FR2778665A1 - New PRAX-1 polypeptide that interact with peripheral benzodiazepine receptor, used to treat e.g. immune, central nervous or endocrine disorders - Google Patents

Abstract

Purified polypeptides (I), designated PRAX-1 (peripheral benzodiazepine receptor associated protein-1) is new. Purified polypeptides (I), designated PRAX-1 (peripheral benzodiazepine receptor associated protein-1) is (i) a 1857 amino acid (aa) sequence (1); (ii) a biologically active derivative of (2), or (iii) a fragment of (2) with biological activity or immunoreactivity similar to (2). Independent claims are also included for the following: (1) nucleic acid (II) encoding (I); (2) cloning and/or expression vector containing (II); (3) host cells transfected with this vector; (4) nucleic acid (IIa), useful as probes and primers, that can hybridize specifically to (II), its complements, or the corresponding mRNA or genes; (5) primer pairs containing (IIa); (6) recombinant production of (I) or its fragments by culturing cells of (c); (7) mono- or poly-clonal antibodies (Ab), or their fragments, chimeras or immunoconjugates, specific for (I); (8) diagnostic kits containing Ab, optionally immobilized, and reagents for detecting complex formation; and (9) pharmaceutical composition containing (I) or an activator/inhibitor of either (I) activity or of any protein-protein interaction that involves PRAX-1.

Description

The invention relates to a novel nucleic acid sequence and

  the corresponding protein sequence.

  The invention also relates to the prophylactic, therapeutic and diagnostic applications thereof. The authors of the present invention have identified the transcript of a new gene as well as the corresponding protein. This PRAX-1 gene was isolated by the double hybrid technique as coding for a protein interacting specifically with the peripheral periphery receptor.

benzodiazepines (PBR).

  The present invention therefore relates to a purified protein

  PRAX-1, or biologically active fragments thereof.

  The invention also relates to isolated nucleic acid sequences encoding said protein or its biologically fragments.

  active and specific oligonucleotides obtained from this sequence.

  It also targets the cloning and / or expression vectors containing at least one of the nucleotide sequences defined above, and the host cells transfected by these cloning and / or expression vectors under conditions allowing replication. and / or the expression of one of said

nucleotide sequences.

  Methods of producing recombinant PRAX-1 proteins or their biologically active fragments by transfected host cells

  are also part of the invention.

  The invention also includes antibodies or derivatives

  antibodies specific for the proteins defined above.

  The invention also relates to any inhibitor or activator of the specific activity of PRAX-1, but also of any protein-protein interaction

involving PRAX-1.

  It also relates to antisense oligonucleotide sequences, specific for the above nucleic acid sequences, which can modulate in

vivo expression of the PRAX-1 gene.

  It relates to oligonucleotide sequences defined from the nucleic acid sequences above, probe or primer usable in

  diagnosis of pathologies where PRAX-1 is involved.

  A final object of the invention is the use in a gene therapy method of a nucleotide sequence according to the invention, in which vectors such as for example inactive viral vectors capable of transferring coding sequences for a protein according to the invention are

  injected into cells deficient for this protein.

  The subject of the present invention is a purified polypeptide whose amino acid sequence is the sequence SEQ ID n 2 or any sequence

  biologically active derived from SEQ ID No. 2.

  In the description of the invention, the definitions are used

  following: - PRAX-1 protein: a polypeptide comprising an amino acid sequence chosen from the sequence SEQ ID No. 2 or any fragment or

  derivative thereof biologically active.

  - derivative: any polypeptide varying from the polypeptide of sequence SEQ ID No. 2 or any molecule resulting from a modification of genetic and / or chemical nature of the sequence SEQ ID No. 2, that is to say obtained by mutation, deletion, addition , substitution and / or chemical modification of a single or a limited number of amino acids, as well as any isoform sequence, that is to say a sequence identical to the sequence SEQ ID No. 2 in one of its modified fragments or sequences containing one or more amino acids in the form of the D enantiomer, said variant, modified or isoform sequences having retained at least one of the properties making them biologically active and / or said sequences being immunologically related to the SEQ sequence

ID # 2.

  - biologically active: capable of interacting with the receptor

  benzodiazepines device.

  - immunologically related: capable of being recognized by the antibodies specific for the polypeptide of sequence SEQ ID No. 2 and / or capable

  to induce antibodies which recognize this polypeptide.

  The manufacture of derivatives can have different objectives, including in particular that of improving its production rates, of increasing its resistance to proteases, of modifying its biological activities or of conferring on it

  new pharmaceutical and / or biological properties.

  Advantageously, the invention relates to a polypeptide comprising the

  domain of interaction with PBR of one of the preceding polypeptides.

  This domain corresponds to the sequence between residue 1564 and residue 1857 of the sequence SEQ ID No. 2. The present invention also relates to the nucleic acid sequences encoding a PRAX-1 protein or fragments or

  biologically active derivatives thereof.

  More preferably, the invention relates to isolated nucleic acid sequences chosen from a) the sequence SEQ ID No. 1, b) the sequence complementary to the sequence SEQ ID No. 1, c) the nucleic acid sequences capable of to specifically hybridize to the sequence SEQ ID No. 1 or to their complementary sequences, or to hybridize specifically to their proximal sequences, d) the sequences derived from the sequences a), b) and c), due to the

  degeneration of the genetic code.

  According to a preferred embodiment, the subject of the invention is the nucleotide sequence SEQ ID n 1 corresponding to the cDNA of the protein

human with sequence SEQ ID No. 2.

  The different nucleotide sequences of the invention can be of artificial origin or not. They may be DNA or RNA sequences, obtained by screening of sequence banks using probes prepared on the basis of the sequence SEQ ID No. 1. Such libraries may be prepared by conventional techniques of biology molecular, known from

one skilled in the art.

  The nucleotide sequences according to the invention can also be prepared by chemical synthesis, or also by mixed methods including chemical or enzymatic modification of sequences obtained by

screening of banks.

  These nucleotide sequences allow the production of nucleotide probes, capable of hybridizing strongly and specifically with a nucleic acid sequence, of a genomic DNA or of a messenger RNA, coding for the polypeptide according to the invention or a biologically fragment active of it. Such probes are also part of the invention. They can be used as an in vitro diagnostic tool for the detection, by hybridization experiments, of specific transcripts of the polypeptides of the invention in biological samples or for the detection of aberrant syntheses or genetic anomalies at the level nucleic acid sequences encoding a polypeptide as defined above such as loss of heterozygosity or genetic rearrangement, resulting from a

  polymorphism, mutations or different splicing.

  To this end, a nucleotide probe according to the invention is brought into contact with a biological sample under conditions allowing the formation of a hybridization complex between the probe and the nucleotide sequence of interest, possibly after a prior step of amplification of said nucleotide sequence; the hybridization complex possibly formed is detected and the nucleotide sequence forming the hybridization complex

  with the probe of the invention is optionally sequenced.

  The nucleotide sequences of the invention can also

  be used for the manufacture of a drug useful in gene therapy.

  The probes of the invention comprise at least 10, preferably at least 16 nucleotides, and at most comprise the entire sequence

  the PRAX-1 gene or its cDNA contained for example in a cosmid.

  Among the shortest probes, that is to say about 10 to 20 nucleotides, the appropriate hybridization conditions correspond to the stringent conditions usually used by those skilled in the art, in particular of high stringency as defined by Sambrook et al. in Molecular Cloning, a laboratory manual, 2nd edition, ColdSpring Harbor Laboratory Press, 1989. The temperature used is preferably between Tm -5 C to Tm -30 C, more preferably between Tm -5 C and Tm -10 C, Tm being the melting temperature, temperature at which 50% of the paired DNA strands separate. Advantageously, the hybridization conditions used are as follows: - temperature: 65 ° C., - hybridization buffer: BSA 1%, SDS 7% 0.5 M NaH 2 PO 4, EDTA 1 mM,

  as described in Example VII.

  Advantageously, these probes are represented by the following oligonucleotides as well as the corresponding complementary nucleotides: SEQIDn 3: GCTCTG TTT GACTATGACCC SEQ ID n 4: AACTTG AAT CCAATGAGC AA SEQ ID n 5: TCT GCT GAC CTG AAA GCT CC SEQ ID n 6 : CCA TCA GGG GGC ACA GGC TC SEQ ID n 7: TGG GCC TTG TAC CCC CAG CT SEQ ID n 8: TCC TCC TGA GGA GCT GCT TC SEQ ID n 9: CCC CTG CCC GGA AGG GCA GC SEQ ID n 10: TTG ACC AAG GTC ACT GAG CC SEQ ID n 11: GGG CCT GCT GGC CCC TGT AA SEQ ID n 12: GCC ATC GGC ATC CTT GTC CC SEQ ID n 13: CCT CTC CGG TCT CTG TTG CCC G SEQ ID n 14: AGG CCC AGG GGG CCT CCG AAG GC SEQ ID n 15: CGC TGC CCG CCT CCT GCT CAT CCT C SEQ ID n 16: TCC TCC AGG GCT GGA GAC TGC CT SEQ ID n 17: GGT TCC AGG ACT TGC TCC TTC CAG AAG SEQ ID n 18: CAG CTC CCT CAC TGG CCA AG SEQ ID n 19: TGG CTC CGG CCA GCC TGC C SEQ ID n 20: TCC GGG TCA CAG GCT ATG CCA T SEQ ID n 21: CC CAG TAC CTA CTG GGC CAC CT SEQ ID n 22: GCT TCC ATA TCT GTG TGA ATG GG SEQ I D n 23: GGT AAG GCA CGG CAG GAG GCT C SEQ ID n 24: GCT GGC TCC TTC TCA CAC TG SEQ ID n 25: GGA GAG CTC ATG GAT GGC CGA AG SEQ ID n 26: CCA GTG AGG TAG AGG AGC AGG TGG SEQ ID no 27: TCC ATT CCC CAG CCT TGC CGG GG SEQ ID no 28: GCT CGA AGA ACA GTG CCG CAG CC SEQ ID no 29: TCG CAA GAG CCA GGG CCT GCA GA SEQ ID no 30: GCTGGAATCGGAGCTCAGCAAG SEQ ID no 31: CCC GTG ATT CTA GGG GAG CCA GAG SEQ ID n 32: GAGTGC AGG GAG CTG CAG GCCA SEQ ID n 33: GGCTGAAGC GGAAGAACG CCGAG SEQ ID n 34: CAA TCT GGA GCT GCT GAG GG SEQ ID n 35: TGAGGTCTGAGGAGAGAGTTCCAAG SE GAG CCA TGG GCA CTG CCC Preferably, the probes of the invention are marked, prior to their use. For this, several techniques are within the reach of those skilled in the art (fluorescent, radioactive, chemiluminescent labeling,

enzymatic, etc.).

  The in vitro diagnostic methods in which these nucleotide probes are used are included in the subject of the present

invention.

  These methods relate, for example, to the detection of abnormal syntheses (eg accumulation of transcripts) or genetic abnormalities, such as loss of heterozygosity and genetic rearrangement, and point mutations in the nucleotide nucleic acid sequences. coding for a PRAX-1 protein, according to the definition given above. The nucleotide sequences of the invention are also useful for the manufacture and use of oligonucleotide primers for specific sequencing, age or amplification reactions according to the so-called technique.

  PCR or any variant thereof (Ligase Chain Reaction (LCR), ...).

  Preferred pairs of primers consist of primers advantageously of at least 16 nucleotides, chosen from the sequence

  nucleotide: SEQ ID No. 1: human sequence of 7033 nucleotides.

  Advantageously, these primers are represented by the following pairs: pair n: sense primer: GCT CTG TTT GAC TAT GAC CC (SEQ ID n 3) antisense primer: AAC TTG AAT CCA ATG AGC AA (SEQ ID n 4) - pair n 2 : sense primer: TCT GCT GAC CTG AAA GCT CC (SEQ ID n 5) antisense primer: TCC TCC TGA GGA GCT GCT TC (SEQ ID n 8) - couple n 3: sense primer: TTG ACC AAG GTC ACT GAG CC (SEQ ID n 10) antisense primer: AAC TTG AAT CCA ATG AGC MAA (SEQ ID n 4) - couple n 4: sense primer: GCT CTG TTT GAC TAT GAC CC (SEQ ID n 3) antisense primer: GCC ATC GGC ATC CTT GTC CC (SEQ ID no 12) - couple 5: sense primer: GCT CTG TTT GAC TAT GAC CC (SEQ ID no 3) antisense primer: TCC TCC TGA GGA GCT GCT TC (SEQ ID no 8)

  "sense" primers ranging from 5 'to 3' and "anti-sense" primers from 3 'to 5'.

