MXPA96001346A - Antigen novedoso of trypanosoma cruzi and the genque codifies for this last and its application for the detection of cha's disease - Google Patents

Abstract

The present invention describes a novel antigen of Trypanosoma cruzi as PTc100, and a coding gene, known as Tc100, which codes for it. According to the invention, the amino acid and nucleotide sequences of TC100 and PTc100 have been determined and described. PTc100 and Tc100, or fragments thereof, modified or unmodified, can be used directly or indirectly to detect Trypanosoma cruzi, or to treat the infection caused by this infection in humans or animals.

Description

REF: 22263 ANTIGEN NOVETJOßO OF TRYPANOSOMA CRTJZI AND THE GENE THAT CODIFIES FOR THIS LAST; AND ITS APPLICATION FOR THE DETECTION OF CHAGAS DISEASE The present invention relates to a novel genetic material, which codes for a novel protein, recognized by anti-Trypanosoma cruzi antisera, and concerns the use of the gene and the protein, particularly for diagnostic, pharmaceutical and therapeutic purposes. Trypanosoma cruzi is a flagellated protozoan parasite, member of the order of inetoplastidae, and of the Trypanosomatidae family, responsible for Chagas disease, which naturally affects millions of people, mainly in Latin America. In the vertebrate host, Trypanosoma cruzi is presented in two forms: a mobile, thanks to its flagellum or try-pomastigote, is not divided; the other non-flagellated, or intracellular amastigote, is multiplied by binary division. The transmission of the protozoan to man is carried out through the intermediation of hematophagous insects of the family of the Reduvidae, during a blood ingestion followed by debris at the site of the bite. The vector insect thus releases the infectious metacyclic trypomastigote forms that, via the blood circulation, will colonize numerous cell types. Trypanosoma cruzi infects cardiac and skeletal muscle cells, glial cells, and those of the mononuclear phagocytic system. After passive penetration into the host cell, the trypo-mastigotic form of the parasite differentiates in an amastigotic manner, is actively divided, and then follows a release of the trypomastigotic forms, thus causing a new cellular invasion. The insects will complete the cycle of the parasite, and ingest, during a blood ingestion, the trypomastigotic forms of the host. The latter differ in epimastigotic forms in the midgut of the vector, and finally in infectious metacyclic trypomastigotes in the hindgut. Two phases are distinguished in Chagas disease: the acute phase and the chronic phase. The acute phase comes after a type of contamination due to transfusion, congenital, or vector, and lasts for a few weeks. This phase is characterized by a large number of parasites circulating in the blood, and corresponds to an exponential division of the protozoan. The acute phase is very often asymptomatic. However, in breast-fed children contaminated by their mother, the acute phase, which is marked by acute heart disease, can be critical. The chronic phase can extend for many years. In certain individuals this phase is asymptomatic. In contrast, other patients have tissue lesions at the level of the heart, or digestive-type manifestations. In any case, the clinical diagnosis must always be confirmed by the tests to show either the antibodies directed against the antigens of the parasite, or the parasite itself. This disease becomes a global problem, due to contamination by blood transfusion. Therefore, it is essential to have diagnostic tests to determine the presence of the parasite in individuals. Different serological tests are available, such as direct agglutination, indirect immunofluorescence (IFA), complement fixation tests (RFC), ELISA (Enzyme Linked Immunosorbent Assay) tests. The Trypanosoma cruzi antigens used for the serological tests come from a total lysate of the non-infecting stage of the parasite, or from partially purified protein fractions. However, these fractions do not allow obtaining antigens in sufficient quantity and quality for the preparation of a reliable serological diagnostic test. In addition, the complexity of the parasite and the antigenic polymorphism of ce-Pa to strain introduce an additional difficulty in the repro-ducibility of the different preparations. Finally there are numerous risks of cross-reactivity with other proto-zoarios, more particularly with the Trypanosoma rangeli, non-pathogenic parasite, and with the Leishmanie family. Another disadvantage of these techniques is the absence of determination of the phase of the disease that allows a treatment of the acute phase. To solve these various problems, it has been considered to develop a serological diagnostic kit, composed of recombinant proteins, which would be specific to Trypanosoma cruzi. Different research groups have examined the expression banks of genomic DNA, or of DNA complementary to Trypanosoma cruzi in the vector lgtll, by the serum of patients afflicted with Chagas disease. The lgtll phage allows the insertion of foreign DNA, of a maximum size of 7 Kb in the EcoRl site located in the lac Z gene, under the control of the lac promoter. The product obtained is a recombinant protein in fusion with beta-galactosidase, inducible by IPTG (isopropyl-thio-beta-D-galactoside). Several genes of Trypanosoma cruzi, which code for proteins recognized by chagasic serums, have been characterized in this way. Among the described recombinant antigens, we can mention the H49 antigen (Paranhos et al., 1994 (1)). However, this antigen does not allow a serological detection sensitivity of 100% of acute or chronic diseases. Thus, it has been considered to associate the H49 antigen with the antigen ARC (Repetitive Cytoplasmic Antigen) (Lafaille et al., (1989) (2)) without solving this problem.

The present inventors have identified and obtained for the first time a novel genetic material, which codes for a novel protein, recognized by anti-Trypanosoma cruzi anti-sera, which makes it possible to obviate the disadvantages mentioned above. The genetic material can be used to produce proteins or polypeptides for the production of diagnostic tests, or for the preparation of vaccine or pharmaceutical compositions, or to be used either as a probe, or for the determination of specific probes, usable in nucleic acid hybridization assays for the detection of Trypanosoma cruzi infections. Likewise, the protein, or any corresponding polypeptide, can be used for the production of parasite-specific antibodies, for diagnostic or passive protection purposes. This gene has been named Te 100 by the Applicant. Accordingly, the present invention relates to a DNA or RNA molecule, consisting of at least one strand, comprising a nucleotide sequence represented in the identifier SEQ ID NO1, or a complementary sequence, or anti-sense, or equivalent to the sequence identified in the identifier SEQ ID NO1, and mainly, a sequence having, for all successions of 100 contiguous monomers, at least 50%, preferably at least 60%, or better still, at least 85% of homology with the referred sequence.

By nucleotide sequence, it is meant either a strand of DNA or its complementary strand, or either an RNA strand or its anti-sense strand, or its corresponding complementary DNAs. The DNA sequence as represented in the identifier SEQ ID NO1 corresponds to the sequence of the messenger RNA, it being understood that the thymine (T) in the DNA is replaced by the uracil (U) in the RNA. According to the invention, two nucleotide sequences are said to be equivalent with respect to each other, or with respect to a reference sequence, if functionally the corresponding biopolymers can play substantially the same role, without being identical, with respect to the application or considered use, or in the technique in which they are involved; Two sequences obtained by the fact of natural variability are mainly equivalent, mainly the spontaneous mutation of the species from which they have been identified, or induced, as well as the homologous sequences, the homology being defined below. By "variability" is meant any modification, spontaneous or induced, of a sequence, mainly by substitution, and / or insertion, and / or deletion of nucleotides and / or nucleotide fragments, and / or extension and / or shortening of the sequence in at least one of its extremities; unnatural variability may result from the genetic engineering techniques used; this variability can be translated into modifications of the entire starting sequence, considered as a reference, and can be expressed by a degree of homology with respect to the reference sequence. Homology characterizes the degree of identity of two compared nucleotide (or peptide) fragments; this is measured by the percentage of identity, which is determined mainly by direct comparison of the nu-cleotidic (or peptide) sequences, with respect to the reference nucleotide (or peptide) sequences. Any nucleotide fragment is called the equivalent of a reference fragment, if it presents a nucleoidic sequence equivalent to the reference sequence; according to the above definition, they are particularly equivalent to a reference nucleotide fragment: a) any fragment capable of hybridizing, at least partially, with the complement of the reference fragment, b) any fragment whose alignment with the reference fragment leads to putting evidence identical contiguous bases, in a more important number than with any other fragment that comes from another taxonomic group, c) any fragment that results or may result from the natural variability of the species, from which it is obtained, d) any fragment that may result from the genetic engineering techniques applied to the reference fragment, e) any fragment, comprising at least 30 contiguous nucleotides, coding for a peptide homologous or identical to the peptide encoded by the reference fragment, f) any different fragment dul reference fragment, by insertion, deletion, substitution of at least one monomer, extension , or shortening at least one of its extremities; for example, every fragment that corresponds to the reference fragment, flanked, at the ends of one of its extremities, by a nucleotide sequence. | a * that does not code for a polypeptide. The invention relates on the other hand to fragments of DNA or