BRPI1003744A2 - recombinant e-ntpdases, use in the production of diagnostic kit for detection of antibodies in leishmaniasis caused by species of the genus leishmania - Google Patents

Abstract

RECOMBINANT E-NTPDases, USE IN THE PRODUCTION OF DIAGNOSTIC KIT FOR DETECTION OF ANTIBODIES IN LEISHMANIOSIS CAUSED BY SPECIES OF THE GENUS Leishmania. This invention relates to proteins of the recombinant E-NTPDases family that can be used as antigens in diagnostic kits for animal and human leishmaniasis. The proteins of the recombinant E-NTPDases family obtained can be used in the production of diagnostic kits for the detection of antibodies in leishmaniasis, using immunological tests. These tests can be applied to detect seropositive individuals, in leishmaniasis control programs or in the laboratory diagnosis of leishmaniasis. These proteins can also be used in prognostic and vaccine assays, in the rational design of chemotherapy for the treatment of leishmaniasis and in the development of monoclonal antibodies to be used in the diagnosis, prognosis or therapy of leishmaniasis.

Description

"RECOMBINANT E-NTPDases, USE IN THE PRODUCTION OF DIAGNOSTIC KIT FOR DETECTION OF ANTIBODIES IN LEISHMANIOSIS CAUSED BY SPECIES OF THE LEISHMANIAN GENRE

Field of Application of the Invention

The proteins from the family of recombinant E-NTPDases obtained, subject to patent application, will be used in the production of diagnostic kits for the detection of antibodies in leishmaniasis, immunological tests. These tests can be applied to detect seropositive individuals in leishmaniasis field epidemiological control programs or in the laboratory diagnosis of leishmaniasis. These proteins may also be used in prognostic assays, vaccines, monoclonal antibody production and rational drug design for leishmaniasis chemotherapy.

State of the Art

Leishmaniasis is known to comprise a set of diseases whose etiological agents are species of the genus Leishmania, which are protists belonging to the Trypanosomatidae family. Transmission occurs through the bite of the female sand fly. Depending on the species of Leishmania involved in the infection and the type of host, leishmaniasis can be classically divided into two clinical forms: visceral and cutaneous or cutaneous. The untreated visceral form is usually fatal, while the cutaneous forms, although rarely evolving into fatality, are mutilating and unpleasant especially when there are multiple lesions. According to the World Health Organization (WHO) (WHO, 2009, http://whqlibdoc.who.int/hq/2007/WHO_CDS_NTD_IDM_2007.3_eng.pdf) leishmaniasis is a zoonotic disease that affects man, a variety of wild and domestic animals. It is an endemic disease in 88 countries on four continents. More than 90% of cutaneous leishmaniasis cases occur in Iran, Afghanistan, Syria, Saudi Arabia, Brazil and Peru, and more than 90% of visceral cases occur in Bangladesh, Brazil, India and Sudan. 2009, http://whqlibdoc.who.int/hq/2007/WHO_CDS_NTD_IDM_2007.3_eng.pdf) The number of cases of cutaneous leishmaniasis (LC) has been growing in Brazil in recent years (1988: 21,800 cases; 1999: 30,550 cases; 2000: 35,000 cases). This fact is related to the economic development and the change of habits and environment that expose the population more to the vectors of the disease. One of the main reservoirs currently described is the dog, which, due to its proximity to humans, actively participates in the process of maintaining leishmaniasis.

Canine leishmaniasis has become the focus of several studies considering the importance of the dog as a domestic animal and its relevance as a reservoir of the disease. However, the dog is not simply a reservoir of parasites, as it also presents the pathology associated with infection in both the cutaneous and visceral form.

One of the main actions employed to control canine leishmaniasis is still the elimination of the infected dog due to failure of antimonotherapy. Dog care is currently prohibited as there is no proven full efficacy with available drugs.

In clinical practice, in the face of suspected infection, dogs undergo several diagnostic tests that are often contradictory and do not always have a correlation with the disease state in the dog, making prognosis and consequent treatment difficult. This type of finding led to the development of more specific techniques based on the amplification of parasite DNA regions by Polymerase-PCR Chain Reaction (Francino, O., Altet, L., Sanchez-Robert, E., Rodriguez, A. , Solano-Gallego, L., Alberola, J., Ferrer, L., Sanchez, A., Roura, X., Veterinary Parasitology 137: 214-221, 2006). Although usually demonstrating high efficiency, the PCR assay has the disadvantage of high cost and flaws in DNA extraction. The diagnosis of the disease in dogs has been widely studied, but even today there is no diagnostic method considered as standard and each laboratory uses its own method of choice (Da Silva ES, Van Der Meide WF, Schoone GJ, Gontijo CMF, SchaIlig HDFH, Brazil RP, Veterinary Research Communications 30: 637-643, 2006). Given this new alternative, more efficient, discriminatory and rapid diagnostic methods are needed.

The approach of recombinant proteins to be used in diagnosis has been the focus of many researchers (S. Farajnia, B. Darbani, H. Babaei, H. Alimohammadian, F. Mahboudi and AM Gavgani, Parasitology 135, 1035-1041, Renato Porrozzi, Marcos V. Santos da Costa, Antonio Teva, Aloísio Falqueto, Adelson L. Ferreira, Claudiney dos Santos, Ana Paula Fernandes, Ricardo T. Gazzinelli, Antonio Campos-Neto and Gabriel Grimaldi, Jr., Clinical And Vaccine Immunology 14: 544-548,2007).

E-NTPDases are a specific family of ecto-nucleotidases, proteins that are generally described as localized or soluble (secreted) ecto membrane proteins whose primary function is the degradation of mainly extracellular, tri and diphosphate nucleotides (Zimmermann , H., Beaudoin, AR, Bollen, M., Belgium, Shaker Publishing; 1-9. 1999.)

Interest in these enzymes is based on literature data suggesting the participation of ecto-nucleotidases, among other things, in virulence, infectivity and purine acquisition in parasites (Barrêdo-Pinho, M., Peres-Sampaio, CE, Chrispim, PPM, Belmont-Firpo, R., Lemos 5. P. P., Martiny, A., Vannier-Santos, A., Meyer-Fernandes, J. Archives of Biochemistry and Biophysics, 391: 16-24, 2001). Recently, studying the experimental infection of C57BL / 6 mice with L. (L.) amazonensis and L. (V.) braziliensis, both causes of cutaneous leishmaniasis, it was found that the animals were able to control L. infection ( V.) braziliensis, but not by L. (L.) amazonensis. The difference in response to infection was related to an anergic state observed in animals infected with L. (L.) amazonensis (Maioli, TU, Erica, T., Arantes, RME, Fietto, JLR, Afonso, LCC, Parasitai Res 94: 207-212, 2004). In addition, it was found that intact L. (L.) amazonensis cells were able to hydrolyze a larger amount of adenosine extracellular nucleotides. Considering that extracellular ATP is a proinflammatory molecule and that one of its degradation products, adenosine has an immunosuppressive effect, this difference could then lead to an anti-inflammatory and immunosuppressive effect, as observed (Maioli, T. U, Erica, T., Arantes, RME, Fietto, JLR, Afonso, LCC, Parasitic Res 94: 207-212, 2004). This was the first evidence of the involvement of ecto-nucleotidases of a parasite in controlling the host immune response.

In the search made with the National Institute of Industrial Property - INPI, patent applications were found in the areas of immunotherapy and immunodiagnosis, but none of these patents use products related to the object of this patent. In this search was found the patent document PI 9406509, which refers to "COMPOUNDS, PROCESS, THEIR PREPARATION, USE OF THE SAME, PHARMACEUTICAL COMPOSITIONS AND PROCESS FOR PROPHYLAXIS AND FOR THERAPEUTIC TREATMENT OF MALARIA AND DISEASES CAUSED BY LEISHMANNEUM POCKYUM, TOXOPYMONAUM, TOXOPHERY "where pyrrol amidines of general formula and the pharmaceutically acceptable salts thereof are described, processes for the preparation and pharmaceutical compositions containing them useful as antiviral and antiparasitic. This document then addresses the use of pharmaceutical formulations derived from pyrrol amidine compounds for treatment and prevention of leishmaniasis. The subject of the patent application in question is different from the patent described in that it uses recombinant protein to be used in diagnosis, prognosis, monoclonal antibody development, vaccination and development of chemotherapy for leishmaniasis. There is no interrelation between them. However, it is important to note that in the case of infectious diseases it is desirable to have alternative methods of diagnosis, prevention and treatment as there is a very wide diversity of response to chemotherapy and vaccination of host populations, as well as different susceptibility to drugs and production of protective immune response to parasites.

Another deposit found was PI 9807332 which deals with "LEISHMANIA ANTIGENS FOR USE IN LEISHMANIASIS THERAPY AND DIAGNOSIS" where compositions and processes for preventing, treating and detecting leishmaniasis and stimulating immune responses in patients are demonstrated. The present invention has no overlap with this patent, although both aim for common applications. The antigens disclosed in PI9807332 are distinct from the antigenic proteins of this patent. The application in the same area is justified as described in the previous item, in addition, the patent itself emphasizes the need to develop alternatives in the diagnosis, treatment and prophylaxis of leishmaniasis that are caused by more than 20 different species of Leishmania.

Deposit No. 2133236A1 made at the Bank of Spain refers to "GEN CHEMICAL FORMED BY DNA SECURITIES CODING THE DETERMINANTS OF L. INFANTUM PROTEINS, APPLICABLE TO ELOLOGICAL DIAGNOSIS CANINEINA PROTEIN YANENIENIS" We describe the antigenic determinants of four L. infantum proteins (LiP2a, LiP2b, LIP2A, LiPO), which were applied in the diagnosis of Leishmaniasis. The objects of this patent are proteins distinct from those being patented in this process, so as previously justified although the application may be the same the antigens are different. In the case of Leishmaniasis, as stated above, it is important to have alternative proteins to be used in diagnosis, prophylaxis and treatment, as there is a great diversity of parasites and hosts.

It is a first object of the present invention to provide proteins from the recombinant E-NTPDase family of three Leishmania species (Leishmania major, Leishmania braziliensis, Leishmania infantum) homologous to their natural protein for use as an antigen in leishmaniasis diagnostic kits. .

It is also an object of the present invention to use said proteins and variants thereof, modified in their amino acid sequences, which retain their antigenic properties, for use in diagnosis, vaccine compounds, antibody development, prognostic markers and rational design of inhibitory drugs. E-NTPDases that can be used in leishmaniasis chemotherapy.

The development of antigenic compounds capable of increasing the efficiency in diagnosis and effectiveness as a vaccine agent has been pursued in the general scope of leishmaniasis research. Recombinant GDPase protein, as seen in Figure 5, points to as a potent antigen. The antigen, when used in ELISA diagnostic tests, was able to satisfactorily discriminate serum from dogs infected with Trypanossoma cruzi from those contaminated with Leishmania sp. Thus investment in this recombinant antigen as a target for diagnostic kit production as well as for other applications is very promising.

Description of the Invention

The first object of the present invention is achieved by recombinantly expressed E-NTPDase family proteins, the sequences being SEQ. ID NO.1, SEQ. ID No.2, SEQ. ID NO.3, SEQ. ID No. 4, SEQ. ID No. 5, SEQ. ID No. 6, SEQ. ID No. 7, SEQ. ID No. 8, SEQ. ID No. 9, SEQ. ID No. 10, SEQ. ID No. 11 and SEQ. ID No. 12 used as antigen.

The second object of the present invention is to use said proteins and variants thereof, modified in their amino acid sequences, which retain their antigenic properties, for diagnostic use, vaccine compounds, prognostic marker, monoclonal antibody production and development of chemotherapeutic agents. the leishmaniases.

The present invention will be described in detail below. Figure 1.1 illustrates the schema of the pET 21b expression vector for Escherichia coli expression system and the DNA expression cassette.