  These primers correspond to the sequences respectively between: - nucleotide n 5091 and nucleotide n 5110 on SEQ ID n 1 for SEQ ID n 3

  - nucleotide n 6700 and nucleotide n 6719 on SEQ ID n 1 for SEQ ID n 4.

  - nucleotide n 5475 and nucleotide n 5494 on SEQ ID n 1 for SEQ ID n 5 - nucleotide n 5939 and nucleotide n 5958 on SEQ ID n 1 for SEQ ID n 8 nucleotide n 6226 and nucleotide n 6245 on SEQ ID n 1 for SEQ ID n 10 - nucleotide n 5188 and nucleotide n 5207 on SEQ ID n 1 for SEQ ID n 12 The nucleotide sequences according to the invention can also be used for the production of PRAX-1 recombinant proteins, according to the

  definition that has been given to this term.

  These proteins can be produced from the nucleotide sequences defined above, according to techniques for the production of recombinant products known to those skilled in the art. In this case, the nucleotide sequence used is placed under the control of signals allowing its

  transcription and its expression in a cellular host.

  An efficient system for producing a recombinant protein requires having a vector, for example of plasmid or viral origin,

and a compatible host cell.

  The cell host can be chosen from prokaryotic systems, such as bacteria, or eukaryotic systems, such as, for example, yeasts, insect or mammalian cells, in particular CHO (Chinese hamster ovary cells) or any other system advantageously available. . A preferred cellular host for the expression of the proteins of the invention consists of the

  E. colt bacteria, in particular strain MC 1061 (Clontech).

  The vector must include a promoter, translation initiation and termination signals, as well as the appropriate regions for transcription regulation. It must be able to be maintained stably in the cell and may possibly have specific signals specifying the secretion of

the translated protein.

  These different control signals are chosen according to the cellular host used. To this end, the nucleotide sequences according to the invention can be inserted into vectors with autonomous replication within the chosen host, or integrative vectors of the chosen host. Such vectors will be prepared according to the methods commonly used by those skilled in the art, and the resulting clones can be introduced into an appropriate host by

  standard methods, such as for example electroporation.

  The cloning and / or expression vectors containing at least one of the nucleotide sequences defined above also form part of

the present invention.

  A cloning and expression vector used is the plasmid pZeoSV2 (+) (Invitrogen, V850-01) which contains both the elements necessary for its use as a cloning vector in E. coli (origin of replication in E. coli and Zeocin resistance gene), and as an expression vector in animal cells (promoter, polyadenylation signal, origin of replication of the SV40 virus), as well as the elements allowing its single-strand copying for the purpose of sequencing ( origin of replication of phage f1). Integration of cDNAs from acid sequences

  nucleic acid of the invention is further described in the examples below.

  According to a preferred embodiment, the proteins of the invention are in the form of fusion proteins, in particular in the form of protein fused with glutathione S-transferase (GST). An expression vector designated in this case is represented by the plasmid vector pGEX-4T-2

(Pharmacia ref-27.4581-01).

  The invention further relates to host cells transfected with these preceding vectors. These cells can be obtained by introducing into host cells a nucleotide sequence inserted into a vector as defined above, then culturing said cells under conditions allowing replication and / or expression of the transfected nucleotide sequence. These cells can be used in a method for producing a recombinant polypeptide of sequence SEQ ID No. 2 or any fragment or

  biologically active derivative thereof.

  The method for producing a polypeptide of the invention in recombinant form is itself included in the present invention, and is characterized in that the transfected cells are cultured under conditions allowing the expression of a recombinant polypeptide of sequence SEQ ID No. 2 or any biologically active fragment or derivative thereof, and which one

  recovers said recombinant polypeptide.

  The purification methods used are known to those skilled in the art. The recombinant polypeptide can be purified from cell lysates and extracts, from the culture medium supernatant, by methods used individually or in combination, such as fractionation, chromatography methods, immunoaffinity techniques using '' specific mono or polyclonal antibodies, etc. A preferred variant consists in producing a recombinant polypeptide fused to a "carrier" protein (chimeric protein). The advantage of this system is that it allows stabilization and a decrease in the proteolysis of the recombinant product, an increase in the solubility during the in vitro renaturation and / or a simplification of the purification when the

  fusion partner has an affinity for a specific ligand.

  Advantageously, the polypeptides of the invention are fused with glutathione S-transferase in the N-terminal position ("GST" Pharmacia system). The fusion product is in this case detected and quantified thanks to the enzymatic activity of GST. The color reagent used is a glutathione acceptor, a substrate for GST. The recombinant product is purified on a chromatography support to which have been previously coupled

glutathione molecules.

  Mono or polyclonal antibodies specifically directed against a PRAX-1 protein according to the definition given above also form part of the invention. Polyclonal antibodies can be obtained from the serum of an animal immunized against the protein, produced for example by genetic recombination according to the method described above, according to the methods

usual operations.

  The monoclonal antibodies can be obtained according to the conventional method of culture of hybridomas described by Kohler and Milstein, Nature, 1975,

256, 495-497.

  Advantageous antibodies are antibodies directed against epitopes located in the region between residue 1564 and residue 1857

for the sequence SEQ ID n 2.

  The antibodies according to the invention can be in the form

  of immunoconjugates or labeled antibodies.

  Furthermore, in addition to their use for the purification of recombinant polypeptides, the antibodies of the invention, in particular the monoclonal antibodies, can also be used for the detection of these

  polypeptides in a biological sample.

  They thus constitute a means of immunocytochemical or immunohistochemical analysis of the expression of the protein PRAX-1 on sections of specific tissues, for example by immunofluorescence, gold labeling, enzyme immunoconjugates. More generally, the antibodies of the invention can be advantageously used in any situation where the expression of a PRAX-1 protein must be observed. The invention therefore also relates to a method of in vitro diagnosis of pathological conditions correlated with an abnormal expression or accumulation of PRAX-1 proteins, from a biological sample, characterized in that at least one contact is made an antibody of the invention with said biological sample, under conditions allowing the possible formation of specific immunological complexes between a PRAX-1 protein and the said antibody or antibodies and in that the specific immunological complexes possibly formed are quantitatively detected. The invention also relates to a kit for the in vitro diagnosis of an abnormal expression or accumulation of the protein PRAX-1 in a biological sample and / or for the measurement of the level of expression thereof in said sample comprising: - at least one antibody specific for the PRAX-1 protein, optionally attached to a support, - means for revealing the formation of specific antigen / antibody complexes between the PRAX-1 protein and said antibody and / or

  means for quantifying these complexes.

  The invention also relates to any diagnostic method by demonstrating in the serum of an individual, autoantibodies directed against the

PRAX-1 protein.

  Such an early diagnosis method is characterized in that a serum sample taken from an individual is brought into contact with a polypeptide of the invention, optionally attached to a support, under conditions allowing the formation of specific immunological complexes between said polypeptide and the autoantibodies possibly present in the serum sample, and in that the specific immunological complexes possibly formed are detected. The invention also relates to a method for determining an allelic variability, a mutation, a deletion, an insertion, a loss of heterozygosity or a genetic abnormality of the PRAX-1 gene. , which can be involved in pathologies, characterized in that it uses at least one nucleotide sequence described above. Among the methods for determining an allelic variability, a mutation, a deletion, an insertion, a loss of heterozygosity or a genetic abnormality of the PRAX-1 gene, a method is preferred comprising at least one step of amplification by PCR of the target nucleic sequence of PRAX-1 capable of exhibiting a polymorphism, a mutation, a deletion or an insertion using pairs of primers of nucleotide sequences defined above, a step during which the amplified products are treated with appropriate restriction enzymes and a step during which at least one of the products of the enzymatic reaction is detected or assayed . The invention also includes pharmaceutical compositions comprising as active ingredient a polypeptide corresponding to the preceding definitions, preferably in soluble form, associated with a

  pharmaceutically acceptable vehicle.

  These pharmaceutical compositions are intended to be administered to subjects having an expression deficient in protein PRAX-1, associated with pathological conditions or else exhibiting a pathological state

  involving PRAX-1 or its receptor.

  The pharmaceutical compositions of the invention may also contain an inhibitor or inactivator of the activity of PRAX-1 and of any protein-protein interaction involving PRAX-1, in particular a

  polypeptide derived from a polypeptide as defined above.

  Preferably, these compositions can be administered by the systemic route, preferably by the intravenous route, by the intramuscular route,

intradermal or orally.

  Their optimal modes of administration, dosages and dosage forms can be determined according to the criteria generally taken into account in establishing a therapeutic treatment adapted to a patient such as for example the patient's age or body weight, the severity of general condition, tolerance to treatment and observed side effects, etc. The invention is illustrated by the following examples for which reference is made to the appended figure which is a schematic representation with a partial restriction map of the plasmid pZeoSV2 (+) containing human PRAX-1 (2H).

EXAMPLE I

  Isolation and identification of PRAX-1 1) Double hybrid strategy The double hybrid technique is a method of cloning by interaction in vivo in the yeast Saccharomyces cerevisiae. The object of this technique is to detect protein-protein interactions in vivo and search for the protein partners of a given protein used as bait. The bait used is defined on the protein sequence of PBR. The definition of the PBR bait must be limited to the hydrophilic regions of the protein according to the constraints of the double hybrid technique. As the PBR protein is strongly hydrophobic, with 5 transmembrane regions, located on the outer mitochondrial membrane, we define the PBR bait from the C-terminal motif of the protein, a region demonstrated cytosolic and accessible to possible protein partners by l existence of an anti-PBR monoclonal antibody directed against this region of the protein. A PBR bait constructed on the basis of this C-terminal motif, by juxtaposition three times of the 14 C-terminal amino acids of PBR was selected. 2) Construction of the PBR bait The PBR bait is a synthetic construction which juxtaposes three times the C-terminal motif of PBR, domain aa156 to 169, separated by glycine linkers: bait protein sequence:

  RDNHGWHGGRRLPE-gly-gly-RDNHGWHGGRRLPE-gly-gly-

RDNHGWHGGRRLPE

or 46 amino acids.

  The nucleotide sequence coding for the PBR bait is cloned

  in the vector pGBT9 (Clontech) at EcoRI and Xhol sites.

  3) Screening of the Fusion Banks The HF7c line is first transformed by the bait plasmid and then by the chosen fusion bank (fetal brain, Matchmaker library, Clontech) according to the method defined by Schiestl et ai. Curr. Genet., 1989, 16, 339-346. The double hybrid screening is carried out on 2,106 cells. The HIS + and LacZ + clones are identified and tested for their specificity by replacing the PBR bait with

  other non-relevant constructions.

  The selection and specificity tests, carried out under the conditions defined in the Matchmaker Library User Manual (Clontech), allow to isolate 4

positive clones.

  4) Identification of PRAX <-1 The insert contained in each of the clones is sequenced with the ABI PRISMTM Dye Terminator cycle sequencing Ready reaction Kit, according to the instructions defined in the protocol of use, on the sequencer Applied Biosystems model 373A . The 4 clones contain an identical 2152bp sequence containing a polyA tail, a STOP codon, unknown in the databases. This sequence was named PRAX-1 for Peripheral benzodiazepine Receptor Associated Protein 1. The sequence isolated by double hybrid encodes a polypeptide of 294 amino acids and is

  incomplete: the ATG codon coding for the initial 5 'methionine is missing.

  Obtaining the complete PRAX-1 sequence is described in Example II.