RNA whose γ-sequence is identical, complementary, anti-sense, or equivalent to any of the following sequences: - that which begins in the niiccleotide 1232 , and is terminated at nucleotide 2207 of SEQ II N01 - that which begins at nucleotide 1232, and is terminated at nucleotide 1825 of SEQ II N01 - and that which begins at nucleotide 1266 and is termed mine in nucleotide 2207, and mainly the fragments of DNA or RNA whose sequence has, for the entire succession of 30 contiguous monomers, at least 50%, preferably at least 60%, or better yet, at least 85% homologous with any of the referred sequences. Another object of the invention is a protein, named PTclOO by the Applicant, which has an apparent molecular mass of approximately 100 kDa, recognized by anti-Trypanosoma cruzi anti-sera, or an immunological equivalent of this protein; and its fragments. The amino acid sequence of this protein is represented in the sequence identifier SEQ ID N02. By immunological equivalent, is meant any polypeptide or peptide capable of being immunologically recognized by the antibodies directed against the PTclOO protein. The invention also relates to any fragment of the PTclOO protein. A particular protein fragment has a sequence starting at the amino acid 323, and terminating at the amino acid 520 of the sequence defined in the identifier SEQ ID NO: 2, the fragment is specifically recognized by the anti-Trypanosoma cruzi antisera; The invention also relates to any immunological equivalent of the aforementioned fragment. The PTclOO protein and protein fragments may include modifications, mainly chemical, that do not alter its inraunogenicide. On the other hand, the present invention also has as object a cassette of functional expression, mainly in a cell that comes from a prokaryotic organism or eu-i., ^ Cariótico, which allows the expression of DNA that codes for the whole or a fragment of the PTclOO protein, in particular a DNA fragment as defined above, placed under the control of the elements necessary for its ex-pressure; Protein and protein fragments are recognized by anti-Trypanosoma cruzi antisera. In a general way, any cell that comes from a prokaryotic or eukaryotic organism can be used within the framework of the present invention. Such cells are known to the specialist. As examples, one can cite the cells that come from a eukaryotic organism, such as the cells that come from a mammal, particularly the OHC cells (0-Vario of Chinese Hamster); The insect cells; the cells that come from a fungus, mainly unicellular, or from a yeast, mainly from strains Pichia, Saccharomyces, Schizosaccharomyces, and very particularly, selected from the group consisting of Saccharomyces cerevisiae, Schizosaccharomyces pombe, Schizosaccharomyces malidevorans, Schizosaccharomyces sloofiae, Schizosaccharomy- 0 ees octosporus. Likewise, among the cells that come from a prokaryotic organism, resources can be had, without this being a limitation, in the cells of a strain of Escherichia coli (E. coli), or in enterobacteria cells. A large number of these cells are commercially available in collections, such as the ATCC (Rockville, MA, USA) and the AFRC (Agriculture &Food Research Council, Norfolk, UK). The cell can be wild type or mutant. Mutations are described in the literature accessible to the specialist. For the purposes of the present invention, an E. coli DH5a cell (sold by the company CLONTECH under the reference: C2007-1) is used. The expression cassette of the invention is intended for the production of the PTclOO protein, or that of the fragments of the protein produced by the E. coli cell mentioned above, and recognized by human antisera. Such antisera come from patients who have contracted a recent or ancient infection with Trypanosoma cruzi, and contain immunoglobulins that specifically recognize PTclOO. Of course, the PTclOO protein can also be recognized by other antibodies, such as, for example, monoclonal or polyclonal antibodies obtained by immunization of several species with the natural protein mentioned above, the recombinant protein, its fragments or peptides. Protein PTclOO means the cytoplasmic antigen of Trypanosoma cruzi natural, or produced mainly by the genetic recombination techniques described in the present application, or any fragment or mutant of this antigen, provided that it is immunologically reactive with antibodies directed against the PTclOO protein of this parasite. Advantageously, such a protein possesses an amino acid sequence having a degree of homology of at least 70%, preferably at least 85% and, most preferably, at least 95% with respect to the sequence identified in the identifier SEQ ID N02. In practice, such equivalence can be obtained by deletion, substitution and / or addition of one or several amino acids of the natural or recombinant protein. It is within the reach of the expert to carry out, by known techniques, these modifications, without affecting the immunological recognition. Within the framework of the present invention, the PTclOO protein can be modified in vitro, mainly by suppression or addition of chemical groups, such as phosphates, sugars or myristic acids, in order to improve its stability, or the presentation of one or several epitopes. . The expression cassette according to the invention allows the production of a PTclOO protein (having an amino acid sequence such as that specified above) and fragments of the protein, fused to an exogenous element that can help its stability, its purification, its production or its recognition. The selection of such exogenous element is within the reach of the expert. It may be mainly a hapten, an exogenous peptide, or a protein. The expression cassette according to the invention comprises the necessary elements for the expression of the DNA fragment in the considered cell. By "elements necessary for expression" is meant the set of elements that allow the transcription of the DNA fragment into menarary RNA (mRNA), and the translation of the latter into protein. The present invention also extends to a vector comprising an expression cassette according to the invention. It can be a viral vector, and mainly a vector derived from a vaculovirus, more particularly intended for expression in insect cells, or a vector derived from an adenovirus, for expression in mammalian cells. It can also be a plasmid vector, of autonomous replication, and in particular, of a multiplying vector. The present invention also relates to a cell that comes from a prokaryotic or eukaryotic organism, comprising an expression cassette, either in an integrated form in the cellular genome, or inserted into a vector. Such a cell has been defined above. Another subject of the present invention is a process for the preparation of a PTclOO protein, or fragments of the protein, according to which: (i) a cell derived from a prokaryotic or eukaryotic organism is cultured under appropriate conditions; "'" comprises the expression cassette according to the invention; and (ii) the expressed protein, which comes from the aforementioned organism, is recovered. The present invention also relates to one or more peptides, whose amino acid sequence corresponds to a part of the PTclOO protein sequence, and which, alone or as a mixture, have a reactivity with all the sera of individuals or animals infected with Trypanosoma cruzi. 0 Peptides can be obtained by chemical synthesis, lysate of the PTclOO protein, or by genetic recombination techniques. The invention also relates to monoclonal or polyclonal antibodies, obtained by immunological reaction of a human or animal organism to an immunogenic agent, constituted by the natural, recombinant PTcllO protein or its fragments, or by a peptide, as defined above. The present invention also relates to a reagent for the detection and / or monitoring of an infection by Trypanosoma cruzi, which comprises, as a reactive substance, a PTclOO protein as defined above, or its fragments, a peptide or a mixture of peptides such as those defined above, or at least one mo-5 notional or polyclonal antibody such as that described above.

] ,. The reagent mentioned above can be fixed directly or indirectly on an appropriate solid support. The solid support may be mainly in the form of a cone, a tube, a well, a ball, or analogous shapes. The term "solid support", as used herein, includes all materials on which a reagent can be immobilized, for use in diagnostic tests. Natural, synthetic materials, chemically modified or not, can be used as solid supports, mainly polysaccharides such as cellulose-based materials, for example paper, cellulose derivatives such as cellulose acetate and nitrocellulose; polymers such as vinyl chloride, polyethylene, polystyrene-5, polyacrylate, or copolymers such as polymers of vinyl chloride and propylene, polymers of vinyl chloride and vinyl acetate; styrene-based copolymers, natural fibers such as cotton, and synthetic fibers such as nylon. Preferably, the solid support is a polystyrene polymer, or a butadiene-styrene copolymer. Advantageously, the support is a polystyrene, or a copolymer based on styrene, comprising between about 10 and 90% by weight of styrene. 