Figure 1.2 - illustrates the DNA insert, in this case the gene gi_ | 154872131, which was cloned between the BamHI and SalI restriction sites.

Figure 2.1 - illustrates the comparison of E-NTPDase sequences of species of the Leishmania genus. The regions marked with yellow frames represent the conserved regions of the E-NTPDases.

Figure 2.2 - Same as Figure 2.1, continued.

Figure 3 - Part A illustrates a 10% polyacrylamide gel (SDS-PAGE) photo showing proteins from a plasmid transfected E. coli BL21 (DE3) lysate containing the Leishmania major GDPase gene expressed at different times. The first column shows a recombinant Trypanosome eruzi NTPDase protein. The next 8 lines show bacterial lysate products after 2 to 4 h of IPTG induction at concentrations of 1 mM, 0.8 mM, 0.6 mM, 0.4 mM, 0.3 mM, 0.2 mM 0.1 mM and without induction respectively. The numbers correspond to those shown by molecular mass marker (kDa). Part B illustrates a Western blot result showing the E. coli lysate major Leishmania GDPase protein reacting with rabbit antibodies immunized with recombinant T. cruzi NTPDase. Lines 1 to 8 show the reaction of GDPase protein with serum from T. cruzi NTPDase immunized rabbits. Line 9 shows no bands when there was no induction of recombinant GDPase protein. The arrow indicates the reaction of the protein with the antibodies showing a band of approximately 78 kDa.

Figure 4 - Illustrates a 10% polyacrylamide gel (SDS-PAGE) photo showing the Leishmania major GDPase protein expressed in E. coli BL21 (DE3) after nickel agarose affinity chromatography purification. Recombinant Leishmania major GDPase protein can be observed in columns 10 and 11 after being recovered in buffer containing 80 mM and 160 mM imidazole respectively. The numbers correspond to those shown by molecular mass marker (kDa). The recombinant Leishmania major GDPase protein has 78.2 kDa.

Figure 5 - Graphic illustrating the ELISA immunological diagnostic result in which the purified Leishmania major recombinant GDPase protein at concentrations of 1.5, 3, 4.5 and 30 μg / ml was used as antigen and the total Leishmania extract was used as a control. sp. Lysate at a concentration of 3 μg / ml, referenced by numbers 1, 2, 3, 4 and 5 respectively. Eight sera from domestic dogs (Canis familiaris) were tested at a 1:80 dilution. Three sera from dogs known to be positive for Leishmania sp. (L +), 3 dog sera known to be negative for Leishmania sp. (L-) and 2 dog sera known to be positive for T. cruzi infection.

Figure 6 - Graphic illustrating the result of immunoassay by ELISA using the recombinant Leishmania major GDPase protein as antigen at a concentration of 1.5 μg / ml. A total of 169 dog sera samples were used, being 117 known to be positive and 52 known to be negative.

Detailed Description of the Invention

The present invention relates to the recombinant E-NTPDase family proteins of Leishmania major, Leishmania braziliensis and Leishmania infantum, obtained from the nucleotide sequence of the coding genes of these proteins with modifications intended for antigenic applications, which following primer preparation steps for amplification of the target genes, amplification of the coding genes of these proteins, and expression vector cloning, is finally expressed and further purified by nickel affinity chromatography. Said recombinant proteins thus purified can be used satisfactorily in ELISA and Western blot immunodiagnosis, as demonstrated by the use of the recombinant L. major GDPase isoform applied to the diagnosis of Canine Leishmaniasis.

The present invention relates to recombinant E-NTPDase family proteins identified as SEQ. ID NO.1, SEQ. ID No.2, SEQ. ID NO.3, SEQ. ID No. 4, SEQ. ID No. 5, SEQ. ID No. 6, SEQ. ID No. 7, SEQ. ID No. 8, SEQ. ID No. 9, SEQ. ID No. 10, SEQ. ID No. 11 and SEQ. ID No. 12 which may be used as antigens in the diagnosis of leishmaniasis.

SEQ. ID No. 2 derived from the L. major GDPase coding gene, has an amino acid identity of 93.74% relative to the natural Leishmania major GDPase protein when compared to the 690 amino acid portions available in GenBank predicted by access gij 154872131. In addition, L. infantum's GDPase (SEQ ID NO: 6) and L. braziliensis (SEQ ID NO: 4) exhibit an identity of 84.49% and 65.75% respectively of the recombinant L. major GDPase. , as shown in Table 1. This high identity shows that they are similar proteins in the same family. This similarity is sufficient for cross-immunological reactivity and it is possible to use all these variants in the same applications described for GDPase, ie diagnosis, prognosis and vaccination for leishmaniasis. The same is true for variants called nucleoside diphosphatases (NTPDases, described as SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 12), which despite having comparatively lower identities (Table 1) have conserved domains, as shown. in Figures 2.1 and 2.2. Thus, recombinant NTPDases are also the subject of this patent application and may be used in the same applications, namely, diagnosis, prognosis, vaccination, monoclonal development and chemotherapy for leishmaniasis.

Table 1 - Relation of Similarity and Identity of the amino acid sequence of the modified recombinant L. major GDPase protein to the other amino acid sequences of the E-NTPDases of different Leishmania species.

<table> table see original document page 9 </column> </row> <table>

Numbers from 1 to 7 horizontally represent the respective vertical proteins. Where the numbers intersect provides the identity in the upper quadrant and similarity in the lower quadrant.

The natural GDPase gene from Leishmania major, Leishmania braziliensis and Leishmania infantum are available from GenBank under the accessions giJ15487213j, XP OO1562788, XP OO1463665 respectively. The major Leishmania gene has a 2073 nucleotide sequence that encodes a 690 amino acid protein. The Leishmania braziliensis gene has a sequence of 2076 nucleotides that encodes a 691 amino acid protein. The Leishmcmia infantum gene has a sequence of 2034 nucleotides that encodes a 677 amino acid protein. The recombinant nucleotide sequences in relation to the natural ones present modifications in the aminoterminal and carboxiterminal portion. To the aminoterminal portion was added 42 base pairs. In the carboxiterminal portion the last three nucleotides (stop codon) were removed and 45 base pairs added. Predicted recombinant proteins from Leishmania major, Leishmania braziliensis and Leishmania infantum, have 719, 720 and 706 amino acids respectively (SEQ. ID NO: 2, SEQ. ID NO: 4 and SEQ. ID NO: 6).

The natural NTPDase gene from Leishmania major, Leishmania braziliensis and Leishmania infantum are available from GenBank under the accessions XP 001681917, XP OO1562178, XM 001464304 respectively. Both species have a sequence of 1278 nucleotides encoding 425 amino acids. The sequence of recombinant nucleotides in relation to the natural one presents modifications in the aminoterminal and carboxiterminal portion. To the aminoterminal portion was added 42 base pairs. In the carboxiterminal portion the last three nucleotides (stop codon) were removed and 45 base pairs were added, except in Leishmania major that only 39 base pairs were added. This difference is due to the use of different restriction sites used in the gene cloning process. Predicted recombinant proteins from Leishmania major, Leishmania braziliensis, and Leishmania infantum have 452, 454, and 454 amino acids respectively (SEQ. ID NO: 8, SEQ. ID NO. 10 and SEQ. ID NO. 12).

The present invention further relates to the use of recombinant GDPase protein in a diagnostic kit for leishmaniasis caused by species of the genus Leishmania, comprising recombinant E-NTPDases, as mentioned above, which are capable of interacting with specific antibodies in serum samples. or blood or other liquids or specimens from individuals being able to indicate infection in these human or animal individuals.

In the following, demonstration experiments will be described which will illustrate the potentiality of the present invention. Briefly, these experiments are based on a sample containing Leishmania major that was subjected to extraction of its genomic material to isolate the specific gene encoding GDPase. Subsequently, PCR amplification of the specific gene was performed and cloned into a cloning and expression vector, which was used to transform Escherichia coli cells that started to express the protozoan recombinant GDPase protein. Leishmania major GDPase protein, after purification, was used as an antigen in ELISA tests on dog sera to screen for IgG class antibodies against Leishmania species. This recombinant Leishmania major GDPase protein, produced in large quantities, can be widely distributed to laboratories throughout the country and thus contribute significantly to the serological diagnosis of leishmaniasis in Brazil or abroad, or be used in kits and applications. immunological and antibody production. All of the processes described herein also extend to the other E-NTPDase isoforms of the other Leishmania species described in the SEQ sequences. ID NO.3, SEQ. ID No. 4, SEQ. ID No. 5, SEQ. ID No. 6, SEQ. ID No. 7, SEQ. ID No. 8, SEQ. ID No. 9, SEQ. ID No. 10, SEQ. ID No. 11 and SEQ. ID No. 12 which may also be used as antigens in the diagnosis of leishmaniasis.

Demonstration Experiments

Isolation and cloning of Leishmattia major GDPase coding region

The strain used for genomic extraction was MHOM / IL / 80 / Friedlin de Leishmcmia major. For extraction of genomic DNA (gDNA) approximately 1x10 ^ 8 cells were harvested by centrifugation at 13,000 χ g for 2 to 3 min, discarding the supernatant was added 500 to 700 μl of phenol equilibrated to the pellet and left at room temperature ( RT) for 5 to 10 min. Subsequently, 200 to 400 μl of sterile 10 mM Tris (pH 8.0) was added and vortexed. The homogenate was centrifuged at 13,000 χ g for 10 to 12 min and the aqueous phase recovered without the interface. To this was added 200 to 400 μl of a phenol: chloroform: isoamyl alcohol mixture (25: 24.1) and vortexed. The homogenate was centrifuged at 13,000 χ g for 10 to 12 min and the aqueous phase recovered without the interface. To this was added 200 to 400 μl of chloroform and vortex homogenized. It is then centrifuged at 13,000 χ g for 10 to 12 min and the aqueous phase recovered as previously described. Then 1/10 volume of a 3 M sodium acetate solution (pH 5.2) and two volumes of 100% ice cold ethanol were homogenized without vortexing. The homogenate was allowed to precipitate for 2 to 3 h in the -80 ° C freezer and then centrifuged at 13,000 χ g for 20 to 30 min at 4 ° C. The supernatant was discarded by adding 250 to 500 μl of 70% cold ethanol to the precipitate and the sample was centrifuged at 13,000 χ g for 5 to 10 min at 4 ° C. The supernatant was discarded and the pellet placed to dry with the vial lid open. Subsequently, the dried DNA pellet was suspended in 50 to 60 μl of 10 mM sterile Tris (pH 8.0). Genomic DNA was stored at -20 ° C and used for isolation of the GDPase gene coding region (gij 154872131) containing 2073 base pairs by polymerase chain reaction (PCR).

For this purpose, the 5'CGGGATCCCATGCCAATGACTGACG3 'direct oligonucleotide was annealed to the Leishmania major genome and inserts a restriction site for BamH I at the 5' end for later insertion into the pET-21b bacterial expression vector and reverse oligonucleotide 5'GCGTCGACGGAGAGAGAGGAGGAGAGAG , which creates a site for Sal I at the end of the coding region. For gene amplification a thermal cycler (Programmable Thermal Controller-PTC-100 ™) was used. The reaction was performed in a total volume of approximately 50 μl using 4 μl of gDNA; 5.0 μl of 1.5 mM MgSO4 buffer (provided by Taq DNA Polymerase manufacturer); 2.4 μl of the 2.5mM dNTPs mix (dCTP, dTTP, dGTP, dATP); 2 μl of oligonucleotides at a pmol / UL concentration; 0.5 μL Taq DNA Polymerase and sterile ultrapure water qsp. The program used consisted of 33 cycles: 94 ° C for 4 min; 94 ° C for 30 sec; 50 ° C for 1 min; 72 ° C for 1 min; 72 ° C for 5 min; 4 ° C until removed from the tubes. The end products of the PCR reactions were analyzed by 1% agarose gel electrophoresis in IX TAE (1 mM EDTA; 40 mM Tris-Acetate; in deionized water) and stained in ethidium bromide solution (Sambrook J., Fritsch EF and Maniatis T., 1989), to verify the formation of a majority band corresponding to the target amplicon size of approximately 2.1 kb. The electrophoresis was performed at approximately 80 V, using as a running buffer TAE. The about 2.1 kb band was purified from the agarose gel using the PureLink Quick Gel Extraction Kit (invitrogen) as per manufacturer's instructions. The TOPO system (Invitrogen1 M) was used for the initial subcloning of the gene.

