FR2824842A1 - New nucleic acid encoding protease inhibitor from Vinca unguiculata, useful e.g. for treating and preventing inflammation - Google Patents

Abstract

Purified and isolated nucleic acid (I) which is: (i) an 857 nucleotide (nt) sequence (S1) fully defined in the specification; (ii) a fragment of (S1) of at least 15 consecutive bp; (iii) a sequence at least 80% identical with (i) or (ii) after optimal alignment; (iv) a sequence that hybridizes under stringent conditions to (i) or (ii); or (v) the complement, or corresponding RNA, of (i)-(iv), is new. Independent claims are also included for the following: (1) purified or isolated nucleic acid (Ia) encoding a polypeptide that includes a fragment of at least 50 amino acids (aa) from a 195 aa sequence (S2), fully defined in the specification; (2) polypeptide (II) that is: (i) sequence (S2); (ii) a fragment of (S2) with at least 5 aa; (iii) a biologically active fragment of (2); or (iv) a polypeptide at least 80% identical with (i)-(iii); (3) cloning and/or expression vectors that contain (I) or encode (II); (4) host cell transformed with the vector of (3); (5) non-human animal, or plant, containing a cell of (4); (6) preparation of recombinant polypeptides (IIa) by culturing cells of (4); (7) (IIa) produced by method (6); (8) poly- or mono-clonal antibodies (Ab) that bind selectively to (II) or (IIa); (9) detecting (II) or (IIa) from reaction with Ab; (10) kit for method (9); (11) DNA chip containing (I); (12) protein chip containing (II), (IIa) or Ab; (13) detecting and/or determining (I); (14) screening compounds for their ability to bind to (II) or (IIa), or to inhibit cysteine protease activity; and (15) screening for compounds that interact, in vitro or in vivo, with (I).

Description

said composition.

  The present invention relates to a new cystatin derived from Vigna unguculata (Cystavine), as well as to its applications in pharmacy, cosmetics and agronomy, in particular for plant transgenesis, for

  production of plants resistant to certain stress, including drought.

  The generic term "Cystatin" is used to designate inhibitors of cysteine proteinases. Cystatins are very potent and reversible inhibitors of proteinases of the cysteine proteinase ("papane-like") class.

  Endoproteases are characterized by the presence of a cysteine residue at the active site.

  Cysteine proteinases include papain, cathepsins B. H. L, viral proteinase A of hepatitis, cysteine proteinases (some caspases) involved in programmed cell death: apoptosis, and cysteines

  plant proteinases some of which are also involved in apoptosis.

  Their inhibitors, cystatin, are a super-family that includes several families: stefines, cystatins, kininogens, cathines (cat L)

  and phytocystatines (Turk and Bode, 1991).

  Stefines consist of a single chain whose molecular weight is about 11 kDa, they do not have disulfide bridges and are not glycosylated. We can name the stefine A and B human, cystatin a and rat

rice orycystatin I and II.

  The cystatins themselves are characterized by the presence of two C terminal disruption bridges, a size of about 1 10 - 1 15 amino acids and a molecular mass of about 13 kDa. For example, mention is made of human Cystatin C

and the egg white cystatin.

  Kininogens have a molecular weight between 68 and 120 kDa in a single protein chain. They strongly inhibit the papam and Cat L and weakly Cat B and H. Their inhibitory property of the papane make them multi-functional inhibitors. they contain several domains (often three) "cystatin-like" whose substrates differ. Their sequence has regions sensitive to proteases and in particular to aspartic proteinase (cathepsin D). The

  kininogens would come from cystatin by triplication of the gene.

  Because of their inhibitory potential for proteases, cystatins are potentially very interesting for various applications involving proteases, and especially for therapeutic applications in the field of diseases related to inflammatory phenomena. Some cosmetic applications

  are also conceivable for these proteins.

  The subject of the present invention is a novel cystatin derived from the Vigna inguiculata species, as well as the corresponding recombinant protein. This

  new cystatin appears to be a double stefine.

  The isolation of this protein was done from a model of resistance to water stress in this plant species. Obtaining a new cystatin is therefore interesting for pharmaceutical and cosmetic applications. In addition, the mode of isolation of the protein makes it possible to predict applications of this protein in plant transgenesis, in order to improve the resistance of plants to drought. Finally, cystatins can also be useful

  in the fight against viruses and insects by transgenic plants containing them.

  Thus, the subject of the present invention is a purified or isolated nucleic acid, characterized in that it comprises a nucleic sequence chosen from the following group of sequences: a) SEQ IDN 1; b) the sequence of a fragment of at least 15 consecutive nucleotides of SEQ ID N 1; c) a nucleic sequence having an identity percentage of at least 80%, after optimal alignment with a sequence defined in a) or b); d) a nucleic sequence hybridizing under conditions of high stringency with a nucleic sequence defined in a) or b); e) the complementary sequence or sequence of RNA

  corresponding to a sequence as defined in a), b), c) or d).

  The nucleic acid sequence according to the invention defined in c) has a percentage of identity of at least 80% after optimal alignment with a sequence as defined in a) or b) above, of 90% accuracy, more

  preferred 95%, most preferably 98/0, or 99%.

  By nucleic acid, nucleic or nucleic acid sequence, polynucleotide, oligonucleotide, polynucleotide sequence, nucleotide sequence, terms which will be used interchangeably in the present

  description means a precise sequence of nucleotides, modified or

  no, allowing to define a fragment or a region of a nucleic acid, with or without non-natural nucleotides, and may also correspond bi to a double-stranded DNA, a single-stranded DNA that transcripts of said DNAs. Thus, the nucleic sequences according to the invention encompass

  also PNA (Peptid Nucleic Acid), or the like.

  It should be understood that the present invention does not relate to nucleotide sequences in their natural chromosomal environment, i.e., in the natural state. These are sequences that have been isolated and / or purified, i.e. they have been taken directly or indirectly, for example by copy, their environment having been at least partially modified. We also hear

  designate the nucleic acids obtained by chemical synthesis.

  Preferably, the sequences according to the invention encode a protein or a polypeptide having an activity of inhibition of the papamus, and / or caspases, in particular caspase 3, and / or calpaine, and / or any other cysteine

vegetable proteinase.

  The fragment according to the invention contains at least 15, 25, 50, 75, 100, 150,

  , 300, 500 consecutive nucleotides of SEQ ID N 1.

  By "percent identity" between two nucleic acid or amino acid sequences within the meaning of the present invention, it is meant to designate a percentage of nocleotides or identical amino acid residues between the two sequences to be compared, obtained after the best alignment, this percentage being purely statistical and the differences between the two sequences being randomly distributed over their entire length. By "best alignment" or "optimal alignment" is meant the alignment for which the percentage of identity determined as hereinafter is the highest. Sequence comparisons between two nucleic acid or amino acid sequences are traditionally performed by comparing these sequences after optimally aligning them, said comparison being made by segment or by "comparison window" to identify and compare the regions. local sequence similarity. The optimal alignment of the sequences for comparison can be realized, besides manually, by means of the local homology algorithm of Smith and Waterman (1981), by means of the local homology algorithm of Neddleman and Wunsch (1970) ), using the Pearson-Lipman (1988) similarity search method, using computer software using these algorithms (GAP, BESTFIT, BLAST P. BLAST N. FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI). In order to obtain the optimal alignment, the BLAST program is used with

  BLOSUM 62 matrix. The PAM or PAM250 matrices can also be used.