EXAMPLE II

  Obtaining the entire sequence and cloning of the cDNA of the human protein PRAX-1 1) Screening of the bank a) The bank The bank selected is a lamda Zap cDNA cortex bank

frontal (Stratagene).

  b) Preparation of the membranes The clones of the library are spread on LB agar medium, 0.2% maltose, MgSO4 10 mM (petri dishes diameter 150) coated with Hybond membranes (Amersham). After one night at 37 ° C., the clones are transferred by contact to new membranes. The latter are treated by depositing them on Whatman 3 mm paper soaked with the following solutions NaOH 0.5 N, NaCI 1.5 M for 2 minutes then Tris HCI 0.5 M pH 8, NaCI 1.5 M for 5 minutes, finally 30 sec on Tris HCI 0, 2M pH 7.5, SSC 2X. The membranes are dried and then incubated in a vacuum oven at 80 ° C. for 2 hours. c) Preparation of the PRAX-1 probes Different PRAX-1 probes are successively used defined on the nucleotide sequence SEQ ID No. 1. The probes are labeled with oE 32p using the RTS Rad Prime DNA labeling system kit (Gibco BRL). The probes are prepared with a specific activity of 5,108 dpm / pg on average. d) Prehybridization and hybridization The membranes prepared in b) are prehybridized for 30 minutes at 42 ° C. in 6 x SSC, 5 x Denhart's, 0.1% SDS and then hybridized overnight in the same buffer supplemented with the probe prepared in c) to Reason to

106 dpm / ml.

  e) Washing and exposure of the membranes The membranes are washed twice at room temperature in

  the buffer 2 x SSC / SDS 0.1% then one hour at 56 C in 6 x SSC / SDS 0.1%.

  Hybrid clones are revealed by autoradiography and selected after

multiple screening.

  2) Sequencing of the clones obtained PRAX-1 The sequential screening of the lambda Zap library made it possible to isolate clones containing the cDNA coding for the whole protein PRAX-1. The sequence is obtained using the ABI PRISMTM Dye Terminator cycle sequencing Ready reaction Kit, according to the instructions defined in the protocol

  of use, on an Applied Biosystems model 373A sequencer.

  The complete nucleotide sequence coding for PRAX-1 is

7033bp, presented in the appendix.

  It codes for a polypeptide of 1857 amino acids, of weight

  molecular 200 kDa. The portion isolated by double hybrid is the C- end

  terminal, of 294 C-terminal amino acids, corresponding on the coding sequence SEQ ID No. 1 to the region 4887 to 7033. The portion of PRAX-1 isolated in the double hybrid screening is referenced PRAX-1 (2H) and corresponds to

  domain extending from amino acids 1564 to 1857 on SEQ ID n 2.

EXAMPLE III

  1) Production of recombinant protein PRAX-1 in E. Co / ia) Construction of the expression plasmid It consists of inserting the coding sequence for the 2H portion of PRAX-1 (294 C-terminal amino acids of PRAX-1) into phase with glutathione S-transferase (GST) in the vector pGEX 4T2 (Pharmacia). For this, the corresponding insert (bp 4887 to 7033) is cloned at the SmaI site in the vector pGEX 4T2. The insert and the plasmid are digested and then purified after migration on 1% agarose gel using the Geneclean kit (Bio1 01). After ligation with 100 ng of insert and 10 ng of dephosphorylated vector and transformation by heat shock, the recombinant E. coli clones are verified by sequencing, age at

  using the Applied Biosystem kit (Perkin Elmer).

  b) Expression and purification of the GST-PRAX- fusion protein

1 (2H)

  This step was carried out using the "Bulk GST purification kit

module "(Pharmacia).

  A recombinant clone was cultured at 37 ° C. in one liter of LB culture medium (bacterotryptone 10 g / l, yeast extract 5 g / l, NaCl 10 g / l) + ampicillin 100 g / ml. At DO6onm 0.8, the expression is induced with 0.5mM IPTG for 2 h at 37 C. After centrifugation, the cell pellet is taken up in cold PBS and then sonicated by ultrasound. After centrifugation at 12000 g, for 10 min at 4 ° C., the supernatant is recovered. The purification is then carried out on a glutathione sepharose 4B affinity chromatography column. Fixation and washing are carried out in PBS buffer and elution is carried out by competition

  with reduced glutathione. The final concentration of GST- fusion protein

  PRAX-1 (2H) is variable between 0.5 and 1 mg / ml depending on the preparations.

  2) Production of PRAX-1 recombinant protein in Jurkat cells a) Construction of the expression plasmid The insert coding for the C-terminal end of PRAX-1 is removed from the

  plasmid of the fetal brain bank pActll-PRAX-1 (2H) by digestion with Bglll.

  This digestion makes it possible to leave in 5 ′ of the insert coding for PRAX1 (2H), the coding sequence for the HA epitope (hemagglutinin). The insert thus digested is cloned into pZeo SV2 + at the BamHI site. The linearized cloning vector and the insert are ligated. Competent "thermal shock" bacteria are transfected by this construction. The construction is checked by sequencing, age (see figure in the appendix). b) Jurkat cells Jurkat cells (human leukemia T) are cultured in RPMI 1640 medium (Gibco) supplemented with 50 mg / ml of gentamycin and

% fetal calf serum (Gibco).

  c) Transformation of Jurkat cells Jurkat cells are transfected by electroporation using the Biorad Gene Pulser device (Biorad), 320V, 250pFr, 5ms. The selection of the transfected clones is carried out with Zéocin 500pg / ml in RPMI 1640 medium, 20% FCS. d) The transfected cells are cultured in RPMI1640 medium, 20% FCS, Zéocin 500 g / ml. The production of recombinant protein HA-PRAX-1 (2H) is verified using the anti-HA antibody (Boerhinger

Mannheim) or anti-PRAX-1.

3C

EXAMPLE IV

  Preparation of specific polyclonal antibodies Immunogenic peptides of 20 amino acids are selected from the protein sequence PRAX-1 SEQ ID No. 2, in the hydrophilic portions of the protein. 5 mg of each KLH coupled peptide are used to immunize 2 rabbits (male weighing approximately 1.5 to 2 kg, New Zealand). Four immunizations were carried out 15 days apart according to the protocol described by Vaitukaitis, Methods in enzymology, 1981, 73, 46. The sera are collected one month after the first immunization then 2 times successively at 1 month and 15 days after interval. Each serum collected is tested in western immunoblotting for the recognition of the PRAX-1 protein. The immune sera are purified on an Affigel affinity column (Biorad). The specificity of recognition of PRAX-1 by the antibodies was verified by displacement of the labeling obtained, in the presence of the corresponding immunogenic peptide. A polyclonal antibody has thus

  been obtained. Its target is the peptide AA1772-1792 on the sequence SEQ ID No. 2.

EXAMPLE V

  Detection of the PRAX-1 protein by western immunoblotting 1) Materials used for immunoblotting a) Cell lines used for immunoblotting The cell lines were cultured, as described in 3 <"Catalog of cell lines and hybridomas , 7th edition, 1992 "of the ATCC (American Type Culture Collection). Human lines: U373 (line

  astrocyte), SHSY5Y (neuroblastoma), NHA (normal astrocytes), Jurkat, Molt-

  4 (T leukemia), Daudi, U937 (B lymphoma), SW480 (colon adenocarcinoma), DU145 (prostate carcinoma), Hacat, SVK14, WS1, NCTC2544, A431, VA ES BJ (keratinocytes). Murine line: J744-A1 (monocytes-macrophage). Line

rat: C62b (Glioma).

  b) E. caoli expressing GST-PRAX-1 (2H) or protein PRAX-1 (2H) translated in vitro The fusion protein GST-PRAX-1 (2H) is prepared as indicated in Example III. PRAX-1 (2H) is prepared in reticulocyte lysate with the TNT T7 / T3 Coupled reticulocyte Lysate system kit (Promega ref. L5010) according to the protocol of use defined in the kit. The plasmid used in this system is a plasmid pBluescript-PRAX-1 (2H) constructed by insertion of the coding sequence for PRAX-1 (2H) (nucleotides 4887 to 7033 on SEQ ID n 1)

  in the vector pBluescript (Stratagene) at the BamHI site.

  2) Preparation of the protein samples from the cell cultures After culture, the cells are washed with PBS and then lysed in the lysis buffer (6M urea, 50mM Tris HCl pH 6.8, 2% SDS, 10% glycerol, mM DTT , 0.1% bromophenol blue). Lysates are sonicated by ultrasound

  at 4 C for 2 min, heated 10 min at 100 C and stored at -20 C.

  3) Western Blot Samples calibrated for their protein content are deposited on 4-12% or 4-20% SDS-PAGE gel and then electrotransfered onto a membrane.

nitrocellulose.

  4) Revelation with anti-PRAX-1 antibody The membrane is incubated for at least 1 h in TBST blocking buffer (10 mM Tris HCI pH 8, 150 mM NaCl, 0.1% Tween 20) added with% milk at temperature ambient. The membrane is successively placed in the presence of the anti-PRAX-1 antibody in the same buffer, for 3 h at room temperature, washed 3 times for 10 min with TBST, then incubated for 45 min with a second anti-immunoglobulin antibody. of rabbit coupled to peroxidase (SIGMA) in TBST 5% milk buffer. After 3 washes in TBST of 15 min, the revelation is carried out using the ECL kit (Amersham) by chemiluminescence. ) Results Figure in annex: The immunoblots obtained with the anti PRAX-1 antibody show that it specifically recognizes the recombinant and endogenous proteins. Analysis of human cell lines shows expression of the PRAX-1 protein detected in all

the lines tested.

Example VI

  Chromosomal localization The chromosomal localization of the PRAX-1 gene was carried out by in situ hybridization according to the procedure described by Heng et a /. Proc. Natl Acad. Sci. USA (1992), 89, 9509-9513 and Chromosoma (1993), 102, 325-332. The cDNA probe used is a 3.5Kb probe defined on the sequence SEQ ID No. 1, comprising the 3 'end of the cDNA isolated by double hybrid. 100 mitoses were analyzed, 73 mitotic images show double spots located on a pair of chromosomes. Results help locate the PRAX-1 gene

  in the q22-q23 region on chromosome 17.

EXAMPLE VII

  Expression of human PRAX-1 mRNA Analysis of expression of PRAX-1 mRNA was performed on a Multiple Tissue Northern Blot membrane and RNA Master Blot (Clontech) carrying poly A + RNA from human tissue separated by electrophoresis or deposited in

dowry.

  The membranes are prehybridized for 1 hour at 65 ° C. in Church buffer (1% BSA, 7% SDS, 0.5 M NaH 2 PO 4, 1 mM EDTA), hybridized in the same buffer containing the cDNA PRAX-1 probe, at 65 ° C. overnight. The membranes are washed for 5 min in 5% SDS buffer, 0.5% BSA, mH NaPO2PO4, lmM EDTA. The markings are revealed by autoradiography after 24 hours of exposure. A PRAX-1 probe of 837 bp is prepared by PCR using the primers SEQ ID no 3 and SEQ ID no 8. The probe purified on gel by geneClean (Bio101) is radiolabeled at a 32 p with the Radprime labeling kit (Gibco)

  under the conditions defined by the kit marking protocol.

  The results show a majority expression of the PRAX-1 transcript in the human brain. The majority visible transcript is 7.5Kb in

  agreement with the size of the cDNA determined after screening of the cDNA libraries.

  The PRAX-1 transcript therefore exhibits strong expression in the central nervous system, with expression varying according to the regions of the brain. On the periphery of the thymus, the pituitary gland are distinguished from other tissues where expression is weaker. Among fetal tissues, the fetal brain gives

  a strong signal, followed by the heart, kidney, thymus and lung.