The amplicon band was suspended in a total volume of approximately 20 μL sterile ultra pure water and 5 to 10 μL of this solution was used for binding at approximately 16 ° C for 10-12 h according to the manufacturer's manual ( TOPO® TA Cloning® Kit for Subcloning - Invitrogen). The ligation mixture (~ 6 μL) containing GDPase TOPO + amplicon vector was added to approximately 100 μL of E. coli TOP 10F 'bacteria-containing solution previously made competent (Sambrook J., Fritsch EF and Maniatis T., Cold Spring Harbor Laboratory Press, 1989). The mixture was incubated on ice for 45 to 60 min, heat shocked (42 ° C) for 1 to 5 min and placed back on ice. 1 to 5 mL of LB-Liquid (1% Tryptone, 0.5% Yeast Extract, 1% NaCl, pH 7.5) was added followed by incubation at 37 ° C for 40 min with shaking of 180 rpm in Shaker TE- 420. The total volume of this mixture was divided into three aliquots and plated on solid LB-Agar media (1% tryptone, 0.5% yeast extract, 1% NaCl, 1.5% select Agar pH 7.5) containing ampicillin (5 to 10 mg / ml "1) and incubated for 12 to 14 h at 37 ° C.

The ampicillin resistant clones were recovered and used to confirm the presence of the plasmid containing the gene of interest by PCR and restriction analysis (using BamH I and Sal I) from the plasmid miniprep extraction plasmid according to (Sambrook J., Fritsch EF and Maniatis T., Cold Spring Harbor Laboratory Press, 1989). One of the isolated and confirmed clones was then subjected to partial automatic sequencing using flanking primers from the Mega-Bace cloning region according to the manufacturer's instructions. Clones containing the construct were stored in LB-liquid medium in 20% glycerol microcentrifuge tubes and kept at -80 ° C.

Plasmid DNA Extraction

Plasmid DNA (pDNA) was extracted by the alkaline lysis method (Sambrook J. Fritsch E.F. and Maniatis T., Coid Spring Harbor Laboratory Press, 1989) as described below. For this, the positive transformation clones with the TOPO vector were grown in individual screw tubes containing 3 mL LB and 30 μl ampicillin (5 to 10 mg / mL "1) for 12 to 14 h at 37 ° C at 180 ° C. At 200 rpm After growth a volume of 1.5 ml of each culture was transferred to microcentrifuge tubes, and the cells were collected by centrifugation (Centrifuge 5415C-EPPENDORF) at 12000 χ g for 1 to 5 min.

The cell pellet was washed with 0.5 to 1 mL STE (0.1 NaCl, 10 mM Tris.Cl, 1 mM EDTA, pH 8.0) to remove endonuclease inhibitors. After centrifugation, ice-cold 200 to 300 μl of solution 1 (25 mM Tris.Cl pH 8.0, 10 mM EDTA and 5 mM D-Glucose) were slowly and slowly suspended and completely pelleted. Then 200 to 300 μl of the freshly prepared Π solution (0.2M NaOH, 1% SDS) were added, and the tubes were slowly inverted for 6 to 10 times and kept on ice for 5 to 10 min. Thereafter, 150 to 300 μl of ice cold solution III (3 M Kac, 2 M Hac pH 8.0) was added and the tubes were slowly inverted 6 to 10 times and kept on ice for 5 min. Then the tubes were centrifuged for 12000 χ g for 5 to 10 min and the supernatants were transferred to other new tubes where 5 μl RNAse A (10 μg / μl) was added and the tubes were kept at 37 ° C in a water bath for 10 minutes. 30 to 50 min. To each tube was added 550 to 700 μί of phenol: chloroform (1: 1), and the tubes were slowly inverted for about 5 to 10 times and centrifuged at 12000 χ g for 5 to 10 min. This procedure was repeated once more. The aqueous phase was transferred to new tubes. Then 550 to 700 μl of chloroform was added and centrifugation was performed again at 12000 χ g for 5 to 10 min. The upper phase was transferred to new tubes, where 550 to 700 μl of cold isopropanol was added. The tubes were slowly inverted and kept at -70 ° C for 10 to 20 min. Then the samples were centrifuged at 12000 g for 5 to 10 min, the supernatant was discarded and the tubes were placed to dry for 30 to 50 min on paper towels. The precipitate was washed with 200 to 400 μl of 70% ice cold ethanol and centrifuged at 12000 χ g for 5 to 10 min. The supernatant was discarded and the tubes were placed to dry for 30 to 50 min. After drying, the plasmid DNA was suspended in 20 to 30 μl of deionized H2O. Clones were selected by restriction analysis using BamH I and Sal I and confirmed by partial sequencing.

Transfer of recombinant GDPase protein coding region to pET-21b prokaryote expression vector

The TOPO amplification vector containing the gene was submitted to BamH I and Sal I digestion. After digestion, the TOPO vector mixture was submitted to 1% agarose gel electrophoresis.

The insert for the entire GDPase coding region was purified with the "PureLink Quick Gel Extraction" kit (invitrogen) and ligated, analogously to the subcloning in TOPO, into the vector pET-21b previously digested with the same enzymes and purified by the same enzyme. same kit. Figure 1.2 shows a schematic of the pET-21b-GDPase construct.

E. coli TOP 10F 'bacteria, previously made competent as described above, were transformed with the ligation product and incubated in LB liquid medium as described above (isolation of the TOPO-containing clone). Clone selection was done by restriction analysis and sequencing. Positive clones were stored in microcentrifuge tubes containing 15 to 30% glycerol and kept at -70 ° C.

Expression of Recombinant GDPase Protein and SDS Gel Analysis Total expression of recombinant proteins was averaged over 250 ml LB. The expression cassette of the pET-21b vector, highlighting the cloning sites and sequence of 6 histidine residues in the carboxy-terminal portion of the recombinant protein, is outlined in Figure 1.2.

Competent bacteria of the E. coli BL21 strain (DE-3) were transformed with the pET-21b-GDPase construct, similar to that performed with the TOPO vector. The Gij Gene 15487213 | encodes an mRNA for the production of a 78.2 kDa protein whose predicted amino acid sequence is described in SEQ. ID No. 2.

A total of 45 extra nucleotides, corresponding to 15 additional amino acids at the carboxy terminal end of the recombinant protein, were added to the final protein by binding the target gene to the pET-21b vector, which encodes 9 binding amino acids between GDPase protein and hexahistidine tail plus 6 histidines. In the aminoterminal portion a total of 14 amino acids were inserted.

Then, a inoculum was pre-inoculated in 5 to 7 mL of LB liquid medium containing 50 µl of ampicillin (5 to 10 mg / mL "1) from the glycerol preserved clone. This pre-inoculum was incubated for 16 at 20 h at 37 ° C. A 2.5 to 5 mL aliquot of culture was collected and inoculated into a total volume of 250 mL LB-liquid containing 2.5 mL ampicillin (5 to 10 mg / mL "). 1). This mixture was incubated at 37 ° C under shaking and cell growth was followed by reading its OD (optical density). For induction of the modified recombinant protein IPTG (isopropyl β-D-thiogalactopyranoside) was used. By the time DOeoo reached about 0.6 to 0.8, approximately 0.3 mM IPTG was added to the tubes of each clone. Incubation was continued for 2 to 4 h at 37 ° C under shaking.

After induction the culture was centrifuged 3000 χ g and the pellet obtained was kept on ice for 15 to 30 min and suspended in lysis buffer (50 mM Tris (pH 8.0), 300 mM NaCl in distilled water), in the proportion of 3 to 5 mL per gram of pellet containing 30 to 50 μί PMSF (100 mg / mL), 50 to 100 μL aprotinin (1 mg / mL), 50 to 100 μl pepstatin (1 mg / mL). Then 1 to 5 mg / ml lysozyme was added and kept on ice for 30 to 50 min.

The cells were then finally disrupted by sonication 6 to 10 times for 10 to 20 seconds at 200 W power with 10 to 20 seconds on ice between each sonication. Then 0.1% Triton was added and the material was centrifuged for 30 to 50 min at 20400 χ g at 4 ° C, and both the pellet (insoluble fraction) and the supernatant (soluble fraction) were stored at -20 ° C. for further purification of the recombinant protein.

To determine the best IPTG concentration to be used as the standard for recombinant protein expression, colonies were inoculated into 4 mL of LB liquid medium containing 40 μL ampicillin (5 to 10 mg / ml / 1) and incubated at 37 ° C. for 12 to 14 h under stirring. An aliquot of 250 to 500 μl of culture was collected and diluted to a total volume of 5 to 10 mL in 11 tubes. This mixture was incubated at 37 ° C under shaking and cell growth was followed by reading its OD600.

When the culture reached DOeoo of approximately 0.6 to 0.8, 1 to 5 mL of the culture was aliquoted (uninduced control), then IPTG at a serial concentration of 0.1 was added to the other tubes; 0.2; 0.3; 0.4; 0.5; 0.6; 0.7; 0.8; 0.9 and 1 mM. Incubation was continued for 2 to 4ha37 ° C under shaking. At the end of this time 1 mL of each culture was collected (induced controls) and DO600 read. Samples (induced and uninduced controls) were centrifuged, the supernatant discarded and the bacterial pellet mixed with SDS-PAGE sample buffer (62.5 mM Tris.HCl pH 6.8, 10% SDS, 0.01% bromophenol blue (ABF), 10% glycerol and 20 mM β-mercaptoethanol). The samples were heated at 100 ° C for 5 to 10 min. Bacterial lysates were analyzed by 10% SDS-PAGE according to Laemmli, U. K., Nature. 227 (259): 680-5, 1970. The small (1mm-thick) Mini-PROTEAN® vertical electrophoresis system was used. The samples were applied in DOeoo 3.0 to 5.0, for a total volume of 20 μl per channel, and after electrophoresis the gel was stained with Comassie blue. The molecular weight standard used was the PageRuler ™ Unstained Protein Ladder (Figure 3, part A).

Purification of Recombinant GDPase Protein and SDS Gel Analysis

For purification of the recombinant protein, supernatant (soluble) samples were employed. These samples were kept under agitation in the presence of 500 to 700 μL of HIS-Selectm Nickel AfFinity Gel resin properly prepared according to the manufacturer in 15 mL tubes for 1 to 3 hours at 4 ° C. Subsequently, the material was passed through a column containing glass wool at the lower end to prevent resin loss, and non-column bound proteins were recovered in the separate volume of resin. Flow was dependent only on atmospheric pressure and was not standardized.