  The percentage identity between two nucleic acid or amino acid sequences is determined by comparing these two optimally aligned sequences, the nucleic acid or amino acid sequence to be compared being able to include additions or deletions by relative to the reference sequence for optimal alignment between these two sequences. The percentage of identity is calculated by determining the number of identical positions for which the nucleotide or amino acid residue is identical between the two sequences, by dividing this number of identical positions by the total number of positions compared and multiplying the number of identical positions. result obtained by 100 to get the

  percentage of identity between these two sequences.

  By nucleic sequences having an identity percentage of at least 80%, preferably 90%, more preferably 98%, after optimal alignment with a reference sequence, is meant nucleic sequences having, compared to the sequence reference nuclei, certain modifications such as in particular a deletion, a truncation, an elongation, a chimeric fusion, and / or a substitution, in particular one-off substitution, whose nocleic sequence has at least 80%, preferably 90%, more preferably 98 %, identity after optimal alignment with the nucleic acid sequence. These are preferably sequences whose complementary sequences are capable of hybridizing specifically with the sequences SEQ ID No. 1 of the invention. Preferably, the specific or highly stringent hydridation conditions will be such as to provide at least 80%, preferably 90%, more preferably 98% identity after optimal alignment between one of the two.

  sequences and the complementary sequence of the other.

  Hybridization under high stringency conditions means that the temperature and ionic strength conditions are chosen such that they allow the maintenance of hybridization between two complementary DNA fragments. By way of illustration, conditions of high stringency of the hybridization step in order to define the polynucleotide fragments described above,

are advantageously the following.

  DNA-DNA or DNA-RNA hybridization is carried out in two steps: (1) prehyDridation at 42 ° C. for 3 hours in phosphate buffer (20 mM, pH 7.5) containing 5 × SSC (1x SSC corresponds to a solution O) 0.1M NaCl + 0.015M sodium citrate), 50% formamide, 7% sodium dodecyl sulfate (SDS), 10 x Denhardt's, 5% dextran sulfate and 1% salmon sperm DNA; (2) hybridization proper overnight at a temperature dependent on the size of the probe (ie: 42 C, for a probe size> 100 nocleotides) followed optionally by non-stringent washes and 2 stringent washes of 15 minutes to 42 C at O. 1x SSC + O.% SDS. The high stringency hybridization conditions described above for a polynucleotide of defined size may be adapted by those skilled in the art for oligonucleotides of larger or smaller size,

  according to the teaching of Sambrook et al., l989.

  By the nucleic acid sequences having an identity percentage of at least 80%, preferably 90%, more preferably 98%, or 99% after optimal alignment with the sequence according to the invention, the variant nucleic acid sequences are also preferred. of SEQ ID N 1, or of its fragments, that is to say

  all the nucleic sequences corresponding to allelic variants, that is,

  that is to say individual variations of the sequence SEQ ID No. 1. These natural sequences correspond to polymorphisms present in mammals, in particular in humans and, in particular, to polymorphisms that can lead to the occurrence of a pathology , such as cell degeneration. Preferably, the present invention relates to variant nucleic sequences in which the mutations lead to a modification of the amino acid sequence of the polypoptide, or to

  its fragments, encoded by the normal sequence of SEQ ID N 1.

  The term "variant nucleic sequence" is intended to mean any RNA or cDNA resulting from a mutation and / or variation of a splice site of the sequence

  genomic nucleic whose cDNA has the sequence SEQ ID N 1.

  The invention relates preferably to a purified or isolated nocleic acid according to the present invention, characterized in that it comprises or consists of the sequence SEQ ID No. 1, its complementary sequence or the sequence of

the RNA corresponding to SEQ ID N 1.

  The invention also relates to a purified or isolated nucleic acid characterized in that it encodes a polypeptide having a continuous moiety of at least 50, 75, more preferably 100, 150, most preferably 175 amino acids of the protein SEQ ID No. 2, and having an activity of inhibition of the papane, and / or caspases, in particular caspase 3, and / or calpane and / or

  any other vegetable cysteine proteinase.

  Preferably, the fragments of SEQ ID No. 1 which comprise the active sites located at amino acids 53 to 57 or 148 to 152 of SEQ ID No. 2 are preferably prepared. A putative site for cleavage with an aspartic proteinase has also been identified with amino acids. 91 to 94 of SEQ ID No. 2. Thus, certain polypeptides that are preferred in the present invention consist of the polypeptides 1-90, or 95-195 of SEQ ID No. 2. These two fragments indeed contain an active site of cysteine inhibition. proteinase. The existence of these two sites, and two polypeptide fragments

  in Cystavine thus allows to define this protein as a double stefine.

  The primers or probes, characterized in that they comprise a sequence of a nucleic acid according to the invention, also form part of the invention. Thus, the present invention also relates to the primers or probes according to the invention which can in particular make it possible to highlight or discriminate the variant nucleic sequences, or to identify the genomic sequence of the genes whose cDNA is represented by SEQ. ID N 1, using in particular an amplification method such as the PCR method, or a related method. The invention also relates to the use of a nucleic acid sequence according to the invention as a probe or primer, for the detection,

  identifying, assaying or amplifying nucleic acid sequence.

  According to the invention, the polynucleotides which can be used as probes or as primer in methods for detecting, identifying, assaying or amplifying nucleic sequences, have a minimum size of 15 bases,

  preferably 20 bases, or more preferably 25 to 30 bases.

  The probes and primers according to the invention may be labeled directly or indirectly with a radioactive or non-radioactive compound by methods well known to those skilled in the art, in order to obtain a detectable and / or quantifiable signal. The unlabeled polynucleotide sequences according to the invention can

  be used directly as a probe or primer.

  The sequences are generally labeled to obtain sequences that can be used for many applications. The labeling of the primers or probes according to the invention is carried out by radioactive elements or by

active non-radioactive molecules.

  Among the radioactive isotopes used, mention may be made of 32p, 33P, 35S, 3H or 25I. The non-radioactive entities are selected from ligands such as biotin, avidin, streptavidin, dioxygenine, haptens, dyes, luminescent agents such as radioluminescent, chemoluminescent agents,

  bioluminescent, fluorescent, phosphorescent.

  The polynucleotides according to the invention can thus be used as primer and / or probe in processes using, in particular, the PCR (polymerase chain amplification) technique (RolLs et al., 1991). This technique requires the choice of oligonucleotide primer pairs flanking the fragment that needs to be amplified. For example, reference can be made to the technique described in U.S. Patent No. 4,683,202. The amplified fragments can be identified, for example after agarose gel or polyacrylamide gel electrophoresis, or after a chromatographic technique such as gel filtration or ion exchange chromatography, and then sequenced. The specificity of the amplification can be controlled by using as primers the nucleotide sequences of the polynucleotides of the invention and as templates, plasmids containing these sequences or the derived amplification products. The amplified nucleotide fragments can be used as reagents in hybridization reactions in order to demonstrate the presence, in a blological sample, of a target nucleic acid of sequence complementary to that of said

  amplified nucleotide fragments.

  The invention also relates to the nucleic acids that can be obtained

  by amplification using primers according to the invention.

