Trp Glu Asn Cys Glu Glu Glu Glu Lys Thr Asn Pro Gly Leu Gln346 Thr Pro Gln Phe Ser Arg Cys His Phe Lys Ser Arg Asn Asp Ser361 Ile Ile His Ile Leu Val Glu Val Thr Thr Ala Pro Gly Thr Val376 His Ser Tyr Leu Gly Ser Pro Phe Trp Ile His Gln Ala Val Arg391 Leu Pro Thr Pro Asn Leu His Trp Arg Glu Ile Ser Ser Gly His 406 Leu Glu Leu Glu Trp Gln His Pro Ser Ser Trp Ala Ala Gln Glu 421 Thr Cys Tyr Gln Leu Arg Tyr Thr Gly Glu Gly His Gln Asp Trp 436 Lys Val Leu Glu Pro Pro Leu Gly Ala Arg Gly Gly Thr  1 Met Pro Ser Trp Ala Leu Phe Met Val Thr Ser Cys Leu Leu Leu 16 Ala Pro Gln Asn Leu Ala Gln Val Ser Ser Gln Asp Val Ser Leu 31 Leu Ala Ser Asp Ser Glu Pro Leu Lys Cys Phe Ser Arg Thr Phe 46 Glu Asp Leu Thr Cys Phe Trp Asp Glu Glu Glu Ala Ala Pro Ser 61 Gly Thr Tyr Gln Leu Leu Tyr Ala Tyr Pro Arg Glu Lys Pro Arg 76 Ala Cys Pro Leu Ser Ser Gln Ser Met Pro His Phe Gly Thr Arg 91 Tyr Val Cys Gln Phe Pro Asp Gln Glu Glu Val Arg Leu Phe Phe106 Pro Leu His Leu Trp Val Lys Asn Val Phe Leu Asn Gln Thr Arg121 Thr Gln Arg Val Leu Phe Val Asp Ser Val Gly Leu Pro Ala Pro136 Fro Ser Ile Ile Lys Ala Met Gly Gly Ser Gln Pro Gly Glu Leu151 Gln Ile Ser Trp Glu Glu Pro Ala Pro Glu Ile Ser Asp Phe Leu166 Arg Tyr Glu Leu Arg Tyr Gly Pro Arg Asp Pro Lys Asn Ser Thr181 Gly Pro Thr Val Ile Gln Leu Ile Ala Thr Glu Thr Cys Cys Pro196 Ala Leu Gln Arg Pro His Ser Ala Ser Ala Leu Asp Gln Ser Pro211 Cys Ala Gln Pro Thr Met Pro Trp Gln Asp Gly Pro Lys Gln Thr226 Ser Pro Ser Arg Glu Ala Ser Ala Leu Thr Ala Glu Gly G ly Ser 241 Cys Leu Ile Ser Gly Leu Gln Pro Gly Asn Ser Tyr Trp Leu Gln256 Leu Arg Ser Glu Pro Asp Gly Ile Ser Leu Gly Gly Ser Trp Gly271 Ser Trp Ser Leu Pro Val Thr Val Asp Leu Pro Gly Asp Ala Val286 Ala Leu Gly Leu Gln Cys Phe Thr Leu Asp Leu Lys Asn Val Thr 301 Cys Gln Trp Gln Gln Gin Asp His Ala Ser Ser Gln Gly Phe Phe 316 Tyr His Ser Arg Ala Arg Cys Cys Pro Arg Asp Arg Tyr Pro Ile 331 Trp Glu Asn Cys Glu Glu Glu Glu Lys Thr Asn Pro Gly Leu Gln346 Thr Pro Gln Phe Ser Arg Cys His Phe Lys Ser Arg Asn Asp Ser361 Ile Ile His Ile Leu Val Glu Val Thr Thr Ala Pro Gly Thr Val376 His Ser Tyr Leu Gly Ser Pro Phe Trp Ile His Gln Ala Val Arg391 Leu Pro Thr Pro Asn Leu His Trp Arg Glu Ile Ser Ser Gly His 406 Leu Glu Leu Glu Trp Gln His Pro Ser Ser Trp Ala Ala Gln Glu 421 Thr Cys Tyr Gln Leu Arg Tyr Thr Gly Glu Gly His Gln Asp Trp 436 Lys Val Leu Glu Pro Pro Leu Gly Ala Arg Gly Gly Thr
Nombre de résidus : 448 Number of residues: 448
Poids moléculaire : 50150 SEQUENCE ID Nº13 Molecular Weight: 50150 SEQUENCE ID Nº13
10 15 | |10 15 | |
1 Ile Leu Leu Leu Ser Tyr Ala Ala Asn Arg Arg Gly Leu Pro Ser1 Ile Leu Leu Leu Ser Tyr Ala Ala Asn Arg Arg Gly Leu Pro Ser
16 Trp Leu Leu Gly Pro Trp Ser Phe Pro Val Thr Val Asp Leu Pro16 Trp Leu Leu Gly Pro Trp Ser Phe Pro Val Thr Val Asp Leu Pro
31 Gly Glu Ala Val Ile Ile Gly Leu Gln Cys Phe Thr Leu Asp Leu31 Gly Glu Ala Val Ile Ile Gly Leu Gln Cys Phe Thr Leu Asp Leu
46 Lys Met Val Thr Cys Gln Trp Gln Gln Gln Asp Arg Thr Ser Ser46 Lys Met Val Thr Cys Gln Trp Gln Gln Gln Asp Arg Thr Ser Ser
61 Gln Gly Phe Phe Arg His Ser Arg Thr Arg Cys Cys Pro Thr Asp61 Gln Gly Phe Phe Arg His Ser Arg Thr Arg Cys Cys Pro Thr Asp
76 Arg Asp Pro Thr Trp Glu Lys Cys Glu Glu Glu Glu Pro Arg Pro76 Arg Asp Pro Thr Trp Glu Lys Cys Glu Glu Glu Glu Pro Arg Pro
91 Gly Ser Gln Pro Ala Leu Val Ser Arg Cys His Phe Lys Ser Arg 06 Asn Asp Ser Val Ile His Ile Leu Val Glu Val Thr Thr Ala Gln91 Gly Ser Gln Pro Ala Leu Val Ser Arg Cys His Phe Lys Ser Arg 06 Asn Asp Ser Val Ile His Ile Leu Val Glu Val Thr Thr Ala Gln
121 Gly Ala Val His Ser Tvr Leu Gly Ser Pro Phe Trp Ile His Gln121 Gly Ala Val His Ser Tvr Leu Gly Ser Pro Phe Trp Ile His Gln
136 Ala Val Leu Leu Pro Thr Pro Ser Leu His Trp Arg Glu Val Ser 51 Ser Gly Arg Leu Glu Leu Glu Trp Gln His Gln Ser Ser Trp Ala136 Ala Val Leu Leu Pro Thr Pro Ser Leu His Trp Arg Glu Val Ser 51 Ser Gly Arg Leu Glu Leu Glu Trp Gln His Gln Ser Ser Trp Ala
166 Ala Gln Glu Thr Cys Tyr Gln Leu Arg Tyr Thr Gly Glu Gly Arg166 Ala Gln Glu Thr Cys Tyr Gln Leu Arg Tyr Thr Gly Glu Gly Arg
181 Glu Asp Trp Lys Val Leu Glu Pro Ser Leu Gly Ala Arg Gly Gly181 Glu Asp Trp Lys Val Leu Glu Pro Ser Leu Gly Ala Arg Gly Gly
1S6 Thr 1S6 Thr
Nombre de résidus : 196 Number of residues: 196
Poids moléculaire : 22304 Molecular Weight: 22304
SEQUENCE ID Nº14 SEQUENCE ID Nº14
5 10 15 | | |5 10 15 | | |
1 Met Ala Cys Ser Thr Leu Pro Lys Ser Pro Lys Asp Lys Ile Asp1 Met Ala Cys Ser Thr Leu Pro Lys Ser Pro Lys Asp Lys Ile Asp
16 Pro Arg Asp Leu Leu Ile Pro Leu Ile Leu Phe Leu Ser Leu Lys16 Pro Arg Asp Leu Leu Ile Pro Leu Ile Leu Phe Leu Ser Leu Lys
31 Gly Ala Arg Ser Ala Ala Pro Gly Ser Ser Pro His Gln Val Tyr31 Gly Ala Arg Ser Ala Ala Pro Gly Ser Ser Pro His Gln Val Tyr
46 Asn Ile Thr Trp Glu Val Thr Asn Gly Asp Arg Glu Thr Val Trp46 Asn Ile Thr Trp Glu Val Thr Asn Gly Asp Arg Glu Thr Val Trp
61 Ala Ile Ser Gly Arg Leu Tyr Val Ser Gly Arg Asp Pro Gly Leu61 Ala Ile Ser Gly Arg Leu Tyr Val Ser Gly Arg Asp Pro Gly Leu
76 Thr Phe Gly Ile Arg Leu Arg Tyr Gln Asn Leu Gly Pro Arg Val76 Thr Phe Gly Ile Arg Leu Arg Tyr Gln Asn Leu Gly Pro Arg Val
91 Pro Ile Gly Pro Asn Pro Val Leu Ala Asp Leu Glu Leu Arg Pro91 Pro Ile Gly Pro Asn Pro Val Leu Ala Asp Leu Glu Leu Arg Pro
106 Arg Ala Arg Tyr Ser Leu Gln Leu Arg Ala Arg Leu Asn Gly Pro106 Arg Ala Arg Tyr Ser Leu Gln Leu Arg Ala Arg Leu Asn Gly Pro
121 Thr Tyr Gln Gly Fro Trp Ser Ala Trp Ser Pro Pro Ala Arg Val121 Thr Tyr Gln Gly Fro Trp Ser Ala Trp Ser Pro Pro Ala Arg Val
126 Ser Thr Gly Ser Glu Thr Ala Trp Ile Thr Leu Val Thr Ala Leu126 Ser Thr Gly Ser Glu Thr Ala Trp Ile Thr Leu Val Thr Ala Leu
151 Leu Leu Val Leu Ser Leu Ser Ala Leu Leu Gly Leu Leu Leu Leu151 Leu Leu Val Leu Ser Leu Ser Ala Leu Leu Gly Leu Leu Leu Leu
166 Lys Trp Gln Phe Pro Ala His Tyr Arg Arg Leu Arg His Ala Leu166 Lys Trp Gln Phe Pro Ala His Tyr Arg Arg Leu Arg His Ala Leu
181 Trp Pro Ser Leu Pro Asp Leu His Arg Val Leu Gly Gln Tyr Leu181 Trp Pro Ser Leu Pro Asp Leu His Arg Val Leu Gly Gln Tyr Leu
196 Arg Asp Thr Ala Ala Leu Ser Pro Ser Lys Ala Thr Val Thr Asp196 Arg Asp Thr Ala Ala Leu Ser Pro Ser Lys Ala Thr Val Thr Asp
211 Ser Cys Glu Glu Val Glu Pro Ser Leu Leu Glu Ile Leu Pro Lys211 Ser Cys Glu Glu Val Glu Pro Ser Leu Leu Glu Ile Leu Pro Lys
226 Ser Ser Glu Ser Thr Pro Leu Pro Leu Cys Pro Ser Gln Pro Gln226 Ser Ser Glu Ser Thr Pro Leu Pro Leu Cys Pro Ser Gln Pro Gln
241 Met Asp Tyr Arg Gly Leu Gln Pro Cys Leu Arg Thr Met Pro Leu241 Met Asp Tyr Arg Gly Leu Gln Pro Cys Leu Arg Thr Met Pro Leu
256 Ser Val Cys Pro Pro Met Ala Glu Thr Gly Ser Cys Cys Thr Thr256 Ser Val Cys Pro Pro Met Ala Glu Thr Gly Ser Cys Cys Thr Thr
271 His Ile Ala Asn His Ser Tyr Leu Pro Leu Ser Tyr Trp Gln271 His Ile Ala Asn His Ser Tyr Leu Pro Leu Ser Tyr Trp Gln