5 The fixation of the reagent on the solid support can be carried out directly or indirectly. Directly, two approximations are possible: either by adsorption of the reagent on the solid support, that is to say by non-covalent bonds (mainly of the hydrogen, Van der Walls or ionic type), either by the establishment of covalent bonds between the reagent and the support. Indirectly, an "anti-active" compound capable of interacting with the reagent can be previously fixed (by adsorption or covalence) on the solid support in such a way that the assembly is immobilized on the solid support. As an example, an anti-PTclOO antibody can be cited, with the proviso that it is immunologically reactive with a part of the protein different from that which is involved in the recognition reaction of the antibodies of the sera; a ligand-receptor system, for example by grafting a molecule such as a vitamin onto the PTclOO protein and immobilizing the corresponding receptor on the solid phase (for example the biotin-streptavidin system). Indirectly, it is also understood the prior graft, or the fusion by genetic recombination of a protein, or a fragment of this protein, or of a polypeptide, to an end of the protein PTclOO, and the immobilization of the latter on the support solid by passive adsorption, or covalence of the grafted or fused protein or polypeptide. The invention also relates to a method for detecting and / or monitoring an infection caused by Trypanosoma cruzi in a biological sample, such as a blood sample from an individual or from an animal susceptible to being infected with Trypanosoma cruzi, characterized in that the sample is contacted and a reagent as defined above, under conditions that allow an eventual immunological reaction, and then the presence of an immune complex with the reagent is detected. As a non-limiting example, mention may be made of the sandwich (sandwich) detection method in one or more steps, such as that described in particular in the patents FR 2 481 318 and FR 2 487 983, which consists in reacting a first monoclonal or polyclonal antibody specific to a investigated antigen, fixed on a solid support, with the sample, and in evidencing the eventual presence of an immune complex thus formed, by a second antibody labeled with any appropriate marker known to the expert, mainly a radioactive isotope, an enzyme, for example peroxidase or alkaline phosphatase, or similar enzymes; by the techniques called competition, well known by experts. The subject of the invention is also an active immunotherapeutic composition, in particular a vaccine preparation comprising, as active principle, a natural, recombinant PTclOO protein or its fragments, or 1. the peptides identified above, the active ingredient is possibly conjugated with a pharmaceutically acceptable carrier, and optionally an excipient and / or an appropriate adjuvant. The present invention also covers a pharmaceutical composition, intended for the treatment or prevention of a Trypanosoma cruzi infection in a man or an animal, comprising a therapeutically effective amount of an expression cassette, a vector, a cell that comes from J Or a prokaryotic or eukaryotic organism as defined above, of a PTclOO protein according to the invention, or of its fragments, or of an antibody of the invention. The present invention also has as object specific probes and baits of T. cruzi, and their use in tests of diagnosis. The term "probe", as used in the present invention, refers to DNA or RNA that comprises at least one strand that has a nucleotide sequence that allows hybridization with nucleic acids that present a nucleotide sequence such as that represented in the identifier SEQ ID NO1, or a complementary sequence, or anti-sense, or equivalent to the referred sequence, and mainly, a sequence having, for all successions of 5 to 100 contiguous monomers, at least 50%, preferably at least 60%, or better yet, at least 85% homology with its fragments, or with a synthetic oligonucleotide allowing such hybridization, unmodified, or comprising one or more modified bases , such as inosine, 5-methyldeoxycytidine, deoxyuridine, 5-dimethylaminodeoxyuridine, 2,6-diaminopurine, 5-bromodesoxyuridine, or any other modified base, and these probes can also be modified. at the sugar level, namely the replacement of at least one deoxyribose by a polyamide (PE NIELSEN et al. (1991) (13)), or at the level of the phosphate group, for example its replacement by esters, particularly selected from among the diphosphate, alkyl and arphosphonate, and phosphorothioate esters The probes may be much shorter than the sequence identified in the identifier SEQ ID NO 1. In practice, such probes comprise at least 5 monomers, advantageously from 8 to 50 monomers, which possess a hybridization specificity under certain conditions to form a hybridization complex with DNA or RNA having a nucleotide sequence as defined above. A probe according to the invention can be used for diagnostic purposes, as a capture and / or detection probe, or for therapy purposes. The capture probe can be immobilized on a solid support, by any appropriate means, that is, directly or indirectly, for example by covalence or passive adsorption. The detection probe is marked by means of a marker selected from the radioactive isotopes, enzymes, selected mainly between peroxidase and alkaline phosphatase, and those capable of hydrolyzing a chromogenic, fluorine or luminescent substrate, chromophore chemical compounds, chromogenic compounds, Fluorescent or luminescent, analogs of a nucleotide base, and biotin. The probes of the present invention used for diagnostic purposes can be applied in all known hybridization techniques, and mainly in techniques called "Stain Transfer" (Maniatis et al. (1982) (14), Southern Spotting ( Southern EM (1975) (15)), Northern Spotting, which is a technique identical to the Southern Spotting technique, but which uses RNA as a target, the sandwich technique (Dunn AR et al. (1977) (16 )).

Advantageously, the sandwich (sandwich) technique is used, which comprises a specific capture probe and / or a specific detection probe, it being understood that the capture probe and the detection probe must have at least a partially different nucleo-thiudic sequence. . Another application of the invention is a therapy probe, to treat infections due to Trypanosoma cruzi, the probe is susceptible to hybridize in vivo on the DNA or RNA of the parasite, to block transcription and / or transcription phenomena and / or of replication. A bait is a probe comprising from 5 to 30 monomers, which possesses a specificity of hybridization under predetermined conditions, for the initiation of an enzymatic polymerization, for example in an amplification technique such as PCR (Polymerase Chain Reaction), in an elongation procedure such as sequencing, in a reverse or analogous transcription method. A probe or bait will preferably comprise a nucleotide sequence selected from the sequences SEQ ID N07, SEQ ID N08, SEQ ID N09, SEQ ID N010, SEQ ID Nol2. The invention also relates to a reagent for detecting and / or identifying Trypanosoma cruzi in a biological sample, comprising at least one probe as defined above, in particular a capture probe and a detection probe, and one and / or another respond to the above definition. The invention thus provides a method for selectively detecting and / or identifying Trypanosoma cruzi in a biological rache, according to which the RNA extracted from the parasite and possibly denatured, or the DNA, extracted denatured, or the DNA obtained from it is exposed. of the reverse transcription of the RNA, at least to a probe as defined above, and the hybridization of the probe is detected. The invention will be better understood by reading the detailed description that follows, made with reference to the appended figures, in which: Figure 1 represents the restriction letter of the TclOO gene, deduced by Southern Spotting of different fragments Obtained after the digestion of Trypanosoma cruzi DNA by restriction endonucleases. Figure 2 is a schematic representation of three translapant regions of the TclOO cDNA. The numbered arrows represent the oligonucleotides used as baits for amplification by PCR. Example 1: Isolation of the Tc50 clone An expression bank has been constructed from genomic DNA fragments of Trypanosoma cruzi. The DNA of T. cruzi, strain G (YOSHIDA, N., (1983) (17), isolated from the metacyclic trypomastigotic tadio, has been digested by the enzyme DNase I. After the selection of the fragments according to their size , these have been linked to synthetic EcoRI adapters, and cloned into the EcoRI site of the lambda gtll vector DNA (Ypung et Davis, 1983 (3); Cotrim et al., 1990) (4). The clone, named Tc50 by the Applicant, has been isolated from the bank by immunological screening, with the help of a mixture of sera from patients suffering from chronic phase Chagas disease. The phage clone Tc50 has been purified, amplified, and the insert has been evidenced by the PCR technique ("Polymerase Chain Reaction"), with the help of the baits: SEQ ID N03 5 '(GGTGGCGACGACTCCTGGAGCCCG) 3' 24, and SEQ ID N04 5 '(TTGACACCAGACCAACTGGTAATG) 3' 24 corresponding, respectively, to the nucleotide sequence of the left and right arms of phage lambda gtll DNA. The Tc50 DNA fragment of 594 base pairs (bp), after EcoRI digestion, has been sub-cloned into the expression vector pGEX (Pharmacia), linearized by EcoRI. The DNA sequencing of clone Tc50 was performed in this same vector, with the help of specific baits, located 3 'and 5' from the cloning site of pGEX, according to the chain termination technique (Sanger et al., 1977 (5)), and according to the provider's protocol (USB-Amersham). The nucleotide sequence of the 594 bp Tc50 fragment, as well as its sequence, deduced in amino acids (198 aa) are represented in the identifiers SEQ ID NO1 and SEQ ID NO2, respectively. The nucleotide sequence of the 594 bp Tc50 fragment begins at nucleotide (nt) 1232, and terminates at nucleotide 1825. The corresponding amino acid sequence begins at the amino acid 323, and is terminated with the amino acid 520 of the SEQ ID N02 Example 2: Expression of the clone Tc50 in Escherichia coli The construction pGEX-Tc50 (198 aa) synthesizes, in the bacterium DH5alpha, a protein fused with the TSG ("Trans-ferase S of Glutathione"), with an apparent molecular mass of 50 kDa , evidenced by SDS-PAGE polyacrylamide gel electrophoresis (SDS: Sodium Dodecyl Sulfate) (Laemmli, 1970 (6)). The reactivity of the protein against human sera infected with Chagas disease has been confirmed by the Western blot technique (Towbin et al., 1979 (7)), with the help of the same mixture of sera infected with Chagas disease of the chronic phase used for screening the bank in lambda gtll. The soluble fraction of the recombinant protein GST-Tc50 obtained after lysate of bacterial extracts by ultrasone has been purified by affinity chromatography on a glutathione agarose column (Sigma), according to the methods of Smith and Johnson, (1988) ( 8). The antigenic properties of the recombinant antigen GST-Tc50 have been tested by ELISA (Voller et al., 1975 (9)). For this, microtiter plates (Maxisorp (trade name), Nunc) were sensitized with 100 ng / ml of the GST-Tc50 antigen in 100 mM NaHCO 3 (pH 9.6). After incubation with patient sera, immunocomplexes have been detected with the help of a goat serum against human IgG, coupled to peroxidase.