After all visible liquid material was removed from the column, manually prepared by immobilizing the affinity resin to a 10 mL syringe, 2 to 4 mL of Wash Buffer 1 (50 mM Tris pH 8.0, 300) was added to it. mM NaCl 2, 5 mM MgCl 2). This procedure was repeated 2 to 4 times. Then a volume of 2-4 ml wash buffer 2 (50 mM Tris pH 8.0, 300 mM NaCl 2, 5 mM Imidazole, 5 mM MgCl 2) was added. This procedure was repeated 2 to 4 times. Then 2 to 4 mL volume of wash buffer 3 (50 mM Tris pH 8.0, 300 mM NaCl 2, 10 mM Imidazole, 5 mM MgCl 2) was added. This procedure was repeated 2 to 4 times. Subsequently the proteins most strongly bound to the resin were eluted in elution buffer. A 1.5 to 3 mL volume of elution buffer 1 (50 mM Tris pH 8.0, 300 mM NaCl 2, 80 mM Imidazole, 5 mM MgCl 2) was added. Upon completion of this elution, a volume of 1 to 3 mL of elution buffer 2 (50 mM Tris pH 8.0, 300 mM NaCl2, 160 mM Imidazole, 5 mM MgCb) and finally a volume of 1 to 3 mL of elution buffer 3 (50 mM Tris pH 8.0, 300 mM NaCl 2, 250 mM Imidazole, 5 mM MgCl 2) proceeded as the first. The elution process 3 was repeated one more time. Samples from all purification steps were collected in microcentrifuge tubes and stored at 4 ° C and analyzed on Comassie blue stained 10% SDS gel. The samples were applied in a total volume of 20 μl (Figure 4).

Western blot

Several samples of the purification process were subjected to 10% SDS-PAGE electrophoresis and transferred under electric current to nitrocellulose membrane under the condition of transfer of 200-250 mA for 2 to 4 h in Mini-PROTEAN ^ 3 Cell vertical transfer system. Assembly Guide. After transfer, the membrane was stained with Ponceau S (Ponceau 0.2% in 1.0% acetic acid) for 10 to 30 min for visualization of protein bands and marking of the region of the molecular mass pattern bands. Then the membrane was bleached with distilled water and placed in blocking solution incubated for 1 to 3 hours at room temperature using TBS-T buffer (0.01 M Tris; 0.14 M NaCl; 0.1% Tween 20). ) containing 5% skimmed milk.

Subsequently the membrane was rapidly washed in TBS buffer (0.01 M Tris; 0.14 M NaCl) and then incubated with the T. cruzi 1.1000 anti-rNTPDase primary antibody diluted in TBS-T buffer by about 16 to 20 h at 100 rpm. Subsequently, the membrane underwent 3 to 6 washes with TBS-T buffer for 5 to 10 min under constant constant agitation. The membrane was then incubated with peroxidase-conjugated rabbit anti-IgG secondary antibody at a dilution of 1: 20,000. The membrane was allowed to stir at 100 to 200 rpm for 1 to 3 hours. Then the membrane went through another 3 to 6 washes with TBS-T buffer for 5 to 10 min under the same stirring conditions. Developing was performed with diaminobenzidine substrate (DAB) in a solution containing 50 mM Tris (pH 7.6); 10 mg DAB; 10 μί H2O2 (20%) in the absence of light for approximately 10 min. After development, the membrane was removed with forceps, the reaction was stopped with water, dried under paper towels and the image was digitalized (Figure 3, part B).

Indirect ELISA Assays Using Recombinant L · major GDPase as Antigen

In ELISA experiments for reactivity analysis of purified recombinant protein 8 sera from previously diagnosed dogs were used, being 3 positive sera from dogs infected with Leishmania sp., 3 negative animal sera and 2 positive sera from dogs infected with T. cruzi.

For rGDPase adsorption, micro-dilution plates were exposed to a solution containing 1.5, 3, 4.5 and 30 μg of purified recombinant GDPase protein in 0.1 M carbonate / bicarbonate buffer pH 9.6 and incubated overnight at 4 ° C. . Non-specific sites were blocked by incubating the plate with blocking solution containing 5% fetal bovine serum (SFB) in PBS for one hour at 37 ° C. Subsequently, the plate was washed 3 to 6 times with PBS-T (0.05% Tween-20 PBS) to remove excess blocking solution. After washes, serum was added at a dilution of 1:80. The reaction containing serum was incubated for 1 to 3 hours at 37 ° C. Washing proceeded in the same manner as described above. The reaction with the peroxidase conjugated dog anti-IgG antibody at the 1: 5000 dilution was incubated for 1 to 3 hours at 37 ° C. The plate was again washed as described above. Reaction of recombinant GDPase protein with antibodies was evidenced by staining with 5 to 10 mg / ml o-phenylenediamine (OPD) and 0.01% H2O2 in citrate phosphate buffer (0.1 M citric acid, 0.2 Sodium phosphate pH 5.0) for 15 to 30 min. The reaction was stopped by the addition of 32 to 40 μl of a 2.5 M H2SO4 solution. The intensity of the reaction, related to the intensity of the staining, was determined by the absorbance reading at 490nm (Labsystems iEMS). Data analysis was performed using the Excell program (Figure 5), which shows positive recognition with higher absorbance reading of sera known to be positive for leishmaniasis in relation to negative sera and dogs with Chagas disease.

Recombinant GDPase was subsequently used in a larger sample assay (Figure 6) showing good specific recognition in most of the positive and negative sera used in the test. Statistical analyzes of these data showed Specificity of 90.3%, Accuracy of 72.1% and Sensitivity of 44.1%.

These results together clearly show the potential use of proteins from the Leishmania E-NTPDase family as antigens for the diagnosis of Leishmnaiosis. LIST OF SEQUENCES

<110> FEDERAL UNIVERSITY OF VIçosa

BLACK GOLD FEDERAL UNIVERSITY

FOUNDATION FOR MINE GENERAL STATE RESEARCH

<120> "RECOMBINANT E-NTPDases, USE IN THE PRODUCTION OF DIAGNOSIS KIT FOR DETECTION OF ANTIBODIES IN LEISHMANIASIS CAUSED BY SPECIES OF THE GENUS Leishmania"

<130> 01 -2010 <160> 12

<210> 1

<211> 2160

<212> DNA

<213> Leishmaniamajor

<400> 1 atggctagca tgactggtgg acagcaaatg ggtcgcggat ccatgccaat gactgacggc 60 cgcgtcccag gcggcaagtt acggaggagc atgagtcgcg tcgttcttgc cttcttcgcc 120 gctgtgcttt tcgtcgtctt tctcacggcg tacgaagtcg gcgttggcac tgccaacccg 180 cttcagtctc ggcacatgca gctcacgcag aacgccgtga agaagagtga ggaaaatcta 240 gtgaactgcc gcgaggtcaa cacggattta agaagtggtg gtgacgtcaa tgtcgcacgg 300 gcgatggcgg agatgaggcg gcagagagag gagctgatga acatcgtcgc gctggagcgt 360 gagcgtgtag tgtcggcgcg tagtctgctg caggtttgcg aggatgagct agcgagcgac 420 ctcagcgtac tcttcggtgt cgctgaccat aacttcactg cgcgcctccg gtcgttggag 480 aaaaagcgga aacacttgga gggtttgcac tcgatgctca acacggaccc gtttggtgca 540 gtgcagctgc gcagcagcag cgaaatccgc gcgctgcagg cggctctctt tcacgagatg 600 cgcgccagca agaagaaagc agaaaacggt gtggcgagcg gcgcggcgtg cgcgaagtct 660 tcggtcaagt actccgtcgt gttcgatatt ggcagcactg gaaatcgtgt tcatgtctac 720 aagtacagag tggcacctgc cacgcgtacc gctgccgcgg ccggcagtga gctcagcgac 780 atcgacctcg tcgaggagtt atttgagcta aatcacaaag cccttagcga gctcgagaat 840 tcggtgcagg a tgcgccgga ggctttatgg gagctcttcg tgaaagccaa ggactttgta 900 ccggcggagc tgcacgcatg cacggcggcc gagttcaagg ctaccgcggg gctgcgcatg 960 ctggggatgg agaaggccaa gccggcattc gcgcgcgcta ccgcaacgag cgaaattctt 1020 acgttctggt tgcgcggcaa cgcatcagtt cgcatcttgg atgcctgcga ggagggccca 1080 atggcgtggc tgacagtaaa ctacttactg ggggcattct ccaggggtgc aacggcaacc 1140 gactcgacgg tggccgtcat cgatctcggc ggcggctcca cgcaaatcgt cttcgaaccc 1200 ggcgagagca ctttccacgg gatgcgcacc gatttccgct acgcggcaac cttgggcagc 1260 cggtcagtga gagcctacca gcacagctac gaaggctacg gcctgcacgc ggccaccaag 1320 aagctgcttt accacataca aggcaagagc caagagaagc cgggcggtgg caccgccacc 1380 aacacagcaa tgacgaccac cacgacagca ccggcaaacg gcggtgacga ggtcctgcct 1440 gtttggaagg ctctgagaaa cctgggtgca gacgggagaa gcgagcgagg cgacatcgtc 1500 accaagagag cgccgccgat gccaccgcca gacgcggagg cgatggaggc gttcccgtgc 1560 ttcgctgtcg gctacgaaga cccactaggg gtgaagaacg tcaagagaaa caatgccgag 1620 gagccggtta tgcccccgaa cttccaggct tgcgcgaacc ttttccgcga tcggttgctg 1680 aagccagtgg ggctg acatg tgaggcagtc aactgcggca tcgctggtgt catgcagcca 1740 ccgctgacca acttcaccgg tgaaatctac gcgttttcgt tcatctttga tctgctggtt 1800 ttggcgaaca gctccctggt gccagcgggg gctgctgtgt cgaaggaaaa gtttgaggtg 1860 aagctgccgg acctagcgaa gatcgcggag ggtcactgcg ccgccttctc cctcacccgt 1920 atcgccgagg cgaccgccaa ggggggcctc ggtagcctaa agccggagta cgagtgtatg 1980 tattactcct acgtgtacgc gcttctccgc tacggttacg aggtgccaga ggaccgcgtg 2040 ctgcacgtgg cgaagaagat ccgcggctac gagaccgcct ggtc.cc.tcgg cgcctcactc 2100 ctctctctta ccgtcgacaa gcttgcggcc gcactcgagc accaccacca ccaccactga 2160

<210> 2 <211> 719 <212> PRT

<213> Leishmaniamajor <400> 2

Met Wing Being Met Thr Gly Gly Gln Gln Met Gly Arg Gly Being Met Pro 15 10 15

Met Thr Asp Gly Arg Val Pro Gly Gly Lys Read Arg Arg Be Met Ser 20 25 30

Arg Val Valu Leu Phe Phe Wing Val Valu Leu Phe Val Val Phe Leu

35 40 45

Thr Wing Tyr Glu Val Gly Val Gly Thr Wing Asn Pro Read Gln Ser Arg 50 55 60

His Met Gln Leu Thr Gln Asn Wing Val Lys Lys Ser Glu Glu Asn Leu 65 70 75 80

Val Asn Cys Arg Glu Val Asn Thr Asp Read Arg Be Gly Gly Asp Val 85 90 95

Asn Val Wing Arg Wing Met Wing Glu Met Wing Arg Arg Gln Arg Glu Glu Leu 100 105 110

Met Asn Ile Val Wing Leu Glu Arg Glu Arg Val Val Ser Wing Arg Ser 115 120 125

Leu Leu Gln Val Cys Glu Asp Glu Leu Wing Be Asp Leu Ser Val Leu 130 135 140

Phe Gly Val Wing Asp His Asn Phe Thr Wing Arg Read Le Arg Be Leu Glu 145 150 155 160

Lys Lys Arg Lys His Leu Glu Gly Leu His Be Met Leu Asn Thr Asp 165 170 175

Pro Phe Gly Wing Val Gln Leu Arg Be Ser Be Ser Glu Ile Arg Wing Leu 180 185 190

Gln Wing Wing Read Phe His Glu Met Arg Wing Wing Be Lys Lys Lys Wing Glu 195 200 205

Asn Gly Val Wing Be Gly Wing Cys Wing Lys Wing Be Ser Val Lys Tyr 210 215 220

Ser Val Val Phe Asp Ile Gly Ser Thr Gly Asn Arg Val His Val Tyr 225 230 235 240

Lys Tyr Arg Val Wing Pro Wing Thr Arg Wing Wing Wing Wing Wing Gly Ser 245 250 255