Nombre de résidus : 284 Number of residues: 284
Poids moléculaire : 31464 Molecular Weight: 31464
SEQUENCE ID N°15 SEQUENCE ID N ° 15
10 20 30 40 10 20 30 40
| | I |
Figure imgf000044_0001
| | I |
Figure imgf000044_0001
AAG ATG CCC TCC TGG GCC CTC TTC ATG GTC ACC TCC TGC CTC CTC CTG GCC CCT MET Pro Ser Trp Ala Leu Phe MET Val Thr Ser Cys Leu Leu Leu Ala Pro AAG ATG CCC TCC TGG GCC CTC TTC ATG GTC ACC TCC TGC CTC CTC CTG GCC CCT MET Pro Ser Trp Ala Leu Phe MET Val Thr Ser Cys Leu Leu Leu Ala Pro
60 70 80 90 100 11060 70 80 90 100 110
I I I I I II I I I I I
CAA AAC CTG GCC CAA GTC AGC AGC CAA GAT GTC TCC TTG CTG GCA TCA GAC TCA CAA AAC CTG GCC CAA GTC AGC AGC CAA GAT GTC TCC TTG CTG GCA TCA GAC TCA
Gln Asn Leu Ala Gln Val Ser Ser Gln Asp Val Ser Leu Leu Ala Ser Asp Sor Gln Asn Leu Ala Gln Val Ser Ser Gln Asp Val Ser Leu Leu Ala Ser Asp Sor
120 130 140 150 120 130 140 150
| | | |
Figure imgf000044_0002
| | | |
Figure imgf000044_0002
GAG CCC CTG A IAG TGT TTC TCC CGA ACA TTT GAG GAC CTC ACT TGC TTC TGG GAT  GAG CCC CTG A IAG TGT TTC TCC CGA ACA TTT GAG GAC CTC ACT TGC TTC TGG GAT
Glu Pro Leu Lys Cys Phe Ser Arg Thr Phe Glu Asp Leu Thr Cys Phe Trp Asp  Glu Pro Leu Lys Cys Phe Ser Arg Thr Phe Glu Asp Leu Thr Cys Phe Trp Asp
170 180 190 200 210 170 180 190 200 210
I I I I I I I I I I
GAG GAA GAG GCA GCG CCC AGT GGG ACA TAC CAG CTG CTG TAT GCC TAC CCG CGG GAG GAA GAG GCA GCG CCC AGT GGG ACA TAC CAG CTG CTG TAT GCC TAC CCG CGG
Glu Glu Glu Ala Ala Pro Ser Gly Thr Tyr Gln Leu Leu Tyr Ala Tyr Pro Arg Glu Glu Glu Ala Ala Pro Ser Gly Thr Tyr Gln Leu Leu Tyr Ala Tyr Pro Arg
SEQUENCE Nº 15 (SUITE) SEQUENCE Nº 15 (CONTINUED)
220 230 240 250 260 270220 230 240 250 260 270
I I I I I II I I I I I
GAG AAG CCC CGT GCT TGC CCC CTG AGT TCC CAG AGC ATG CCC CAC TTT GGA ACC GAG AAG CCC CGT GCT TGC CCC CTG AGT TCC CAG AGC ATG CCC CAC TTT GGA ACC
Glu Lys Pro Arg Ala Cys Pro Leu Ser Ser Gln Ser MET Pro His Phe Gly Thr  Glu Lys Pro Arg Ala Cys Pro Leu Ser Ser Gln Ser MET Pro His Phe Gly Thr
280 290 300 310 280 290 300 310
| | | |
Figure imgf000045_0001
| | | |
Figure imgf000045_0001
CGA TAC GTG TGC CAG TTT CCA GAC CAG GAG GAA GTG CGT CTC TTC TTT CCG CTG CGA TAC GTG TGC CAG TTT CCA GAC CAG GAG GAA GTG CGT CTC TTC TTT CCG CTG
Arg Tyr Val Cys Gln Phe Pro Asp Gln Glu Glu Val Arg Leu Phe Phe Pro Leu  Arg Tyr Val Cys Gln Phe Pro Asp Gln Glu Glu Val Arg Leu Phe Phe Pro Leu
330 340| 350 360 3 330 340 | 350 360 3
| | |
Figure imgf000045_0002
Figure imgf000045_0003
| | |
Figure imgf000045_0002
Figure imgf000045_0003
CAC CTC TGG GTG AAG AAT GTG TTC CTA AAC CAG ACT CGG ACT CAG CGA GTC CTC CAC CTC TGG GTG AAG AAT GTG TTC CTA AAC CAG ACT CGG ACT CAG CGA GTC CTC
His Leu Trp Val Lys Asn Val Phe Leu Asn Gln Thr Arg Thr Gln Arg Val Leu  His Leu Trp Val Lys Asn Val Phe Leu Asn Gln Thr Arg Thr Gln Arg Val Leu
39|0 400 410 420 39 | 0 400 410 420
| | |
Figure imgf000045_0004
| | |
Figure imgf000045_0004
TTT GTG GAC AGT GTA GGC CTG CCG GCT CCC CCC AGT ATC ATC AAG GCC ATG GGT  TTT GTG GAC AGT GTA GGC CTG CCG GCT CCC CCC AGT ATC ATC AAG GCC ATG GGT
Phe Val Asp Ser Val Gly Leu Pro Ala Pro Pro Ser Ile Ile Lys Ala MET Gly Phe Val Asp Ser Val Gly Leu Pro Ala Pro Pro Ser Ile Ile Lys Ala MET Gly
SEQUENCE N° 15 (SUITE) SEQUENCE N ° 15 (CONTINUED)
440 450 460 470 480  440 450 460 470 480
| | | | |  | | | | |
GGG AGC CAG CCA GGG GAA CTT CAG ATC AGC TGG GAG GAG CCA GCT CCA GAA ATC  GGG AGC CAG CCA GGG GAA CTT CAG ATC AGC TGG GAG GAG CCA GCT CCA GAA ATC
Gly Ser Gln Pro Gly Glu Leu Gln Ile Ser Trp Glu Glu Pro Ala Pro Glu Ile  Gly Ser Gln Pro Gly Glu Leu Gln Ile Ser Trp Glu Glu Pro Ala Pro Glu Ile
490 500 510 520 530 490 500 510 520 530
| | | | |
Figure imgf000046_0001
| | | | |
Figure imgf000046_0001
AGT GAT TTC CTG AGG TAC GAA CTC CGC TAT GGC CCC AGA GAT CCC AAG AAC TCC AGT GAT TTC CTG AGG TAC GAA CTC CGC TAT GGC CCC AGA GAT CCC AAG AAC TCC
Ser Asp Phe Leu Arg Tyr Glu Leu Arg Tyr Gly Pro Arg Asp Pro Lys Asn Ser  Ser Asp Phe Leu Arg Tyr Glu Leu Arg Tyr Gly Pro Arg Asp Pro Lys Asn Ser
550 560 570 580 590 | | | | | 550 560 570 580 590 | | | | |
ACT GGT CCC ACG GTC ATA CAG CTG ATT GCC ACA GAA ACC TGC TGC CCT GCT CTG  ACT GGT CCC ACG GTC ATA CAG CTG ATT GCC ACA GAA ACC TGC TGC CCT GCT CTG
Thr Gly Pro Thr Val Ile Gln Leu Ile Ala Thr Glu Thr Cys Cys Pro Ala Leu  Thr Gly Pro Thr Val Ile Gln Leu Ile Ala Thr Glu Thr Cys Cys Pro Ala Leu
600 610 620 630 640 600 610 620 630 640
| | | | |
Figure imgf000046_0002
| | | | |
Figure imgf000046_0002
CAG AGG CCT CAC TCA GCC TCT GCT CTG GAC CAG TCT CCA TGT GCT CAG CCC ACA CAG AGG CCT CAC TCA GCC TCT GCT CTG GAC CAG TCT CCA TGT GCT CAG CCC ACA
Gln Arg Pro His Ser Ala Ser Ala Leu Asp Gln Ser Pro Cys Ala Gln Pro Thr  Gln Arg Pro His Ser Ala Ser Ala Leu Asp Gln Ser Pro Cys Ala Gln Pro Thr
660 670 680 690 660 670 680 690
| | | |
Figure imgf000046_0003
| | | |
Figure imgf000046_0003
ATG CCC TGG CAA GAT GGA CCA AAG CAG ACC TCC CCA AGT AGA GAA GCT TCA GCT ATG CCC TGG CAA GAT GGA CCA AAG CAG ACC TCC CCA AGT AGA GAA GCT TCA GCT
MET Pro Trp Gln Asp Gly Pro Lys Gln Thr Ser Pro Ser Arg Glu Ala Ser Ala MET Pro Trp Gln Asp Gly Pro Lys Gln Thr Ser Pro Ser Arg Glu Ala Ser Ala
SEQUENCE N° 15 (SUITE) SEQUENCE N ° 15 (CONTINUED)
710 720 730 740 750  710 720 730 740 750
| | | | |  | | | | |
CTG ACA GCA GAG GGT GGA AGC TGC CTC ATC TCA GGA CTC CAG CCT GGC AAC TCC  CTG ACA GCA GAG GGT GGA AGC TGC CTC ATC TCA GGA CTC CAG CCT GGC AAC TCC
Leu Thr Ala Glu Gly Gly Ser Cys Leu Ile Ser Gly Leu Gln Pro Gly Asn Ser  Leu Thr Ala Glu Gly Gly Ser Cys Leu Ile Ser Gly Leu Gln Pro Gly Asn Ser
760 770 780 790 800 760 770 780 790 800
| | | | |
Figure imgf000047_0001
| | | | |
Figure imgf000047_0001
TAC TGG CTG CAG CTG CGC AGC GAA CCT GAT GGG ATC TCC CTC GGT GGC TCC TGG TAC TGG CTG CAG CTG CGC AGC GAA CCT GAT GGG ATC TCC CTC GGT GGC TCC TGG
Tyr Trp Leu Gln Leu Arg Ser Glu Pro Asp Gly Ile Ser Leu Gly Gly Ser Trp  Tyr Trp Leu Gln Leu Arg Ser Glu Pro Asp Gly Ile Ser Leu Gly Gly Ser Trp
820 830 840 350 860820 830 840 350 860
I I I I II I I I I
GGA TCC TGG TCC CTC CCT GTG ACT GTG GAC CTG CCT GGA GAT GCA GTG GCA CTT GGA TCC TGG TCC CTC CCT GTG ACT GTG GAC CTG CCT GGA GAT GCA GTG GCA CTT
Gly Ser Trp Ser Leu Pro Val Thr Val Asp Leu Pro Gly Asp Ala Val Ala Leu  Gly Ser Trp Ser Leu Pro Val Thr Val Asp Leu Pro Gly Asp Ala Val Ala Leu
870 880 89|0 900 910 870 880 89 | 0 900 910
| | | |
Figure imgf000047_0002
| | | |
Figure imgf000047_0002
GGA CTG CAA TGC TTT ACC TTG GAC CTG AAG AAT GTT ACC TGT CAA TGG CAG CAA GGA CTG CAA TGC TTT ACC TTG GAC CTG AAG AAT GTT ACC TGT CAA TGG CAG CAA
Gly Leu Gln Cys Phe Thr Leu Asp Leu Lys Asn Val Thr Cys Gln Trp Gln Gln  Gly Leu Gln Cys Phe Thr Leu Asp Leu Lys Asn Val Thr Cys Gln Trp Gln Gln