  Other amplification techniques for the target nucleic acid can advantageously be used as an alternative to PCR (PCR-like) using a pair of nucleotide sequence primers according to the invention. By PCR-like is meant all methods implementing direct or indirect reproductions of nucleic acid sequences, or in which the labeling systems have been amplified, these techniques are of course known. In general it is the amplification of the DNA by a polymerase; when the original sample is an RNA, it is first necessary to perform a reverse transcription. There are currently many methods for this amplification, such as for example the SDA technique (Strand Displacement Amplification) or strand displacement amplification technique (Walker et al., 1992), the TAS (Transcription-based Amplification System) technique. described by Kwoh et al. (1989), the 3SR technique (Self-Sustained Sequence Replication) described by Guatelli et al. (1990), NASBA (Nucleic Acid Sequence Based Amplification) technique described by Kievitis et al. (1991), the TMA (Transcription Mediated Amplification) technique, the LCR (Ligase Chain Reaction) technique described by Landegren et al. (1988), the Repair Chain Reaction (RCR) technique described by Segev (1992), the CPR (Cycling Probe Reaction) technique described by Duck et al. (1990), the Q-beta-replicase amplification technique described by Miele et al.

  (1983). Some of these techniques have since been perfected.

  In the case where the target polynucleotide to be detected is an mRNA, it is advantageous to use, prior to carrying out an amplification reaction with the aid of the primers according to the invention or the implementation of a method detection using the probes of the invention, a reverse transcriptase enzyme to obtain a cDNA from the mRNA contained in the biological sample. The cDNA obtained will then serve as a target for the primers or the probes

  implemented in the amplification or detection method according to the invention.

  The probe hybridization technique can be performed in a variety of ways (Matthews et al., 1988). The most general method is to immobilize the nucleic acid extracted from cells of different tissues or cells in culture on a support (such as nitrocellulose, nylon, polystyrene) and to incubate, under well defined conditions, the target nucleic acid immobilized with the probe. After hybridization, the excess probe is removed and the hybrid molecules formed are detected by the appropriate method (measurement of radioactivity, fluorescence

  or enzymatic activity related to the probe).

  According to another embodiment of the nucleic probes according to the invention, the latter can be used as capture probes. In this case, a probe, called a "capture probe", is immobilized on a support and serves to capture by specific hybridization the target nucleic acid obtained from the biological sample to be tested and the target nucleic acid is then detected by a second probe, called a "detection probe", marked by an element easily

detectable.

  Among the nucleic acid fragments of interest, the antisense oligonucleotides, that is to say whose structure ensures, by hybridization with the target sequence, an inhibition of the expression of the corresponding product, should be mentioned in particular. We must also mention the sense oligonucleotides which, by interaction with proteins involved in the regulation of the expression of the product

  corresponding, will induce either an inhibition or an activation of this expression.

  Such oligonucleotides can be used as therapeutics and drugs to regulate inflammation and tumor repression phenomena. The use of its sense or antisense sequences can also be implemented in vitro, to define new means of model and study.

  resistance to stress, especially water.

  The present invention also relates to an isolated polypeptide characterized in that it comprises a polypeptide chosen from: a) a polypeptide of sequence SEQ ID N 2; b) a fragment of at least 5 consecutive amino acids of a polypeptide defined in a); c) a biologically active fragment of a polypeptide defined in a); d) a polypeptide having at least 80% identity with the

polypoptide of a), b) or c).

  By "polypeptide" is meant, in the sense of the present invention, to designate

proteins or peptides.

  A fragment according to the invention preferably has at least 5, 7, 8, 10,

  , 20, 25, 50 consecutive amino acids of SEQ ID N 2.

  By "biologically active fragment" is meant a fragment having the same biological activity as the peptide fragment from which it is deduced, with preterence in the same order of magnitude (within a factor of 10). It preferably has at least 5, 7, 8, 10, 15, 20, 25, 50 consecutive amino acids of SEQ ID No. 2. In particular, the fragments containing active sites 53-57 and / or 148- 152 of SEQ ID N 2, as well as fragments containing amino acids 1-90

or 95-195 of SEQ ID N 2.

  Preferably, a protein or a polypeptide according to the invention has an activity of inhibition of the papane, and / or caspases, in particular caspase 3, and / or

  1 5 of calpame and / or any other plant cysteine proteinase.

  Preferably a polypeptide according to the invention is a polypeptide consisting of the sequence SEQ ID N 2 (corresponding to the protein encoded by the Cystavine gene) or of a sequence having at least 80% identity with SEQ ID N

2 after optimal alignment.

  The polypeptide sequence has an identity percentage of at least% after optimal alignment with the sequences SEQ ID No. 2 or amino acids 1-90 or 95-195 of SEQ ID No. 2, preferably 90 or 95%, more preferably 98%, or 99%, and preferably has inhibitory activity of the papamus, and / or caspase 3, and / or calpane and / or any other plant cysteine proteinase. By polypeptide whose amino acid sequence having an identity percentage of at least 80%, preferably 90%, more preferably 98%, after optimal alignment with a reference sequence, is meant to designate polypeptides presenting certain modifications with respect to the polypoptide of retention, such as in particular one or more deletions, truncations, a

  elongation, chimeric fusion, and / or one or more substitutions.

  Among the polypoptides whose amino acid sequence has an identity percentage of at least 80%, preferably 90%, more preferably 98%, after optimal alignment with the sequence SEQ ID N 2 or with one of its fragments according to the invention, the variant polypeptides encoded by the variant nucleic sequences as defined above, in particular the polypeptides whose amino acid sequence has at least one mutation corresponding in particular to a truncation, deletion, substitution and or addition of at least one amino acid residue with respect to the sequence SEQ ID No. 2 or with one

  of his fragments. The invention also includes glycosylated polypeptides.

  The present invention also relates to cloning and / or expression vectors comprising a nucleic acid or coding for a polypeptide according to the invention. Such a vector may also contain the elements necessary for

  expression and optionally secretion of the polypeptide in a host cell.

  Such a host cell is also an object of the invention. Such a vector can be

  replicative or intogrative in the host cell.

  The vectors characterized in that they comprise a promoter and / or regulator sequence according to the invention also form part of the invention. Said vectors preferably include a promoter, translation initiation and termination signals, as well as suitable transcriptional regulatory regions. They must be able to be stably maintained in the cell and may possibly have particular signals

  specifying the location and / or secretion of the translated protein.

  These different control signals are chosen according to the cellular host used. For this purpose, the nucleic acid sequences according to the invention can be inserted into autonomously replicating vectors within the host.

  chosen, or integrative vectors of the chosen host.

  Among the systems with autonomous replication, preterence is used according to the host cell, plasmid or viral type systems, the viral vectors possibly being adenoviruses (Perricaudet et al., 1992), retroviruses, lentiviruses, poxvirus or herpesviruses (Epstein et al., 1992). The person skilled in the art knows the technologies that can be used for each of these systems. When it is desired to integrate the sequence into the chromosomes of the host cell, it is possible to use, for example, plasmid or viral type systems; such viruses are, for example, retroviruses (Temin, 1986), or AAVs

(Carter, 1993).

  Among the non-viral vectors, naked polynucleotides such as naked DNA or bare RNA according to the technique developed by the company VICAL, bacterial artificial chromosomes (BACs), artificial chromosomes of yeast (BAC) are preferred ( YAC, yeast artificial chromosome) for expression in yeast, artificial chromosome (MAC), for expression in murine cells and, preferably, artificial chromosomes of humans (HAC, human artificial

  chromo some) for expression in human cells.