Glu Leu Ser Asp Ile Asp Leu Val Glu Glu Leu Phe Glu Leu Asn His 260 265 270

Lys Wing Read Glu Wing Read Glu Wing Asn Be Val Gln Asp Wing Pro Glu Wing 275 280 285

Leu Trp Glu Leu Phe Val Lys Wing Lys Asp Phe Val Pro Glu Wing Leu 290 295 300

His Wing Cys Thr Wing Glu Wing Phe Lys Wing Thr Wing Gly Leu Arg Met 305 310 315 320

Read Gly Met Glu Lys Wing Asn Glu Ile Read Gly Wing Gly Ile Arg Wing Arg 325 330 335

Tyr Arg Asn Glu Thr Phe Trp Read Arg Gly Asn Wing Ser Val Arg Ile 340 345 350

Leu Asp Cys Wing Glu Glu Gly Pro Met Trp Wing Leu Thr Val Asn Tyr 355 360 365

Leu Leu Gly Wing Phe Be Arg Gly Wing Thr Wing As Thr Be Val Val 370 375 380

Val Ile Asp Wing Read Gly Gly Gly Ser Thr Thr Gln Ile Val Phe Glu Pro 385 390 395 400

Gly Glu Being Thr Phe His Gly Met Arg Thr Asp Phe Arg Tyr Wing 405 410 415

Thr Read Gly Be Arg Be Val Arg Wing Tyr Gln His Be Tyr Glu Gly 420 425 430

Tyr Gly Read His Wing Ward Thr Lys Lys Read Leu Read Tyr His Ile Gln Gly 435 440 445

Lys Be Gln Glu Lys Pro Gly Gly Gly Thr Wing Thr Asn Thr Wing Met 450 455 460

Thr Thr Thr Thr Thr Pro Wing Asn Wing Gly Gly Asp Glu Val Leu Pro 465 470 475 480

Val Trp Lys Wing Read Arg Asn Read Le Gly Wing Asp Gly Arg Be Glu Arg 485 490 495

Gly Asp Ile Val Thr Lys Arg Pro Wing Pro Mer Pro Star Pro Asp Wing 500 505 510

Glu Wing Met Glu Wing Phe Pro Cys Phe Wing Val Gly Tyr Glu Asp Pro 515 520 525

Leu Gly Val Lys Asn Val Lys Arg Asn Asn Asn Wing Glu Glu Pro Val Met 530 535 540 Pro Pro 545

Lys Pro

Val met

Be Phe

Gly Wing 610

Read Wing 625

Ile wing

Tyr glu

Tyr glu

Gly Tyr 690

Val asp

705

Asn phe

Val gly

Gln Pro 580

Ile Phe 595

Wing wing

Lys ile

Glu Wing

Cys Met 660

Val Pro 675

Glu thr

Lys Leu

Gln Wing 550

Read Thr 565

Pro leu

Asp Leu

Val ser

Glu Wing 630

Thr Wing 645

Tyr tyr

Glu Asp

Trp wing

Wing Wing 710

Cys Wing

Cys glu

Thr asn

Leu Val 600

Lys Glu 615

Gly his

Lys gly

To be tyr

Arg Val 680

Ser Leu 695

Wing Leu

Asn leu

Val Wing 570

Phe Thr 585

Lcu Ala Lys Phe

Cys Wing

Gly Leu 650

Val Tyr 665

Read His Gly Wing Glu His

Phe Arg 555

Asn cys

Gly Glu

Asn ser

Glu Val 620

Phe Wing 635

Gly ser

Wing Leu

Val Wing

Ser Leu 700

His his

715

Asp Arg

Gly ile

Ile Tyr 590

Ser Leu 605

Lys Leu

To be read

Read lys

Read Arg 670

Lys Lys 685

Leu Ser His His

Leu Leu 560

Gly Wing 575

Phe Wing

Val pro

Pro Asp

Thr Arg 640

Pro Glu 655

Tyr Gly Ile Arg Read His Thr

<210> 3

<211> 2166

<212> DNA

<213> Leishmaniabraziliensis

<400> 3

atggctagca tgactggtgg acagcaaatg ttcacagctg gcaagccgct gagaggcatg atgcttgttg cctttgtcat cactgcgtac cagtcccgtc acatacagct cgcgcagaac ggctgccgcg aggccaatgc gaatttaaag atcgcggaga tggcacggca gaaagcggag cgtgtcgtgt cggcgcgtag tttgttgcag cgtacactct tcggcaccgc ccaccgcaac agacgggctc atctgaagtc cgagcacgag

ggtcgcggat ccatggcagt ggttggcggc 60

ggccgcatcg tccttggcct cttcgccgtc 120

caagtcggcg ttagcaccgc caacccacgt 180

gccgtggcga agagcgaggc aatgctgact 240

aacagcggta gtgtcaaggg tgcacaagcg 300

ctgacgagta ctgtcgcact ggagcgtgag 360

gcgtgcgagg atgggctggc aagcgaacac 420

accacggcgc acctcctgtg gttgaaacag 480

aagctcaccg agggtccact cggtgtagtc 540 gagccgcgcc gcagcagcgg catccgcgca cctagcactg gtttagctgg aaacggtgtg gccaagtact ccgttgtgtt cgatattggc tacagggtga accctctcac gcagatctct gacctcgtcg gggagctgtt cgagctgaat gtgcaggatg ccccggaagc cttgtgggag gcagagttgc acgcatgcac accaatcgag gggttggaga aagccacaga gattcttgct ttctggctgc gcggcagcgc gccagtccgc gcgtggctga cggtgaactt tttactaggg tcgacggcgg ctatcattga catcggtggc gaagacacgt tctacaaaat gcgcgcagac tcggtgaaag tgtatcagca cagctacgaa ctgctttttc atattcaagg caagcgccaa gccacgacga cggcacggac cagcggcgat cgcaacgtga gcgccaatga cagcaaagac gcgccgccga aaactctacc gtggctacag tgcttcgctg tcggctacga ggaccagctc ggagaacccg ctaggcaccc tgacttccag ctgaaaccgg tggggctgac atgcgaggag ccgccgctgg ccaacttcac cggtgacatc gccatggcga acaactctct agcgccagtg gtgaagctgc ctgacctggc aaagattggg cggattgccg aggcgaccgc aaagggcggc atgtattact cctacacgta cgcgctgctc gtgctgcacg tggcaaagaa gatcagcggc ctcatctctg tcacctaagt cgacaagctt cactga

ctgcaggcgg ctctccttca agaaatacac 600 gagaaccgca aggcgtgcgt ggacgctgtg 660 agcactggca atcgtgtcca tgtctacaag 720 gcctcagcgc ctgatgagct cagcaaaatc 780 tacaaagccc ttagtgagct caacaatccg 840 cttttcgcga aggccaagaa cttcgtgccg 900 ttcaaggcta ctgcgggact gcgcatgctg 960 gagattcgtg ctcgctaccg caaggaaacg 1020 atcttggatt cccacgagga gggcctgatg 1080 acatttgcca ggaacactga ggcaacagcc 1140 ggctccacgc agatcgtctt cgagcctggt 1200 gtgcgtggct cggcaacgtt gggcggccgg 1260 ggctacggtc tgcacgcagc caccaaggcg 1320 gagaggccgg gcggcggctc cgctacccgc 1380 gacagcaccc catccgtcgg gaatggtgtc 1440 gaagaagacg aaaacatgat caccgacaca 1500 ttggatacgg aggcggtgga cgcattcccc 1560 ggggtgcgga acactaagag aaacgacgcc 1620 gcttgcgcga accttttccg cgaccggttg 1680 gtcaactgcg gcgtcgcggg tgtcttccag 1740 tacgcgtttt catttctttt tgatcttctg 1800 ggggctgcag tatcgaacga taagttcgag 1860 gagcgtcact gcgcggcctt ctccctcacc 1920 ctcggtagtc tgaagccaga gtacgagtgc 1980 cgatatgggt acgaggtgcc ggagggccgc 2040 tacgagaccg cctggccctt gggtgcctca 2100 gcggccgcac tcgagcac ca ccaccaccac 2160

2166

<210> 4 <211> 720 <212> PRT

<213> Leishmania braziliensis <400> 4

Met Wing Be Met Thr Gly Gly Gln Gln Met Gly Arg Gly Be Met Wing 1 5 10 15

Val Val Gly Gly Phe Thr Wing Gly Lys Pro Read Arg Gly Met Gly Arg 20 25 30

Ile Val Leu Gly Leu Phe Ala Val Met Leu Val Ala Phe Val Ile Thr 35 40 45

Wing Tyr Gln Val Gly Val Ser Thr Wing Asn Pro Arg Gln Ser Arg His 50 55 60

Ile Gln Leu Wing Gln Asn Wing Val Wing Lys Ser Glu Wing Met Leu Thr 65 70 75 80

Gly Cys Arg Glu Wing Asn Wing Asn Leu Lys Asn Ser Gly Ser Val Lys 85 90 95

Gly Wing Gln Wing Ile Wing Glu Met Wing Arg Gln Lys Wing Glu Leu Thr 100 105 110

Ser Thr Val Wing Leu Glu Arg Glu Arg Val Val Ser Wing Arg Ser Leu 115 120 125

Leu Gln Wing Cys Glu Asp Gly Leu Wing Wing Be Glu His Arg Thr Leu Phe 130 135 140

Gly Thr Wing His Arg Asn Thr Thr Wing His Leu Leu Trp Leu Lys Gln 145 150 155 160

Arg Arg Wing His Leu Lys Be Glu His Glu Lys Leu Thr Glu Gly Pro 165 170 175

Read Gly Val Val Glu Pro Arg Arg Be Ser Gly Ile Arg Wing Read Le Gln 180 185 190

Wing Wing Read Leu Gln Glu Ile His Pro To Be Thr Gly Read Wing Gly Asn 195 200 205

Gly Val Glu Asn Arg Lys Wing Cys Val Asp Wing Val Val Wing Lys Tyr Ser 210 215 220

Val Val Phe Asp Ile Gly Be Thr Gly Asn Arg Val His Val Tyr Lys 225 230 235 240

Tyr Arg Val Asn Pro Read Thr Gln Ile Be Wing Be Wing Pro Asp Glu 245 250 255

Leu Ser Lys Ile Asp Leu Val Gly Glu Leu Phe Glu Leu Asn Tyr Lys 260 265 270

Wing Read Ser Glu Read Asn Asn Pro Val Gln Asp Wing Pro Glu Wing Leu 275 280 285

Trp Glu Leu Phe Wing Lys Wing Lys Wing Asn Phe Val Pro Glu Wing Leu His 290 295 300 Cys Wing Thr Pro Ile Glu Phe Lys Wing Wing Gly Leu Arg Met Leu 305 310 315 320

Gly Leu Glu Lys Wing Thr Glu Ile Leu Glu Wing Ile Arg Wing Arg Tyr 325 330 335

Arg Lys Glu Thr Phe Trp Leu Arg Gly Ser Wing Pro Val Arg Ile Leu 340 345 350

Asp Be His Glu Glu Gly Leu Met Wing Trp Leu Thr Val Asn Phe Leu 355 360 365

Leu Gly Thr Phe Wing Arg Asn Thr Glu Wing Thr Wing Be Thr Wing Wing 370 375 380

Ile Ile Asp Ile Gly Gly Gly Gly Thr Thr Gln Ile Val Phe Glu Pro Gly 385 390 395 400

Glu Asp Thr Phe Tyr Lys Met Arg Wing Asp Val Arg Gly Ser Wing Thr 405 410 415

Read Gly Gly Arg Be Val Lys Val Tyr Gln His Be Tyr Glu Gly Thr 420 425 430

Gly Leu His Wing Ala Thr Lys Wing Leu Leu Phe His Ile Gln Gly Lys 435 440 445

Arg Gln Glu Arg Pro Gly Gly Gly Be Wing Thr Thr Wing Wing Thr Thr Thr 450 455 460