930 940 950 960 930 940 950 960
| | | |
Figure imgf000047_0003
| | | |
Figure imgf000047_0003
CAG GAC CAT GCT AGC TCC CAA GGC TTC TTC TAC CAC AGC AGG GCA CGG TGC TGC  CAG GAC CAT GCT AGC TCC CAA GGC TTC TTC TAC CAC AGC AGG GCA CGG TGC TGC
Gln Asp His Ala Ser Ser Gln Gly Phe Phe Tyr His Ser Arg Ala Arg Cys Cys  Gln Asp His Ala Ser Ser Gln Gly Phe Phe Tyr His Ser Arg Ala Arg Cys Cys
980 990 1000 1010 1020 980 990 1000 1010 1020
| | | | |  | | | | |
CCC AGA GAC AGG TAC CCC ATC TGG GAG AAC TGC GAA GAG GAA GAG AAA ACA AAT  CCC AGA GAC AGG TAC CCC ATC TGG GAG AAC TGC GAA GAG GAA GAG AAA ACA AAT
Pro Arg Asp Arg Tyr Pro Ile Trp Glu Asn Cys Glu Glu Glu Glu Lys Thr Asn Pro Arg Asp Arg Tyr Pro Ile Trp Glu Asn Cys Glu Glu Glu Glu Lys Thr Asn
SEQUENCE Nº 15 (SUITE) SEQUENCE Nº 15 (CONTINUED)
1030 1040 1050 1060 1070 10801030 1040 1050 1060 1070 1080
I I I I I II I I I I I
CCA GGA CTA CAG ACC CCA CAG TTC TCT CGC TGC CAC TTC AAG TCA CGA AAT GAC CCA GGA CTA CAG ACC CCA CAG TTC TCT CGC TGC CAC TTC AAG TCA CGA AAT GAC
Pro Gly Leu Gln Thr Pro Gln Phe Ser Arg Cys His Phe Lys Ser Arg Asn Asp  Pro Gly Leu Gln Thr Pro Gln Phe Ser Arg Cys His Phe Lys Ser Arg Asn Asp
1090 1100 1110 1120 1090 1100 1110 1120
| | | |
Figure imgf000048_0003
| | | |
Figure imgf000048_0003
AGC ATT ATT CAC ATC CTT GTG GAG GTG ACC ACA GCC CCG GGT ACT GTT CAC AGC AGC ATT ATT CAC ATC CTT GTG GAG GTG ACC ACA GCC CCG GGT ACT GTT CAC AGC
Ser Ile Ile His Ile Leu Val Glu Val Thr Thr Ala Pro Gly Thr Val His Ser  Ser Ile Ile His Ile Leu Val Glu Val Thr Thr Ala Pro Gly Thr Val His Ser
1140 1150 1160 1170 1180 1140 1150 1160 1170 1180
| | | | |
Figure imgf000048_0001
| | | | |
Figure imgf000048_0001
TAC CTG GGC TCC CCT TTC TGG ATC CAC CAG GCT GTG CGC CTC CCC ACC CCA AAC TAC CTG GGC TCC CCT TTC TGG ATC CAC CAG GCT GTG CGC CTC CCC ACC CCA AAC
Tyr Leu Gly Ser Pro Phe Trp Ile His Gln Ala Val Arg Leu Pro Thr Pro Asn  Tyr Leu Gly Ser Pro Phe Trp Ile His Gln Ala Val Arg Leu Pro Thr Pro Asn
1200 1210 1220 1230 1200 1210 1220 1230
| | | |
Figure imgf000048_0002
| | | |
Figure imgf000048_0002
TTG CAC TGG AGG GAG ATC TCC AGT GGG CAT CTG GAA TTG GAG TGG CAG CAC CCA  TTG CAC TGG AGG GAG ATC TCC AGT GGG CAT CTG GAA TTG GAG TGG CAG CAC CCA
Leu His Trp Arg Glu Ile Ser Ser Gly His Leu Glu Leu Glu Trp Gln His Pro  Leu His Trp Arg Glu Ile Ser Ser Gly His Leu Glu Leu Glu Trp Gln His Pro
1250 1260 1270 1280 1290 1250 1260 1270 1280 1290
| | | | |  | | | | |
TCG TCC TGG GCA GCC CAA GAG ACC TGT TAT CAA CTC CGA TAC ACA GGA GAA GGC  TCG TCC TGG GCA GCC CAA GAG ACC TGT TAT CAA CTC CGA TAC ACA GGA GAA GGC
Ser Ser Trp Ala Ala Gln Glu Thr Cys Tyr Gln Leu Arg Tyr Thr Gly Glu Gly Ser Ser Trp Ala Ala Gln Glu Thr Cys Tyr Gln Leu Arg Tyr Thr Gly Glu Gly
SEQUENCE N° 15 (SUITE) SEQUENCE N ° 15 (CONTINUED)
1300 1310 1320 1330 1340  1300 1310 1320 1330 1340
| | | | |
Figure imgf000049_0001
| | | | |
Figure imgf000049_0001
CAT CAG GAC TGG AAG GTG CTG GAG CCG CCT CTC GGG GCC CGA GGA GGG ACC CTG CAT CAG GAC TGG AAG GTG CTG GAG CCG CCT CTC GGG GCC CGA GGA GGG ACC CTG
His Gln Asp Trp Lys Val Leu Glu Pro Pro Leu Gly Ala Arg Gly Gly Thr Leu 1360 1370 1380 1390  His Gln Asp Trp Lys Val Leu Glu Pro Pro Leu Gly Ala Arg Gly Gly Thr Leu 1360 1370 1380 1390
| | | |
Figure imgf000049_0002
GAG CTG CGC CCG CGA TCT CGC TAC CGT TTA CAG CTG CGC GCC AGG CTC AAC GGC | | | |
Figure imgf000049_0002
GAG CTG CGC CCG CGA TCT CGC TAC CGT TTA CAG CTG CGC GCC AGG CTC AAC GGC
Glu Leu Arg Pro Arg Ser Arg Tyr Arg Leu Gln Leu Arg Ala Arg Leu Asn Gly 1410 1420 1430 1440 1450 0 | | | | |
Figure imgf000049_0003
CCC ACC TAC CAA GGT CCC TGG AGC TCG TGG TCG GAC CCA ACT AGG GTG GAG ACC Glu Leu Arg Pro Arg Ser Arg Tyr Arg Leu Gln Leu Arg Ala Arg Leu Asn Gly 1410 1420 1430 1440 1450 0 | | | | |
Figure imgf000049_0003
CCC ACC TAC CAA GGT CCC TGG AGC TCG TGG TCG GAC CCA ACT AGG GTG GAG ACC
Pro Thr Tyr Gln Gly Pro Trp Ser Ser Trp Ser Asp Pro Thr Arg Val Glu Thr 1470 1480 1490 1500  Pro Thr Tyr Gln Gly Pro Trp Ser Ser Trp Ser Asp Pro Thr Arg Val Glu Thr 1470 1480 1490 1500
| | | |
Figure imgf000049_0004
| | | |
Figure imgf000049_0004
GCC ACC GAG ACC GCC TGG ATC TCC TTG GTG ACC GCT CTG CAT CTA GTG CTG GGC  GCC ACC GAG ACC GCC TGG ATC TCC TTG GTG ACC GCT CTG CAT CTA GTG CTG GGC
Ala Thr Glu Thr Ala Trp Ile Ser Leu Val Thr Ala Leu His Leu Val Leu Gly 1520 1530 1540 1550 1560  Ala Thr Glu Thr Ala Trp Ile Ser Leu Val Thr Ala Leu His Leu Val Leu Gly 1520 1530 1540 1550 1560
| | | | |  | | | | |
CTC AGC GCC GTC CTG GGC CTG CTG CTG CTG AGG TGG CAG TTT CCT GCA CAC TAC  CTC AGC GCC GTC CTG GGC CTG CTG CTG CTG AGG TGG CAG TTT CCT GCA CAC TAC
Leu Ser Ala Val Leu Gly Leu Leu Leu Leu Arg Trp Gln Phe Pro Ala His Tyr  Leu Ser Ala Val Leu Gly Leu Leu Leu Leu Arg Trp Gln Phe Pro Ala His Tyr
1570 1580 1590 1600 1610 1570 1580 1590 1600 1610
| | | | |
Figure imgf000049_0005
| | | | |
Figure imgf000049_0005
AGG AGA CTG AGG CAT GCC CTG TGG CCC TCA CTT CCA GAC CTG CAC CGG GTC CTA AGG AGA CTG AGG CAT GCC CTG TGG CCC TCA CTT CCA GAC CTG CAC CGG GTC CTA
Arg Arg Leu Arg His Ala Leu Trp Pro Ser Leu Pro Asp Leu His Arg Val Leu Arg Arg Leu Arg His Ala Leu Trp Pro Ser Leu Pro Asp Leu His Arg Val Leu
SEQUENCE N° 15 (SUITE) SEQUENCE N ° 15 (CONTINUED)
1630 1640 1650 1660 1670  1630 1640 1650 1660 1670
I I I I I I I I I I
GGC CAG TAC CTT AGG GAC ACT GCA GCC CTG AGC CCG CCC AAG GCC ACA GTC TCA GGC CAG TAC CTT AGG GAC ACT GCA GCC CTG AGC CCG CCC AAG GCC ACA GTC TCA
Gly Gln Tyr Leu Arg Asp Thr Ala Ala Leu Ser Pro Pro Lys Ala Thr Val Ser Gly Gln Tyr Leu Arg Asp Thr Ala Ala Leu Ser Pro Pro Lys Ala Thr Val Ser
1680 1690 1700 1710 1720 1680 1690 1700 1710 1720
I l I I I
Figure imgf000050_0001
I l III
Figure imgf000050_0001
GAT ACC TGT GAA GAA GTG GAA CCC AGC CTC CTT GAA ATC CTC CCC AAG TCC TCA GAT ACC TGT GAA GAA GTG GAA CCC AGC CTC CTT GAA ATC CTC CCC AAG TCC TCA
Asp Thr Cys Glu Glu Val Glu Pro Ser Leu Leu Glu Ile Leu Pro Lys Ser Ser Asp Thr Cys Glu Glu Val Glu Pro Ser Leu Leu Glu Ile Leu Pro Lys Ser Ser
1740 1750 1760 1770 1780 1740 1750 1760 1770 1780
I I I I I I I I I I
GAG AGG ACT CCT TTG CCC CTG TGT TCC TCC CAG GCC CAG ATG GAC TAC CGA AGA GAG AGG ACT CCT TTG CCC CTG TGT TCC TCC CAG GCC CAG ATG GAC TAC CGA AGA
Glu Arg Thr Pro Leu Pro Leu Cys Ser Ser Gln Ala Gln MET Asp Tyr Arg Arg  Glu Arg Thr Pro Leu Pro Leu Cys Ser Ser Gln Ala Gln MET Asp Tyr Arg Arg
1790 1800 1810 1820 1830 1790 1800 1810 1820 1830
I I I I I I I I I I
TTG CAG CCT TCT TGC CTG GGG ACC ATG CCC CTG TCT GTG TGC CCA CCC ATG GCT TTG CAG CCT TCT TGC CTG GGG ACC ATG CCC CTG TCT GTG TGC CCA CCC ATG GCT
Leu Gln Pro Ser Cys Leu Gly Thr MET Pro Leu Ser Val Cys Pro Pro MET Ala Leu Gln Pro Ser Cys Leu Gly Thr MET Pro Leu Ser Val Cys Pro Pro MET Ala