  Such vectors are prepared according to the methods commonly used by those skilled in the art, and the resulting clones can be introduced into a suitable host by standard methods, such as, for example, lipofection, electroporation, heat shock. , the transformation after permeabilization

  chemical membrane, cell fusion. The invention furthermore comprises the host cells, in particular the cells

  encaryotes and prokaryotes, transformed by the vectors according to the invention as well as transgenic plants and animals, preferably mammals, except humans, comprising one of said transformed cells according to the invention. These animals can be used as models, for the study of the etiology of

  inflammatory and / or immune diseases, or for the study of cancers.

  Among the cells that can be used within the meaning of the present invention, mention may be made of bacterial cells (Olins and Lee, 1993), but also yeast cells (Buckholz, 1993), as well as animal cells, in particular cell cultures. mammals (Edwards and Aruffo, 1993), and in particular Chinese hamster ovary (CHO) cells. Insect cells may also be mentioned in which processes using, for example, baculoviruses may be used (Luckow, 1993). Plant cells, in particular monocotyledonous or dycotyledonous plant cells (angiosperms) or gymnosperms, are also preferably mentioned. These include tobacco cells, bananas, rice, alfalfa, alfalfa, cassava, wheat, cotton, soybeans, beans, cabbage, tomatoes, maize, potatoes, beetroot ... It is however more interesting to produce the protein. from the transgenic complete plant regenerated from these plant cells. A plant cell of interest is also a cell of seaweed, including microscopic algae. Among the mammals according to the invention, animals such as rodents, in the form of mice, rats or rabbits, are described which express a polypeptide.

according to the invention.

  Among the mammals according to the invention, animals such as mice, rats or rabbits are also claimed, characterized in that the gene encoding the protein of sequence SEQ ID No. 2, or whose sequence is encoded by the gene. in these animals, is not functional, is invalidated or

least one mutation.

  These transgenic animals are obtained, for example, by homologous recombination on embryonic stem cells, transfer of these stem cells to embryos, selection of the affected chimeras at the level of the lines.

  reproducers, and growth of said chimeras.

  The transgenic animals according to the invention can thus overexpress the gene coding for the protein according to the invention, or express said gene in which a mutation is introduced. These transgenic animals can also overexpress, under-express the intrinsic homologous gene corresponding to Cystavine. It is even possible that this homologous gene is silenced by a mutation in

  the genome of these transgenic animals.

  These transgenic animals, in particular mice, are obtained for example by transfection of a copy of this gene under the control of a strong promoter of

  ubiquitous or selective nature of a tissue type, or after viral transcription e.

  Alternatively, the transgenic animals according to the invention can be rendered deficient for the gene coding for the polypeptide of the sequence SEQ ID No. 2, or its homologous gene, by inactivation using the LOXP / CRE recombinase system (Rohlmann et al. , 1996) or any other inactivation system of

the expression of this gene.

  The invention also relates to a plant, characterized in that it contains one (or more) nucleotide sequence (s) recombinant (s) according to the invention, integrated (s) stably in its genome, said plant selected from rapeseed, tobacco, maize, peas, tomatoes, carrots, wheat, barley, potatoes, soya beans, sunflowers, lettuce, rice, alfalfa, beet, vine, cotton, cabbage, or any other gymnosperm or mono or dicotyledonous angiosperm. The invention also relates to a plant extract or part of a plant, in particular leaves and / or fruits and / or seeds and / or plant cells, genetically transformed, derived from said plant according to the invention. Preferably, said nucleotide sequence according to the invention is integrated in a

  stable in the genome of said plant, at a locus that is not the natural locus.

  Advantageously, the recombinant nucleotide sequences according to the invention contain one (or more) sequence (s) coding for a peptide responsible for addressing the recombinant polypeptides in a specific compartment of the plant cell, in particular in the endoplasmic reticulum, the apparatus Golgi, mitochondria, chloroplasts or in storage vacuoles or vacuol es lysosomal es, or bi in same outside the cell, d years

  the pectocellulosic wall or in the extracellular space also called apoplasm.

  Among the transcription terminators that can be used for the transformation of plant cells in the context of the present invention, mention may be made of the polyA 35S terminator of the moss virus (CaMV), or the polyA NOS terminator. , which corresponds to the 3 'non-coding region of the gene of the

  nopaline synthase of the Ti plasmid of Agrobacterium tumetaciens nopaline strain.

  Among the transcription promoters that can be used for the transformation of plant cells in the context of the present invention, mention may be made of the 35S promoter (P35S), or advantageously the double constitutive 35S promoter (Pd35S) of CaMV, these promoters allowing the expression of the recombinant polypeptides of the invention in the whole of the plast obtained from transformed cells according to the invention, and are described in the article by Kay et al., (1987, Science, 236, 1299-1302), the PCRU promoter of the radiot cruciferin gene permitting the expression of the recombinant polypeptides of the invention only in the seeds (or seeds) of the plant obtained from cells transformed according to the invention, and described in Depigny-This et al., (1992, Plant Mol Mol., 20, 467-479), the PGEAI and PGEA6 promoters corresponding to the 5 'non-coding region of the seed bank, GEAI and GEAI, respectively, of Arabidopsis thaliana (Gaubier et al., 1993, Mol. Gen. Genet., 238, 409-418), and allowing a specific expression in the seeds, the super promoter PSP (Ni M et al., 1995), consisting of the fusion of a triple repetition of an element transcriptional activator of the Agrobacterium tumefaciens octopine synthase gene promoter, a transcriptional activator element of the mannopine synthase gene promoter and the Agrobacterium tumefaciens mannopine synthase promoter, the rice actin promoter followed by the intron rice actin (PAR-IAR) contained in plasmid pAct1-F4 described by McElroy et al. (1991, Mol Gen Genet, 231, 150-160), the HMGW promoter (High Molecular Weight Glutenine) of barley (Anderson et al., 1989, TAG, 77, 689-700), the promoter of the zein gene of but (pyzein) contained in the plasmid py63 described in Reina et al. (1990, Nucleic Acid Rescarch, 18, 6426), and allowing the expression in the albumen of corn seeds. The skilled person also knows the promoters allowing the expression

  Specifies genes in the leaves or roots of the plant.

  The sequences coding for an addressing peptide used in the context of the present invention may be of plant, human or animal origin, and there may be mentioned: the nucleotide sequence of 69 nucleotides coding for the prepeptide (signal peptide) of 23 amino acid of sporamin A in sweet potato, this signal peptide allowing the entry of the recombinant polypeptides of the invention into the secretory system of the transformed plant cells according to the invention (namely mainly in the endoplasmic reticulum), the sequence 42-nucleotide nucleotide coding for the N-terminal 14 amino acid vacuolation address propeptid of sporamin A in sweet potato, allowing the accumulation of the recombinant polypeptides of the invention in the vacuoles of transformed plant cells according to the invention, or the nucleotide sequence of 111 nucleotides encoding the 37 amino acid prepropeptide of sporamin A consisting of the N-terminal portion to the C-terminal portion of the 23 amino acids of the aforementioned signal poptide followed by the 14 amino acids of the aforementioned propeptide, this prepropeptide allowing the entry of recombinant polypeptides of the invention into the secretion system and their accumulation in the vacuoles of the transformed plant cells according to the invention (Murakami et al., 1986, Plant Mol.