Wing Arg Thr Be Gly Asp Asp Be Thr Pro Be Val Gly Asn Gly Val 465 470 475 480

Arg Asn Val Be Wing Asn Asp Be Lys Asp Glu Glu Asp Glu Asn Met 485 490 495

Ile Thr Asp Thr Wing Pro Pro Lys Thr Pro Leu Pro Trp Leu Gln Leu Asp 500 505 510

Thr Glu Wing Val Asp Wing Phe Pro Cys Wing Phe Wing Val Gly Tyr Glu Wing 515 520 525

Gln Leu Gly Val Arg Asn Thr Lys Arg Asn Asp Wing Gly Glu Pro Wing 530 535 540

Arg His Pro Asp Phe Gln Cys Wing Asn Leu Phe Arg Asp Arg Leu 545 550 555 560

Leu Ile Pro Val Gly Leu Thr Cys Glu Glu Val Asn Cys Gly Val Wing 565 570 575

Gly Val Phe Gln Pro Pro Read Wing Asn Phe Thr Gly Asp Ile Tyr Wing 580 585 590

Phe Ser Phe Leu Phe Asp Leu Leu Wing Met Wing Asn Asn Ser Leu Wing 595 600 605

Pro Val Gly Wing Val Wing Val Asn Asp Lys Phe Glu Val Lys Leu Pro 610 615 620 Asp Leu Wing Lys Ile Gly Glu Arg His Cys Wing Phe Ser Leu Thr 625 630 635 640

Arg Ile Wing Glu Wing Thr Wing Lys Gly Gly Leu Gly Ser Leu Lys Pro 645 650 655

Glu Tyr Glu Cys Met Tyr Tyr Ser Tyr Thr Tyr Wing Leu Read Arg Tyr 660 665 670

Gly Tyr Glu Val Pro Glu Gly Arg Val Read His Val Wing Lys Lys Ile 675 680 685

Ser Gly Tyr Glu Thr Wing Trp Pro Read Gly Wing Ser Read Ile Ser Val 690 695 700

Thr Val Asp Lys Leu Wing Wing Wing Leu Glu His His His His His 705 710 715 720

<210> 5

<211> 2124

<212> DNA

<213> Leishmaniainfantum

<400> 5

atggctagca tgactggtgg acagcaaatg ggtcgcggat ccatgagtcg cgtctttgtt 60 gccttcttcg ctgctgtgct tttcgtcgtc tttctcaccg cgtacgaagt cggcgttggc 120 accgccaacc cgcttcagtc gcggcacatg cagctcacgc agaacgcagt gaagaagagt 180 gaggcaaatc tagtgaactg ccgcgaggtc aacgcggatt taaagagcgg tggtggcgtc 240 aatgccgcac aggcgattgc ggagatgagg cggcagagag aggagctgat gaacatcgtc 300 gcgctggagc gtgagcgtgt agtgtcggcg cgtagtctgc tgcaggtttg cgaggatgag 360 ctagccagcg acctcagtgt actcttcggt gtcgccgacc acaacttcac tgcgcgcatc 420 cggtcgttgg aggaaaagcg gagggtttgc actcgatgct caacacgacc gaaacatttg 480 ccgtttggtg cagtggagct gcgccgcagc agcgaaatcc gtgcactgca ggcggctctc 540 tttcacgaga tgcgcgccag caagaagaaa gcagaaaacg gtgtggcgaa cggcgaagcg 600 tgcacgaaga cttcggacaa gtactccgtc gtgttcgaca tcggcagcac tggaaatcgt 660 gtccatgtct acaagtacag agtggcccct gccacgcata ccgctgctgc ggccggcagt 720 gagctcagcg acatcgacct cgtcgaggag ttgtttgagc taaatcacaa agcccttagc 780 gagctcgata atccggtgca ggatgcgccg gaagccttat gggagctctt catgaaagcc 840 aagtactttg taccggcgga gctgcacgca tgcacggcag tcgagttcaa ggctaccgcg 900 ggactgcgca tgctggggat ggagaaggcc accgaaattc ttgacgggat tcgcgcgcgc 960 taccgcaacg aaacgttctg gttgcgcggc aatgcaccgg ttcgcatctt ggatgcctgc 1020 gaggagggcc caatggcgtg gctcacggta aactacttac tgggggtatt ctccaggggt 1080 acaaaggcaa ccgcctcgac ggtggccgtc atcgacctcg gaggcggctc cacgcagatc 1140 gtcttcgaac ccggcgagag cgcgttccac gggatgcgca ccgatttgcg ctactcggca 1200 accttgggca gccggtctgt gagtgcctac caacacagct acgaaggcta cggcctgcac 1260 gcggccacca aggagc. TGCT tttccacatt caaggcaaga gccaagagaa gccgggaggc, 1320 ggtaccacca ccagcacagc aacgacgacc accacgacaa caccggcaaa cagcggcgac 1380 aaggctctgt ctgtttggaa cgttctggga aacctgggtg cagacgggag cagcgagcga 1440 gacgacatcg tcaccaagag agcgccgccg atgccaccac cgccactgcc ggacgcggag 1500 gcggtggagg cgttcccctg cttcgctgtc ggctacgaag acccgctagg ggtgaagaac 1560 atcaagaaaa acaataccgg ggagccggct atgcccccga acttccaggc ttgcgcgaac 1620 cttttccgcg atcggctgct aaagccagtg gggctgacat gtgaggcggc caactgcggc 1680 atcgctggtg tcatgcagcc a ccgctgacc aacttcaccg gggaaatcta cgtgttttcg 1740 ttcatctttg atctgctggc cttggcgaac agctccctgg tgccagcggg ggctgccgtg 1800 tcaagggaaa agtttgaggt gaagctgccg gacctagcga cgattgcgga gggtcactgc 1860 gccgccttct ccctcacccg tatcgccgag gcgaccgcca aggagggcct cggtagcctg 1920 aagccggagt acgagtgcat gtattactcc tacgtgtacg cgcttctccg ctacgggtac 1980 gaggtgccag aggatcgcgt gctgcacgtg gtgaagaaga tccgcggcta cgagaccgcc 2040 tggtccctcg gcgcctcact cctctctctt acctaagtcg acaagcttgc ggccgcactc 2100 gagcaccacc accaccacca CTGA 2124

<210> 6 <211> 706 <212> PRT

<213> Leishmania infantum <400> 6

Met Wing Be Met Thr Gly Gly Gln Gln Met Gly Arg Gly Be Met Ser 15 10 15

Arg Val Phe Val Wing Phe Phe Wing Wing Val Leu Phe Val Val Phe Leu 20 25 30

Thr Wing Tyr Glu Val Gly Val Gly Thr Wing Asn Pro Read Gln Ser Arg 35 40 45

His Met Gln Leu Thr Gln Asn Wing Val Lys Lys Ser Glu Wing Asn Leu 50 55 60

Val Asn Cys Arg Glu Val Asn Wing Asp Leu Lys Ser Gly Gly Gly Val 65 70 75 80 Asn Wing