The results are presented in the appended table, and show that all of the human sera infected with Chagas disease tested react specifically with the recombinant protein. No cross-reactivity was observed in 7 sera from patients suffering from cutaneous or visceral leishmaniasis. Example 3: Identification of the natural protein of T. cruzi, which presents the antigenic determinants of the clone Tc50 The evidence of the natural protein of T. cru-zi has been made after the immunopurification of a mixture of human sera infected with Chagas disease, on the corresponding recombinant protein, named by the Applicant PTc50. The monoclonal polyclonal antibody eluate obtained has been used as a probe, in Western blotting, on total protein extracts of the different stages of the parasite. The selected antibodies have specifically reacted with a protein of apparent molecular mass of 100 KDa, termed PTclOO by the Applicant, expressed in all tested strains of the parasite. Example 4: Molecular Analysis of the TclOO gene - Southern stains In order to establish the restriction letter of the TclOO gene (Figure 1), the T. cruzi nuclear DNA, strain G, was digested with different restriction endonucleases (BamHI, EcoRI, HindIII, PstI, PvuII, Sacl, BamHI / EcoRI, BamHI / PvuII, EcoRI / HindIII, EcoRI / PstI, EcoRI / PvuII, EcoRI / SacI, PstI / Sacl, PstI / PvuII, PvuII / SacI, PvuII / HindlII) , it was separated on agarose gel, and then transferred on a nylon filter, according to the Southern technique. Hybridization of the Southern blots was performed with the Te4 DNA of 594 bp, which is a fragment of the TclOO DNA described above, marked isotopically with 32P, by random incorporation (Amersham) -Cloning of a TclOO genomic fragment of 3500 bp According to the results obtained in the Southern spotting, the genomic DNA of T. cruzi, strain G, was digested with the enzyme EcoRI, and then separated on an agarose gel. The EcoRI restriction fragments of approximately 3500 bp (Figure 1) were cloned in the lambda gtll vector (Huynh et al., 1984 (10)) linearized by EcoRI. The phage clone containing the 3500 bp TclOO genomic insert was isolated with the aid of the 594 bp isotopically labeled probe described above. A fragment of 1041 bp, located in the 3 'region of the 3500 bp TclOO genomic insert was sequenced. This sequencing, or determination of the sequence, was carried out progressively, with the help of the following baits: SEQ ID N05 5 '(TCGGGCACTGACGCGGCG) 3' 18 SEQ ID N06 5 '(CTTATGAGTATTTCTTCCAGGGTA) 3' 24 The bait SEQ ID N05 is located in the previously sequenced part of the fragment of 594 bp of Tc50. The bait SEQ ID N06 corresponds to the bait of the phage lambda gtlO. This fragment of 1041 bp, beginning at nucleotide 1403, and ending at nucleotide 2443 of ls SEQ ID NOl, presents an open reading frame, in phase with the fragment sequence of 594 bp of Tc50. Example 5: Cloning of the TclOO cDNA The cDNA was synthesized from total RNA of T. cruzi epimas-tigotes, strain G. The TclOO cDNA was amplified by the PCR technique, in three different fragments: a fragment A, corresponding to the 5 'region of 1459 bp, a B fragment, corresponding to the central region of 942 bp, a C fragment, corresponding to the 3' region of 1406 bp of the TclOO cDNA, as schematically depicted in Figure 2. -Cloning of fragment A of cDNA TclOO The total cDNA synthesized by reverse transcriptase of VMA ("avian myeloblastosis virus"), with the help of random hexanucleotides (Boehringer Mannheim), was amplified, by PCR, thanks to the pair of baits following: SEQ ID N07 5 '(AACGCTATTATTAGAACAGTT) 3' 21, and SEQ ID N08 5 '(TGCAGCAGCGGCAGAAGT) 3' 18 SEQ ID N07 corresponds to a part of the consensus sequence of 35 nucleotides, present at 5 'of the mRNAs in the trypanosomatidos, and called "Leader spliced" ( Parsons et al. 1984 (11)).

SEQ ID N08 corresponds to the sequence complementary to a part of the predetermined sequence of the 594 bp fragment, and starts at nucleotide 1442, to be terminated with nucleotide 1459 of SEQ ID NO1, according to the numbering of the strand it encodes. After verification in Southern blotting, with the aid of the previously described isotopically labeled 594 bp probe, the 1459 bp cDNA fragment corresponding to the 5 'region of TclOO was cloned into the plasmid called pCRII (tradename) (Invitrogen), and the sequence was determined. The sequence represented in the identifier SEQ ID NO1 starts at nucleotide 1, and ends at nucleotide 1459. -Cloning of fragment B of cDNA TclOO The total cDNA of T. cruzi was amplified by PCR, with the help of baits: SEQ ID N09: 5 '(CAGCCGACGGTAGCTGCGTCCT) 3' 22, and SEQ ID N010: 5 '(ACATAATGGCCTCGTTCACAC) 3' 21 The sequence ID N09 corresponding to a part of the predetermined 594 bp sequence of the TclOO gene, starts at the nucleotide 1266, and terminated at nucleotide 1287 of SEQ ID NOl. The sequence SEQ ID N010 corresponds to the sequence complementary to a part of the sequence previously described of 1041 bp of the TclOO gene. This sequence SEQ ID N010 starts at nucleotide 2187, and ends at nucleotide 2207 of SEQ ID NO1, according to the numbering of the strand it encodes. The obtained fragment, of a length of 942 bp, was cloned in plasmid pCRII, and its sequence was determined. The sequence represented in the identifier SEQ ID NO1 starts at nucleotide 1266, and ends at nucleotide 2207. -Cloning of fragment C of the TclOO cDNA In order to isolate the 3 'part of the TclOO cDNA, the T-total cDNA of T cruzi was synthesized, with the help of the oligo (dT) hybrid bait. fi adapter: SEQ ID Noli: 5 '(GACTCGCTGCAGATCGATTTTTTTTTTTTTTTTT) 3' 34, according to the AREC protocol ("Rapid Amplification of cDNA Ends") (Frohman et al., 1988 (12)). The 3 'region of the TclOO cDNA was amplified by the adapter bait, and the following pair of primers: SEQ ID N012: 5' (CGAAGAGACCATGAACAACTT) 3 '21, and SEQ ID N013: 5' (GACTCGCTGCAGATCGAT) 3 '18 The sequence SEQ ID N012 corresponds to a part of the 1041 bp sequence previously described for the TclOO gene, which starts at nucleotide 1997, and ends at nucleotide 2017. The sequence SEQ ID N011 corresponds to the arbitrary sequence of the adapter represented in SEQ ID N011 After control in Southern blotting, with the aid of the 1041 bp isotopically-labeled fragment described above, the 3 'fragment of the TclOO cDNA, which is 1423 bp long, was cloned into pCRII, and its sequence was determined. The sequence represented in the identifier SEQ ID NO1 starts at nucleotide 1997, and ends at nucleotide 3402. The complete TclOO cDNA, with a size of 3402 bp, was completely sequenced (its sequence was determined). It has an open reading frame of 2745 bp, and the deduced amino acid sequence is 915. The methionine codon is at position 266, and the terminus codon at position 3011.