  Biol., 7, 343-355 and Matsuoka et al, 1991 Proc. Natl. Acad. Sci. USA, 88, 834-838).

  It is also possible to mention the carboxyterminal propeptide of the barley lectin described in particular in the articles by Schroeder et al., 1993 (Plant Physiol., 101, 451 458), and Bednarek et al., 1991 (Plant Cell, 3, 11951206). ), and the PRS (Pathogenesis Related Protein, Cornelissen et al., 1986) allowing the secretion, or the sequences coding for the peptides KDEL, SEKDEL and HDEL and allowing addressing

in the endoplasmic reticulum.

  The cells, plants and mammals according to the invention can be used in a method for producing a polypeptide according to the invention, as described herein.

  below, and can also be used as an analytical model.

  The transformed cells, plants or mammals as described above can also be used as models to study the interactions between the polypeptides according to the invention, and the chemical or protic compounds, directly or indirectly involved in the activities of the cells. polypeptides according to the invention, in order to study the various mechanisms and

interactions involved.

  They may in particular be used for the selection of products interacting with the polypeptides according to the invention, in particular the protein of sequence SEQ ID No. 2 or its variants according to the invention, as a cofactor, or an inhibitor, in particular a competitive inhibitor, or having agonist activity or

  antagonist of the activity of the polypeptides according to the invention.

  The invention also relates to the use of a cell, a plant, a mammal or a polypoptide according to the invention for the screening of chemical or biochemical compounds that can interact directly or indirectly with the polypeptides according to the invention. invention, and / or capable of modulating

  the expression or activity of these polypeptides.

  Thus, the invention relates to a method for screening compounds capable of binding to a polypoptide of sequence SEQ ID No. 2, characterized in that it comprises the steps of placing a polypeptide, a cell, in contact with each other. , a mammal, a plant according to the invention, with a candidate compound and for detecting the formation of a complex between said candidate compound and said polypeptide. The invention also relates to screening methods allowing the identification of compounds that decrease or inhibit the ability of a polypeptide according to the invention to inhibit the activities of cytein proteinase, and in particular of calpain, caspases (in particular caspase 3 ) or papalne, in which an experimental protocol is used to demonstrate the aforementioned inhibitory activity, and the said activity is examined in the presence

or in the absence of the test compound.

  In the same way, the invention also relates to a method for screening compounds capable of interacting in vitro or in vivo with a nucleic acid according to the invention, by using a nucleic acid, a cell, a plant or a mammal according to the invention. invention, and by detecting the formation of a complex between

  candidate compounds and noceleic acid according to the invention.

  The compounds thus selected are also objects of the invention.

  Such a compound according to the invention may be a compound having a chemical structure (of the small organic molecule type), a lipid, a sugar, a protein, a peptide, a hybrid protein-lipid, protein-sugar, poptide-lipid compound, or peptide-sugar, a protein or peptide to which branches have been added

chemical.

  Among the chemical compounds envisaged, they may contain one or more rings, aromatic or not, as well as several residues of any kind

  (in particular lower alkyl, that is to say having between 1 to 6 carbon atoms).

  The invention also relates to the use of a compound interacting with a polypeptide or a nucleic acid according to the invention, for the preparation of a medicament intended in particular for the treatment of cancer or of a disease

  inflammatory, characterized in that it is identified by a method according to the invention.

  The invention also relates to the use of a compound which decreases or inhibits the ability of Cystavine to inhibit cytein proteinase activities, and in particular calpane, caspases (in particular caspase 3) or the papam, for the preparation of a medicament intended in particular for the treatment of cancer or of an inflammatory disease, characterized in that it is identified by a

process according to the invention.

  The contemplated drugs can also be used for the treatment of other

  diseases, such as those mentioned below.

  The compounds according to the invention can also be used for the preparation of

compositions for cosmetic use.

  The invention also relates to the use of a nucleic acid sequence

  according to the invention for the synthesis of recombinant polypeptides.

  The method for producing a polypeptide of the invention in recombinant form, itself included in the present invention, is characterized in that the transformed cells, in particular the cells or plants of the present invention, are cultured in conditions allowing the expression of a recombinant polypoptide encoded by a nucleic acid sequence according to the invention, and that said recombinant polypeptide is recovered. A most preferred method is

  in the use of E. coli for the production of the recombinant protein.

  The recombinant polypeptides, characterized in that they are capable of being obtained by said production method, also form part of the invention. The recombinant polypeptides obtained as indicated above, can be both in glycosylated and non-glycosylated form and can

  present or not the natural tertiary structure.

  The sequences of the recombinant polypeptides can also be

  modified to improve their solubility, especially in aqueous solvents.

  Such modifications are known to those skilled in the art, for example the deletion of hydrophobic domains or the substitution of amino acids.

  hydrophobic by hydrophilic amino acids.

  These polypeptides can be produced from the above-described nocinoic acid sequences according to recombinant polypeptide production techniques known to those skilled in the art. In this case, the nucleic acid sequence used is placed under the control of signals allowing its expression

in a cellular host.

  An efficient system for producing a recombinant polypeptide requires

  to have a vector and a host cell according to the invention.

  These cells can be obtained by introducing into a host cell a nucleotide sequence inserted into a vector as defined above, and then culturing said cells under conditions allowing replication.

  and / or the expression of the transected nucleotide sequence.

  The methods used for the purification of a recombinant polypeptide are known to those skilled in the art. The recombinant polypeptide can be purified from lysates and cell extracts, from the supernatant of the culture medium, by methods used individually or in combination, such as fractionation, chromatography methods, immunoaffinity techniques using Monoclonal or specific polyclonal antibodies, His-Tag, etc. The polypeptides according to the present invention can also be obtained by chemical synthesis using one of the many known peptide syntheses, for example techniques using solid phases (see in particular Stewart et al., 1984) or techniques using partial solid phases, for example

  condensation of fragments or by synthesis in conventional solution.

  Polypeptides obtained by chemical synthesis and which may comprise

  corresponding non-natural amino acids are also included in the invention.

  The mono- or polyclonal antibodies or their fragments, chimeric or immunoconjugated antibodies, characterized in that they are capable of specifically recognizing a polypeptide according to the invention, form part of the invention. Specific polyclonal antibodies can be obtained from a serum of an animal immunized against the polypeptides according to the invention, in particular produced by genetic recombination or by peptide synthesis, according to the modes

usual procedures.

  In particular, we note the interest of specifically recognizing antibodies.

  certain polypeptides, variants, or their immunogenic fragments, according to the invention.

  The mono- or polyclonal anti-bodies or their chimeric or immunoconjugated anti-body fragments, characterized in that they are capable of specifically recognizing the polypeptide of sequence SEQ ID N 2 are

particularly prerequisites.

  Specific monoclonal antibodies can be obtained according to the

  conventional method of hybridoma culture described by Kohler and Milstein (1975).

  The antibodies according to the invention are, for example, chimeric antibodies, humanized antibodies, Fab or F (ab ') 2 fragments. They can also be in the form of immunoconjugates or labeled antibodies so

  to obtain a detectable and / or quantifiable signal.

  The invention also relates to methods for detecting and / or purifying a polypeptide according to the invention, characterized in that they use an antibody according to the invention. The invention further comprises purified polypeptides, characterized in that

  that they are obtained by a method according to the invention.