Met asn

Leu Leu

Phe Gly 130

Glu Lys 145

Pro phe

Gln Wing

Asn gly

Ser Val 210

Lys Tyr 225

Glu Leu Lys Wing

Read trp

His Wing 290

Read Gly 305

Tyr arg

Read asp

Leu Leu

Val 370 Wing

Gly Glu 385

Gln Wing

Ile Val 100

Gln Val 115

Val Wing

Arg lys

Gly Wing

Wing Leu 180

Val Wing 195

Val phe

Arg val

Be asp

Read Ser 260

Glu Leu 275

Cys thr

Met glu

Asn glu

Cys 340 Wing

Gly Val 355

Ile Asp Ser Ala

Ala Ile 85

Wing Leu

Cys glu

Asp His

His Leu 150

Val Glu 165

Phe his

Asn gly

Asp Ile

Pro 230 Wing

Ile Asp 245

Glu Leu

Phe Met

Val Wing

Lys Wing 310

Thr Phe 325

Glu Glu

Phe Ser

Read gly

Phe His 390

Glu Wing

Glu Arg

Asp Glu 120

Asp Phe 135

Glu Gly

Read arg

Glu Met

Glu Wing 200

Gly Ser 215

Thr wing

Read val

Asp Asn

Lys Wing 280

Glu Phe 295

Thr glu

Trp Leu

Gly pro

Arg Gly 360

Gly Gly 375

Gly met

Met Arg 90

Glu Arg 105

Read Wing

Thr wing

Read his

Arg Ser 170

Arg Wing 185

Cys thr

Thr gly

His thr

Glu Glu 250

Pro Val 265

Lys tyr

Lys Wing

Ile leu

Arg Gly 330

Met Wing 345

Thr Lys Ser Thr Arg Thr

Arg Gln

Val Val

Be asp

Arg Ile 140

Ser Met 155

Be glu

Be lys

Lys thr

Asn Arg 220

Wing Wing 235

Read phe

Gln asp

Phe val

Thr Wing 300

Asp Gly 315

Asn wing

Trp Leu

Thr wing

Gln Ile 380

Asp Leu

395

Arg glu

Be Wing 110

Read Ser 125

Arg Ser Read Asn

Ile arg

Lys Lys 190

Ser Asp 205

Val his

Wing wing

Glu Leu

Pro 270 Wing

Pro Wing 285

Gly Leu

Ile arg

Pro val

Thr Val 350

Ser 365 wing

Val Phe Arg Tyr

Glu Leu 95

Arg Ser

Val leu

Read Glu

Thr Thr 160

Wing Leu 175

Glu Wing

Lys tyr

Val tyr

Gly Ser 240

Asn his 255

Glu Wing

Glu Leu

Arg Met

Arg 320 Wing

Arg Ile 335

Asn tyr

Thr val

Glu Pro

Ser Wing 400 Thr Leu

Tyr gly

Lys Ser

Thr Thr 450

Val Trp 465

Asp Asp

Pro Asp

Glu Asp

Pro Wing 530

Arg Leu 545

Ile wing

Tyr val

Read val

Leu Pro 610

Read Thr 625

Lys Pro

Arg tyr

Lys ile

Ser Leu 690 His His 705

Gly ser

Read his 420

Gln Glu 435

Thr thr

Asn val

Ile val

Glu 500 Wing

Pro Leu 515

Met pro

Read lys

Gly val

Phe Ser 580

Pro Wing 595

Asp Leu

Arg Ile

Glu Tyr

Gly Tyr 660

Arg Gly 675

Thr val

Arg Ser 405

Wing wing

Lys Pro

Thr thr

Read Gly 470

Thr Lys 485

Val Wing

Gly val

Pro asn

Pro Val 550

Met Gln 565

Phe Ile

Gly Wing

Thr wing

Arg Glu 630

Glu Cyr 645

Glu Val Tyr Glu Asp Lys

Val ser

Thr lys

Gly Gly 440

Pro Wing 455

Asn leu

Arg Wing

Glu Wing

Lys Asn 520

Phe Gln 535

Gly Leu

Pro pro

Phe asp

Val Wing 600

Ile Wing 615

Thr wing

Met tyr

Pro glu

Thr Wing 680

Read Wing 695

Tyr 410 wing

Glu Leu 425

Gly thr

Asn ser

Gly Wing

Pro Pro 490

Phe Pro 505

Ile lys

Cys Wing

Thr cys

Read Thr 570

Leu Leu 585

To be Arg

Glu Gly

Wing Lys

Tyr Ser 650

Asp Arg 665

Trp ser

Wing wing

Gln his

Read phe

Thr thr

Gly Asp 460

Asp Gly 475

Met pro

Cys Phe

Lys asn

Asn Wing 540

Glu Wing 555

Asn phe

Wing Leu

Glu Lys

His Cys 620

Glu Gly 635

Tyr val

Val leu

Read gly

Read Glu 700

To be tyr

His Ile 430

Ser Thr 445

Lys Wing

To be to be

Pro pro

Val Wing 510

Asn thr 525

Read phe

Asn Wing

Thr gly

Wing Asp 590

Phe Glu 605

Wing wing

Read gly

Tyr wing

His Val 670

Ser 685 wing

His his

Glu Gly 415

Gln gly

Thr wing

Read Ser

Glu Arg 480

Pro Leu 495

Gly tyr

Gly Glu

Arg Asp

Cys Gly 560

Glu Ile 575

To be to be

Val lys

Phe Ser

Ser Leu 640

Leu Leu 655

Val lys

Leu Leu

His His <210> 7

<211> 1362

<212> DNA

<213> Leishmania major

<400> 7

atggctagca tgactggtgg acagcaaatg ggtcgcggat ccatgcgtcc gtactcctcg 60 gtgcggcgca tgactcaaca atcgaagcgc ctgcgcgtcg ccagcaccct tgtgcttagc 120 gcgcttgtta tcttcggttt cctcgtctac catcaaagcc ctttgttctc cccatgtgac 180 tcggcgtacg cgaatgtata cgacgtcgtc atcgacgctg gcagcactgg ctcacgtgtg 240 catgtatttc agtacgagcg cggccgcacc ggtttcgtac ttctgagaga gcgcttcaaa 300 cgagcagagc cggatctgtc ctctttcgcc accgacctgg acggcgccaa ggcgtcgctt 360 gaggggcttc tgcgctttgc ggatacggtg gttccgcaga gctaccagaa gtgcacctct 420 gtcactctca aggccaccgc cggccttagg ctcctacctg agtccgccca gcaggcgctg 480 ctggacgttg cccagcacac actcaacgca tctccatttc agtctcgcgg cgcctccatc 540 atctctggcg ctcaagaggg cgtctacggg tggctgacgg tgaactacct gctggacaga 600 ctcgacacgg acgttgccac cgtcgcgacc atcgatatgg gaggcgcctc gacacaggtc 660 gtcttcgaga cgacgcccac gtctggagaa tggctgccct tcaactatgc ctaccagctg 720 cgcacaccaa agcgcacgat cgccatgtat cagcacagct acctcggcct tgggatgaac 780 gaggcgaaga agaggcttat gacgttgttc gccgaagcga acgggacgtt gtctttcccg 840 tgcttcccga gagggtatac gaagcgcgta aacggtgtgg aacttcgaaa cagtgacgcc 900 acggacttta acgcgtgtgc aggactattt cgcgaacatg tcataacaac gtcgacctgc 960 aagtttgatg cctgtggcgc ccgcggcgtg ccgcagccgc tgtttccgtc aaggcggcat 1020 cccatctacg ccttctccta cttttacaac cgcctctacg acttcctcaa ggaggggagc 1080 caggtctacg tctcctcgta caaggaggtc gggcaagagg tgtgccaccg ggaatctgcg 1140 aggcggacca ccgcccctga ggaaactgcc tgcatggaac tggcgtacat gtacagcttc 1200 ttgacgtacg ggctagggct tagtgacgcc acggttctca cggtgcccaa ccgcatcgag 1260 ggtatggcgg tttcctggtc tcttggctcc tctctctcct tcctgctcaa gatggaatga 1320 aagcttgcgg ccgcactcga gcaccaccac ca ca ca ca ca ct ga 1362

<210> 8

<211> 452

<212> PRT

<213> Leishmania major <400> 8

Met Wing Be Met Thr Gly Gly Gln Gln Met Gly Arg Gly Be Met Arg 1 5 10 15

Pro Tyr Be Val Arg Arg Met Thr Gln Gln Be Lys Arg Leu Arg 20 25 30

Val Wing Ser Thr Leu Val Wing Le Ser Wing Le Val Ile Phe Gly Phe Leu 35 40 45

Val Tyr His Gln Be Pro Read Phe Be Pro Cys Asp Be Wing Tyr Wing 50 55 60

Asn Val Tyr Asp Val Val Ile Asp Wing Gly Ser Tyr Gly Ser Arg Val 65 70 75 80

His Val Phe Gn Tyr Glu Arg Gly Arg Thr Gly Phe Val Leu Leu Arg 85 90 95

Glu Arg Phe Lys Arg Wing Glu Pro Asp Read Be Ser Phe Wing Thr Asp 100 105 110

Read Asp Gly Wing Lys Wing Be Read Glu Gly Read Leu Arg Phe Wing Asp 115 120 125

Thr Val Val Pro Gln Be Tyr Gln Lys Cys Thr Be Val Thr Leu Lys 130 135 140

Wing Thr Wing Wing Gly Leu Arg Leu Leu Pro Glu Ser Wing Gln Gln Wing Leu 145 150 155 160

Leu Asp Val Wing Gln His Thr Leu Asn Wing Ser Pro Phe Gln Ser Arg 165 170 175

Gly Wing Be Ile Ile Be Gly Wing Gln Glu Gly Val Tyr Gly Trp Leu 180 185 190

Thr Val Asn Tyr Leu Read Asp Arg Leu Asp Thr Asp Val Wing Thr Val 195 200 205

Wing Thr Ile Asp Met Gly Gly Wing Being Thr Gln Val Val Phe Glu Thr 210 215 220

Thr Pro Thr Be Gly Glu Trp Leu Pro Phe Asn Tyr Wing Tyr Gln Leu 225 230 235 240

Arg Thr Pro Lys Arg Thr Ile Wing Met Tyr Gln His Ser Tyr Leu Gly 245 250 255

Read Gly Met Asn Glu Wing Lys Lys Arg Read Met Met Thr Read Phe Wing Glu 260 265 270

Wing Asn Gly Thr Read Be Phe Pro Cys Phe Pro Arg Gly Tyr Thr Lys 275 280 285

Arg Val Asn Gly Val Glu Leu Arg Asn Ser Asp Wing As Thr Thr Phe Asn 290 295 300

Cys Wing Gly Wing Read Phe Arg Glu His Val Ile Thr Thr Be Thr Cys 305 310 315 320 Lys Phe Asp Wing

Ser Arg Arg His 340

Tyr Asp Phe Leu 355

Glu Val Gly Gln 370

Pro Glu Glu Wing 385

Read Thr Tyr Gly

Asn Arg Ile Glu 420

Ser Phe Leu Leu 435

His His His His 450

Cys Gly Wing Arg 325

Pro Ile Tyr Wing

Lys Glu Gly Ser 360

Glu Val Cys His 375

Thr Cys Met 390 Wing

Leu Gly Leu Ser 405

Gly Met Wing Val

Lys Met Glu Lys 440

Gly Val Pro Gln 330

Phe Ser Tyr Phe 345

Gln Val Tyr Val

Arg Glu Ser Wing 380

Glu Leu Wing Tyr 395

Asp Wing Thr Val 410

Ser Trp Ser Leu 425

Read Wing Wing Wing

Pro Leu Phe Pro 335

Tyr Asn Arg Leu 350

Ser Ser Tyr Lys 365

Arg Arg Thr Thr

Met Tyr Ser Phe 400

Read Thr Val Pro 415

Gly Ser Ser Leu 430

Read Glu His His 445

<210> 9

<211> 1368

<212> DNA

<213> Leishmania braziliensis

<400> 9

atggctagca tgactggtgg acagcaaatg ggtcgcggat ccatgcgacc gtactcctca 60 gtgcggcgga tgactcaaca gtcgaaccga ctgcgcgtca tcggcgtact cgtgcttagc 120 gcattcgttc tcttcggctt tgtcatttac tctgaaagcc cttggttctc tccttgcaac 180 tcgccatact cgaatgtcta cgacatcgta atcgacgccg gcagtaccgg ctcgcgcgta 240 catgtgtttc agtacgagcg cagtagcact ggtgtcatac ttctgagaga gcgcttcaaa 300 cggatagagc cgggactgtc ttcatttgcc accgatcagg aaggcgctaa gcagtcgctt 360 gcggggctcc tgcgcttcgc tgaaaaggca gttccacgaa gctaccagag gtgcacttcc 420 gtaaccctca aggccaccgc cggcctccgg cttctccctg aagccgatca gcaggtgttg 480 ctggatgctg gtctctggtg cccagcagac ctcaagaggg cctcaaggca cgtctatggg tttccgtttc tggctcacgg agtctcgtgg tgaactacct cgcctccatc ccttaacagg 540 600 ctcgacaagg agggcgccac cgtcgcgacc atagacatgg ggggtgcctc gacgcaggtg 660 gtgttcgaga cgaagttcac gtctggagag tggctgccct tcaactacgc ccaccagctg 720 cgcacgccaa agcgcaccat cgccatgtat cagcacagct acctaggcct tgggctcaac 780 gaggcaaaga agacactgat gacgttgttc gccaaagtga acgggacatc 840 ccctttttca

tgcttcccga gaaggcatac ggaacacctg aatggtgtgg aacttcgaaa cggcgattcc 900

acggacttcg acgtttgtgt aaatcttttt cgagagcacg tcataacgaa gccgatctgc 960

aggtttgatg cctgtggcgc tcgcggcgtg ccgcagccgc cgctgccgtc gaagcagcat 1020

ccaatttacg ccttttctta cttctacgac cgtctctacc acttccgcag tgaaggattc 1080

ccggtatacg tctcgtcgta caaggagctc gggcgggagg tgtgtcagcg agagtctgca 1140

gaccacacca ccacccccaa ggaaacgacc tgcatggagc tggcatactt gtacagcttc 1200

ttgacgcacg ggctagggct tagcgacgac agaaccctcg aggttcctaa ccgcatagag 12 60

ggtatcgcgg tctcctggtc tctaggctgc tctctgtcct tcgtgctcaa gatggaatga 1320

gtcgacaagc ttgcggccgc actcgagcac caccaccacc accactga 1368

<210> 10 <211> 454 <212> PRT

<213> Leishmania braziliensis <400> 10

Met Wing Be Met Thr Gly Gly Gln Gln Met Gly Arg Gly Be Met Arg 1 5 10 15

Pro Tyr Ser Be Val Arg Arg Met Thr Gln Gln Be Glu Arg Leu Arg 20 25 30

Val Ile Gly Val Leu Val Leu Ser Arg Phe Val Leu Phe Gly Phe Val 35 40 45

Ile Tyr Be Glu Be Pro Trp Phe Be Pro Cys Asn Be Pro Tyr Be 50 55 60

Asn Val Tyr Asp Ile Val Ile Asp Wing Gly Be Thr Gly Be Arg Val 65 70 75 80

His Val Phe Gln Tyr Glu Arg Ser Be Thr Gly Val Ile Leu Leu Arg 85 90 95

Glu Arg Phe Lys Arg Ile Glu Pro Gly Leu Be Ser Phe Wing Thr Asp 100 105 110

Gln Glu Gly Wing Lys Gln Being Read Wing Gly Read Leu Read Arg Phe Wing Glu 115 120 125

Lys Wing Val Pro Arg Be Tyr Gln Arg Cys Thr Be Val Thr Leu Lys 130 135 140

Wing Thr Wing Wing Gly Leu Arg Leu Leu Pro Glu Wing Asp Gln Gln Val Leu 145 150 155 160

Read Asp Wing Gln Wing Gln Thr Read Lys Wing Phe Pro Phe Gln Ser Arg 165 170 175 Gly Wing Ser Ile Val Ser Gly Wing Gln Glu Gly Val Tyr Gly Trp Leu 180 185 190