LIST OF SEQUENCES

(1) GENERAL INFORMATION:

  (i) DEPOSITOR: (A) NAME: Sanofi (B) STREET: 32-34 rue Marbeuf

(C) CITY: PARIS

(E) COUNTRY: FRANCE

(F) POSTAL CODE: 75008

  (ii) TITLE OF THE INVENTION: PRAX-1 polypeptide (iii) NUMBER OF SEQUENCES: 2 (iv) FORM READABLE BY COMPUTER: (A) TYPE OF MEDIUM: Floppy disk (B) COMPUTER: IBM PC compatible

  (C) OPERATING SYSTEM: PC-DOS / MS-DOS

  (D) SOFTWARE: PatentIn Release # 1.0, Version # 1.25 (EPO)

  (2) INFORMATION FOR SEQ ID NO: 1:

  (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 7033 base pairs (B) TYPE: nucleic acid (C) NUMBER OF STRANDS: simple (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: cDNA (vi) ORIGIN : (A) ORGANIZATION: Homo sapiens (ix) ADDITIONAL CHARACTERISTICS:

(A) NAME / KEY: CDS

(B) LOCATION: 198..5768

  (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1:

  GGCTCCAGCT CCAGCAGCCA TGGCGCCTTG TGCAGCCTCC TTTGTGGGTG GAGAAGAGCT 60

  CAGCCAACCT TGGATGGAGC GTCGGTGAAG CAAGAGCTCA GAGGGGAGAG GGGCTGGCCT 120

  GGCATGACCG TCGCCAGTCC CCCCACCTGG CTGGGTGATG TCCCCCGGCC CGGGTTCTGG 180

  GGCCCCTTGG CAGTACC ATG GAG CAA CTG ACA ACC CTC CCA CGG CCT GGG 230

  Met Glu Gln Leu Thr Thr Leu Pro Arg Pro Gly

1 5 10

  GAC CTT GGA GCC ATG GAG CCA TGG GCA CTG CCC ACC TGG CAT AGC TGG 278

  Asp Leu Gly Ala Met Glu Pro Trp Ala Leu Pro Thr Trp His Ser Trp

20 25

  ACT CCA GGT CGA GGG GGT GAA CCT AGC AGT GCA GCC CCA AGC ATC GCT 326

  Thr Pro Gly Arg Gly Gly Glu Pro Ser Ser Ala Ala Pro Ser Ile Ala

35 40

  GAT ACT CCT CCG GCA GCT CTG CAG CTT CAA GAA CTG AGG TCT GAG GAG 374

  Asp Thr Pro Pro Ala Ala Leu Gln Leu Gln Glu Leu Arg Ser Glu Glu

50 55

  AGT TCC AAG CCC AAA GGA CAC GGG AGC TCC AGG CCC GTG GGG GGA ACT 422

  Ser Ser Lys Pro Lys Gly His Gly Ser Ser Arg Pro Val Gly Gly Thr

65 70 75

  GAC CCT GAA GGA GCA CAG GCT TGT CTG CCC AGC CTG GGC CAG CAA GCA 470

  Asp Pro Glu Gly Ala Gln Ala Cys Leu Pro Ser Leu Gly Gln Gln Ala

85 90

  TCC AGC TCT GGA CCC GCC TGC CAG AGG CCA GAG GAT GAG GAA GTG GAG 518

  Ser Ser Ser Gly Pro Ala Cys Gln Arg Pro Glu Asp Glu Glu Val Glu

100 105

  GCT TTC CTG AAG GCC AAG CTG AAT ATG AGC TTT GGG GAC AGG CCC AAT 566

  Ala Phe Leu Lys Ala Lys Leu Asn Met Ser Phe Gly Asp Arg Pro Asn

115 120

  CTG GAG CTG CTG AGG GCC CTG GGG GAG CTG CGG CAG CGC TGT GCC ATC 614

* Leu Glu Leu Leu Arg Ala Leu Gly Glu Leu Arg Gln Arg Cys Ala Ile

130 135

  CTT AAG GAG GAA AAC CAG ATG CTG AGG AAG AGC AGC TTC CCT GAG ACA 662

  Leu Lys Glu Glu Asn Gln Met Leu Arg Lys Ser Ser Phe Pro Glu Thr

145 150 155

  GAA GAG AAG GTG CGG AGG CTG AAG CGG AAG AAC GCC GAG CTG GCG GTC 710

  Glu Glu Lys Val Arg Arg Leu Lys Arg Lys Asn Ala Glu Leu Ala Val

165 170

  ATT GCC AAG CGC CTG GAG GAG AGG GCC CGA AAG CTG CAG GAA ACG AAC 758

  Ile Ala Lys Arg Leu Glu Glu Arg Ala Arg Lys Leu Gln Glu Thr Asn

180 185

  CTG AGG GTG GTG AGT GCC CCC TTG CCC CGG CCG GGG ACC AGC TTG GAG 806

  Leu Arg Val Val Ser Ala Pro Leu Pro Arg Pro Gly Thr Ser Leu Glu

195,200

  TTG TGT CGG AAG GCC CTA GCC CGC CAG CGA GCC CGG GAC CTC AGT GAG 854

  Leu Cys Arg Lys Ala Leu Ala Arg Gln Arg Ala Arg Asp Leu Ser Glu

205 210 215

  ACA GCC AGT GCA CTG CTG GCC AAG GAC AAG CAG ATT GCT GCC TTG CAG 902

  Thr Ala Ser Ala Leu Leu Ala Lys Asp Lys Gin Ile Ala Ala Leu Gln

220 225 230 235

  CGG GAG TGC AGG GAG CTG CAG GCC AGG CTC ACT CTG GTG GGC AAG GAG 950

  Arg Glu Cys Arg Glu Leu Gln Ala Arg Leu Thr Leu Val Gly Lys Glu

240 245 250

  GGT CCC CAG TGG CTC CAC GTG CGG GAC TTC GAT CGG CTG CTG CGC GAG 998

  Gly Pro Gln Trp Leu His Val Arg Asp Phe Asp Arg Leu Leu Arg Glu

255 260 265

  TCC CAG CGG GAG GTG CTG CGG CTG CAG AGG CAG ATC GCG CTG CGC AAC 1046

  Ser Gln Arg Glu Val Leu Arg Leu Gln Arg Gln Ile Ala Leu Arg Asn

270,275,280

  CAG CGG GAG ACG CTC CCG CTC CCG CCG TCC TGG CCC CCG GGC CCT GCT 1094

  Gln Arg Glu Thr Leu Pro Leu Pro Pro Ser Trp Pro Pro Gly Pro Ala

285 290 295

  CTC CAG GCC AGA GCA GGG GCG CCT GCT CCC GGG GCC CCG GGA GAG GCC 1142

  Leu Gin Ala Arg Ala Gly Ala Pro Ala Pro Gly Ala Pro Gly Glu Ala

300 305 310 315

  ACG CCC CAG GAG GAT GCG GAC AAC CTA CCC GTG ATT CTA GGG GAG CCA 1190

  Thr Pro Gln Glu Asp Ala Asp Asn Leu Pro Val Ile Leu Gly Glu Pro

320 325 330

  GAG AAA GAG CAG AGG GTG CAG CAG CTG GAA TCG GAG CTC AGC AAG AAG 1238

  Glu Lys Glu Gln Arg Val Gln Gln Leu Glu Ser Glu Leu Ser Lys Lys

335 340 345

  CGG AAG AAA TGC GAG AGC CTG GAG CAG GAA GCC CGG AAA AAG CAG AGG 1286

  Arg Lys Lys Cys Glu Ser Leu Glu Gln Glu Ala Arg Lys Lys Gln Arg

350 355 360

  CAA TGT GAG GAG CTG GAA CTG CAG CTG AGA CAA GCG CAG AAT GAG AAT 1334

  Gln Cys Glu Glu Leu Glu Leu Gln Leu Arg Gln Ala Gln Asn Glu Asn

365 370 375

  GCC CGC CTG GTG GAG GAG AAC TCC CGG CTC AGT GGG AGA GCC ACA GAG 1382

  Ala Arg Leu Val Glu Glu Asn Ser Arg Leu Ser Gly Arg Ala Thr Glu

380 385 390 395

  AAG GAG CAG GTG GAG TGG GAG AAT GCG GAG CTG AGG GGC CAG CTC CTG 1430

  Lys Glu Gln Val Glu Trp Glu Asn Ala Glu Leu Arg Gly Gln Leu Leu

400 405 410

  GGG GTG ACA CAG GAG AGG GAC TCA GCC CTT CGC AAG AGC CAG GGC CTG 1478

  Gly Val Thr Gln Glu Arg Asp Ser Ala Leu Arg Lys Ser Gln Gly Leu

415 420 425

  CAG AGC AAG CTG GAG AGC CTG GAG CAA GTG CTG AAG CAC ATG CGG GAG 1526

  Gln Ser Lys Leu Glu Ser Leu Glu Gln Val Leu Lys His Met Arg Glu

430 435 440

  GTG GCC CAG CGG CGG CAG CAG CTG GAG GTG GAG CAT GAA CAG GCT CGG 1574

  Val Ala Gin Arg Arg Gln Gin Leu Glu Val Glu His Glu Gin Ala Arg

445 450 455

  CTC AGC CTA CGG GAG AAG CAG GAG GAG GTC CGG AGA CTG CAG CAG GCC 1622

  Leu Ser Leu Arg Glu Lys Gin Glu Glu Val Arg Arg Leu Gln Gln Ala

460 465 470 475

  CAG GCT GAA GCC CAG AGG GAA CAT GAA GGA GCC GTG CAG CTG CTG GAG 1670

  Gln Ala Glu Ala Gin Arg Glu His Glu Gly Ala Val Gin Leu Leu Glu

480 485 490

  TCT ACC TTG GAT TCC ATG CAG GCC CGG GTT CGA GAG CTC GAA GAA CAG 1718

  Ser Thr Leu Asp Ser Met Gin Ala Arg Val Arg Glu Leu Glu Glu Gln

495,500 505

  TGC CGC AGC CAA ACC GAG CAG TTC AGC CTC CTG GCA CAA GAA CTC CAG 1766

  Cys Arg Ser Gln Thr Glu Gln Phe Ser Leu Leu Ala Gln Glu Leu Gln

510 515 520

  GCT TTC CGC CTG CAC CCG GGC CCC TTG GAT CTG CTC ACA TCT GCC CTG 1814

  Ala Phe Arg Leu His Pro Gly Pro Leu Asp Leu Leu Thr Ser Ala Leu

525 530 535

  GAC TGT GGG AGC CTT GGA GAC TGC CCA CCA CCC CCC TGC TGC TGC TCC 1862

  Asp Cys Gly Ser Leu Gly Asp Cys Pro Pro Pro Pro Cys Cys Cys Ser

540 545 550 555

  ATT CCC CAG CCT TGC CGG GGG TCT GGC CCC AAA GAC CTT GAC CTC CCG 1910

  Ile Pro Gln Pro Cys Arg Gly Ser Gly Pro Lys Asp Leu Asp Leu Pro

560 565 570

  CCG GGC TCC CCT GGG CGC TGC ACC CCA AAG TCT TCC GAG CCT GCC CCT 1958

  Pro Gly Ser Pro Gly Arg Cys Thr Pro Lys Ser Ser Glu Pro Ala Pro

575 580 585

  GCC ACT CTC ACT GGG GTC CCT CGA AGG ACA GCC AAG AAG GCA GAG TCT 2006

  Ala Thr Leu Thr Gly Val Pro Arg Arg Thr Ala Lys Lys Ala Glu Ser

590 595 600

  CTC TCC AAC TCC TCC CAC TCC GAG TCC ATC CAC AAC AGC CCC AAG TCA 2054

  Leu Ser Asn Ser Ser His Ser Glu Ser Ile His Asn Ser Pro Lys Ser