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

  biological (especially for ELISA or similar tests).

  They thus constitute a means of immunocytochemical or immunohistochemical analysis of the expression of the polypeptides according to the invention, in particular the polypeptide of sequence SEQ ID No. 2 or one of its variants, for example by

  immunofluorescence, gold labeling, enzymatic immunoconjugates.

  They can, in particular, make it possible to highlight an expression

  abnormal of these polypeptides in tissues or biological samples.

  More generally, the antibodies of the invention can be advantageously used in any situation where the expression of a

  polypeptide according to the invention, normal or mutated, must be observed.

  The antibodies can be obtained directly from human serum, or from animals immunized with polypeptides according to the invention, and then

"humanized".

  Thus, a method for detecting a polypeptide according to the invention in a biological sample, comprising the steps of bringing the biological sample into contact with an antibody according to the invention and of demonstrating the antigen-antibody complex formed is also an object of the invention, as well as a kit permitting to implement such a method. Such a kit contains in particular: a) a monoclonal or polyclonal antibody according to the invention; b) optionally reagents for the constitution of a medium conducive to the immunological reaction; c) reagents for the detection of the antigenic complex

  antibody produced during the immunological reaction.

  The polypeptides or antibodies according to the invention can be used in the treatment of inflammatory diseases, cancers, or for combating the effects of aging in a mammal, in particular in humans. Glomerular proteinuria, bone resorption, metabolism of metastases, tumor invasion, myocardial infarction (including tissue damage), Duchenne's disease, inflammatory disease or inflammatory phenomena ( for example, ankylosing spondylitis, psoriatic arthritis, rheumatoid arthritis, granulomatous inflammatory disease, such as Blau Syndrome or Crohn's disease), muscular dystrophy, Alzeimer's disease, sclerosis

  multiple, viral diseases (including ocular), degenerative diseases.

  The polypoptides or the antibodies according to the invention can be used as such or in the preparation of a medicament for the treatment of the aforementioned diseases. The analysis can be performed by sequencing all or part of the gene, or by other methods known to those skilled in the art. In particular, it is possible to use methods based on PCR, for example PCR-SSCP which makes it possible to detect

punctual mutations.

  The invention also relates to a DNA chip containing a sequence according to the invention. Such a chip can be used to perform the analysis of the presence and / or expression of the nocleic acid according to the invention by fixing a probe according to the invention corresponding to one of the SEQ ID sequences.

  N 1 on a DNA chip and hybridization on these microplates.

  Similarly, a protein chip containing an amino acid sequence according to the invention is also an object of the invention. Such a protein chip enables the study of the interactions between the polypeptides according to the invention and other proteins or chemical compounds, and can thus be useful for the screening of compounds interacting with the polypeptides according to the invention. The protein chips according to the invention can also be used to detect the presence of antibodies directed against the polypetides according to the invention in the serum of patients. Can also

  implement a protein chip containing an antibody according to the invention.

  The invention also relates to the use of plants, plant extracts or parts of plants according to the invention, and / or of polypeptides according to the invention, for the preparation of pharmaceutical, medical, odontological, cosmetic or biotechnological compositions, as well as a pharmaceutical, medical, odontological, cosmetic or biotechnological composition, characterized in that it comprises plants, plant extracts, plant parts or polypeptides according to the invention The invention also relates to a detection method and / or or assaying and / or isolating a nucleic acid according to the invention in a biological sample, comprising the following steps of contacting a probe according to the invention with a biological sample and detecting and / or assaying and / or isolating the hybrid formed between said polynucleotide and the nucleic acid of

the biological sample.

  The agent capable of specifically detecting a nucleic acid encoding the Cystavine protein is advantageously an oligonucleotide probe according to the invention, which may be formed of DNA, RNA or PNA, modified or otherwise. The modifications may include radioactive or fluorescent labeling, or be due to modifications in the bonds between the bases (phosphorothioates, or methylphosphonates, for example). The person skilled in the art knows the protocols

  to isolate a specific DNA sequence.

  Those skilled in the art can implement such a method, and can in particular use a reagent kit comprising: a) a polynucleotide according to the invention, used as a probe; b) reagents necessary for carrying out a hybridization reaction between said probe and the nucleic acid of the biological sample; c) the reagents necessary for detecting and / or assaying the hybrid formed between said probe and the nucleic acid of the biological sample;

  which is also an object of the invention.

  Such a kit may also contain positive or negative controls

  to ensure the quality of the results obtained.

  However, in order to detect and / or dose and / or isolate a nucleic acid according to the invention, one skilled in the art can also carry out an amplification step

  using primers chosen from the sequences according to the invention.

  The invention also relates to a compound characterized in that it is ..chos parm a) a nucleic acid according to the invention; b) a polypeptide according to the invention; c) a vector according to the invention; d) a cell according to the invention; e) an antibody according to the invention; f) a compound according to the invention, as a medicament, as well as one of these compounds for the prevention and / or treatment of an inflammatory disease, a cancer, or for combating

  the effects of aging in a mammal, especially in humans.

  The subject of the invention is also the use of a nucleic acid according to the invention for the production of a transgenic plant having improved properties of resistance to ablotic stress, in particular water stress, and to biotic stresses such as those induced by viruses and insects, compared to the non-transgenic plant. The nucleic acid according to the invention can also be used to produce transgenic plants which are more resistant to freezing, or to extreme conditions of salinity. The nucleic acids according to the invention may also be

  used to fight cell senescence.

  The nucleic acid according to the invention can be introduced into the plant either via a replicative vector in the cells or via an integrative vector in the genome of the cell. The transgenic plates are as described above. Preferably, the cystatin according to the invention will

  expressed in the leaves of the plant thus transformed.

  Indeed, the work of the present invention indicate for the first time that the gene according to the invention is strongly involved in the adaptive response of plants to drought: the resistant plants stimulate the expression of the gene encoding cystatin Cystavine which n is not the case of sensitive plants. There is indeed a correlation between the regulation of the gene and the membrane resistance and the

resistance to the fields of plants.

* Cystavine is therefore involved in the cellular protection during stress via the modulation of the activity of foliar cysteine proteinases. Consequently, a transgenesis strategy aimed at inducing or overexpressing the transgene according to the invention via a stress-inducible and leaf-specific promoter can lead to obtaining plants that are more resistant to drought-induced water stress, high temperatures, gel, salinity, cellular senescence,

  insect and / or virus resistance.

  Studies reported by the present invention show that Cystavine inhibits caspase activity. Members of the caspase family are cysteine proteases that have specific cleavage of aspartate. They play a significant role in both inflammation (Leung et al., 2000, J. Medicinal Chem., 43: 305-341) and apoptosis. Caspase inhibitors are very important tools because they inhibit the induction of apoptosis in various tumor cells (Schlogel et al., 1996, J. Biol Chem 271: 1841) and in the cells.

  normal (Zaks et al., 1999, J Immunol 162: 3273-9).

EXAMPLES

  Example 1 Characterization and Cloning of the cDNA Encoding a Novel Putative Cstatin The cDNA was characterized after screening of an expression library of

  Cowpea bean leaf (Vigna unguiculata Vu, Cowpea).