The TclOO gene of Trypanosoma cruzi codes for the novel protein PTclOO, with a theoretical molecular mass of 100 kDa. Of course, since the DNA sequence of the gene has been fully identified, it is possible to produce the corresponding DNA entirely by chemical synthesis, and then insert the DNA into commercially available DNA vectors, using known techniques of technology relating to genetic recombination .

TABLE Illness Serums DO (492 nm) Detection threshold = 0.320 CHAGAS DISEASE 1 1.358 (+) 2 1.278 (+) 3 0.328 (+) 4 0.404 (+) 5 1.378 (+) 6 1.059 (+) 7 0.895 (+) 8 1.791 (+) 9 1.635 (+) 10 1.427 (+) 11 1.009 (+) 12 1.743 (+) 13 0.530 (+) 14 1.035 (+) 15 0.461 (+) CUTANEOUS LEISHMANIOSIS 16 0.291 (") LEISHMANIOSIS 17 0.071 (-) VISCERAL (Kala- 18 0.081 (") chance) 19 0.279 (") 20 0.098 (") 21 0.067 (-) 22 0.125 (-) NUMBER OF SEQUENCES: 13 1 INFORMATION FOR SEQ ID NO: 1 li SEQUENCE CHARACTERISTICS: LONG LENGTH: 3402 liB TYPE: acid nucleus liC NUMBER OF HEBRAS: double liD CONFIGURATION: linear lii TYPE OF MOLECULE: complementary DNA T. cruzi IviA ORGANISM: T. cruzi IviB CEPA: G IviD DEVELOPMENT STAGE: epimastigote IVIIA LIBRARY: IviiB CLON: TclOO lxi DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1 20 30 40 SO 60 AACGCTATTA TTAGAACAGT TTCTGTACTA TATTGTCATT TGGGGAGGGG GGAAAGGGGG 70 80 90 100 110 120 GAAGTACTTG CCGTTTTGTG TGGGTGACGA GACAACACAC ATCGAGCGGG AAG'AAAAAA 130 140 150 160 170 180 AAAAGGAAAT AAATTAAATT AAATTATTTG TTCTTTGAAT AGGCAAAGAA GAAGAAGAAG 190 200 210 220 230 240 AAAAGGTGCT GGGGAGGGAG GAGAAAGCGA CACACACACA AAGGAATTGC AAAAAAAAAA 250 260 270 280 290 300 GGAAATAACA ACGCAAGGCG CGGACATGAC CGTGACGGTG GATTTGTTCA ATCATGCGAA 330 310 320 340 350 360 GCCGAGCAAC AATGAGGGCC GCGTGTGGTC TGTGGACGCC GCGACATTTA ACGAGGTGCC 370 380 390 400 410 420 TGAGGCGCAG CGTGTGCTGG CGGATTCGCA GTTTTATCTT GCCTACACCA TGAAGCGGCG 430 440 450 460 470 480 TCACGTGCTG CGTGTGGTGA AGCGCTCGAA CCTTTTGAAG GGCACCGTGC GGGCACACTC 490 500 510 520 530 540 AAAGCCCATT CATGCGGTGA AGTTTGTGAA TTACCGCAGT AACGTCGCAG CATCGGCTGG 550 560 570 580 590 600 GAAGGGGGAG TTCTTCGTGT GGGTTGTGAC GGATGAAACG GAGGCGAGCA ACGGCAAGCC 610 620 630 640 650 660 GGATCTCGCA GCCCGCCTCA CAGTGAAGGT GTACTTTAAG CTTCAGGATC CTGTCACAAT 670 680 690 700 710 720 TCCATGCTTT TCTTTCTTTA TCAACGCCGA GAGTCAGCGG CCTGATCTGC TTGTCCTTTA 730 740 750 760 770 780 CGAAACGCAG GCGGCAATTC TTGACAGCTC CTCCCTCATT GAGCGCTTTG ACGTGGAATC 790 800 810 820 830 840 ACTGGAGGCA ACACTACAGC GGAATTGCAC AACCCTGCGA ACCCTGACTC AACCGGTTAG_850_860 870 880 890 900 TGAGAACAGT TTATGCTCCG TTGGCTCTGG CGGATGGTTC ACCTTTACCA CGGAACCAAC 910 920 930 940 950 960 AATGGTAGCG GCATGCACAT TACGAAACCG CAGCACTCCA TCATGGGCGT GTTGCGAGGG 1000 1010 970 980 990 1020 TGAGCCAGTG AAGGCATTGC ATCTCCTTGA CGCAACCGTT GAGGAAAATG TCAGTGTTCT 1030 1040 1050 1060 1070 1080 CGTGGCCGCA TCTACAAAAG GGGTGTACCA ATGGCTCCTT ACGGGTGTAG CAGAACCAAA 1090 1100 1110 1120 1130 1140 CTTGTTGCGC AAGTTTGTCA TTGATGGATC TATTGTCGCG ATGGAAAGCT CACGAGAAAC 1150 1160 1170 1180 1190 1200 GTTTGCCGTG TTTGACGACA GGAAGCAGCT GGCGCTGGTG AACATGCATT CCCCTCATAA 1210 1220 1230 1240 1250 1260 CTTTACCTGC ACACACTACA TGATGCCTTG TCAGGTACAG CGTAACGGCT TTTGCTTCAA 1270 1280 1290 1300 1310 1320 TCGTACAGCC GACGGTAGCT GCGTCCTGGC TGACATGTCG AATCGATTGA CGATCTTCCA 1330 1340 1350 1360 1370 1380 TCTCCGGTCC TCCCGCAGGG AAGAACAGCA GCCAGGCCAA AAAACATCGG TAGTGGCGAC 1390 1400 1410 1420 1430 1440 GGCGAAACCG GGGTGTGTGT CCTCGGGCAC TGACGCGGCG AGTAGCAGTC ATACCAATAC 1450 1460 1470 1480 1490 1500 GACTTCTGCC GCTGCTGCAT CCCCTGCATC ACCCCCTGTT TCAGCGCCAG CGAAGGCAGC 1510 1520 1530 1540 1550 1560 CGCGCCTCCT GCCGCGGCGC GATCGGCTGA GCCGCACGTG GGGAGCAAGA TCATTGCTAA 1570 1580 1590 1600 16-10. 1620 TCTAGTGAAT CAGCTGGGGA TTAATGTCAC CCAAAGGAGC GTCGTCAGCA CTGGAGCGCC 1630 1640 1650 1660 1670 1680 GGCCACGACG AGGTCTACGG CGGTGACGTC CACGACTACC GCCCCGCAGC GAACAAGTCC 1690 1700 1710 1720 1730 1740 ATACGGGCAC AATGGCCGAC CTGTGACGGC TGGATTGGTG GCAGCTAATA GTGGTGCCAG 1750 1760 1770 1780 1790 1800 CGCGGCCTCG TCTCCCACAG CCGCGGCGAA ACCAACAGGA GAAGAAAAGG CCTCCGCGGC 1810 1820 1830 1840 1850 1860 ATGTGAAACG AGCTCCGTGG CGATAAATGC GACACGCCCG GCGCTTCACA ACGCCTCTCT 1870 1880 1890 1900 1910 1920 CCCGCAGGCG CCAACGGATG GCGTTTTGGC GGCAGCAGTA TACCAGTCGG AGGGCGAGGT 1930 1940 1950 1960 1970 1980 TCATCAGTCG CTGGAGCGGC TGGAGTCCGT CATAACCAAC ACGTCTCGGG TTCTGAAGTT 1990 2000 2010 2020 2030 2040 GCTCCCTGAC ACCATTCGAA GAGACCATGA ACAACTTCTG AATCTGGGTT TAGAGGCACA 2050 2060 2070 2080 2090 2100 GATGACAGAG CTGCAGCAGA GCCGTCCAAC ACCGCAAACA CAGCCGAGAG ACACAACCTC 2110 2120 2130 2140 2150 2160 CGCGAAATCA TCCGTGTTTG AGACGTACAC CCTTGTTCTC ATTGCGGATT CCCTCTCTCG 2170 2180 2190 2200 2210 2220 CAACATCACG AAGGGGGTGA AGC GTGGTGT GAACGAGGCC ATTATGTTGC ATCTCGACCA 2230 2240 2250 2260 2270 2280 TGAGGTGCGG CACGCCATAG GGAACCGGCT TCGGCAAACA CAAAAGAACA TCATCAAGAG 2290 2300 2310 2320 2330 2340 CCGCCTCGAT GAAGCGTTGA AGGAAAGCAC TACACAGTTT ACGGCTCAAT TGACGCAAAC 2350 2360 2370 2380 2390 2400 GGTGGAGAAT CTGGTGAAGC GCGAGCTTGC CGAGGTGCTT GGTAGCATCA ACGGCTCCCT 2410 2420 2430 2440 2450 2460 CACTTCTCTC GTGAAGGAAA ATGCCTCATT ACAGAAAGAC TTGAATTCCA TAATGTCTAG_2470_2480 2490 2500 2510 2520 TGGGGTGTTG GATGAAATGC GTCGTATGCG GGAAGAGCTG TGCACATTGC GAGAGTCCGT 2530 2540 2550 2560 2570 2580 TGCGAAGCGG AAGGCAACAA TGCCAGATTC TTCTCTTCAC GCCACGAGCT CCTTTCAAGG 2590 2600 2610 2620 2630 2640 AAGAAGGTCT GCGCCCGAGA CAATTCTTGC AACCGCGTTA TCGATGGTGC GAGAGCAGCA 2650 2660 2670 2680 2690 2700 ATACCGTCAG GGACTGGAAT ACATGTTGAT GGCTCAGCAG CCCTCTCTCC TCCTGCGGTT 2710 2720 2730 2740 2750 2760 CCTCAGCATA CTTACAAGGG AAAACGAAAA CGCCTACTCG GAACTTATTG AAAATGTAGA 2770 2780 2790 2800 2810 2820 GACGCCGAAT GACGTGTGGT GTTCGGTTCT GTTGCAACTC ATAGAGGCCG CGGCGACGGA 2830 2840 2850 2860 2880 28.70 GGCTGAGAAG GAGGTGGTTG TTGGCGTCGC CATTGATATT CTCTCCGAGC GCGATCAAAT 2890 2900 2910 2920 2930 2940 TGCTCAGAAC GGCGCACTCG GCTCGAAACT CACCACCGCC ATGCGAGCCT TTGAGCGACA 2950 2960 2970 2980 2990 3000 GGCAAGGTCG GAGACAACGA GCAGGTCATT CTTGCAATGC CTGAAGAACC TGGAAAAGCT 3010 3020 • 3030 3040 3050 3060 TCTGCAATCA TGATAATAAA AAGAACTCAA CGAATACAGT TGTTGATTAT TAAGGAAGGG 3070 3080 3090 3100 3110 3120 AAAAGAGAGA AAGAGAGAGA GAGAGAGAGA AATGTAATGG GCGTTTAGTT ACGGTAGAAA 3130 3140 3150 3160 3170 3180 GAAAACGTGT GGATAAGAAG GAGGGGTTTT GTGTGCGACC AGGAATTACT GGGGAACGCT 3190 3200 3210 3220 3230 3240 GCTACACGGC GGAATCGACC ATTTTATTAT TATTATTATT GTCTTTAGTA TTATGTTTTT 3250 3260 3270 3280 3290 3300 TCTTGTGTGT