SEQUENCE N° 15 (SUITE) 1840 1850 1360 1870 1380 1890SEQUENCE N ° 15 (CONTINUED) 1840 1850 1360 1870 1380 1890
| I I I I | GAG TCA GGG TCC TGC TGT ACC ACC CAC ATT GCC AAC CAT TCC TAC CTA CCA CTA | I I I I | GAG TCA GGG TCC TGC TGT ACC ACC CAC ATT GCC AAC CAT TCC TAC CTA CCA CTA
Glu Ser Gly Ser Cys Cys Thr Thr His Ile Ala Asn His Ser Tyr Leu Pro Leu 1900 1910  Glu Ser Gly Ser Cys Cys Thr Thr His Ile Ala Asn His Ser Tyr Leu Pro Leu 1900 1910
I |  I |
AGC TAT TGG CAG CAG CCT TGAGGACAGG CTCCTCACTC CCAGTTCCCT GGACAGAGCT  AGC TAT TGG CAG CAG CCT TGAGGACAGG CTCCTCACTC CCAGTTCCCT GGACAGAGCT
Ser Tyr Trp Gln Gln Pro AAACTCTCGA GACTTCTCTG TGAACTTCCC TACCCTACCC CCACAACACA AGCACCCCAG ACCTCACCTC CATCCCCCTC TGTCTG Ser Tyr Trp Gln Gln Pro AAACTCTCGA GACTTCTCTG TGAACTTCCC TACCCTACCC CCACAACACA AGCACCCCAG ACCTCACCTC CATCCCCCTC TGTCTG
SEQUENCE ID Nº16 SEQUENCE ID Nº16
1350 1360 1370 1380 1390 1350 1360 1370 1380 1390
I I I I I I I I I I
CTG GAG CTG CGC CCG CGA TCT CGC TAC CGT TTA CAG CTG CGC GCC CTG GAG CTG CGC CCG CGA TCT CGC TAC CGT TTA CAG CTG CGC GCC
Leu Glu Leu Arg Pro Arg Ser Arg Tyr Arg Leu Gln Leu Arg Ala  Leu Glu Leu Arg Pro Arg Ser Arg Tyr Arg Leu Gln Leu Arg Ala
1400 1410 1420 1430 1400 1410 1420 1430
| | | |  | | | |
AGG CTC AAC GGC CCC ACC TAC CAA GGT CCC TGG AGC TCG TGG TCG  AGG CTC AAC GGC CCC ACC TAC CAA GGT CCC TGG AGC TCG TGG TCG
Arg Leu Asn Gly Pro Thr Tyr Gln Gly Pro Trp Ser Ser Trp Ser  Arg Leu Asn Gly Pro Thr Tyr Gln Gly Pro Trp Ser Ser Trp Ser
1440 1450 1460 1470 1480 1440 1450 1460 1470 1480
I I I l I I I I l I
GAC CCA ACT AGG GTG GAG ACC GCC ACC GAG ACC GCC TGG ATC TCC GAC CCA ACT AGG GTG GAG ACC GCC ACC GAG ACC GCC TGG ATC TCC
Asp Pro Thr Arg Val Glu Thr Ala Thr Glu Thr Ala Trp Ile Ser  Asp Pro Thr Arg Val Glu Thr Ala Thr Glu Thr Ala Trp Ile Ser
1490 1500 1510 1520 1490 1500 1510 1520
I I I I I I I I
TTG GTG ACC GCT CTG CAT CTA GTG CTG GGC CTC AGC GCC GTC CTG TTG GTG ACC GCT CTG CAT CTA GTG CTG GGC CTC AGC GCC GTC CTG
Leu Val Thr Ala Leu His Leu Val Leu Gly Leu Ser Ala Val Leu  Leu Val Thr Ala Leu His Leu Val Leu Gly Leu Ser Ala Val Leu
1530 1540 1550 1560 1570 1530 1540 1550 1560 1570
I I I I I I I I I I
GGC CTG CTG CTG CTG AGG TGG CAG TTT CCT GCA CAC TAC AGG AGA GGC CTG CTG CTG CTG AGG TGG CAG TTT CCT GCA CAC TAC AGG AGA
Gly Leu Leu Leu Leu Arg Trp Gln Phe Pro Ala His Tyr Arg Arg  Gly Leu Leu Leu Leu Arg Trp Gln Phe Pro Ala His Tyr Arg Arg
1580 1590 1600 1610 1580 1590 1600 1610
| | | |  | | | |
CTG AGG CAT GCC CTG TGG CCC TCA CTT CCA GAC CTG CAC CGG GTC  CTG AGG CAT GCC CTG TGG CCC TCA CTT CCA GAC CTG CAC CGG GTC
Leu Arg His Ala Leu Trp Pro Ser Leu Pro Asp Leu His Arg Val  Leu Arg His Ala Leu Trp Pro Ser Leu Pro Asp Leu His Arg Val
1620 1630 1640 1650 1660 1620 1630 1640 1650 1660
I I I I I I I I I I
CTA GGC CAG TAC CTT AGG GAC ACT GCA GCC CTG AGC CCG CCC AAG CTA GGC CAG TAC CTT AGG GAC ACT GCA GCC CTG AGC CCG CCC AAG
Leu Gly Gln Tyr Leu Arg Asp Thr Ala Ala Leu Ser Pro Pro Lys 1670 1680 1690 1700  Leu Gly Gln Tyr Leu Arg Asp Thr Ala Ala Leu Ser Pro Pro Lys 1670 1680 1690 1700
| | | |  | | | |
GCC ACA GTC TCA GAT ACC TGT GAA GAA GTG GAA CCC AGC CTC CTT  GCC ACA GTC TCA GAT ACC TGT GAA GAA GTG GAA CCC AGC CTC CTT
Ala Thr Val Ser Asp Thr Cys Glu Glu Val Glu Pro Ser Leu Leu SEQUENCE N° 16 (SUITE) Ala Thr Val Ser Asp Thr Cys Glu Glu Val Glu Pro Ser Leu Leu SEQUENCE N ° 16 (CONTINUED)
1710 1720 1730 1740 1750 1710 1720 1730 1740 1750
| | | | | | | | | |
GAA ATC CTC CCC AAG TCC TCA GAG AGG ACT CCT TTG CCC CTG TGT GAA ATC CTC CCC AAG TCC TCA GAG AGG ACT CCT TTG CCC CTG TGT
Glu Ile Leu Pro Lys Ser Ser Glu Arg Thr Pro Leu Pro Leu Cys  Glu Ile Leu Pro Lys Ser Ser Glu Arg Thr Pro Leu Pro Leu Cys
1760 1770 1780 1790 1760 1770 1780 1790
| | | | | | | |
TCC TCC CAG GCC CAG ATG GAC TAC CGA AGA TTG CAG CCT TCT TGC TCC TCC CAG GCC CAG ATG GAC TAC CGA AGA TTG CAG CCT TCT TGC
Ser Ser Gln Ala Gln MET Asp Tyr Arg Arg Leu Gln Pro Ser Cys  Ser Ser Gln Ala Gln MET Asp Tyr Arg Arg Leu Gln Pro Ser Cys
1800 1810 1820 1830 1840 1800 1810 1820 1830 1840
| | | | | | | | | |
CTG GGG ACC ATG CCC CTG TCT GTG TGC CCA CCC ATG GCT GAG TCA CTG GGG ACC ATG CCC CTG TCT GTG TGC CCA CCC ATG GCT GAG TCA
Leu Gly Thr MET Pro Leu Ser Val Cys Pro Pro MET Ala Glu Ser 1850 1860 1870 1880  Leu Gly Thr MET Pro Leu Ser Val Cys Pro Pro MET Ala Glu Ser 1850 1860 1870 1880
| | | |  | | | |
GGG TCC TGC TGT ACC ACC CAC ATT GCC AAC CAT TCC TAC CTA CCA  GGG TCC TGC TGT ACC ACC CAC ATT GCC AAC CAT TCC TAC CTA CCA
Gly Ser Cys cys Thr Thr His Ile Ala Asn His Ser Tyr Leu Pro  Gly Ser Cys cys Thr Thr His Ile Ala Asn His Ser Tyr Leu Pro
1890 1900 1890 1900
| |  | |
CTA AGC TAT TGG CAG  CTA AGC TAT TGG CAG
Leu Ser Tyr Trp Gln Leu Ser Tyr Trp Gln
10 20 30 4010 20 30 40
ATC CTA CT IG CTC AGC TACI GCA GCC AAC CIGA CGG GGT CTIC C ATC CTA CT IG CTC AGC TACI GCA GCC AAC CIGA CGG GGT CTIC C
Ile Leu Leu Leu Ser Tyr Ala Ala Asn Arg Arg Gly Leu P  Ile Leu Leu Leu Ser Tyr Ala Ala Asn Arg Arg Gly Leu P
60 70 80 90 60 70 80 90
I I
GGA CCC TGG TCC TTC CCT GTG ACT GTG GAT CTT CCA GGA G GGA CCC TGG TCC TTC CCT GTG ACT GTG GAT CTT CCA GGA G
Gly Pro Trp Ser Phe Pro Val Thr Val Asp Leu Pro Gly G  Gly Pro Trp Ser Phe Pro Val Thr Val Asp Leu Pro Gly G
120 130 140 150120 130 140 150
GGA CTT CAG TGC TTT ACC TTG GAT CTG AAG ATG GTC ACC T GGA CTT CAG TGC TTT ACC TTG GAT CTG AAG ATG GTC ACC T
Gly Leu Gln Cys Phe Thr Leu Asp Leu Lys MET Val Thr C  Gly Leu Gln Cys Phe Thr Leu Asp Leu Lys MET Val Thr C
170 180 190 200 170 180 190 200
I I I I I I I I
CAA GAC CGC ACT AGC TCC CAA GGC TTC TTC CGT CAC AGC A CAA GAC CGC ACT AGC TCC CAA GGC TTC TTC CGT CAC AGC A
Gln Asp Arg Thr Ser Ser Gln Gly Phe Phe Arg His Ser A  Gln Asp Arg Thr Ser Ser Gln Gly Phe Phe Arg His Ser A
220 230 240 250 26220 230 240 250 26
I I I II I I I
CCC ACA GAC AGG GAC CCC ACC TGG GAG AAA TGT GAA GAG GA CCC ACA GAC AGG GAC CCC ACC TGG GAG AAA TGT GAA GAG GA
Pro Thr Asp Arg Asp Pro Thr Trp Glu Lys Cys Glu Glu Gl Pro Thr Asp Arg Asp Pro Thr Trp Glu Lys Cys Glu Glu Gl
SEQUENCE N° 17 (SUITE) SEQUENCE N ° 17 (CONTINUED)
280 290 300 310 320 | | | | |280 290 300 310 320 | | | | |
GGA TCA CAG CCC GCT CTC GTC TCC CGC TGC CAC TTC AAG TCA CGA AAT GAC AGT GGA TCA CAG CCC GCT CTC GTC TCC CGC TGC CAC TTC AAG TCA CGA AAT GAC AGT
Gly Ser Gln Pro Ala Leu Val Ser Arg Cys His Phe Lys Ser Arg Asn Asp Ser Gly Ser Gln Pro Ala Leu Val Ser Arg Cys His Phe Lys Ser Arg Asn Asp Ser
330 340 350 360 370 330 340 350 360 370
| | | | |
Figure imgf000055_0001
| | | | |
Figure imgf000055_0001
GTT ATT CAC ATC CTT GTA GAG GTG ACC ACA GCG CAA GGT GCC GTT CAC AGC TAC GTT ATT CAC ATC CTT GTA GAG GTG ACC ACA GCG CAA GGT GCC GTT CAC AGC TAC