605 610 615

  TGC CCT ACA CCT GAG GTG GAC ACA GCC AGT GAG GTA GAG GAG CTG GAG 2102

  Cys Pro Thr Pro Glu Val Asp Thr Ala Ser Glu Val Glu Glu Leu Glu

620 625 630 635

  GCA GAC AGT GTC TCC CTG CTC CCA GCT GCG CCA GAG GGC AGC CGG GGA 2150

  Ala Asp Ser Val Ser Leu Leu Pro Ala Ala Pro Glu Gly Ser Arg Gly

640 645 650

  GGA GCC AGG ATC CAG GTC TTC CTA GCA CGT TAT AGC TAC AAC CCC TTT 2198

  Gly Ala Arg Ile Gln Val Phe Leu Ala Arg Tyr Ser Tyr Asn Pro Phe

655,660,665

  GAG GGT CCC AAT GAG AAT CCA GAA GCA GAG CTT CCG CTG ACA GCT GGC 2246

  Glu Gly Pro Asn Glu Asn Pro Glu Ala Glu Leu Pro Leu Thr Ala Gly

670 675 680

  GAG TAC ATC TAC ATC TAT GGC AAC ATG GAT GAG GAT GGC TTT TTT GAA 2294

  Glu Tyr Ile Tyr Ile Tyr Gly Asn Met Asp Glu Asp Gly Phe Phe Glu

685 690 695

  GGA GAG CTC ATG GAT GGC CGA AGG GGC CTG GTC CCT TCC AAT TTT GTA 2342

  Gly Glu Leu Met Asp Gly Arg Arg Gly Leu Val Pro Ser Asn Phe Val

700 705 710 715

  GAG CGT GTG TCG GAT GAT GAC CTC CTG ACC TCC CTC CCT CCA GAG CTG 2390

  Glu Arg Val Ser Asp Asp Asp Leu Leu Thr Ser Leu Pro Pro Glu Leu

720,725,730

  GCC GAT TTG TCC CAC AGC TCA GGC CCT GAA CTC AGT TTC CTG AGT GTA 2438

  Ala Asp Leu Ser His Ser Ser Gly Pro Glu Leu Ser Phe Leu Ser Val

735 740 745

  GGT GGG GGT GGC AGC AGT AGC GGG GGC CAA AGC AGT GTG AGA AGG AGC 2486

  Gly Gly Gly Gly Ser Ser Ser Gly Gly Gln Ser Ser Val Arg Arg Ser

750 755 760

  CAG CCC AGA CCT GAG GAG GAG GAT GCA GGG GAC GAG CTC AGT CTG AGC 2534

  Gln Pro Arg Pro Glu Glu Glu Asp Ala Gly Asp Glu Leu Ser Leu Ser

765,770,775

  CCA TCA CCG GAG GGC CTG GGC GAG CCT CCT GCC GTG CCT TAC CCC CGC 2582

  Pro Ser Pro Glu Gly Leu Gly Glu Pro Pro Ala Val Pro Tyr Pro Arg

780,785 790,795

  CGT CTG GTG GTC CTC AAG CAG CTG GCC CAC AGC GTG GTG CTG GCC TGG 2630

  Arg Leu Val Val Leu Lys Gln Leu Ala His Ser Val Val Leu Ala Trp

800 805 810

  GAG CCG CCT CCT GAG CAA GTG GAG CTA CAC GGC TTC CAT ATC TGT GTG 2678

  Glu Pro Pro Pro Glu Gln Val Glu Leu His Gly Phe His Ile Cys Val

815 820 825

  AAT GGG GAG CTG CGA CAG GCC CTG GGG CCT GGG GCG CCA CCC AAG GCC 2726

  Asn Gly Glu Leu Arg Gln Ala Leu Gly Pro Gly Ala Pro Pro Lys Ala

830 835 840

  GTG CTG GAG AAC CTG GAC CTG TGG GCC GGG CCC CTT CAC ATT TCT GTC 2774

  Val Leu Glu Asn Leu Asp Leu Trp Ala Gly Pro Leu His Ile Ser Val

845 850 855

  CAG GCC CTG ACT AGC CGG GGC AGC TCT GAC CCA CTG CGC TGT TGC TTG 2822

  Gln Ala Leu Thr Ser Arg Gly Ser Ser Asp Pro Leu Arg Cys Cys Leu

860,865 870,875

  GCG GTG GGT GCC CGG GCC GGA GTG GTG CCC AGC CAG CTG CGG GTC CAT 2870

  Ala Val Gly Ala Arg Ala Gly Val Val Pro Ser Gln Leu Arg Val His

880 885 890

  CGG TTG ACA GCC ACA TCT GCT GAG ATC ACC TGG GTG CCC GGC AAT AGC 2918

  Arg Leu Thr Ala Thr Ser Ala Glu Ile Thr Trp Val Pro Gly Asn Ser

895 900 905

  AAC TTG GCC CAT GCC ATC TAC CTC AAT GGG GAA GAA TGC CCA CCT GCC 2966

  Asn Leu Ala His Ala Ile Tyr Leu Asn Gly Glu Glu Cys Pro Pro Ala

910 915 920

  AGC CCC AGT ACC TAC TGG GCC ACC TTC TGC CAC TTA CGG CCT GGC ACA 3014

  Ser Pro Ser Thr Tyr Trp Ala Thr Phe Cys His Leu Arg Pro Gly Thr

925 930 935

  CCC TAT CAG GCC CAA GTG GAG GCT CAG CTC CCA CCC CAA GGG CCC TGG 3062

  Pro Tyr Gln Ala Gln Val Glu Ala Gln Leu Pro Pro Gln Gly Pro Trp

940 945 950 955

  GAA CCA GGC TGG GAG AGG CTG GAG CAG CGG GCT GCC ACC CTG CAG TTC 3110

  Glu Pro Gly Trp Glu Arg Leu Glu Gln Arg Ala Ala Thr Leu Gln Phe

960 965 970

  ACC ACA CTC CCA GCA GGC CCA CCT GAT GCC CCT CTG GAT GTG CAG ATC 3158

  Thr Thr Leu Pro Ala Gly Pro Pro Asp Ala Pro Leu Asp Val Gln Ile

975 980 985

  GAG CCT GGG CCC TCC CCT GGG ATC TTG ATC ATC AGT TGG CTC CCA GTC 3206

  Glu Pro Gly Pro Ser Pro Gly Ile Leu Ile Ile Ser Trp Leu Pro Val

990 995 1000

  ACC ATC GAT GCT GCT GGC ACA TCC AAC GGT GTC CGG GTC ACA GGC TAT 3254

  Thr Ile Asp Ala Ala Gly Thr Ser Asn Gly Val Arg Val Thr Gly Tyr

1005 1010 1015

  GCC ATC TAC GCT GAT GGG CAG AAG ATC ATG GAG GTG GCC TCA CCC ACG 3302

  Ala Ile Tyr Ala Asp Gly Gln Lys Ile Met Glu Val Ala Ser Pro Thr

1020 1025 1030 1035

  GCA GGC AGT GTA CTG GTG GAG TTG TCC CAG CTG CAG CTG CTG CAG GTG 3350

  Ala Gly Ser Val Leu Val Glu Leu Ser Gln Leu Gln Leu Leu Gln Val

1040 1045 1050

  TGT CGT GAG GTG GTC GTG CGC ACC ATG TCG CCC CAC GGG GAG TCG GCG 3398

  Cys Arg Glu Val Val Val Arg Thr Met Ser Pro His Gly Glu Ser Ala

1055 1060 1065

  GAC TCC ATC CCG GCT CCT ATC ACT CCC GCC CTG GCT CCG GCC AGC CTG 3446

  Asp Ser Ile Pro Ala Pro Ile Thr Pro Ala Leu Ala Pro Ala Ser Leu

1070 1075 1080

  CCA GCC CGA GTC TCC TGC CCC TCA CCG CAC CCA AGC CCA GAG GCC AGA 3494

  Pro Ala Arg Val Ser Cys Pro Ser Pro His Pro Ser Pro Glu Ala Arg

1085 1090 1095

  GCG CCC CTT GCT TCA GCC TCC CCA GGG CCT GGA GAC CCC AGC TCT CCT 3542

  Ala Pro Leu Ala Ser Ala Ser Pro Gly Pro Gly Asp Pro Ser Ser Pro

1100 1105 1110 1115

  CTC CAG CAC CCT GCT CCC CTT GGA ACT CAA GAG CCT CCA GGA GCA CCC 3590

  Leu Gln His Pro Ala Pro Leu Gly Thr Gln Glu Pro Pro Gly Ala Pro

1120 1125 1130

  CCT GCA AGC CCT TCC AGA GAG ATG GCA AAA GGG TCC CAC GAG GAC CCT 3638

  Pro Ala Ser Pro Ser Arg Glu Met Ala Lys Gly Ser His Glu Asp Pro

1135 1140 1145

  CCA GCA CCT TGC TCC CAG GAG GAG GCT GGG GCA GCA GTG CTG GGC ACC 3686

  Pro Ala Pro Cys Ser Gln Glu Glu Ala Gly Ala Ala Val Leu Gly Thr

1150 1155 1160

  TCA GAG GAG AGG ACA GCC AGC ACA TCT ACC CTG GGT GAG AAG GAC CCT 3734

  Ser Glu Glu Arg Thr Ala Ser Thr Ser Thr Leu Gly Glu Lys Asp Pro

1165 1170 1175

  GGC CCC GCA GCT CCC TCA CTG GCC AAG CAG GAG GCC GAG TGG ACT GCA 3782

  Gly Pro Ala Ala Pro Ser Leu Ala Lys Gln Glu Ala Glu Trp Thr Ala

1180 1185 1190 1195

  GGA GAG GCC TGT CCG GCC TCC AGC TCC ACC CAG GGA GCA CGG GCC CAG 3830

  Gly Glu Ala Cys Pro Ala Ser Ser Ser Thr Gln Gly Ala Arg Ala Gln

1200 1205 1210

  CAG GCG CCA AAT ACC GAG ATG TGC CAA GGA GGA GAC CCA GGG TCT GGG 3878

  Gln Ala Pro Asn Thr Glu Met Cys Gln Gly Gly Asp Pro Gly Ser Gly

1215 1220 1225

  CTG AGG CCC AGG GCT GAG AAG GAG GAC ACA GCA GAG CTT GGG GTT CAT 3926

  Leu Arg Pro Arg Ala Glu Lys Glu Asp Thr Ala Glu Leu Gly Val His

1230 1235 1240

  CTG GTG AAC TCC CTC GTG GAC CAC GGC CGC AAC TCA GAC CTG TCA GAC 3974

  Leu Val Asn Ser Leu Val Asp His Gly Arg Asn Ser Asp Leu Ser Asp

1245 1250 1255

  ATC CAG GAG GAA GAG GAA GAG GAG GAG GAG GAG GAG GAA GAG GAG CTG 4022

  Gln Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Leu

1260 1265 1270 1275

  GGT TCC AGG ACT TGC TCC TTC CAG AAG CAG GTT GCT GGC AAC AGC ATC 4070

  Gly Ser Arg Thr Cys Ser Phe Gln Lys Gln Val Ala Gly Asn Ser Ile

1280 1285 1290

  AGG GAG AAT GGG GCC AAG TCC CAG CCC GAC CCC TTT TGT GAG ACT GAC 4118

  Arg Glu Asn Gly Ala Lys Ser Gln Pro Asp Pro Phe Cys Glu Thr Asp

1295 1300 1305

  AGC GAT GAG GAG ATC TTG GAG CAG ATC CTG GAG CTG CCC CTC CAG CAG 4166

  Ser Asp Glu Glu Ile Leu Glu Gln Ile Leu Glu Leu Pro Leu Gln Gin

1310 1315 1320

  TTC TGT AGC AAG AAG CTC TTT AGC ATC CCG GAG GAG GAG GAA GAG GAA 4214

  Phe Cys Ser Lys Lys Leu Phe Ser Ile Pro Glu Glu Glu Glu Glu Glu