  The DNA probe required for the screening was obtained by PCR (Polymerase Chain Reaction) amplification using dogenere oligonucleotide primers deduced from two consensus sequences determined by alignment of three published plant cystatin sequences (BLAST) via Infobiogen (SEQ ID N 3

and SEQ ID N 4).

  The cDNA was prepared from 2 mg of Vu leaf mRNA (Amersham kit) and then purified (phenol, chloroform, Oncor Appligen). Amplification of the probe was performed by PCR using purified cDNA as template, degenerate primers, Goldstar Red DNA polymerase (Eurogenetec), corresponding reaction buffer (Eurogenetec), dNTPs (5mM, Master Mix, Eurogentec). The amplification of the DNA was carried out by means of a thermocycler

(Mastercycler 5330, Eppendorf).

  The amplified DNAs were separated by electrophoresis (Mupid, Eurogentec)

on 1% agarose gel (Promega).

  A 160 bp probe was amplified, excised from the gel and then purified (QuiaexII, Quiagen). The DNA was cloned into phagemid pBluscriptIISK (Stratagene). Competent bacteria (Epicurian coli XL1 blue, Stratagene) were transformed with the recombinant plasmids. Twelve clones were selected on the basis of the size of the insert and then depending on the efficiency of their PCR amplification. The plasmid corresponding to the selected clone was purified (Qula Prep Spin kit

  Miniprep, Quiagen) and the insert was sequenced.

  The sequence obtained proved to be characteristic of a portion of plant cystatin. After excision of the plasmid insert, the probe is thus available to screen a Vu leaf cDNA library made in the

  phagemid Zip Lox (Gibco BRL), in the laboratory.

  To do this, the probe was labeled with 32p (Megaprime Kit, Amersham), purified by chromatography (Nick columns, Pharmacia), incubated with the fingerprints which were autoradiographed (OMAT X film, Kodak). Screening of the library was able to select a clone from which (lysis sequence) the recombinant phage DNA was amplified and introduced into E. coli DH 1 OB (Gibco BRL) bacteria capable of recombined state to transform

  (circularization) phages in plasmids.

  The recombinant plasmid DNA was extracted from the bacteria and purified (kit

  Wizard Plus Midiprep, Quiagen) then sequenced (ESGS).

  The nucleotide sequence obtained is that of a new cystatin (SEQ ID No. 1). Under these conditions it was decided to try to obtain the protein in vitro by

  expression in a heterologous system.

  Amino acid sequence of Cystavine (S1EQ ID N 2).

  MATATVTLGGITDVPGAANSVEIANLARFAVDDHNKK

  QNGVLEFVRVISAKQQVVSGILYYITLEAKDGETKKV

  YKTKVWVREWLNPKEVQEFNLVTDSAIGTKDGGVGD

  VP SDTLHIENLARFAVDQYNKNENANLEFVRVIDAKE

  QV VEGFIYYITLEAKDGESKNVYEAKVWERSWLNSIE

L L F K P V D V A V

  Example 2 Expression of Cystatin in E. coli 2.1-PCR Construction

  E coli was chosen because of the efficiency of its expression system.

  The cystatin cDNA was prepared by PCR so as to eliminate the starting codon of the ORF and to insert on both sides two restriction sites, BamHI and SalI, allowing it to be inserted into the plasmidial vector. PQE30 (QIA Expressionist, Quiagen) contains the same restriction sites, the ATG of initiation

  of translation and coding for 6 Histidines of His-tag.

  The vector is prepared (cleavage by SalI and BamHI, dephosphorylation by CIAP (Calf Intestinal Alkaline Phosphatase, Promega and purification) and the ligation is carried out (T4 DNA ligase, USB) The recombinant plasmid obtained is subcloned in order to preserve the construction in E coli (X11Blue (Subcloning-Grade

competent Cells, Stratagene).

  2.2 Expression of cystatin The recombinant vector pQE30 is used (after extraction of the bacterium Xll Blue, purification and assay) to transform E. coli M15 bacteria (QIA expressionist kit, Quiagen). The bacteria are cultured in liquid medium (LB medium, ampicillin) and the induction of the expression of cystatin is initiated by adding IPTG (lmM). The bacteria are then collected by centrifugation (JE-21ME, Bekman), the proteins are extracted and analyzed by automatic electrophoresis on acrylamide gel under denaturing conditions (SDS-PAGE, Phast system, Pharmacia). A majority kes band was obtained, including the molecular mass

  corresponds to the deduced mass of the putative cystatin cDNA sequence.

  EXAMPLE 3 Role of the VuCystl Gene and Activity of the Cystatin Cystavine 3.1Expression (transcripts) of the YuCystl Enum The study of the expression of the gene by Northern blotting or RT-PCR (reverse transcriptase polymerase chain reaction) was carried out in the case of plants whose sensitivity to drought (experimental or natural) differs. 4 batches of plants were studied: well watered control plants (T), plants subjected to a controlled water deficit. The results obtained show that the gene is not expressed in control plots, whereas transcripts accumulate as a function of stress intensity. This reaction is. in addition, correlated with the degree of susceptibility of the plant to drought in the field. Thus the VuCystl gene (coding for Cystavine) is expressed in response to the water constraint, all the more since it is severe and all the more so since the plant is sensitive. It therefore appears that the VuCystl cDNA is a transgene. candidate for transgenic plant breeding with a strategy for drought resistance. Knowing that most other ablotic stress: gel, salinity, floading, high temperatures induce cellular stress on the cell scale, VuCystl is a candidate transgene for the improvement of plants for resistance.

to these stresses too.

  3.2- Activity of cystatin Cvstavine The activity of the recombinant protein Cystavine was tested in three

  substrates: the papamus, the caspase, the calpame.

  Cysinvin, Inl: 'ibiteur de la papane: The principle of the activity test is as follows: this anti-cysteine proteinase must block the hydrolysis reaction which occurs between a cysteine proteinase

  plant, papaya (Sigma) and its substrate azocasein.

  The reaction medium is as follows: Tris buffer X5 200 μl (0.5 M Tris, 25 mM Bercercaptoethanol, pH 6.0), 1% azocasein (Sigma), papain 250 ng per liter (Sigma), purified VuCYST1 1.17 μl / lll, H2O qsp 5001. The reaction is developed at 37 ° C. for 15 min and then the proteins are precipitated by treatment with 10% trichloroacetic acid for 30 min at 5 ° C. The determination of the amino acids released

  is carried out spectrophotometrically by measuring the absorbance (A) at 340.

  The results obtained are as follows: Tests (3 independent experiments, SD <5%) (A) 340nm Without enzyme + azocasein (substrate) 0.338 Cystavine + azocasein 0.336 Papame (cysteine proteinase) + azocasein 0.554 Papaine + Cystavine + azocasein (1) 0.343 Papain + Cystavine + azocasein (21l) 0.325 The results obtained clearly show that cysteine proteinase (papain) hydrolyzes azocasein (substrate), the release of amino acids leads to the isolation of the chromogenic group and therefore at the increase of the absortance A for \ 340nm. On the other hand, this phenomenon is totally inhibited in the presence of the inhibitor Cystavine. In conclusion, the cystatin of Yigna unguiculata Cystavine produced by

  Expression of cDNA in E. colz is well functional.

  Cystavine inhibitor of caspase 3 The test was carried out using the kit "Caspase-3 Assay Kit colorimetric

  (Sigma). The substrate used is "Ac-DEVD-pNa" (acetyl-Asp-Glu-Val-Asp o-

  pnitrosanilide). Cystavine is used at 24 mg / ml.