Val Ile His Ile Leu Val Glu Val Thr Thr Ala Gln Gly Ala Val His Ser Tyr Val Ile His Ile Leu Val Glu Val Thr Thr Ala Gln Gly Ala Val His Ser Tyr
390 400 410 420 390 400 410 420
I | | |
Figure imgf000055_0002
I | | |
Figure imgf000055_0002
CTG GGC TCC CCT TTT TGG ATC CAC CAG GCT GTG CTC CTT CCC ACC CCG AGC CTG  CTG GGC TCC CCT TTT TGG ATC CAC CAG GCT GTG CTC CTT CCC ACC CCG AGC CTG
Leu Gly Ser Pro Phe Trp Ile His Gln Ala Val Leu Leu Pro Thr Pro Ser Leu Leu Gly Ser Pro Phe Trp Ile His Gln Ala Val Leu Leu Pro Thr Pro Ser Leu
SEQUENCE N° 17 (SUITE) SEQUENCE N ° 17 (CONTINUED)
440 450 460 470 480  440 450 460 470 480
| | | | |  | | | | |
CAC TGG AGG GAG GTC TCA AGT GGA AGG CTG GAG TTG GAG TGG CAG CAC CAG TCA  CAC TGG AGG GAG GTC TCA AGT GGA AGG CTG GAG TTG GAG TGG CAG CAC CAG TCA
His Trp Arg Glu Val Ser Ser Gly Arg Leu Glu Leu Glu Trp Gln His Gln Ser  His Trp Arg Glu Val Ser Ser Gly Arg Leu Glu Leu Glu Trp Gln His Gln Ser
490 500 510 520 530 490 500 510 520 530
| | | | |
Figure imgf000056_0001
| | | | |
Figure imgf000056_0001
TCT TGG GCA GCT CAA GAG ACC TGC TAC CAG CTC CGG TAC ACG GGA GAA GGC CGT TCT TGG GCA GCT CAA GAG ACC TGC TAC CAG CTC CGG TAC ACG GGA GAA GGC CGT
Ser Trp Ala Ala Gln Glu Thr Cys Tyr Gln Leu Arg Tyr Thr Gly Glu Gly Arg  Ser Trp Ala Ala Gln Glu Thr Cys Tyr Gln Leu Arg Tyr Thr Gly Glu Gly Arg
550 560 570 580 590 | | | | | 550 560 570 580 590 | | | | |
GAG GAC TGG AAG GTG CTG GAG CCA TCT CTC GGT GCC CGG GGA GGG ACC CTA GAG  GAG GAC TGG AAG GTG CTG GAG CCA TCT CTC GGT GCC CGG GGA GGG ACC CTA GAG
Glu Asp Trp Lys Val Leu Glu Pro Ser Leu Gly Ala Arg Gly Gly Thr Leu Glu  Glu Asp Trp Lys Val Leu Glu Pro Ser Leu Gly Ala Arg Gly Gly Thr Leu Glu
600 610 620 630 640 600 610 620 630 640
| | | | |
Figure imgf000056_0002
| | | | |
Figure imgf000056_0002
CTG CGC CCC CGA GCT CGC TAC AGC TTG CAG CTG CGT GCC AGG CTC AAC GGC CCC CTG CGC CCC CGA GCT CGC TAC AGC TTG CAG CTG CGT GCC AGG CTC AAC GGC CCC
Leu Arg Pro Arg Ala Arg Tyr Ser Leu Gln Leu Arg Ala Arg Leu Asn Gly Pro  Leu Arg Pro Arg Ala Arg Tyr Ser Leu Gln Leu Arg Ala Arg Leu Asn Gly Pro
660 670 680 690 660 670 680 690
| | | |
Figure imgf000056_0003
| | | |
Figure imgf000056_0003
ACC TAC CAA GGT CCC TGG AGC GCC TGG TCT CCC CCA GCT AGG GTG TCC ACG GGC  ACC TAC CAA GGT CCC TGG AGC GCC TGG TCT CCC CCA GCT AGG GTG TCC ACG GGC
Thr Tyr Gln Gly Pro Trp Ser Ala Trp Ser Pro Pro Ala Arg Val Ser Thr Gly  Thr Tyr Gln Gly Pro Trp Ser Ala Trp Ser Pro Pro Ala Arg Val Ser Thr Gly
710 720 730 740 750 710 720 730 740 750
| | | | |  | | | | |
TCC GAG ACT GCT TGG ATC ACC TTG GTG ACT GCT CTG CTC CTG GTG CTG AGC CTC  TCC GAG ACT GCT TGG ATC ACC TTG GTG ACT GCT CTG CTC CTG GTG CTG AGC CTC
Ser Glu Thr Ala Trp Ile Thr Leu Val Thr Ala Leu Leu Leu Val Leu Ser Leu Ser Glu Thr Ala Trp Ile Thr Leu Val Thr Ala Leu Leu Leu Val Leu Ser Leu
SEQUENCE N° 17 (SUITE) SEQUENCE N ° 17 (CONTINUED)
760 770 780 790 800  760 770 780 790 800
| | | | |
Figure imgf000057_0001
| | | | |
Figure imgf000057_0001
AGT GCC CTT CTG GGC CTA CTG CTG CTA AAG TGG CAA TTT CCT GCG CAC TAC AGG AGT GCC CTT CTG GGC CTA CTG CTG CTA AAG TGG CAA TTT CCT GCG CAC TAC AGG
Ser Ala Leu Leu Gly Leu Leu Leu Leu Lys Trp Gln Phe Pro Ala His Tyr Arg 820 830 840 850  Ser Ala Leu Leu Gly Leu Leu Leu Leu Lys Trp Gln Phe Pro Ala His Tyr Arg 820 830 840 850
| | | |
Figure imgf000057_0002
AGA CTG AGG CAT GCT TTG TGG CCC TCG CTT CCA GAC CTA CAC CGG GTC CTA GGC | | | |
Figure imgf000057_0002
AGA CTG AGG CAT GCT TTG TGG CCC TCG CTT CCA GAC CTA CAC CGG GTC CTA GGC
Arg Leu Arg His Ala Leu Trp Pro Ser Leu Pro Asp Leu His Arg Val Leu Gly 870 880 890 900 910  Arg Leu Arg His Ala Leu Trp Pro Ser Leu Pro Asp Leu His Arg Val Leu Gly 870 880 890 900 910
| | | | |
Figure imgf000057_0003
CAG TAC CTC AGA GAC ACT GCA GCC CTA AGT CCT TCT AAG GCC ACG GTT ACC GAT | | | | |
Figure imgf000057_0003
CAG TAC CTC AGA GAC ACT GCA GCC CTA AGT CCT TCT AAG GCC ACG GTT ACC GAT
Gln Tyr Leu Arg Asp Thr Ala Ala Leu Ser Pro Ser Lys Ala Thr Val Thr Asp 930 940 950 960  Gln Tyr Leu Arg Asp Thr Ala Ala Leu Ser Pro Ser Lys Ala Thr Val Thr Asp 930 940 950 960
| | | |
Figure imgf000057_0004
| | | |
Figure imgf000057_0004
AGC TGT GAA GAA GTG GAA CCC AGC CTC CTG GAA ATC CTC CCT AAA TCC TCA GAG  AGC TGT GAA GAA GTG GAA CCC AGC CTC CTG GAA ATC CTC CCT AAA TCC TCA GAG
Ser Cys Glu Glu Val Glu Pro Ser Leu Leu Glu Ile Leu Pro Lys Ser Ser Glu  Ser Cys Glu Glu Val Glu Pro Ser Leu Leu Glu Ile Leu Pro Lys Ser Ser Glu
980 990 1000 1010 1020 980 990 1000 1010 1020
| | | | |  | | | | |
AGC ACT CCT TTA CCT CTG TGT CCC TCC CAA CCT CAG ATG GAC TAC AGA GGA CTG  AGC ACT CCT TTA CCT CTG TGT CCC TCC CAA CCT CAG ATG GAC TAC AGA GGA CTG
Ser Thr Pro Leu Pro Leu Cys Pro Ser Gln Pro Gln MET Asp Tyr Arg Gly Leu Ser Thr Pro Leu Pro Leu Cys Pro Ser Gln Pro Gln MET Asp Tyr Arg Gly Leu
SEQUENCE N° 17 (SUITE) SEQUENCE N ° 17 (CONTINUED)
1030 1040 1050 1060 1070 1080 l l l l l I CAA CCT TGC CTG CGG ACC ATG CCC CTG TCT GTG TGT CCA CCC ATG GCT GAG ACG 1030 1040 1050 1060 1070 1080 l l l l l CAA CCT TGC CTG CGG ACC ATG CCC CTG TCT GTG TGT CCA CCC ATG GCT GAG ACG
Gln Pro Cys Leu Arg Thr MET Pro Leu Ser Val Cys Pro Pro MET Ala Glu Thr  Gln Pro Cys Leu Arg Thr MET Pro Leu Ser Val Cys Pro Pro MET Ala Glu Thr
1090 1100 1110 1120 1130 | | | | | 1090 1100 1110 1120 1130 | | | | |
GGG TCC TGC TGC ACC ACA CAC ATT GCC AAC CAC TCC TAC CTA CCA CTA AGC TAT  GGG TCC TGC TGC ACC ACA CAC ATT GCC AAC CAC TCC TAC CTA CCA CTA AGC TAT
Gly Ser Cys Cys Thr Thr His Ile Ala Asn His Ser Tyr Leu Pro Leu Ser Tyr 1140  Gly Ser Cys Cys Thr Thr His Ile Ala Asn His Ser Tyr Leu Pro Leu Ser Tyr 1140
|  |
TGG CAG CAG CCC TGAAGGCAGT CCCCATGCTA CTGCAGACCT ATACATTCCT ACACACTACC  TGG CAG CAG CCC TGAAGGCAGT CCCCATGCTA CTGCAGACCT ATACATTCCT ACACACTACC
Trp Gin Gin Pro ---  Trp Gin Gin Pro ---
TTATCCATCC TCAACACCAT CCATTCTGTT GCCACCCCAC TCCCCCTCTG GCTTTATAAC TTATCCATCC TCAACACCAT CCATTCTGTT GCCACCCCAC TCCCCCTCTG GCTTTATAAC
ACTGATCACT CCAAGATGGC TGCTCACAAA TCCAGAGCTC TGTCTCTGCA G ACTGATCACT CCAAGATGGC TGCTCACAAA TCCAGAGCTC TGTCTCTGCA G
SEQUENCE ID Nº 18 SEQUENCE ID Nº 18
310 320 330 340 310 320 330 340
| | | |  | | | |
CTA GAG CTG CGC CCC CGA GCT CGC TAC AGC TTG CAG CTG CGT GCC  CTA GAG CTG CGC CCC CGA GCT CGC TAC AGC TTG CAG CTG CGT GCC
Leu Glu Leu Arg Pro Arg Ala Arg Tyr Ser Leu Gln Leu Arg Ala Leu Glu Leu Arg Pro Arg Ala Arg Tyr Ser Leu Gln Leu Arg Ala
350 360 370 380 390 | | | | |350 360 370 380 390 | | | | |
AGG CTC AAC GGC CCC ACC TAC CAA GGT CCC TGG AGC GCC TGG TCT AGG CTC AAC GGC CCC ACC TAC CAA GGT CCC TGG AGC GCC TGG TCT
Arg Leu Asn Gly Pro Thr Tyr Gln Gly Pro Trp Ser Ala Trp Ser Arg Leu Asn Gly Pro Thr Tyr Gln Gly Pro Trp Ser Ala Trp Ser
400 410 420 430 400 410 420 430
| | | |  | | | |