1325 1330 1335

  GAG GAG GAC GAG GAG GAG GAG AAG TCT GGG GCA GGC TGT TCT TCC CGA 4262

  Glu Glu Asp Glu Glu Glu Glu Lys Ser Gly Ala Gly Cys Ser Ser Arg

1340 1345 1350 1355

  GAC CCT GGC CCG CCT GAA CCT GCA TTG CTG GGG CTG GGC TGT GAC AGT 4310

  Asp Pro Gly Pro Pro Glu Pro Ala Leu Leu Gly Leu Gly Cys Asp Ser

1360 1365 1370

  GGT CAG CCC CGA AGA CCT GGC CAG TGT CCC TTG TCT CCT GAG TCC TCC 4358

  Gly Gln Pro Arg Arg Pro Gly Gln Cys Pro Leu Ser Pro Glu Ser Ser

1375 1380 1385

  AGG GCT GGA GAC TGC CTG GAA GAC ATG CCT GGA TTA GTT GGT GGA AGC 4406

  Arg Ala Gly Asp Cys Leu Glu Asp Met Pro Gly Leu Val Gly Gly Ser

1390 1395 1400

  AGC CGG AGG AGA GGA GGG GGC TCC CCT GAG AAG CCC CCA AGC CGC AGG 4454

  Ser Arg Arg Arg Gly Gly Gly Ser Pro Glu Lys Pro Pro Ser Arg Arg

1405 1410 1415

  CGG CCT CCA GAT CCC CGA GAA CAC TGC AGC CGA CTT CTC AGC AAC AAT 4502

  Arg Pro Pro Asp Pro Arg Glu His Cys Ser Arg Leu Leu Ser Asn Asn

1420 1425 1430 1435

  GGG CCC CAG GCC TCT GGA CGA CTG GGC CCC ACA CGG GAG AGG GGT GGC 4550

* Gly Pro Gin Ala Ser Gly Arg Leu Gly Pro Thr Arg Glu Arg Gly Gly

1440 1445 1450

  CTC CCC GTA ATT GAG GGC CCC AGG ACT GGA CTA GAG GCT AGC GGG AGA 4598

  Leu Pro Val Ile Glu Gly Pro Arg Thr Gly Leu Glu Ala Ser Gly Arg

1455 1460 1465

  GGC CGG CTG GGC CCT TCC CGG AGG TGC TCC CGT GGC CGG GCG CTG GAG 4646

  Gly Arg Leu Gly Pro Ser Arg Arg Cys Ser Arg Gly Arg Ala Leu Glu

1470 1475 1480 CCT GGC CTG GCC AGC TGC CTT TCC CCC AAG TGC TTG GAA ATC AGC ATT 4694

  Pro Gly Leu Ala Ser Cys Leu Ser Pro Lys Cys Leu Glu Ile Ser Ile

1485 1490 1495

  GAA TAT GAT TCG GAG GAT GAG CAG GAG GCG GGC AGC GGG GGC ATC AGC 4742

  Glu Tyr Asp Ser Glu Asp Glu Gln Glu Ala Gly Ser Gly Gly Ile Ser

1500 1505 1510 1515

  ATC ACC AGC TCC TGC TAC CCT GGA GAT AGG GAG GCC TGG GGC ACA GCA 4790

  Ile Thr Ser Ser Cys Tyr Pro Gly Asp Arg Glu Ala Trp Gly Thr Ala

1520 1525 1530

  ACT GTA GGA AGG CCC AGG GGG CCT CCG AAG GCC AAT TCA GGC CCC AAG 4838

  Thr Val Gly Arg Pro Arg Gly Pro Pro Lys Ala Asn Ser Gly Pro Lys

1535 1540 1545

  CCC TAC CCA CGC CTC CCA GCC TGG GAG AAA GGG GAA CCA GAG CGG AGA 4886

  Pro Tyr Pro Arg Leu Pro Ala Trp Glu Lys Gly Glu Pro Glu Arg Arg

1550 1555 1560

  GGC CGC AGT GCG ACG GGC AGA GCC AAG GAG CCA CTC TCC CGG GCA ACA 4934

  Gly Arg Ser Ala Thr Gly Arg Ala Lys Glu Pro Leu Ser Arg Ala Thr

1565 1570 1575

  GAG ACC GGA GAG GCC AGA GGG CAG GAC GGC TCT GGG CGG AGG GGC CCC 4982

  Glu Thr Gly Glu Ala Arg Gly Gln Asp Gly Ser Gly Arg Arg Gly Pro

1580 1585 1590 1595

  CAG AAG AGA GGT GTC CGA GTC CTC AGG CCA AGC ACT GCA GAG CTA GTC 5030

  Gln Lys Arg Gly Val Arg Val Leu Arg Pro Ser Thr Ala Glu Leu Val

1600 1605 1610

  CCT GCG AGG AGC CCC TCA GAA ACA CTG GCT TAC CAG CAC CTA CCC GTC 5078

  Pro Ala Arg Ser Pro Ser Glu Thr Leu Ala Tyr Gln His Leu Pro Val

1615 1620 1625

  AGG ATC TTT GTG GCT CTG TTT GAC TAT GAC CCC GTG TCA ATG TCG CCC 5126

  Arg Ile Phe Val Ala Leu Phe Asp Tyr Asp Pro Val Ser Met Ser Pro

1630 1635 1640

  AAT CCT GAT GCT GGA GAA GAA GAG CTT CCC TTC CGA GAG GGT CAG ATC 5174

  Asn Pro Asp Ala Gly Glu Glu Glu Leu Pro Phe Arg Glu Gly Gln Ile

1645 1650 1655

  CTG AAG GTG TTT GGG GAC AAG GAT GCC GAT GGC TTC TAC CAG GGC GAA 5222

  Leu Lys Val Phe Gly Asp Lys Asp Ala Asp Gly Phe Tyr Gln Gly Glu

1660 1665 1670 1675

  GGT GGG GGC CGG ACA GGC TAC ATT CCC TGC AAC ATG GTG GCT GAG GTG 5270

  Gly Gly Gly Arg Thr Gly Tyr Ile Pro Cys Asn Met Val Ala Glu Val

1680 1685 1690

  GCT GTG GAC AGC CCT GCT GGG AGA CAG CAA CTG CTC CAG CGG GGT TAT 5318

  Ala Val Asp Ser Pro Ala Gly Arg Gln Gln Leu Leu Gln Arg Gly Tyr

1695 1700 1705

  TTG TCC CCA GAT ATT CTC CTT GAG GGC TCA GGG AAT GGT CCG TTT GTG 5366

  Leu Ser Pro Asp Ile Leu Leu Glu Gly Ser Gly Asn Gly Pro Phe Val

1710 1715 1720

  TAC TCC ACA GCC CAC ACA ACT GGG CCT CCT CCC AAG CCC CGC CGC TCC 5414

  Tyr Ser Thr Ala His Thr Thr Gly Pro Pro Pro Lys Pro Arg Arg Ser

1725 1730 1735

  AAG AAA GCT GAG TCG GAA GGC CCT GCC CAG CCC TGT CCA GGC CCC CCT 5462

  Lys Lys Ala Glu Ser Glu Gly Pro Ala Gln Pro Cys Pro Gly Pro Pro

1740 1745 1750 1755

  AAG CTG GTC CCC TCT GCT GAC CTG AAA GCT CCC CAC TCC ATG GTG GCT 5510

  Lys Leu Val Pro Ser Ala Asp Leu Lys Ala Pro His Ser Met Val Ala

1760 1765 1770

  GCA TTT GAC TAC AAC CCC CAG GAG AGT TCC CCC AAT ATG GAC GTG GAG 5558

  Ala Phe Asp Tyr Asn Pro Gln Glu Ser Ser Pro Asn Met Asp Val Glu

1775 1780 1785

  GCA GAG CTG CCC TTC CGG GCA GGG GAT GTC ATT ACT GTG TTT GGG GGC 5606

  Ala Glu Leu Pro Phe Arg Ala Gly Asp Val Ile Thr Val Phe Gly Gly

1790 1795 1800

  ACG GAC GAT GAC GGT TTC TAC TAT GGG GAA TTA AAT GGA CAA AGG GGC 5654

  Thr Asp Asp Asp Gly Phe Tyr Tyr Gly Glu Leu Asn Gly Gln Arg Gly

1805 1810 1815

  CTG GTT CCA TCC AAC TTC CTG GAG GGC CCT GGG CCT GAG GCA GGC GGC 5702

  Leu Val Pro Ser Asn Phe Leu Glu Gly Pro Gly Pro Glu Ala Gly Gly

1820 1825 1830 1835

  CTG GAC AGG GAA CCC AGG ACA CCC CAG GCT GAG AGT CAG AGA ACG AGG 5750

  Leu Asp Arg Glu Pro Arg Thr Pro Gln Ala Glu Ser Gln Arg Thr Arg

1840 1845 1850

  AGG AGA AGA GTC CAG TGC TAGATGGAGA TAGATATATG TAGAGAGAGC 5798

Arg Arg Arg Val Gln Cys

  AACATGACTG GGGCTGCACC ACACAAGGGT CCCCAGGGCC CCCAGGTGGG CCTTGTACCC 5858

  CCAGCTCTGG CAGCGCCCCC AGGATTGAAC GTGGGGAGCC CCAGGGCAGA AGCGAGAAGG 5918

  TGTGGGTTTT TTTCCAAGGG GAAGCAGCTC CTCAGGAGGA TGGGCTCTGG GAAGAAGGAG 5978

  TGAAGCTGTG GCCCATCCTG CAGGCAGAAG AGGCCCTGAG AGGCCCCCAG ATCACTGTTT 6038

  TTGCTGAGCA GGGAAGGCTC AGAATGGGCC CAGAGCCCCC GTTCTTTTGC GTTAACACTG 6098

  TGGCATTCAG AGGGAATCAA AGAGCCTTGG TGAAGGTCAG AGCTAAATGG CTCCTTAAGG 6158

  ATGAACTTTT TAGAGAAGCA CCTTCCTCCT ACAGGAGGGG AAACTGAGCC CACAGTGAGT 6218

  ATGTAACTTG ACCAAGGTCA CTGAGCCAGG ACTGGACCCA GGACCCTCGC GTCCTGGTCC 6278

  ACCCACCTCG TCTACTAGTG TCCCACAGTG CTGCGCTAGT CCCTTTTGCC ACCCTTCCCA 6338

  GTCCCAGGAC GGGCCCTGGA GGGAGAAAGG AGCCTGTGCC CCCTGATGGC TCTGGCTGTC 6398

  CTGATCCTGT CTTCCCTCCC CTGAAGGAAA GTTTGCACTG GATTTTATTG GAGCCCCATC 6458

  TCCCCAGCGG GCAGGCGGGC GGAGCCTGTA TATATGTATA TACTCAGTGC CTCAGTTCAG 6518

  CTTCCTCCAC CTCGCTTCCA CTGCACAGGC CCAGGAAGGA GAAAGGCCAA GCCAAAGTGG 6578

  GCCCCACCCT GCCCCCGTCG TGCTCCATCC TTCCCTGCCG GGGCCTGCTG GCCCCTGTAA 6638

  GGTCCCGCCC CCAAAGACCC TGGGGCCAGC GGGGCCGAAA GCGGAGTTGG GTTTGCTTAT 6698

  TTTGCTCATT GGATTCAAGT TCTTTTGCAT AGTTTTTTTC TAACCCCTGT TGGAGTCCAG 6758

  GGGCTGGAGA AAAGGACAGA TTTATGCAGC TATTTTCATA CATTCCCTGT TCAGAGTGGG 6818

  GTAGGGGTTT TCCGCCGTTA CCCGATCCAA TCCATCCCCC ACCCTTTGAG GGGTGAGTGT 6878

  GTCTTTGCAT GTTTCCTTTG CTGTGGTGGG AGATAGTTTG ACTGAACCCC CACCTTGACC 6938

  TTGGTCTCCA GGGGTGTGGA ATGGTGGGGG AATTTGTTTA AAAAGACATT TTATTATAAT 6998

  AAAGTCTATT TTCACAAAAA AAAAAAAAAA AAAAA 7033

  (2) INFORMATION FOR SEQ ID NO: 2:

  (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 1857 amino acids (B) TYPE: amino acid (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: protein

  (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2:

  Met Glu Gln Leu Thr Thr Leu Pro Arg Pro Gly Asp Leu Gly Ala Met

1 5 10 15

  Glu Pro Trp Ala Leu Pro Thr Trp His Ser Trp Thr Pro Gly Arg Gly

25 30

  Gly Glu Pro Ser Ser Ala Ala Pro Ser Ile Ala Asp Thr Pro Pro Ala

40 45

  Ala Leu Gin Leu Gln Glu Leu Arg Ser Glu Glu Ser Ser Lys Pro Lys

55 60

  Gly His Gly Ser Ser Arg Pro Val Gly Gly Thr Asp Pro Glu Gly Ala

70 75 80

  Gln Ala Cys Leu Pro Ser Leu Gly Gln Gln Ala Ser Ser Ser Gly Pro

90 95

  Ala Cys Gln Arg Pro Glu Asp Glu Glu Val Glu Ala Phe Leu Lys Ala

105 110

  Lys Leu Asn Met Ser Phe Gly Asp Arg Pro Asn Leu Glu Leu Leu Arg

120 125

  Ala Leu Gly Glu Leu Arg Gln Arg Cys Ala Ile Leu Lys Glu Glu Asn

135 140

  Gln Met Leu Arg Lys Ser Ser Phe Pro Glu Thr Glu Glu Lys Val Arg

150 155 160

  Arg Leu Lys Arg Lys Asn Ala Glu Leu Ala Val Ile Ala Lys Arg Leu

170 175

  Glu Glu Arg Ala Arg Lys Leu Gln Glu Thr Asn Leu Arg Val Val Ser

185 190

  Ala Pro Leu Pro Arg Pro Gly Thr Ser Leu Glu Leu Cys Arg Lys Ala

200 205

  Leu Ala Arg Gln Arg Ala Arg Asp Leu Ser Glu Thr Ala Ser Ala Leu

210 215 220

  Leu Ala Lys Asp Lys Gln Ile Ala Ala Leu Gln Arg Glu Cys Arg Glu

225 230 235 240

  Leu Gln Ala Arg Leu Thr Leu Val Gly Lys Glu Gly Pro Gln Trp Leu

245 250 255

  His Val Arg Asp Phe Asp Arg Leu Leu Arg Glu Ser Gin Arg Glu Val

260 265 270

  Leu Arg Leu Gln Arg Gin Ile Ala Leu Arg Asn Gln Arg Glu Thr Leu

275 280 285

  Pro Leu Pro Pro Ser Trp Pro Pro Gly Pro Ala Leu Gin Ala Arg Ala

290 295 300

  Gly Ala Pro Ala Pro Gly Ala Pro Gly Glu Ala Thr Pro Gln Glu Asp

305 310 315 320

  Ala Asp Asn Leu Pro Val Ile Leu Gly Glu Pro Glu Lys Glu Gin Arg

325 330 335

  Val Gln Gln Leu Glu Ser Glu Leu Ser Lys Lys Arg Lys Lys Cys Glu

340 345 350

  Ser Leu Glu Gln Glu Ala Arg Lys Lys Gln Arg Gln Cys Glu Glu Leu

355 360 365

  Glu Leu Gln Leu Arg Gln Ala Gln Asn Glu Asn Ala Arg Leu Val Glu

370 375 380

  Glu Asn Ser Arg Leu Ser Gly Arg Ala Thr Glu Lys Glu Gln Val Glu

385 390 395 400

  Trp Glu Asn Ala Glu Leu Arg Gly Gln Leu Leu Gly Val Thr Gln Glu

405 410 415

  Arg Asp Ser Ala Leu Arg Lys Ser Gln Gly Leu Gln Ser Lys Leu Glu

420 425 430

  Ser Leu Glu Gln Val Leu Lys His Met Arg Glu Val Ala Gln Arg Arg

435 440 445

  Gln Gln Leu Glu Val Glu His Glu Gln Ala Arg Leu Ser Leu Arg Glu

450 455 460

  Lys Gln Glu Glu Val Arg Arg Leu Gln Gln Ala Gln Ala Glu Ala Gln

465 470 475 480

  Arg Glu His Glu Gly Ala Val Gln Leu Leu Glu Ser Thr Leu Asp Ser

485 490 495

  Met Gln Ala Arg Val Arg Glu Leu Glu Glu Gln Cys Arg Ser Gln Thr

500 505 510

  Glu Gln Phe Ser Leu Leu Ala Gln Glu Leu Gln Ala Phe Arg Leu His

515 520 525

  Pro Gly Pro Leu Asp Leu Leu Thr Ser Ala Leu Asp Cys Gly Ser Leu

530 535 540

  Gly Asp Cys Pro Pro Pro Pro Cys Cys Cys Ser Ile Pro Gln Pro Cys

545 550 555 560

  Arg Gly Ser Gly Pro Lys Asp Leu Asp Leu Pro Pro Gly Ser Pro Gly

565,570,575

  Arg Cys Thr Pro Lys Ser Ser Glu Pro Ala Pro Ala Thr Leu Thr Gly

580 585 590

  Val Pro Arg Arg Thr Ala Lys Lys Ala Glu Ser Leu Ser Asn Ser Ser

595 600 605

  His Ser Glu Ser Ile His Asn Ser Pro Lys Ser Cys Pro Thr Pro Glu

610 615 620

  Val Asp Thr Ala Ser Glu Val Glu Glu Leu Glu Ala Asp Ser Val Ser

625 630 635 640

  Leu Leu Pro Ala Ala Pro Glu Gly Ser Arg Gly Gly Ala Arg Ile Gln

645 650 655

  Val Phe Leu Ala Arg Tyr Ser Tyr Asn Pro Phe Glu Gly Pro Asn Glu

660 665 670

  Asn Pro Glu Ala Glu Leu Pro Leu Thr Ala Gly Glu Tyr Ile Tyr Ile

675 680 685

  Tyr Gly Asn Met Asp Glu Asp Gly Phe Phe Glu Gly Glu Leu Met Asp

690 695 700

  Gly Arg Arg Gly Leu Val Pro Ser Asn Phe Val Glu Arg Val Ser Asp

705,710,715,720

  Asp Asp Leu Leu Thr Ser Leu Pro Pro Glu Leu Ala Asp Leu Ser His

725 730 735

  Ser Ser Gly Pro Glu Leu Ser Phe Leu Ser Val Gly Gly Gly Gly Ser

740 745 750

  Ser Ser Gly Gly Gln Ser Ser Val Arg Arg Ser Gln Pro Arg Pro Glu

755,760,765

  Glu Glu Asp Ala Gly Asp Glu Leu Ser Leu Ser Pro Ser Pro Glu Gly

770,775,780

  Leu Gly Glu Pro Pro Ala Val Pro Tyr Pro Arg Arg Leu Val Val Leu

785,790,795,800

  Lys Gln Leu Ala His Ser Val Val Leu Ala Trp Glu Pro Pro Pro Glu

805 810 815

  Gln Val Glu Leu His Gly Phe His Ile Cys Val Asn Gly Glu Leu Arg

820 825 830

  Gln Ala Leu Gly Pro Gly Ala Pro Pro Lys Ala Val Leu Glu Asn Leu

835 840 845

  Asp Leu Trp Ala Gly Pro Leu His Ile Ser Val Gin Ala Leu Thr Ser

850 855 860

  Arg Gly Ser Ser Asp Pro Leu Arg Cys Cys Leu Ala Val Gly Ala Arg

865 870 875 880

  Ala Gly Val Val Pro Ser Gln Leu Arg Val His Arg Leu Thr Ala Thr

885 890 895

  Ser Ala Glu Ile Thr Trp Val Pro Gly Asn Ser Asn Leu Ala His Ala

900 905 910

  Ile Tyr Leu Asn Gly Glu Glu Cys Pro Pro Ala Ser Pro Ser Thr Tyr

915 920 925

  Trp Ala Thr Phe Cys His Leu Arg Pro Gly Thr Pro Tyr Gln Ala Gln

930 935 940

  Val Glu Ala Gln Leu Pro Pro Gln Gly Pro Trp Glu Pro Gly Trp Glu

945 950 955 960

  Arg Leu Glu Gln Arg Ala Ala Thr Leu Gln Phe Thr Thr Leu Pro Ala

965,970,975

  Gly Pro Pro Asp Ala Pro Leu Asp Val Gln Ile Glu Pro Gly Pro Ser

980 985 990

  Pro Gly Ile Leu Ile Ser Trp Leu Pro Val Thr Ile Asp Ala Ala

995 1000 1005

  Gly Thr Ser Asn Gly Val Arg Val Thr Gly Tyr Ala Ile Tyr Ala Asp

1010 1015 1020

  Gly Gln Lys Ile Met Glu Val Ala Ser Pro Thr Ala Gly Ser Val Leu

1025 1030 1035 1040

  Val Glu Leu Ser Gln Leu Gln Leu Leu Gln Val Cys Arg Glu Val Val

1045 1050 1055

  Val Arg Thr Met Ser Pro His Gly Glu Ser Ala Asp Ser Ile Pro Ala

1060 1065 1070

  Pro Ile Thr Pro Ala Leu Ala Pro Ala Ser Leu Pro Ala Arg Val Ser

1075 1080 1085

  Cys Pro Ser Pro His Pro Ser Pro Glu Ala Arg Ala Pro Leu Ala Ser

1090 1095 1100

  Ala Ser Pro Gly Pro Gly Asp Pro Ser Ser Pro Leu Gin His Pro Ala

1105 1110 1115 1120

  Pro Leu Gly Thr Gln Glu Pro Pro Gly Ala Pro Pro Ala Ser Pro Ser

1125 1130 1135

  Arg Glu Met Ala Lys Gly Ser His Glu Asp Pro Pro Ala Pro Cys Ser

1140 1145 1150

* Gln Glu Glu Ala Gly Ala Ala Val Leu Gly Thr Ser Glu Glu Arg Thr

1155 1160 1165

  Ala Ser Thr Ser Thr Leu Gly Glu Lys Asp Pro Gly Pro Ala Ala Pro

1170 1175 1180

  Ser Leu Ala Lys Gln Glu Ala Glu Trp Thr Ala Gly Glu Ala Cys Pro

1185 1190 1195 1200

  Ala Ser Ser Ser Thr Gln Gly Ala Arg Ala Gln Gln Ala Pro Asn Thr

1205 1210 1215

  Glu Met Cys Gln Gly Gly Asp Pro Gly Ser Gly Leu Arg Pro Arg Ala

1220 1225 1230

  Glu Lys Glu Asp Thr Ala Glu Leu Gly Val His Leu Val Asn Ser Leu

1235 1240 1245

  Val Asp His Gly Arg Asn Ser Asp Leu Ser Asp Ile Gln Glu Glu Glu

1250 1255 1260

  Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Leu Gly Ser Arg Thr Cys

1265 1270 1275 1280

  Ser Phe Gln Lys Gln Val Ala Gly Asn Ser Ile Arg Glu Asn Gly Ala

1285 1290 1295

  Lys Ser Gln Pro Asp Pro Phe Cys Glu Thr Asp Ser Asp Glu Glu Ile

1300 1305 1310

  Leu Glu Gln Ile Leu Glu Leu Pro Leu Gln Gln Phe Cys Ser Lys Lys

1315 1320 1325

  Leu Phe Ser Ile Pro Glu Glu Glu Glu Glu Glu Glu Glu Asp Glu Glu

1330 1335 1340

  Glu Glu Lys Ser Gly Ala Gly Cys Ser Ser Arg Asp Pro Gly Pro Pro

1345 1350 1355 1360

  Glu Pro Ala Leu Leu Gly Leu Gly Cys Asp Ser Gly Gln Pro Arg Arg

1365 1370 1375

  Pro Gly Gln Cys Pro Leu Ser Pro Glu Ser Ser Arg Ala Gly Asp Cys

1380 1385 1390

  Leu Glu Asp Met Pro Gly Leu Val Gly Gly Ser Ser Arg Arg Arg Gly

1395 1400 1405

  Gly Gly Ser Pro Glu Lys Pro Pro Ser Arg Arg Arg Pro Pro Asp Pro

1410 1415 1420

  Arg Glu His Cys Ser Arg Leu Leu Ser Asn Asn Gly Pro Gin Ala Ser

1425 1430 1435 1440

  Gly Arg Leu Gly Pro Thr Arg Glu Arg Gly Gly Leu Pro Val Ile Glu

1445 1450 1455

  Gly Pro Arg Thr Gly Leu Glu Ala Ser Gly Arg Gly Arg Leu Gly Pro

1460 1465 1470

  Ser Arg Arg Cys Ser Arg Gly Arg Ala Leu Glu Pro Gly Leu Ala Ser

1475 1480 1485

  Cys Leu Ser Pro Lys Cys Leu Glu Ile Ser Ile Glu Tyr Asp Ser Glu

1490 1495 1500

  Asp Glu Gln Glu Ala Gly Ser Gly Gly Ile Ser Ile Thr Ser Ser Cys

1505 1510 1515 1520

  Tyr Pro Gly Asp Arg Glu Ala Trp Gly Thr Ala Thr Val Gly Arg Pro

1525 1530 1535

  Arg Gly Pro Pro Lys Ala Asn Ser Gly Pro Lys Pro Tyr Pro Arg Leu

1540 1545 1550

  Pro Ala Trp Glu Lys Gly Glu Pro Glu Arg Arg Gly Arg Ser Ala Thr

1555 1560 1565

  Gly Arg Ala Lys Glu Pro Leu Ser Arg Ala Thr Glu Thr Gly Glu Ala

1570 1575 1580

  Arg Gly Gln Asp Gly Ser Gly Arg Arg Gly Pro Gln Lys Arg Gly Val

1585 1590 1595 1600

  Arg Val Leu Arg Pro Ser Thr Ala Glu Leu Val Pro Ala Arg Ser Pro

1605 1610 1615

  Ser Glu Thr Leu Ala Tyr Gln His Leu Pro Val Arg Ile Phe Val Ala

1620 1625 1630

  Leu Phe Asp Tyr Asp Pro Val Ser Met Ser Pro Asn Pro Asp Ala Gly

1635 1640 1645

  Glu Glu Glu Leu Pro Phe Arg Glu Gly Gln Ile Leu Lys Val Phe Gly

1650 1655 1660

  Asp Lys Asp Ala Asp Gly Phe Tyr Gln Gly Glu Gly Gly Gly Arg Thr

1665 1670 1675 1680

  Gly Tyr Ile Pro Cys Asn Met Val Ala Glu Val Ala Val Asp Ser Pro

1685 1690 1695

  Ala Gly Arg Gln Gln Leu Leu Gln Arg Gly Tyr Leu Ser Pro Asp Ile

1700 1705 1710

  Leu Leu Glu Gly Ser Gly Asn Gly Pro Phe Val Tyr Ser Thr Ala His

1715 1720 1725

  Thr Thr Gly Pro Pro Pro Lys Pro Arg Arg Ser Lys Lys Ala Glu Ser

1730 1735 1740

  Glu Gly Pro Ala Gln Pro Cys Pro Gly Pro Pro Lys Leu Val Pro Ser

1745 1750 1755 1760

  Ala Asp Leu Lys Ala Pro His Ser Met Val Ala Ala Phe Asp Tyr Asn

1765 1770 1775

  Pro Gln Glu Ser Ser Pro Asn Met Asp Val Glu Ala Glu Leu Pro Phe

1780 1785 1790

  Arg Ala Gly Asp Val Ile Thr Val Phe Gly Gly Thr Asp Asp Asp Gly

1795 1800 1805

  Phe Tyr Tyr Gly Glu Leu Asn Gly Gln Arg Gly Leu Val Pro Ser Asn

1810 1815 1820

  Phe Leu Glu Gly Pro Gly Pro Glu Ala Gly Gly Leu Asp Arg Glu Pro