GTGTGTGTGT GTTTGTGTGT GTGCGGTTAT TTTGTATCCG TTTGCTCCCG 3310 3320 3330 3340 3350 3360 CCCCTGCCCC CCATCACCCG AGGAGAAAGT AGAATAAGAC ACATACGATT GTTGTTTTTG 3370 3380 3390 3400 TTATCCTTAA AAGGAAGAGA GACCAAAAAA AAAAAAAAAA AA 1 INFORMATION FOR SEQ ID NO: 2 li CHARACTERISTICS OF THE SEQUENCE: lia LENGTH: 915 liB TYPE: protein liC NUMBER OF HEBRAS: simple lvi ORIGIN: T. cruzi IviA ORGANIZATION: T. cruzi IviB CEPA: G lvii IMMEDIATE SOURCE: IVIIA LIBRARY: IviiB CLON: TclOO lxi DESCRIPTION OF THE SEQUENCE SEQ ID NO: 2 2 INFORMATION FOR SEQ ID NO: 2 2i CHARACTERISTICS OF THE SEQUENCE: 2iA LENGTH: 915 amines 2iB TYPE: proteic 2iC NUMBER OF HEBRAS: simple 2VI ORIGIN: T. cruzi 2viA ORGANIZATION: T. cruzi 2viB CEPA: G 2vii IMMEDIATE SOURCE: 2VIiA LIBRARY: 2viiB CLON: TclOO 2xi DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2 10 15 20 Met Thr Val Thr Val Asp Leu Phe Asn Nis Ala Lys Pro Ser Asn Aßn Glu Gly Arg Val 30 35 40 Trp Ser Val Asp Wing Wing Thr Phe Asn Glu Val Pro Glu Wing Gln Arg Val Leu Wing Asp 45 '55 60 Being Gln Phe Tyr Leu Wing Tyr Thr Met Lys Arg Arg His Val Leu Arg Val Val Lys Arg 65 70 75 80 Be Asn Leu Leu Lys Gly Thr Val Arg Ala His Ser Lys Pro lie His Wing Val Lys Phe 85 90 95 100 Val Asn Tyr Arg Ser Asn Val Wing Wing Being Wing Gly Lys Gly Glu Phe Phe Val Trp Val 105 110 115 120 Val Thr Asp Glu Thr Glu Wing Being Asn Gly Lys Pro Asp Leu Wing Wing Arg Leu Thr Val 125 130 135 140 Lys Val Tyr Phe Lys Leu Glp Asp Pro Val Thr He Pro Cys Phe Ser Phe Pne Lie Asn 145 150 155 160 Wing Glu Ser Gln Arg Pro Asp Leu Leu Val Leu Tyr Glu Thr Gln Wing Wing He Leu Asp 165 170 175 180 Being Ser Leu He Glu Arg Phe Asp Val Glu Ser Leu Glu Ala Thr Leu Gln Arg Asn 185 190 195 200 Cys Thr Thr Leu Arg Thr Leu Thr Gln Pro Val Ser Glu Asn Ser Leu Cys Ser Val Gly 205 210 215 220 Be Gly Gly Trp Phe Thr Phe Thr Thr Gtu Pro Thr Met Val Wing Wing Cys Thr Leu Arg 225 230 235 240 Asn Arg Ser Thr Pro Ser Trp Wing Cys Cys Glu Gly Glu Pro Val Lys Wing Leu His Leu 24S 250 255 260 Leu Asp Ala Thr al Glu Glu Asn Val Ser Val Leu Val Ala Ala Ser Thr Lys Gly Val 265 270 275 280 Tyr Gln Trp Leu Leu Thr Gly Val Wing Glu Pro Asn Leu Leu Arg Lys Phe Val He Asp 285 290 295 300 Gly Ser He Val Wing Met Glu Ser Ser Arg Glu Thr Phe Wing Val Phe Asp Asp Arg Lys 305 310 315 320 Gln Leu Ala Leu Val Asn Met His Ser Pro His Asn Phe Thr Cys Thr His Tyr Met Het 325 330 335 340 Pro Cys Gln Val Gln Arg Asn Gly Phe Cys Phe Asn Arg Thr Wing Asp Gly Ser Cys Val 345 350 355 360 Leu Ala Asp Met Ser Asn Arg Leu Thr He Phe His Leu Arg Ser Ser Arg Arg Glu Glu 36S 370 375 380 Gln Gln Pro Gly Gln Lys Thr Ser Val Val Wing Thr Wing Lys Pro Gly Cys Val Ser Ser 385 390 395 400 Gly Thr Asp Ala Ala Ser Ser Ser His Thr Asn Thr Thr Ser Ala Ala Ala Ala Ser Ser 405 410 415 420 Pro Wing Pro Pro Wing Pro Wing Pro Wing Lys Wing Wing Pro Pro Wing Wing Wing Arg Ser 425 430 435 440 Wing Glu Pro His Val Gly Ser Lys He He Wing Asn Leu Val Asn Gln leu Gly lie Asn 445 450 455 460 Val Thr Gln Arg Ser Val Val Ser Thr Gly Wing Pro Wing Thr Thr Arg Ser Thr Wing Val 465 470 475 480 Thr Ser Thr Thr Thr Wing Pro Gln Arg Thr Ser Pro Tyr Gly His Asn Gly Arg Pro Val 485 490 495 500 Thr Ala Gly Leu Val Ala Ata Asn Ser Gly Ala Ser Ala Ala Ser Ser Pro Thr Ala Ala 505 510 515 520 Wing Lys Pro Thr Gly Glu Glu Lys Wing Being Wing Wing Cys Glu Thr Ser Ser Val Wing He 525 530 535 540 Asn Ala Thr Arg Pro Ata Leu His Asn Ata Ser Leu Pro Gtn Ala Pro Thr Asp Gly Val 545 550 555 560 Leu Ala Ala Ala Val Tyr Gln Ser Glu Gly Glu Val His Gln Ser Leu Glu Arg Leu Glu 565 c? 0 575 580 Ser Val lie Thr Asn Thr Ser Arg Val LÍU Lys Leu Leu Pro Asp Thr He Arg Arg Asp 585 590 595 600 His Glu Gln Leu Leu Asn Leu Gly Leu Glu Wing Gln Met Thr Glu Leu Gln Gln Ser Arg 605 610 615 620 Pro Thr Pro Gln Thr Gln Pro Arg Asp Thr Ser Ser Ala Lys Ser Ser Val Phe Glu Thr 625 630 635 60 Tyr Thr Leu Val Leu lie Wing Asp Ser Leu Ser Arg Asn lie Thr Lys Gly Val Lys Arg 645 650 655 660 Gly Val Asn Glu Ala He Met Leu His Leu Asp His Glu Val Arg His Ala He Gly Asn 665 670 675 680 Arg Leu Arg Gln Thr Gln Lys Asn He He Lys Ser Arg Leu Asp Glu Wing Leu Lys Glu 685 690 695 700 Be Thr Thr Gln Phe Thr Wing Gln Leu Thr Gln Thr Val Glu Asn Leu Val Lys Arg Glu 705 710 715 720 Leu Ala Glu Val Leu Gly Ser He Asn Gly Ser Leu Thr Ser Leu Val Lys Glu Asn Ala 725 730 735 740 Ser Leu Gln Lys Glu Leu Asn Ser He Met Ser Ser Gly Val Leu Asp Glu Met Arg Arg 745 750 755 760 Het Arg Glu Glu Leu Cys Thr Leu Arg Glu Ser Val Wing Lys Arg Lys Wing Thr Met Pro 765 770 775 780 Asp Being Ser Leu His Wing Thr Ser Being Phe Gln Gly Arg Arg Being Wing Pro Glu Thr He 785 790 795 800 Leu Ala Thr Ala Leu Ser Met Val Arg Glu Gln Gln Tyr Arg Gln Gly Leu Glu Tyr Met 805 810 815 820 Leu Met Ala Gln Gln Pro Ser Leu Leu Leu Arg Phe Leu Ser He Leu Thr Arg Glu Asn 825 830 835 840 Glu Asn Wing Tyr Ser Glu Leu He Glu Asn Val Gtu Thr Pro Asn Asp Val Trp Cys Ser 845 850 855 860 Val Leu Leu Gln Leu He Glu Ala Ala Ala Thr Glu Ala Glu Lys Glu Val Val Val Gly 865 870 875 880 Val Ala He Asp He Leu Ser Glu Arg Asp Gln He Wing Gln Asn Gly Wing Leu Gly Ser 885 890 895 900 Lys Leu Thr Thr Wing Met Arg Wing Phe Glu Arg Gln Wing Arg Ser Glu Thr Thr Ser Arg 905 910 915 Ser Phe Leu Gln Cys Leu Lys Asn Leu Glu Lys Leu Leu Gln Ser 1 INFORMATION FOR SEQ ID NO: 3 li SEQUENCE CHARACTERISTICS: liA LENGTH: 24 bases liB TYPE: nucleic acid liC NUMBER OF HEBRAS: simple liD CONFIGURATION: linear lii TYPE OF MOLECULE: DNA (phage) liii HYPOTHETICAL: no liv ANTI-SENSE: no lv TYPE OF FRAGMENT: lvi ORIGIN: synthesis oligonucleotide lvii IMMEDIATE SOURCE: bioMérieux S. A. lxi DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3 GGTGGCGACGACTCCTGGAGCCCG 24 1 INFORMATION FOR SEQ ID NO: 4 li SEQUENCE CHARACTERISTICS: LY LENGTH: 24 bases liB TYPE: nucleic acid liC NUMBER OF HEBRAS: simple liD CONFIGURATION: linear lii TYPE OF MOLECULE: DNA (phage) liii HYPOTHETICAL: no liv ANTI-SENSE: yes lv TYPE OF FRAGMENT: lvi ORIGIN: synthesis oligonucleotide lvii IMMEDIATE SOURCE: bioMérieux S. A. lxi DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4 TTGACACCAGACCAACTGGTAATG 24 1 INFORMATION FOR SEQ ID NO: 5 li SEQUENCE CHARACTERISTICS: LONG LENGTH: 18 LiB bases TYPE: liC nucleic acid NUMBER OF HEBRAS: simple liD CONFIGURATION: linear lii TYPE OF MOLECULE: DNA (genomic) liii HYPOTHETICAL: no liv ANTI-SENSE: no lv TYPE OF FRAGMENT: lvi ORIGIN: synthesis oligonucleotide lvii IMMEDIATE SOURCE: bioMérieux S. A. lxi DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 5 TCGGGCACTGACGCGGCG 18 1 INFORMATION FOR SEQ ID NO: 6 li SEQUENCE CHARACTERISTICS: LONG LENGTH: 24 bases liB TYPE: nucleic acid liC NUMBER OF HEBRAS: simple liD CONFIGURATION: linear lii TYPE OF MOLECULE: DNA (phage lambda gtlO) liii HYPOTHETICAL: no liv ANTI-SENSE: yes lv TYPE OF FRAGMENT: lvi ORIGIN: synthesis oligonucleotide lvii IMMEDIATE SOURCE: nioMérieux S. A. lxi DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6 CTTATGAGTATTTCTTCCAGGGTA 24 1 INFORMATION FOR SEQ ID NO: 7 li SEQUENCE CHARACTERISTICS: LiA LENGTH: 21 LiB bases TYPE: liC nucleic acid NUMBER OF HEBRAS: simple liD CONFIGURATION: linear lii TYPE OF MOLECULE: cDNA liii HYPOTHETICAL: no liv ANTI-SENSE: no lv TYPE OF FRAGMENT: lvi ORIGIN: synthesis oligonucleotide lvii IMMEDIATE SOURCE: bioMérieux S. A. lxi DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 7 AACGCTATTATTAGAACAGTT 21 1 INFORMATION FOR SEQ ID NO: 8 li SEQUENCE CHARACTERISTICS: LONG LENGTH: 18 liB bases TYPE: liC nucleic acid NUMBER OF HEBRAS: simple liD CONFIGURATION: linear lii TYPE OF MOLECULE: DNA (genomic) liii HYPOTHETICAL: no liv ANTI-SENSE: yes iv TYPE OF FRAGMENT: lvi ORIGIN: synthesis oligonucleotide lvii IMMEDIATE SOURCE: bioMérieux S. A. lxi DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 8 TGCAGCAGCGGCAGAAGT 18 /, '1 INFORMATION FOR SEQ ID NO: 9 li SEQUENCE CHARACTERISTICS: liA LENGTH: 22 liB bases TYPE: nucleic acid 5 liC NUMBER OF HEBRAS: simple liD CONFIGURATION: linear lii TYPE OF MOLECULE: DNA (genomic) liii HYPOTHETICAL: no 0 liv ANTI-SENSE: no lv TYPE OF FRAGMENT: lvi ORIGIN: synthesis oligonucleotide lvii IMMEDIATE SOURCE: bioMérieux S. A. lxi DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 9 0 CAGCCGACGGTAGCTGCGTCCT 22 1 INFORMATION FOR SEQ ID NO: 10 li SEQUENCE CHARACTERISTICS: liA LENGTH: 21 liB bases TYPE: liC nucleic acid NUMBER OF HEBRAS: simple liD CONFIGURATION: linear lii TYPE OF MOLECULE: DNA (genomic) liii HYPOTHETICAL: no liv ANTI-SENSE: yes lv TYPE OF FRAGMENT: lvi ORIGIN: synthesis oligonucleotide lvii IMMEDIATE SOURCE: bioMérieux S. A. lxi DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 10 ACATAATGGCCTCGTTCACAC 21 1 INFORMATION FOR SEQ ID NO: 11 li SEQUENCE CHARACTERISTICS: LONG LENGTH: 34 bases liB TYPE: nucleic acid liC NUMBER OF HEBRAS: simple liD CONFIGURATION: linear lii TYPE OF MOLECULE: cDNA liii HYPOTHETICAL: no liv ANTI-SENSE: yes lv TYPE OF FRAGMENT: lvi ORIGIN: synthesis oligonucleotide lvii IMMEDIATE SOURCE: bioMérieux S. A. lxi DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 11 GACTCGCTGCAGATCGATTTTTTTTTTTTTTTTT 34 1 INFORMATION FOR SEQ ID NO: 12 li SEQUENCE CHARACTERISTICS: LiA LENGTH: 21 LiB bases TYPE: liC nucleic acid NUMBER OF HEBRAS: simple liD CONFIGURATION: linear lii TYPE OF MOLECULE: DNA (genomic) liii HYPOTHETICAL: no liv ANTI-SENSE: no lv TYPE OF FRAGMENT: lvi ORIGIN: synthesis oligonucleotide lvii IMMEDIATE SOURCE: bioMérieux S. A. lxi DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 12 CGAAGAGACCATGAACAACTT 21 1 INFORMATION FOR SEQ ID NO: 13 li SEQUENCE CHARACTERISTICS: liA LENGTH: 18 liB bases TYPE: nucleic acid liC NUMBER OF HEBRAS: simple liD CONFIGURATION: linear lii TYPE OF MOLECULE: DNA liii HYPOTHETICAL: no LV ANTI-SENSE: yes lv TYPE OF FRAGMENT: Ivi ORIGIN: synthesis oligonucleotide lvii IMMEDIATE SOURCE: bioMérieux S. A. lxi DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 13 GACTCGCTGCAGATCGAT 18 BIBLIOGRAPHICAL REFERENCES The classic techniques of molecular biology have been carried out according to the protocols cited in: "Molecular cloning, a laboratory manual". Maniatis T., Fritsch, E. F. & Sambrook, J., Second Edition. Cold Spring Harbor Laboratory Press (New York) (1989). 1. Paranhos-Baccala, G., Santos, M., Cotrim, P., Rassi, A., Jolivet, M., Camargo, M. E. & Da Silveira, J. F. Detection of antibodies in sera from Chagas Disease patients using Trypanosoma cruzi immunodominant recombinant antigen. Parasite Immunology (1994): 16: 165-169. 2. Lafaille, J. J., Linss, J., Krieger, M.A., Padron, T.S., De Souza, W. & Goldenberg, S. Structure and expression of two Trypanosoma cruzi genes encoding antigenic proteins bearing repetitive epitopes. Molecular and Biochemical Para-sitology. (1989). 35: 127-136. 3. Young, R. A. & Davis, R. W. Efficient Isolation of genes by using antibody probes. Proc. Nati Acad. Sci. USA. (1983). 80: 1194-1198. 4. Cotrim, P.C., Paranhos, G., Mortara, R.A., Wan-derley, J., Rassi, A., Camargo, M.E. & Da Silveira, J. F. Expression in Escherichia coli of a dominant immunogen of Trypanosoma cruzi recognized by human chagasic sera. Journal of Clinical Microbiology. ( 1990 ). 28 (3): 519-524. 5. Sanger, F., Nicklen, S. & Coulson, A. R. DNA sequen-r < - cing with chain-terminating inhibitors. Proc. Nati Acad. Sci. USA. (1877). 74: 5463-5467. 6. Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T. Nature (1970). 227: 680-685. 7. Towbin, H., Staehlin, T. & Gordon, J. Electrophoretic transfer of proteins from polyacry lamide gels to. nitrocellulo- se sheets: procedure and some applications. Proc. Nati Acad. Sci. USA. (1979). 76: 4350-4354. 8. Smith, D. B. & Johnson, K. S. Single step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S transferase. Gene. (1988). 67: 31-40. 9. Voller, A., Draper, C, Bidwell, D. E. & Bartlett, A. Microplate enzyme-linked immunosorbent assay for Chagas design. Lancet (1975). 1 .: 426-428. 10. Huynh, T. V., Young, R. A. & Davis, R. W. DNA Cloning: A practical approach. (1984) (Ed. D. Glover) 49-78. IRL. Oxford 11. Parsons, M., Nelson, R.G., Watkins, K. P. & Agabian, N. Trypanosoma mRNAs share a common 5 'Spliced Leader sequence. Cell. ( 1984 ). 38: 309-316. 12. Frohman, M.A., Dush, M.K. & Martin, G. R. Rapid production of full length cDNA from rare transcripts: amplifications using a single gene specific oligonucleotide primer. Proc. Nati Acad. Sci. USA. (1984) 85: 8998-9002. 13. Nielsen, P. E., et al., Science, 254, 1497-1500 (1991). 14. Maniatis et al., Molecular Cloning, Cold Spring Harbor, (1982). 15. Southern, E.M., J. Mol. Biol., 98, 503, (1975). 16. Dunn, A.R., Hassel, J.A., Cell, L2, 23 (1977). 17. Yoshida, N., Surface antigens of metacyclic trypo-mastigotes of Trypanosoma cruzi, Infection and Immunity, 40, 836-839, (1983).

Claims (37)

1. CLAIMS 1. A molecule of ADf1 or RNA, characterized in that it is constituted by at least one strand, comprising an identical, complementary, anti-sense nucleotide sequence, or equivalent to the sequence identified in the identifier SEQ ID NOl, and mainly a sequence that presents, for all successions of 100 contiguous monomers, at least 50%, preferably at least 60%, or better yet, at least 85% homology with the referred sequence.