CCC CCA GCT AGG GTG TCC ACG GGC TCC GAG ACT GCT TGG ATC ACC  CCC CCA GCT AGG GTG TCC ACG GGC TCC GAG ACT GCT TGG ATC ACC
Pro Pro Ala Arg Val Ser Thr Gly Ser Glu Thr Ala Trp Ile Thr Pro Pro Ala Arg Val Ser Thr Gly Ser Glu Thr Ala Trp Ile Thr
440 450 460 470 480 | | | | |440 450 460 470 480 | | | | |
TTG GTG ACT GCT CTG CTC CTG GTG CTG AGC CTC AGT GCC CTT CTG TTG GTG ACT GCT CTG CTC CTG GTG CTG AGC CTC AGT GCC CTT CTG
Leu Val Thr Ala Leu Leu Leu Val Leu Ser Leu Ser Ala Leu Leu  Leu Val Thr Ala Leu Leu Leu Val Leu Ser Leu Ser Ala Leu Leu
490 500 510 520 490 500 510 520
| | | |  | | | |
GGC CTA CTG CTG CTA AAG TGG CAA TTT CCT GCG CAC TAC AGG AGA  GGC CTA CTG CTG CTA AAG TGG CAA TTT CCT GCG CAC TAC AGG AGA
Gly Leu Leu Leu Leu Lys Trp Gln Phe Pro Ala His Tyr Arg Arg  Gly Leu Leu Leu Leu Lys Trp Gln Phe Pro Ala His Tyr Arg Arg
530 540 550 560 570 | | | | |530 540 550 560 570 | | | | |
CTG AGG CAT GCT TTG TGG CCC TCG CTT CCA GAC CTA CAC CGG GTC CTG AGG CAT GCT TTG TGG CCC TCG CTT CCA GAC CTA CAC CGG GTC
Leu Arg His Ala Leu Trp Pro Ser Leu Pro Asp Leu His Arg Val  Leu Arg His Ala Leu Trp Pro Ser Leu Pro Asp Leu His Arg Val
580 590 600 610 580 590 600 610
| | | |  | | | |
CTA GGC CAG TAC CTC AGA GAC ACT GCA GCC CTA AGT CCT TCT AAG  CTA GGC CAG TAC CTC AGA GAC ACT GCA GCC CTA AGT CCT TCT AAG
Leu Gly Gln Tyr Leu Arg Asp Thr Ala Ala Leu Ser Pro Ser Lys  Leu Gly Gln Tyr Leu Arg Asp Thr Ala Ala Leu Ser Pro Ser Lys
620 630 640 650 660 | | | | |620 630 640 650 660 | | | | |
GCC ACG GTT ACC GAT AGC TGT GAA GAA GTG GAA CCC AGC CTC CTG GCC ACG GTT ACC GAT AGC TGT GAA GAA GTG GAA CCC AGC CTC CTG
Ala Thr Val Thr Asp Ser Cys Glu Glu Val Glu Pro Ser Leu Leu SEQUENCE Nº 18 ( SUITE) Ala Thr Val Thr Asp Ser Cys Glu Glu Val Glu Pro Ser Leu Leu SEQUENCE Nº 18 (CONTINUED)
670 680 690 700 670 680 690 700
| | | |  | | | |
GAA ATC CTC CCT AAG TCC TCA GAG AGC ACT CCT TTA CCT CTG TGT  GAA ATC CTC CCT AAG TCC TCA GAG AGC ACT CCT TTA CCT CTG TGT
Glu Ile Leu Pro Lys Ser Ser Glu Ser Thr Pro Leu Pro Leu Cys Glu Ile Leu Pro Lys Ser Ser Glu Ser Thr Pro Leu Pro Leu Cys
710 720 730 740 750 | | | | |710 720 730 740 750 | | | | |
CCC TCC CAA CCT CAG ATG GAC TAC AGA GGA CTG CAA CCT TGC CTG CCC TCC CAA CCT CAG ATG GAC TAC AGA GGA CTG CAA CCT TGC CTG
Pro Ser Gln Pro Gln MET Asp Tyr Arg Gly Leu Gin Pro Cys Leu Pro Ser Gln Pro Gln MET Asp Tyr Arg Gly Leu Gin Pro Cys Leu
760 770 780 790 760 770 780 790
| | | |  | | | |
CGG ACC ATG C ICC CTG TCT GTG TGT CCA CCC ATG GCT GAG ACG GGG  CGG ACC ATG C ICC CTG TCT GTG TGT CCA CCC ATG GCT GAG ACG GGG
Arg Thr MET Pro Leu Ser Val Cys Pro Pro MET Ala Glu Thr Gly  Arg Thr MET Pro Leu Ser Val Cys Pro Pro MET Ala Glu Thr Gly
800 810 820 830 840 | | | | |800 810 820 830 840 | | | | |
TCC TGC TGC ACC ACA CAC ATT GCC AAC CAC TCC TAC CTA CCA CTA TCC TGC TGC ACC ACA CAC ATT GCC AAC CAC TCC TAC CTA CCA CTA
Ser Cys Cys Thr Thr His Ile Ala Asn His Ser Tyr Leu Pro Leu Ser Cys Cys Thr Thr His Ile Ala Asn His Ser Tyr Leu Pro Leu
850 850
|  |
AGC TAT TGG CAG AGC TAT TGG CAG
Ser Tyr Trp Gln Ser Tyr Trp Gln
SEQUENCE ID Nº19 SEQUENCE ID Nº19
500 510 520 530 540 500 510 520 530 540
| | | | | | | | | |
TGG CAA TTT CCT GCG CAC TAC AGG AGA CTG AGG CAT GCT TTG TGG TGG CAA TTT CCT GCG CAC TAC AGG AGA CTG AGG CAT GCT TTG TGG
Trp Gln Phe Pro Ala His Tyr Arg Arg Leu Arg His Ala Leu Trp  Trp Gln Phe Pro Ala His Tyr Arg Arg Leu Arg His Ala Leu Trp
550 560 570 580 550 560 570 580
| | | | | | | |
CCC TCG CTT CCA GAC CTA CAC CGG GTC CTA GGC CAG TAC CTC AGA CCC TCG CTT CCA GAC CTA CAC CGG GTC CTA GGC CAG TAC CTC AGA
Pro Ser Leu Pro Asp Leu His Arg Val Leu Gly Gln Tyr Leu Arg  Pro Ser Leu Pro Asp Leu His Arg Val Leu Gly Gln Tyr Leu Arg
590 600 590,600
| |  | |
GAC ACT GCA GCC CTA AGT CCT ASD Thr Ala Ala Leu Ser Pro GAC ACT GCA GCC CTA AGT CCT ASD Thr Ala Ala Leu Ser Pro
SEQUENCE ID Nº 20 SEQUENCE ID Nº 20
M A C S T L P K S P K D K I D P R D L L 20 1 ATGGCGTGTTCAACGCTCCCAAAATCCCCTAAAGATAAGATTGACCCGCGGGACCTCCTA M A C S T L P K S P K D K I D P R D L L 20 1 ATGGCGTGTTCAACGCTCCCAAAATCCCCTAAAGATAAGATTGACCCGCGGGACCTCCTA
I P L I L F L S L K G A R S A A P G S S 40 61 ATCCCCTTAATTCTCTTCCTGTCTCTCAAAGGGGCCAGATCCGCAGCACCCGGCTCCAGC  I P L I L F L S L K G A R S A A P G S S 40 61 ATCCCCTTAATTCTCTTCCTGTCTCTCAAAGGGGCCAGATCCGCAGCACCCGGCTCCAGC
P H Q V Y N I T W E V T N G D R E T V W 60 121 CCTCACCAGGTCTACAACATTACCTGGGAAGTGACCAATGGGGATCGGGAGACAGTATGG  P H Q V Y N I T W E V T N G D R E T V W 60 121 CCTCACCAGGTCTACAACATTACCTGGGAAGTGACCAATGGGGATCGGGAGACACTTGG
A I S G R L Y V S G R D P G L T F G I R 80 181 GCAATATCAGGACGTCTTTATGTCΓCTGGGCGGGACCCGGGGCTTACTTTCGGGATCCGA  A I S G R L Y V S G R D P G L T F G I R 80 181 GCAATATCAGGACGTCTTTATGTCΓCTGGGCGGGACCCGGGGCTTACTTTCGGGATCCGA
L R Y Q N L G P R V P I G P N P V L A D 100 241 CTCAGATATCAAAATCTAGGACCTCGGGTCCCGATAGGACCGAACCCCGTCCTGGCAGAC  L R Y Q N L G P R V P I G P N P V L A D 100 241 CTCAGATATCAAAATCTAGGACCTCGGGTCCCGATAGGACCGAACCCCGTCCTGGCAGAC
L E L R P R A R Y S L Q L R A R L N G P 120 L E L R P R A R Y S L Q L R A R L N G P 120
301 CTAGAGCTGCGCCCCCGAGCTCGCTACAGCTTGCAGCTGCGTGCCAGGCTCAACGGCCCC301 CTAGAGCTGCGCCCCCGAGCTCGCTACAGCTTGCAGCTGCGTGCCAGGCTCAACGGCCCC
T Y Q G P W S A W S P P A R V S T G S E 140 361 ACCTACCAAGGTCCCTGGAGCGCCTGGTCTCCCCCAGCTAGGGTGTCCACGGGCTCCGAG T Y Q G P W S A W S P P A R V S T G S E 140 361 ACCTACCAAGGTCCCTGGAGCGCCTGGTCTCCCCCAGCTAGGGTGTCCACGGGCTCCGAG
T A W I T L V T A L L L V L S L S A L L 160 421 ACTGCTTGGATCACCTTGGTGACTGC-TCTGCTCCTGGTGCTGAGCCrCAGTGCCCTTCTG T A W I T L V T A L L L V L S L S A L L 160 421 ACTGCTTGGATCACCTTGGTGACTGC-TCTGCTCCTGGTGCTGAGCCrCAGTGCCCTTCTG
G L L L L X W Q F P A H Y R R L R H A L 180 481 GGCCTACrGCTGCTAAAGTGGCAATTTCCTGCGCACTACAGGAGACTGAGGCATGCTTTGG L L L L X W Q F P A H Y R R L R H A L 180 481 GGCCTACrGCTGCTAAAGTGGCAATTTCCTGCGCACTACAGGAGACTGAGGCATGCTTTG
W P S L P D L H R V L G Q Y L R D T A A 200 541 TGGCCCTCGCTTCCAGACCTACACCGGGTCCTAGGCCAGTACCTCAGAGACACTGCAGCCW P S L P D L H R V L G Q Y L R D T A A 200 541 TGGCCCTCGCTTCCAGACCTACACCGGGTCCTAGGCCAGTACCTCAGAGACACTGCAGCC
L S P S K A T V T D S C E E V E P S L L 220 601 CTAAGTCCTTCTAAGGCCACGGTTACCGATAGCTGTGAAGAAGTGGAACCCAGCCTCCTGL S P S K A T V T D S C E E V E P S L L 220 601 CTAAGTCCTTCTAAGGCCACGGTTACCGATAGCTGTGAAGAAGTGGAACCCAGCCTCCTG
E I L P X S S E S T P L P L C P S Q P Q 240 661 GAAATCCTCCCTAAGTCCTCAGAGAGCACTCCTTTACCTCTGTGTCCCTCCCAACCTCAGE I L P X S S E S T P L P L C P S Q P Q 240 661 GAAATCCTCCCTAAGTCCTCAGAGAGCACTCCTTTACCTCTGTGTCCCTCCCAACCTCAG
M D Y R G L Q P C L R T M P L S V C P P 260 721 ATGGACTACAGAGGACTGCAACCTTGCCTGCGGACCATGCCCCTGTCTGTGTGTCCACCCM D Y R G L Q P C L R T M P L S V C P P 260 721 ATGGACTACAGAGGACTGCAACCTTGCCTGCGGACCATGCCCCTGTCTGTGTGTCCACCC