  Trials (3 independent experiments SD <5%) (A) n405 nm Caspase-3 + AcDEVD-pNa (substrate) 0.060 Caspase-3 + Cystavine 20 + Ac-DEVD-pNa (substrate) 0.052 Caspase-3 + Cystavine 40 + Ac -DEVD-pNa (substrate) 0.043 Caspase-3 + Cystavine 50 + Ac-DEVD-pNa (substrate) + 0.025 preincubation Caspase-3 / Cystavine 15 min

  These results show that Cystavine is a caspase inhibitor.

  The effectiveness of the inhibition depends on the concentration of cystatin and a pre-

  incubation between the cysteine proteinase (Caspase) and the inhibitor before the addition of the substrate of the enzyme. An adjustment is in progress to determine the parameters of the inhibition in order to optimize the Cystavine irhilireur test of the calparne Preliminary kinetics of enzymatic inhibition kinetics were carried out using a test adapted to animal cystatins. Cystavine inhibition of 40% calpame activity was achieved without any optimization of the parameters. Under these conditions it is likely that the optimization of the parameters will increase the effectiveness of the inhibition of calpam by Cystavine.

Claims (27)

claims   Purified or isolated nucleic acid, characterized in that it comprises a nucleic sequence chosen from the following group of sequences: a) SEQIDN 1; b) the sequence of a fragment of at least 15 consecutive nucleotides of SEQ ID No. 1; c) a nucleic sequence having an identity percentage of at least 80%, after optimal alignment with a sequence defined in a) or b); d) a nucleic sequence hybridizing under conditions of high stringency with a nucleic sequence defined in a) or b); e) the complementary sequence or the RNA sequence   corresponds to a sequence as defined in a), b), c) or d).   2. Purified or isolated nucleic acid according to claim 1, characterized in that it comprises or consists of a sequence selected from the group consisting of SEQ ID No. 1, the sequence complementary to SEQ ID No. 1 and the sequence RNA corresponding to SEQ ID N l.   Purified or isolated nucleic acid characterized in that it encodes a polypeptide having a continuous fragment of at least 50 amino acids of the protein SEQ ID N 2.   4. Isolated polypeptide characterized in that it comprises a polypeptide chosen from: a) a polypeptide of sequence SEQ ID N 2; b) a fragrnent of at least 5 consecutive amino acids of a polypeptide defined in a); c) a biologically active fragrnent of a polypeptide defined in a); d) a polypeptide having at least 80% identity with the polypeptide of a), b) or c).   5. Cloning and / or expression vector comprising a nocleic acid   according to one of claims 1 to 3 or coding for a polypeptide according to claim 4.   56. Host cell characterized in that it is transformed by a vector according to claim 5.   7. Animal, except man, characterized in that it comprises a cell according to claim 6.   Plant, characterized in that it comprises a cell according to claim 6.   9. Use of a nucleic acid sequence according to one of   claims 1 to 3 as probe or primer, for detection and / or   amplification of nucleic acid sequences.   10. In vitro use of a nucleic acid according to one of claims 1   to 3 as sense or antisense oligonucleotide.   11. Use of a nucleic acid sequence according to one of   Claims 1 to 3 for the production of a recombinant polypeptide.   12. Process for obtaining a recombinant polypeptide, characterized in that a cell according to claim 6 is cultivated under conditions allowing   expressing said polypeptide and recovering said recombinant polypeptide.   13.The recombinant polypeptide characterized in that it is obtained by a   process according to claim 12.   14. Monoclonal or polyclonal antibody characterized in that it binds   selectively a polypoptide according to one of claims 4 or 13.   15. Method for detecting a polypeptide according to one of claims 4,   or 13, characterized in that it comprises the following steps: a) contacting a biological sample with an antibody according to claim 14; b) demonstration of the antigen-antibody complex formed. 16. A kit of reagents for carrying out a process according to claim 15, characterized in that it comprises: a) a monoclonal or polyclonal antibody according to claim 14; b) optionally reagents for the constitution of a medium conducive to the immunological reaction; c) reagents for the detection of the antigenic complex   antibody produced during the immunological reaction.   17. DNA chip characterized in that it contains a nucleic sequence   according to one of claims 1 to 3.   18. A protein chip characterized in that it contains a polypeptide according to   one of claims 4 or 13, or an antibody according to claim 14.   19. A method for detecting and / or assaying a nucleic acid according to one of   Claims 1 to 3 in a biological sample, characterized in that   comprises the following steps: a) contacting a polynucleotide according to one of claims 1 to 3, marked;   b) detecting and / or assaying the hybrid formed between said   polynucleotide and the nucleic acid of the biological sample.   20. A method for detecting and / or assaying a nucleic acid according to one of   claims I to 3 in a biological sample, characterized in that   comprises a step of amplifying the nucleic acids of said biological sample with the aid of primers chosen from nucleic acids according to one of the Claims 1 to 2.   21. Method for screening compounds capable of binding to a polypoptide   according to one of claims 4 or 13, characterized in that it comprises the steps   for bringing into contact a polypeptide according to one of claims 4 or 13, of a   A cell according to claim 6, an animal according to claim 7, a plant according to claim 8, with a candidate compound and detection of the formation   a complex between said candidate compound and said polypeptide.   22. Method for screening compounds capable of interacting in vitro or in vitro   with a nucleic acid according to one of claims 1 to 3, characterized in that   comprises the steps of contacting a nucleic acid according to one of   1 to 3, of a cell according to claim 6, of an animal according to   claim 7, a plant according to claim 8, with a candidate compound and for detecting the formation of a complex between said candidate compound and said nucleic acid.   23. A method of screening compounds that decrease or inhibit the ability of a   polypeptide according to one of claims 4 or 13 to inhibit cyteine activities   proteinase, comprising the step of examining said activity of inhibiting activity   cysteine proteinase of said polypeptide in the presence or absence of the test compound.   24. Use of a compound interacting with a polypeptide according to one of   claims 4 or 13 for the preparation of a medicament intended in particular   treatment of cancer or inflammatory disease, characterized in that it is   identified by a process according to claim 21.   25. Use of an interacting compound in vitro or in vivo with an acid   nucleic acid according to one of claims 1 to 3 for the preparation of a medicament   intended in particular for the treatment of cancer or of an inflammatory disease,   characterized in that it is identified by a method according to claim 22.   26. Use of a compound decreasing or inhibiting the efficacy of a   polypeptide according to one of claims 4 or 13 to inhibit cyteine activities   proteinase, for the preparation of a medicament intended in particular for the treatment of the aneer or of an inflammatory disease, characterized in that it is identified by a process according to claim 23. 27. A compound characterized in that it is chosen from   a) a nucleic acid according to one of claims 1 to 3;   b) a polypeptide according to one of claims 4 or 13,   c) a vector according to claim 5; d) a cell according to claim 6; e) an antibody according to claim 14; as mediately.   28. Compound according to claim 27, for the prevention and / or treatment of an inflammatory disease, an eruption, or for combating the effects   aging in a mammal, especially in humans.   29. Use of a nueletic acid according to one of claims 1 to 3 for the production of a transgenic plant having improved properties of abiotic stress resistance, particularly water stress, and stress