Thr Val Asn Tyr Leu Leu Asn Arg Leu Asp Lys Glu Gly Wing Thr Val 195 200 205

Wing Thr Ile Asp Met Gly Gly Wing Being Thr Gln Val Val Phe Glu Thr 210 215 220

Lys Phe Thr Be Gly Glu Trp Leu Pro Phe Asn Tyr Wing His Gln Leu 225 230 235 240

Arg Thr Pro Lys Arg Thr Ile Wing Met Tyr Gln His Ser Tyr Leu Gly 245 250 255

Leu Gly Leu Asn Glu Wing Lys Lys Thr Leu Met Thr Leu Phe Wing Lys 260 265 270

Val Asn Gly Thr Be Pro Phe Be Cys Phe Pro Arg Arg His Thr Glu 275 280 285

His Leu Asn Gly Val Glu Leu Arg Asn Gly Asp Ser Thr Asp Phe Asp 290 295 300

Val Cys Val Asn Leu Phe Arg Glu His Val Ile Thr Lys Pro Ile Cys 305 310 315 320

Arg Phe Asp Cys Wing Gly Wing Arg Gly Val Pro Gln Pro Pro Leu Pro 325 330 335

Ser Lys Gln His Pro Ile Tyr Ala Phe Ser Tyr Phe Tyr Asp Arg Leu 340 345 350

Tyr His Phe Arg Be Glu Gly Phe Pro Val Tyr Val Be Ser Tyr Lys 355 360 365

Glu Leu Gly Arg Glu Val Cys Gln Arg Glu Ser Wing Asp His Thr Thr 370 375 380

Thr Pro Lys Glu Thr Thr Cys Met Glu Leu Wing Tyr Leu Tyr Ser Phe 385 390 395 400

Leu Thr His Gly Leu Gly Leu Be Asp Asp Arg Thr Leu Glu Val Pro 405 410 415

Asn Arg Ile Glu Gly Ile Wing Val Ser Trp Ser Leu Gly Cys Ser Leu 420 425 430

Ser Phe Val Leu Lye Met Glu Val Asp Lys Leu Wing Wing Wing Leu Glu 435 440 445

His His His His His 450

<210> 11

<211> 1368

<212> DNA

<213> Leishmania infantum <400> 11 atggctagca tgactggtgg acagcaaatg ggtcgcggat ccatgcgacc gtactcctcg 60 gtgcggcgca tgactcaaca gtcgaagcga ctgcgcatcg ccggcaccct tgtgcttagc 120 gcgcttgtta tcttcggttt cctcgtttac tatcaaagcc ctctgctctc cccatgtgac 180 tcggcgtacg cgaatgtata cgacgtcgtc atcgacgccg gcagcactgg ctcacgtgtg 240 catgtgttcc aatacgagcg cggccgcacc ggtctcgtac ttctgagaga gcgcttcaaa 300 cgagtagagc cgggtctatc ctcttttgcc accgacccag acggcgccaa ggagtcgctt 360 gcggggctgc tgcgcttcgc ggataaggtg gtcccgcaaa gctaccagaa gtgcaccttc 420 gtcaccctca aggccaccgc cggccttcgg ctcctacctg agtccgtcca gcaggtgctg 480 ctggacgctg cccagcacac actcaacgca tctccatttc agtctcgtgg tgcctccatc 540 atctctggcg ctcaagaggg cgtctacggg tggctgacgg tgaactacct gctgaacagg 600 ctcgacacgg acgttgccac cgtcgcgacc atcgacatgg gaggtgcctc gacacaggtc 660 gtcttcgaga cgacgcccac gtctggagaa tggctgccct tcaactatgc ccaccagctg 720 cgcacaccaa agcgcacgat caccatgtat cagcacagct atctcggcct tgggatgaac 780 gaggcgaaga agaagcttat gatgtcattc gccgaagcga acgggacg tc gtctttcccg 840 tgcttcccga gagggtatgc gaagcgcctg aacgatgtgg aacttcgaaa cagtgacgcc 900 acggactttg acgcgtgtgc aggactattt cgcgaacatg tcataacaaa gacgacctgc 960 aagtttgatg cctgtggcgc ccgcggcgtg ccgcagccgc tgtttccgtc aaggcggcat 1020 ctcatctacg ccttctccta cttttacgac cgactctacc acttcagcaa ggaggggagc 1080 ccggtctacg tctcgtcgta caaggaggtc gggcaagagg tgtgccaccg ggaatctgcg 1140 aggcggacca ccgcccctga ggaaacggcc tgcatggagc tggcgtacat gtacagcttc 1200 ttgacatacg gcctagggct tagtgacgcc acggctctca cggtgcccaa ccgcatcgag 1260 ggtatggcgg tttcctggtc tcttggctcc tctctctcct tcctgctcaa gatggaatga 1320 gtcgacaagc ttgcggccgc actcgagcac caccaccacc accactga 1368

<210> 12 <211> 454 <212> PRT

<213> Leishmaniainfantum <400> 12

Met Wing Be Met Thr Gly Gly Gln Gln Met Gly Arg Gly Be Met Arg 1 5 10 15 Pro Tyr Be Ser Val Arg Arg Met Thr Gln Gln Be Lys Arg Leu Arg 20 25 30

Ile Wing Gly Thr Leu Val Leu Ser Wing Leu Val Ile Phe Gly Phe Leu 35 40 45

Val Tyr Tyr Gln Be Pro Read Read Be Pro Cys Asp Be Wing Tyr Wing 50 55 60

Asn Val Tyr Asp Val Val Ile Asp Wing Gly Be Thr Gly Be Arg Val 65 70 75 80

His Val Phe Gln Tyr Glu Arg Gly Arg Thr Gly Leu Val Leu Leu Arg 85 90 95

Glu Arg Phe Lys Arg Val Glu Pro Gly Leu Be Ser Phe Wing Thr Asp 100 105 110

Pro Asp Gly Wing Lys Glu Being Read Wing Gly Read Leu Arg Phe Wing Asp 115 120 125

Lys Val Val Pro Gln Ser Tyr Gln Lys Cys Thr Phe Val Thr Read Lys 130 135 140

Wing Thr Wing Wing Gly Leu Arg Leu Leu Pro Glu Ser Val Gln Gln Val Leu 145 150 155 160

Read Asp Wing Wing Gln His Thr Read Asn Wing Wing Pro Phe Gln Be Arg 165 170 175

Gly Wing Be Ile Ile Be Gly Wing Gln Glu Gly Val Tyr Gly Trp Leu 180 185 190

Thr Val Asn Tyr Leu Read Asn Arg Leu Asp Thr Asp Val Wing Thr Val 195 200 205

Wing Thr Ile Asp Met Gly Gly Wing Being Thr Gln Val Val Phe Glu Thr 210 215 220

Thr Pro Thr Be Gly Glu Trp Leu Pro Phe Asn Tyr Wing His Gln Leu 225 230 235 240

Arg Thr Pro Lys Arg Thr Ile Thr Met Tyr Gln His Ser Tyr Leu Gly 245 250 255

Read Gly Met Asn Glu Wing Lys Lys Lys Read Met Met Ser Phe Wing Glu 260 265 270

Wing Asn Gly Thr Be Being Phe Pro Cys Phe Pro Arg Gly Tyr Wing Lys 275 280 285

Arg Asu Asn Asp Val Glu Asu Arg Asn Be Asp Wing Asp Thr Asp Phe Asp 290 295 300

Cys Wing Gly Wing Read Phe Arg Glu His Val Ile Thr Lys Thr Thr Cys 305 310 315 320

Lys Phe Asp Cys Wing Gly Arg Wing Gly Val Pro Gln Pro Read Phe Pro 325 330 335

Ser Arg Arg His Leu Ile Tyr Ala Phe Ser Tyr Phe Tyr Asp Arg Leu 340

345

350

Tyr His Phe Ser Lys Glu Gly Ser Pro Val Tyr Val Ser Ser Tyr Lys 355 360 365

Glu Val Gly Gln Glu Val Cys His Arg Glu Ser Wing Arg Arg Thr Thr 370 375 380

Pro Wing Glu Glu Thr Wing Cys Met Glu Read Arg Tyr Met Tyr Ser Phe 385 390 395 400

Leu Thr Tyr Gly Leu Gly Leu Be Asp Wing Thr Wing Leu Thr Val Pro 405 410 415

Asn Arg Ile Glu Gly Met Wing Val Ser Trp Ser Leu Gly Ser Ser Leu 420 425 430

Ser Phe Leu Leu Lys Met Glu Val Asp Lys Leu Wing Wing Wing Leu Glu 435 440 445

His His 450

His His His His

Claims (8)

1. "Leishmanian RECOMBINANT E-NTPDases characterized in that they are homologous to natural proteins obtained from the nucleotide sequence of the coding genes for these proteins, including SEQ. ID NO. 1, SEQ. ID. NO. 2, SEQ. ID. No. 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 9 10, SEQ ID NO: 11 and SEQ ID NO: 12, and any distinct variation of said sequences, provided that they are intended to retain their antigenic properties; "Leishmania major RECOMBINANT E-NTPDase" according to claim 1, characterized by SEQ. ID NO 1 and SEQ. ID No. 2, derived from the Leishmania major GDPase coding gene, having an amino acid identity of 93.74% relative to the natural Leishmania major GDPase protein, with 42 base pairs being added to the aminoterminal portion and the carboxy terminal portion removed. the last three nucleotides (stop codon) and added 45 base pairs, having 719 amino acids; "Leishmania braziliensis RECOMBINANT E-NTPDase" according to Claim 1, characterized by SEQ. ID No. 3 and SEQ. ID No. 4, derived from the Leishmania braziliensis GDPase coding gene, having an amino acid identity of 65.75% relative to the recombinant Leishmania major GDPase protein, with 42 base pairs being added to the aminoterminal portion and the carboxy terminal portion removed. the last three nucleotides (stop codon) and added 45 base pairs, having 720 amino acids; "Leishmania infantum RECOMBINANT E-NTPDase" according to claim 1, characterized by SEQ. ID No. 5 and SEQ. ID No. 6, derived from the Leishmania infantum GDPase coding gene, having an amino acid identity of 84.49% relative to the Leishmania major recombinant GDPase protein, with 42 base pairs being added to the aminoterminal portion and the carboxy terminal portion removed. the last three nucleotides (stop codon) and added 45 base pairs, having 706 amino acids; "Leishmania major RECOMBINANT NTPDase" according to claim 1, characterized by SEQ. ID No. 7 and SEQ. ID No. 8, derived from the Leishmania major NTPDase coding gene, with an amino acid identity of 23.09% relative to the natural Leishmania major GDPase protein, with 42 base pairs being added to the aminoterminal portion and the carboxy terminal portion removed. the last three nucleotides (stop codon) and added 39 base pairs, having 452 amino acids; "Leishmania braziliensis RECOMBINANT NTPDase" according to claim 1, characterized by SEQ. ID No. 9 and SEQ. ID No. 10, derived from the Leishmania braziliensis NTPDase coding gene, with an amino acid identity of 23.09% relative to the natural Leishmania major GDPase protein, with 42 base pairs being added to the aminoterminal portion and the carboxiterminal portion removed. the last three nucleotides (stop codon) and added 45 base pairs, having 454 amino acids; "Leishmania infantum RECOMBINANT NTPDase" according to claim 1, characterized by SEQ. ID No. 11 and SEQ. ID No. 12, derived from the Leishmania infantum NTPDase coding gene, having an amino acid identity of 22.13% relative to the natural Leishmania major GDPase protein, with 42 base pairs being added to the aminoterminal portion and the carboxiterminal portion removed. the last three nucleotides (stop codon) and added 45 base pairs, having 454 amino acids; 8. "USE OF DEFINING RECOMBINANT E-NTPDases IN CLAIMS 1 to 7", characterized in that it is used in the production of a diagnostic kit for detection of antibodies in leishmaniasis caused by species of the genus Leishmaniay in vaccine compounds, in the development of monoclonal antibodies, in prognostic markers in the rational design of E-NTPDase inhibitor drugs that can be used in leishmaniasis chemotherapy.