M A E T G S C C T T H I A N H S Y L P L 280 781 ATGGCrGAGACGGGGTCCTGCTGCACCACACACATTGCCAACCACTCCTACCTACCACTAM A E T G S C C T T H I A N H S Y L P L 280 781 ATGGCrGAGACGGGGTCCTGCTGCACCACACACATTGCCAACCACTCCTACCTACCACTA
S Y W Q - 284S Y W Q - 284
841 AGCTATTGGCAGTAGTCCTGAAGGCCAGTCCCCATGCRACTGCAGACCTATACATTCCTAC 901 ACACTACCTTATCCATCGACCTCTAGGCCTAGTAAGAGATAGTATGGCCAAATTAAGAGA 961 GAGACTCACTCAGAGACAAAAACTATTTGAGTCGAGCCAAGGATGGTTCGAAGGATTGTT841 AGCTATTGGCAGTAGTCCTGAAGGCCAGTCCCCATGCRACTGCAGACCTATACATTCCTAC 901 ACACTACCTTATCCATCGACCTCTAGGCCTAGTAAGAGATAGTATGGCCAAATTAAGAGA 961 GAGACTCACTCAGAGACAAAAACTATTTGAGTCGAGCCTAGGATGATT
1021 TAACAGATCCCCCTGGTTTACCACGTTAATATCCACCATCATGGGGCCTCTCATTATACT1021 TAACAGATCCCCCTGGTTTACCACGTTAATATCCACCATCATGGGGCCTCTCATTATACT
1081 CCTACTAATTCTGCCTTTTTGGACCCTGCCATTCTTAATTGA 1081 CCTACTAATTCTGCCTTTTTGGACCCTGCCATTCTTAATTGA
1141 CAGGATCTCAGTAGTCCAGGCTTTAGTCCTGACTCAACAATACCACCAGCTAAAACCACT 1141 CAGGATCTCAGTAGTCCAGGCTTTAGTCCTGACTCAACAATACCACCAGCTAAAACCACT
1201 AGAATACGAGCCATGA 1201 AGAATACGAGCCATGA

Claims

REVENDICATIONS
1/ Polypeptide, caractérisé soit en ce qu'il correspond à l'enchainement d'acides aminés désigné par SEQ ID NO1 dans la liste des séquences, soit en ce qu'il comprend l'enchaînement SEQ ID NO 1 ou un fragment de cet enchaînement dès lors que le polypeptide répond à l'une au moins des conditions suivantes :  1 / Polypeptide, characterized either in that it corresponds to the chain of amino acids designated by SEQ ID NO1 in the list of sequences, or in that it comprises the chain SEQ ID NO 1 or a fragment of this linking as soon as the polypeptide meets at least one of the following conditions:
a) il est capable lorsqu'il est produit à partir du génome du retrovirus MPLV, de provoquer et/ou de favoriser in vitro et in vivo la prolifération des lignées cellulaires hématopoiétiques,  a) it is capable, when produced from the genome of the MPLV retrovirus, of causing and / or promoting in vitro and in vivo the proliferation of hematopoietic cell lines,
b) il intervient in vitro ou in vivo, dans la différenciation cellulaire de lignées cellulaires hématopoiétiques, lorsqu'il est produit à partir du génome du retrovirus MPLV,  b) it intervenes in vitro or in vivo, in the cell differentiation of hematopoietic cell lines, when it is produced from the genome of the MPLV retrovirus,
c) il est susceptible d'intervenir in vivo dans une fonction de récepteur de facteur de croissance hématopoiétique, soit au niveau de la fixation d'un ligand, soit au niveau de la transmission d'un signal, d) il est reconnu par des anticorps dirigés contre l'enchaînement d'acides aminés représenté par la séquence SEQ ID NO1,  c) it is capable of intervening in vivo in a hematopoietic growth factor receptor function, either at the level of the binding of a ligand, or at the level of the transmission of a signal, d) it is recognized by antibodies directed against the chain of amino acids represented by the sequence SEQ ID NO1,
ou encore en ce qu'il s'agit d'une séquence d'acides aminés présentant une homologie d'au moins 80% de préférence 88% avec le fragment représenté par la SEQor in that it is an amino acid sequence having a homology of at least 80%, preferably 88%, with the fragment represented by SEQ
ID NO 2, contenu dans l'enchaînement d'acides aminésID NO 2, contained in the chain of amino acids
SEQ ID NO 1. SEQ ID NO 1.
2/ Polypeptide selon la revendication 1, caractérisé en ce qu'il comprend un enchaînement d'acides aminés WSXWS dans lequel X est un acide aminé quelconque et de préférence correspond à l'arginine ou à la serine.  2 / A polypeptide according to claim 1, characterized in that it comprises a chain of amino acids WSXWS in which X is any amino acid and preferably corresponds to arginine or serine.
3/ Polypeptide selon la revendication 1, caractérisé en ce qu'il s'agit du fragment identifié sous la référence 3 / Polypeptide according to claim 1, characterized in that it is the fragment identified under the reference
SEQ ID NO3. SEQ ID NO3.
4/ Polypeptide selon l'une quelconque des revendications 1 à 3, caractérisé en ce qu'il répond à l'un des enchaînements d'acides aminés identifiés sous les références suivantes : SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11. 4 / Polypeptide according to any one of claims 1 to 3, characterized in that it responds to one of the sequences of amino acids identified under the following references: SEQ ID NO 2, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11.
5/ Polypeptide selon l'une quelconque des revendications 1 à 4, caractérisé en ce qu'il est codé par une séquence nucléotidique capable d'hybrider dans des conditions stringentes avec une des sondes Sacl - PstI ou PstI - PstI de la séquence identifiée par la référence SEQ ID NO 2 ou encore avec l'une de ces deux sondes. 5 / A polypeptide according to any one of claims 1 to 4, characterized in that it is encoded by a nucleotide sequence capable of hybridizing under stringent conditions with one of the Sacl - PstI or PstI - PstI probes of the sequence identified by the reference SEQ ID NO 2 or with one of these two probes.
6/ Polypeptide selon l'une quelconque des revendications 1 à 3, caractérisé en ce qu'il répond à l'un des enchaînements d'acides aminés identifiés sous les références suivantes : SEQ ID NO 12, SEQ ID NO 13. 7/ Récepteur de facteur de croissance, caractérisé en ce qu'il comprend la SEQ ID NO 1 selon la revendication 1 ou en ce qu'il comprend une forme soluble de cette séquence.  6 / A polypeptide according to any one of claims 1 to 3, characterized in that it responds to one of the amino acid sequences identified under the following references: SEQ ID NO 12, SEQ ID NO 13. 7 / Receiver growth factor, characterized in that it comprises SEQ ID NO 1 according to claim 1 or in that it comprises a soluble form of this sequence.
8/ Protéine de fusion, caractérisée en ce qu'elle comprend un polypeptide selon l'une quelconque des revendications 1 à 5, en association avec un enchaînement d'acides aminés déterminé, notamment avec la gp70 du MPLV, en particulier en ce qu'il s'agit de la séquence d'acides aminés SEQ ID NO 14, ou encore avec la protéine codée par le gène gag ou avec une glycoprotéine exogène par exemple une glycoprotéine d'un virus différent d'un retrovirus, tel que VSV.  8 / fusion protein, characterized in that it comprises a polypeptide according to any one of claims 1 to 5, in association with a determined amino acid sequence, in particular with the gp70 of MPLV, in particular in that it is the amino acid sequence SEQ ID NO 14, or else with the protein encoded by the gag gene or with an exogenous glycoprotein, for example a glycoprotein from a virus different from a retrovirus, such as VSV.