1825 1830 1835 1840

  Arg Thr Pro Gln Ala Glu Ser Gln Arg Thr Arg Arg Arg Arg Val Gln

1845 1850 1855

Cys

Claims (32)

  1. Purified polypeptide, comprising an amino acid sequence chosen from: a) the sequence SEQ ID No. 2; b) a biologically active sequence derived from SEQ ID No. 2; and c) a fragment of the sequence SEQ ID No. 2 having a biological activity of the same nature as that of the polypeptide of sequence SEQ ID No. 2,   or immunologically related to the polypeptide of sequence SEQ ID No. 2.   2. Polypeptide according to claim 1, characterized in that it comprises the sequence between: residue 1564 and residue 1857 of SEQ ID # 2.   3. Polypeptide according to claim 1, characterized in that it   results from an alternative splicing of the messenger RNA of the corresponding gene.   4. Polypeptide according to any one of the claims   above, characterized in that it is a recombinant polypeptide produced   in the form of a fusion protein.   5. Isolated nucleic acid sequence encoding a   A polypeptide according to any one of the preceding claims.   6. Isolated nucleic acid sequence according to claim, characterized in that it is chosen from: a) the sequence SEQ ID n 1; b) the sequence complementary to the sequence SEQ ID No. 1; c) the nucleic acid sequences capable of hybridizing specifically to the sequence SEQ ID No. 1, or to their complementary sequences, or to hybridizing specifically to their proximal sequences. d) the sequences derived from the sequences a), b) and c), due to the degeneration of the genetic code, mutation, deletion, insertion, a   alternative splicing or allelic variability.   7. Cloning and / or expression vector containing a   nucleic acid sequence according to any one of claims 5 and 6.   8. Vector according to claim 7, characterized in that it is plasmid pZeoSV2 +.   9. Host cell transfected with a vector according to claim 7 or 8. 10. transfected host cell according to claim 9,   characterized in that it is E. coi MC 1061.   11. Nucleotide sequence usable as probe or primer characterized in that it hybridizes specifically with any of the   sequences according to claims 5 and 6 or their complementary sequences   or the corresponding messenger RNAs or the corresponding genes.   12. Nucleotide sequence according to claim 11 usable as probe or primer, characterized in that it comprises at least 16 nucleotides. 13. Nucleotide sequence according to claim 12 usable as a probe, characterized in that it comprises the entire sequence   of the gene coding for one of the polypeptides of claim 1.   14. Nucleotide probe or primer chosen from the following oligonucleotides or their complementary: SEQ ID n 3: GCT CTG TTT GAC TAT GAC CC SEQ IDn 4: AACTTG AATCCAATGAGC AA SEQ ID n 5: TCT GCT GAC CTG AAA GCT CC SEQ ID n 6 : CCA TCA GGG GGC ACA GGC TC SEQ ID n 7: TGG GCCTTGTAC CCC CAG CT SEQ ID n 8: TCC TCC TGA GGA GCT GCT TC SEQ ID n 9: CCC CTG CCC GGAAGG GCA GC SEQ ID n 10: TTG ACC AAG GTC ACT GAG CC SEQ ID no 11: GGG CCT GCT GGC CCC TGT AA SEQ ID no 12: GCC ATC GGC ATC CTT GTC CC SEQ ID no 13: CCT CTC CGG TCT CTG TTG CCC G SEQ ID no 14: AGG CCC AGG GGG CCT CCG MAAG GC SEQ ID n 15: CGC TGC CCG CCT CCT GCT CAT CCT C SEQ ID n 16: TCC TCC AGG GCT GGA GAC TGC CT SEQ ID n 17: GGT TCC AGG ACT TGC TCC TTC CAG AAMG SEQ ID n 18: CAG CTC CCT CAC TGG CCAAG SEQ ID no 19: TGG CTC CGG CCA GCC TGC C SEQ ID no 20: TCC GGG TCA CAG GCT ATG CCA T SEQ ID no 21: CC CAG TAC CTA CTG GGC CAC CT SEQ ID no 22: GCT TCC ATA TCT GTG TGA ATG GG SEQ ID n 23: GGT MAAG GCA CGG CAG GAG GCT C SEQ ID n 24: GCT GGC TCC C TC TCA CAC TG SEQ ID n 25: GGA GAG CTC ATG GAT GGC CGA AG SEQ ID n 26: CCA GTG AGG TAG AGG AGC TGG SEQ ID n 27: TCC ATT CCC CAG CCT TGC CGG GG SEQ ID n 28: GCT CGA AGA ACA GTG CCG CAG CC SEQ ID n 29: TCG CMAA GAG CCA GGG CCT GCA GA SEQ ID n 30: GCT GGA ATC GGA GOT CAG CMAA G SEQ ID n 31: CCC GTG ATT CTA GGG GAG CCA GAG SEQ ID No. 32: GAG TGC AGG GAG CTG CAG GCC A SEQ ID No. 33: GGC TGA AGC GGA AGA ACG CCG AG SEQ ID No. 34: CMAA TCT GGA GCT GCT GAG GG SEQ ID No. 35: TGA GGT CTG AGG AGA GTT CCA AG SEQ ID # 36: ATG GAG CCA TGG GCA CTG CCC 15. Use of a sequence according to any one of   claims 5 and 6 for the manufacture of oligonucleotide primers for   specific sequencing, age or amplification reactions according to the PCR technique or any variant thereof. 16. Couples of nucleotide primers chosen from the following sequences: - couple n 1 sense primer: GCT CTG TTT GAC TAT GAC CC (SEQ ID n 3) antisense primer: MAAC TTG AAT CCA ATG AGC AA (SEQ ID n 4) - couple n 2: sense primer: TCT GCT GAC CTG AAA GCT CC (SEQ ID n 5) antisense primer: TCC TCC TGA GGA GCT GCT TC (SEQ ID n 8) - couple n 3: sense primer: TTG ACC AAG GTC ACT GAG CC (SEQ ID n 1 0) antisense primer: AAC TTG AAT CCA ATG AGC MAA (SEQ ID n 4) - couple n 4: sense primer: GCT CTG TTT GAC TAT GAC CC (SEQ ID n 3) antisense primer: GCC ATC GGC ATC CTT GTC CC (SEQ ID no 12) - pair 5: sense primer: GCT CTG TTT GAC TAT GAC CC (SEQ ID no 3) antisense primer: TCC TCC TGA GGA GCT GCT TC (SEQ ID no 8) 17. Use of a sequence according to any one of   claims 5 and 6 for the manufacture of a medicament for use in therapy   gene. 18. Use of a sequence according to any one of   Claims 5 and 6 for the production of nucleotide probes or primers   diagnostic, or antisense sequences usable for the manufacture of a   drug usable in gene therapy.   19. Use of nucleotide primers according to any one   of claims 11 and 12 for sequencing.   20. Use of a probe or primer according to any one   claims 11 to 16, as an in vitro diagnostic tool for detection,   by hybridization procedures, of nucleic acid sequences encoding   for a polypeptide according to any one of claims 1 to 4, in   biological samples, or for the detection of outliers or genetic abnormalities.   21. In vitro diagnostic method for the detection of aberrant syntheses or genetic abnormalities in the nucleic acid sequences coding for a polypeptide according to any one of   Claims 1 to 4, characterized in that it comprises:   - contacting a nucleotide probe according to one   any of claims 11 to 16 with a biological sample in   conditions allowing the formation of a hybridization complex between said probe and said nucleotide sequence, optionally after a prior step of amplification of said nucleotide sequence; - detection of the hybridization complex possibly formed; possibly sequencing, age of the nucleotide sequence   forming the hybridization complex with the probe of the invention.   22. Use of a nucleic acid sequence according to one   any of claims 5 and 6 for the production of a polypeptide   recombinant according to any one of claims 1 to 4.   23. Method for producing a recombinant protein PRAX-1 or fragment or biologically active derivative thereof or fragment immunologically related thereto, characterized in that cultured transfected cells according to claim 9 or 10 in conditions allowing the expression of a recombinant polypeptide of sequence SEQ ID No. 2 or fragment or biologically active derivative thereof, and that said recovery is obtained recombinant polypeptide.   24. Mono or polyclonal antibodies or their fragments, chimeric or immunoconjugate antibodies, characterized in that they are specifically   raised against a polypeptide according to any one of claims 1 to 4.   25. Use of the antibodies according to the preceding claim, for the purification or the detection of a polypeptide according to any one of   Claims 1 to 4 in a biological sample.   26. A method of in vitro diagnosis of pathologies correlated with an abnormal expression or accumulation of PRAX-1 proteins, from a biological sample, characterized in that at least one antibody according to claim 24 is brought into contact with said biological sample, under conditions allowing the formation of specific immunological complexes between a PRAX-1 protein and said antibody or antibodies and in that the   specific immunological complexes possibly formed.   27. Kit for the in vitro diagnosis of an abnormal expression or accumulation of PRAX-1 proteins in a biological sample and / or for measuring the level of expression of these in said sample comprising: - at least one antibody according to claim 24, optionally fixed on a support, - means for revealing the formation of specific antigen / antibody complexes between a PRAX-1 protein and said antibody   and / or means for quantifying these complexes.   28. Method for determining an allelic variability, a mutation, a deletion, an insertion, a loss of heterozygosity or a genetic abnormality in the PRAX-1 gene, characterized in that it implements at least one nucleotide having a nucleotide sequence according to one   any of claims 5 or 6.   29. Pharmaceutical composition comprising, as a principle   active, a polypeptide according to any one of claims 1 to 4.   30. Pharmaceutical composition according to the preceding claim, characterized in that it comprises a polypeptide according to the claim 2.   31. Pharmaceutical composition containing an inhibitor or an activator of the activity of PRAX-1 and of any protein-protein interaction making intervene PRAX-1.   32. Pharmaceutical composition containing a derived polypeptide   of a polypeptide according to any one of claims 1 to 4 characterized in   what it is an inhibitor or activator of PRAX-1, or any interaction   protein-protein involving PRAX-1.