2. 2. A fragment of DNA or RNA, characterized in that it comprises an identical, complementary, antisense nucleotide sequence, or equivalent to the sequence starting at nucleotide 1232, and terminating at nucleotide 2207 of SEQ ID NOl, and principally, a sequence having, for any succession of 30 contiguous monomers, at least 50%, preferably at least 60%, or better still, at least 85% homology with the referred sequence.
3. 3. A fragment of DNA or RNA, characterized in that it comprises an identical, complementary, antisense nucleotide sequence, or equivalent to the sequence starting at nucleotide 1232 and ending at nucleotide 1825 of the sequence SEQ ID NOl , and mainly, a sequence that has, for all successions, 30 contiguous monomers, at least 50%, preferably at least 60%, or better yet, at least 85% homology with the referred sequence. Ir "
4. 4. A fragment of DNA or RNA, characterized in that it comprises an identical, complementary, anti-sense nucleotide sequence, or equivalent to the sequence starting at nucleotide 1266, and terminating at nucleotide 2207 of SEQ ID NOl , and mainly, a sequence that has, for all succesions of 30 contiguous monomers, at least 50%, preferably at least 60%, or better yet, at least 85% homology with the sequence referred to
5. 5. A protein substance, recognized by anti-sera or anti Trypanosoma cruzi, characterized in that it is selected from the group comprising: -a protein of apparent molecular mass of approximately 100 kDa, recognized by anti-Trypanosoma cruzi antisera 5 -all immunological equivalent of the aforementioned protein and any fragment of the referred protein
6. 6. A protein substance, according to claim 5, characterized in that it is selected from the group comprising: 0 - a protein having a sequence of amino acids, identified in the identifier SEQ ID N02 - all fragment of the referred protein.
7. 7. The protein fragment according to claim 6, characterized in that it comprises a sequence of 5 amino acids starting at amino acid 323, and ending at amino acid 520 of SEQ ID NO: 2, or any immunological equivalent of the reference fragment.
8. 8. A cassette for functional expression in a cell derived from a prokaryotic or eukaryotic organism, characterized in that it allows the expression of DNA encoding a protein fragment according to claim 7, or of a protein according to any of the claims 5 or 6.
9. 9. The expression cassette according to claim 8, characterized in that it is functional in the cells that come from a eukaryotic organism, mainly the cells that come from a mammal, the cells of insects, the cells that they come from a fungus, and the cells that come from a yeast.
10. 10. The expression cassette according to claim 8, characterized in that it is functional in the cells that come from a prokaryotic organism, mainly the cells that come from an E. coli strain, or the cells that come from Enterobacteriaceae.
11. 11. The expression cassette according to claim 10, characterized in that the cell that comes from a prokaryotic organism is a cell of the E. coli strain DH5a,
12. 12. The expression cassette according to any of claims 8 to 11, characterized in that the DNA encodes a protein recognized by anti-Trypanosoma cruzi anti-sera., which has the sequence identified in sequence identifier SL 'ID N02, which starts at amino acid +1, and ends at amino acid +915, or all immunological equivalent of the referred protein.
13. 13. The expression cassette according to any of claims 8 to 11, characterized in that the DNA encodes a Trypanosoma cruzi specific protein, having the sequence identified in the sequence identifier SEQ ID N02, which starts at amino acid 323 , and is terminated at amino acid 520.
14. 14. A vector, characterized in that it comprises an expression cassette according to any of claims 8 to 13.
15. 15. The cell that comes from a eukaryotic or prokaryotic organism, characterized in that it comprises an expression cassette according to any one of claims 8 to 13, or a vector according to claim 14.
16. 16. The method of preparing a protein according to claim 5 or 6, or of a protein fragment according to with claim 7, the method is characterized in that: (i) a cell that is derived from a cell is cultured under appropriate conditions. a prokaryotic or eukaryotic organism according to claim 15, and (ii) the protein produced by the cell originating from the dead organ is recovered.
17. 17. A peptide, characterized in that it comprises a peptide sequence belonging to the protein according to claim 5 or 6, or a protein fragment according to claim 7, and because it has a reactivity with sera from individuals or animals infected by T . cruzi
18. 18. A peptide composition, characterized in that it comprises a mixture of peptides, according to claim 17.
19. 19. Monoclonal or polyclonal antibodies, characterized in that they are obtained by immunological reaction of a human or animal organism with an immunogenic agent constituted by a protein according to claims 5 or 6, or with a protein fragment according to claim 7, or a peptide according to claim 17,
20. 20. A reagent for the detection and / or monitoring of a trypanosoma cruzi infection, characterized in that it comprises, as a reactive substance, at least one antibody according to claim 19.
21. 21. A reagent for detection and / or the surveillance of an infection by Trypanosoma cruzi, characterized in that it comprises, as a reactive substance, a protein according to claim 5 or 6, or a protein fragment according to claim 7, or a peptide according to claim 1. 17, or a peptide composition according to claim 18.
22. 22. A means for detecting and / or monitoring an infection by trypanosoma cruzi, characterized in that a reagent according to any of claims 20 or 21, fixed on a solid support, compatible with the reagent.
23. 23. A method for detecting and / or monitoring a trypanosoma cruzi infection in a biological sample, such as a blood sample from an individual or from an animal susceptible to having been infected with trypanosoma cruzi, the method is characterized in that the sample and a reagent are contacted in accordance with claims 20 or 21, under conditions that allow an eventual immunological reaction, and the presence of an immune complex with the reagent referred to is then detected.
24. 24. A probe for the identification of Trypanosoma cruzi, characterized in that it comprises a nucleotide sequence that allows hybridization with a nucleic acid whose nucleotide sequence corresponds to all or part of the sequence represented in the identifier SEQ ID NO1, or of the complementary sequence, anti-sense, or equivalent to the referred sequence, and mainly a sequence that presents, for all succession of 5 to 100 contiguous monomers, at least 50%, preferably at least 60%, or better still, at least 85% of the homology with the reference sequence
25. 25. The probe according to claim 24, characterized in that it comprises a sequence of 5 to 100, in particular of eight to 50 nucleotides,
26. 26. a therapy probe, for the treatment of infections due to Trypanosoma cruzi, characterized in that it comprises a nucleotide sequence that allows hybridization with a nucleic acid whose nucleotide sequence corresponds to all or part of the sequence represented in the identifier SEQ ID NO1, or of the complementary sequence, antisense, or equivalent to the referred sequence, and mainly a sequence having, for any succession of 5 to 100 contiguous monomers, at least 50%, of preference at least 60%, or better yet, at least 85% homology with the referred sequence.
27. 27. A bait for the amplification of a nucleotide sequence, characterized in that it comprises a nucleotide sequence that allows hybridization with a nucleic acid whose nucleotide sequence corresponds to all or part of the sequence represented in the identifier SEQ ID NOl, or of the complementary sequence , anti-sense, or equivalent to the referred sequence, and mainly a sequence that presents, for every sequence of 5 to 100 contiguous monomers, at least 50%, preferably at least 60%, or better yet, at least 85% of homology with the referred sequence.
28. 28. The bait of conformity with claim 27, characterized in that it comprises a nucleotide sequence of at least five nucleotides.
29. 29. The bait according to claim 28, characterized in that it has a nucleotide sequence selected from SEQ ID N07, SEQ ID N08, SEQ ID N09, SEQ ID NO.lO and SEQ ID N012.
30. 30. A pharmaceutical composition for preventing or treating infections due to Trypanosoma cruzi, characterized in that it comprises, as an active ingredient, a therapeutically active amount of a protein substance according to claim 5 or 6., of a protein fragment according to claim 7, of a catheter according to any of claims 8 to 13, of a vector according to claim 14, of a cell according to claim 15, or of an antibody according to claim 19.
31. 31. A reagent for detecting and / or identifying Trypanosin-nia cruzi in a biological sample, characterized in that it comprises a so-called capture probe and / or a detection probe according to claims 24 or 25, the capture probe and the detection probe, if both are present, respectively have at least partially different nucleotide sequences.
32. 32. The reagent according to claim 31, characterized in that the capture probe is fixed on a solid support, directly or indirectly.
33. 33. The reagent according to claim 31, characterized in that the detection probe is labeled by means of a marker selected from the radioactive isotopes, enzymes selected mainly between peroxidase and alkaline phosphatase, and those capable of hydrolyzing a chromogenic substrate, Fluorinated or luminescent, chromophoric chemical compounds, fluoride, luminescent compounds, nucleotide base analogs, and biotin.
34. 34. The reagent according to claim 31, characterized in that it comprises at least one bait according to any of claims 27 to 29.
35. 35. A method for detecting and / or identifying Trypanosoma cruzi in a biological sample, characterized in that the extracted and denatured DNA of Trypanosoma cruzi is exposed, or the DNA obtained by reverse transcription of the RNA, at least to a probe according to claim 24 or 25, and then the hybridization of the probe is detected.
36. 36. A method of detecting and / or identifying Trypanosoma cruzi in a biological sample, characterized in that the RNA extracted and optionally denatured Trypanosoma cruzi is exposed to at least one probe according to claim 24 or 25, and then detects the hi-bridation of the probe.
37. 37. A method according to claim 35 or 36, characterized in that, before exposing the DNA or RNA to the probe, an amplification of the DNA or RNA is carried out, in the presence of an appropriate enzymatic system, and at least with a bait according to any of claims 27 to 29.