9/ Séquence nucléotidique, caractérisée en ce qu'elle code pour un polypeptide selon l'une quelconque des revendications 1 à 8.  9 / Nucleotide sequence, characterized in that it codes for a polypeptide according to any one of claims 1 to 8.
10/ Séquence nucléotidique, caractérisée en ce qu'elle correspond à l'une quelconque des séquences nucléotidiques SEQ ID NO 15, NO 16, NO 17, NO 18, NO 19, NO 20 ou à un enchaînement complémentaire de ces séquences, ou à une séquence capable d'hybrider avec l'une des séquences ci-dessus dans des conditions stringentes. 11/ Hôte cellulaire recombinant, caractérisé en ce qu'il comprend une séquence nucléotidique selon l'une quelconque des revendications 9 ou 10, intégrée dans son génome. 10 / Nucleotide sequence, characterized in that it corresponds to any one of the nucleotide sequences SEQ ID NO 15, NO 16, NO 17, NO 18, NO 19, NO 20 or to a complementary sequence of these sequences, or to a sequence capable of hybridizing with one of the above sequences under stringent conditions. 11 / Recombinant cell host, characterized in that it comprises a nucleotide sequence according to any one of claims 9 or 10, integrated into its genome.
12/ Hôte cellulaire selon la revendication 11, caractérisé en ce qu'il s'agit d'un virus, notamment du virus MPLV, d'une bactérie, d'une cellule d'insecte ou de mammifère telle que COS ou CHO.  12 / cell host according to claim 11, characterized in that it is a virus, in particular the MPLV virus, a bacterium, an insect or mammalian cell such as COS or CHO.
13/ Vecteur recombinant, caractérisé en ce qu'il comprend une séquence nucléotidique selon l'une quelconque des revendications 9 ou 10, sous le contrôle des séquences régulatrices nécessaires à son expression dans un hôte cellulaire déterminé, ce vecteur pouvant par exemple être le virus vaccine, le baculovirus.  13 / Recombinant vector, characterized in that it comprises a nucleotide sequence according to any one of claims 9 or 10, under the control of the regulatory sequences necessary for its expression in a determined cellular host, this vector can for example be the virus vaccinia, baculovirus.
14/ Sonde nucléotidique comprenant au moins 9 nucléotides, capable d'hybrider dans des conditions stringentes avec une séquence nucléotidique selon l'une quelconque des revendications 9 ou 10, cette sonde étant le cas échéant marquée. 14 / Nucleotide probe comprising at least 9 nucleotides, capable of hybridizing under stringent conditions with a nucleotide sequence according to any one of claims 9 or 10, this probe being optionally labeled.
15/ Anticorps polyclonaux ou monoclonaux, caractérisés en ce qu'ils reconnaissent un enchaînement d'acides aminés selon l'une quelconque des revendications 1 à 6. 16/ Procédé pour la détection in vitro d'une expression anormale d'un polypeptide selon l'une quelconque des revendications 1 à 6, comprenant :  15 / Polyclonal or monoclonal antibodies, characterized in that they recognize a chain of amino acids according to any one of claims 1 to 6. 16 / Method for the in vitro detection of an abnormal expression of a polypeptide according to l any of claims 1 to 6, comprising:
la mise en contact d'un échantillon biologique susceptible de contenir le polypeptide recherché, avec des anticorps selon la revendication 16,  bringing a biological sample capable of containing the desired polypeptide into contact with antibodies according to claim 16,
- la détection d'une réaction immunologique du type antigène-anticorps. - the detection of an immunological reaction of the antigen-antibody type.
17/ Procédé pour la détection in vitro, de l'expression anormale d'une séquence nucléotidique selon l'une quelconque des revendications 9 ou 10, comprenant : a) la mise en contact d'un échantillon biologique susceptible de contenir la séquence nucléotidique recherchée, utilisée comme matrice, avec une amorce nucléotidique capable de s'hybrider avec la séquence nucléotidique recherchée, en présence des 4 différents nucléosides triphosphates, et d'un agent de polymérisation, dans des conditions d'hybridation telles que pour chaque séquence nucléotidique ayant hybride avec une amorce, un produit d'elongation de chaque amorce, complémentaire de la matrice est synthétisé; 17 / A method for the in vitro detection of the abnormal expression of a nucleotide sequence according to any one of claims 9 or 10, comprising: a) contacting a biological sample capable of containing the desired nucleotide sequence , used as a template, with a nucleotide primer capable of hybridizing with the sequence nucleotide sought, in the presence of the 4 different nucleoside triphosphates, and of a polymerization agent, under hybridization conditions such as for each nucleotide sequence having hybridized with a primer, an elongation product of each primer, complementary to the matrix is synthesized;
b) la séparation de la matrice et du produit d'elongation obtenu, ce dernier pouvant alors également se comporter comme une matrice;  b) separation of the matrix and of the elongation product obtained, the latter then also being able to behave like a matrix;
c) la répétition de l'étape a) de façon à obtenir une quantité détectable des séquences nucléotidiques recherchées;  c) repeating step a) so as to obtain a detectable quantity of the nucleotide sequences sought;
d) la détection du produit d'amplification des séquences nucléotidiques.  d) detection of the amplification product of the nucleotide sequences.
18/ Procédé pour la détection de l'affinité d'une molécule pour un polypeptide selon l'une quelconque des revendications 1 à 5, caractérisé par :  18 / A method for detecting the affinity of a molecule for a polypeptide according to any one of claims 1 to 5, characterized by:
- la mise en contact de la molécule testée avec un hôte cellulaire préalablement modifié par une séquence nucléotidique selon l'une quelconque des revendications 9 ou 10, dans des conditions permettant l'expression de cette séquence de façon à obtenir un polypeptide selon l'une quelconque des revendications 1 à 5 comportant au moins un site susceptible d'interagir avec la molécule testée, exposé à la surface de l'hôte cellulaire, - bringing the tested molecule into contact with a cellular host previously modified by a nucleotide sequence according to any one of claims 9 or 10, under conditions allowing the expression of this sequence so as to obtain a polypeptide according to one any one of claims 1 to 5 comprising at least one site capable of interacting with the test molecule, exposed on the surface of the cell host,
- la détection de la formation d'un complexe entre la molécule testée et le polypeptide. - detecting the formation of a complex between the molecule tested and the polypeptide.
19/ Médicament caractérisé en ce qu'il comprend un polypeptide selon l'une quelconque des revendications 1 ou 6 sous forme soluble, en combinaison avec un véhicule pharmaceutique acceptable.  19 / Medicament, characterized in that it comprises a polypeptide according to any one of claims 1 or 6 in soluble form, in combination with an acceptable pharmaceutical vehicle.
PCT/FR1990/000762 1990-10-19 1990-10-19 Polypeptide of a growth factor receptor family, application in the diagnosis and treatment of myeloproliferative diseases WO1992007074A1 (en)

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PCT/FR1990/000762 WO1992007074A1 (en) 1990-10-19 1990-10-19 Polypeptide of a growth factor receptor family, application in the diagnosis and treatment of myeloproliferative diseases
JP2515169A JPH06501915A (en) 1990-10-19 1990-10-19 Polypeptides of the growth factor receptor family and their application for the diagnosis and treatment of myeloproliferative disorders
CA002094261A CA2094261C (en) 1990-10-19 1990-10-19 Polypeptide of a growth factor receptor family, application in the diagnosis and treatment of myeloproliferative diseases
US08/078,311 US5925750A (en) 1990-10-19 1990-10-19 Nucleic acid encoding polypeptide of a growth factor receptor family

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PCT/FR1990/000762 WO1992007074A1 (en) 1990-10-19 1990-10-19 Polypeptide of a growth factor receptor family, application in the diagnosis and treatment of myeloproliferative diseases
CA002094261A CA2094261C (en) 1990-10-19 1990-10-19 Polypeptide of a growth factor receptor family, application in the diagnosis and treatment of myeloproliferative diseases

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995019992A1 (en) * 1994-01-21 1995-07-27 Amgen Inc. Compositions and methods using unbound mpl receptor for stimulating platelet production
US5766581A (en) * 1994-03-31 1998-06-16 Amgen Inc. Method for treating mammals with monopegylated proteins that stimulates megakaryocyte growth and differentiation
US5795569A (en) * 1994-03-31 1998-08-18 Amgen Inc. Mono-pegylated proteins that stimulate megakaryocyte growth and differentiation
WO1998053318A1 (en) * 1997-05-20 1998-11-26 The Johns Hopkins University A novel thrombopoietin signaling defect in polycythemia vera platelets
US6852313B1 (en) 1989-10-16 2005-02-08 Amgen Inc. Method of stimulating growth of melanocyte cells by administering stem cell factor
US7144731B2 (en) 1989-10-16 2006-12-05 Amgen Inc. SCF antibody compositions and methods of using the same

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Journal of Virology, vol. 61, no. 2, February 1987, American Society for Microbiology, J.F. Penciolelli et al.: "Genetic analysis of myeloproliferative leukemia virus, a novel acute leukemogenic replication -defective retrovirus", pages 579-583 *

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6852313B1 (en) 1989-10-16 2005-02-08 Amgen Inc. Method of stimulating growth of melanocyte cells by administering stem cell factor
US6967029B1 (en) 1989-10-16 2005-11-22 Amgen Inc. Method for increasing hematopoietic progenitor cells by stem cell factor
US7144731B2 (en) 1989-10-16 2006-12-05 Amgen Inc. SCF antibody compositions and methods of using the same
WO1995019992A1 (en) * 1994-01-21 1995-07-27 Amgen Inc. Compositions and methods using unbound mpl receptor for stimulating platelet production
US5498599A (en) * 1994-01-21 1996-03-12 Amgen Inc. Methods for stimulating platelet production
US5766581A (en) * 1994-03-31 1998-06-16 Amgen Inc. Method for treating mammals with monopegylated proteins that stimulates megakaryocyte growth and differentiation
US5795569A (en) * 1994-03-31 1998-08-18 Amgen Inc. Mono-pegylated proteins that stimulate megakaryocyte growth and differentiation
WO1998053318A1 (en) * 1997-05-20 1998-11-26 The Johns Hopkins University A novel thrombopoietin signaling defect in polycythemia vera platelets
WO1998053320A1 (en) * 1997-05-20 1998-11-26 The Johns Hopkins University A novel thrombopoietin signaling defect in polycythemia vera platelets
US6027902A (en) * 1997-05-20 2000-02-22 Johns Hopkins University Thrombopoietin signaling defect in polycythemia vera platelets
US6150120A (en) * 1997-05-20 2000-11-21 The Johns Hopkins University Methods to assay a thrombopoietin signaling defect in polycythemia vera platelets

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