ES2325701B1 - USE OF THE NCBSR4 GENE FOR THE DIAGNOSIS AND PREVENTION OF NEOSPOROSIS AND AS A MARKER FOR THE ANALYSIS OF PATHOGENICS. - Google Patents

Abstract

Use of the Nc BSR4 gene for the diagnosis and prevention of neosporosis and as a marker for the analysis of pathogenesis.

The present invention relates to the use of the Nc BSR4 gene and the Nc BSR4 protein, expression vectors and host cells containing them, for the diagnosis and prevention of neosporosis and, as a specific marker of the bradyzoite phase of N Caninum , for the analysis of the pathogenesis or the efficacy of vaccines against the establishment of chronic infection in the intermediate host. It also refers to the use of the Nc BSR4 gene promoter for the expression of heterologous genes that allows the study of molecular mechanisms that determine the conversion of tachyzoite stage to bradyzoite and vice versa.

Furthermore, the present invention relates to the development of monoclonal antibodies and monospecific polyclonal sera against the Nc BSR4 protein and its recombinant form, which represent an alternative for the serological diagnosis of neosporosis and chronic infection by N. caninum in tissues animals.

Description

Use of the Nc BSR4 gene for the diagnosis and prevention of neosporosis, and as a marker for the analysis of pathogenesis.

Object of the invention

The present invention is established in the field of animal health and refers, as expressed in the statement of this specification, to the diagnosis, analysis of the pathogenesis and prevention of the disease caused by the protozoan parasite Neospora caninum . More specifically, the invention relates to the polynucleotide molecule corresponding to the Nc BSR4 gene of N. caninum , its messenger RNA and the antigen it encodes, NcBSR4, this being a specific protein of the bradyzoite stage, as well as the oligonucleotides, the vectors recombinant, transformed host cells, and recombinantly expressed proteins, their use as reagents for the diagnosis of the disease, its application to the development of molecular techniques that allow to study the pathogenesis of the disease, in addition to its usefulness for vaccine production

Background Neosporosis

N. caninum is a protozoan parasite belonging to the phylum Apicomplexa that includes other important pathogenic parasites such as Toxoplasma gondii with which it is closely related. N. caninum has been described since 1989 as a producer of abortion and neonatal mortality in cattle (Thilsted and Dubey. 1989. J. Vet. Diagn. Invest. 1, 205-209), although it is capable of infecting a broad spectrum of mammalian species (Dubey JP 2003. Korean J Parasitol. 41, 1-16; Moore DP 2005. Vet. Parasitol. 127, 87-97).

Bovine neosporosis is considered a parasitic disease of cosmopolitan distribution and one of the most frequent causes of reproductive failure (Dubey JP 2003. J. Parasitol. 89, S42-S56). The most important clinical manifestation of infection in pregnant females is abortion and usually takes place between the third and ninth month of pregnancy, being more frequent around 5-6 months. Congenitally infected calves born alive may present with neuromuscular problems, although the most common is the birth of clinically healthy calves (Buxton et al . 2002. Trends Parasitol. 18, 546-552). Likewise, neosporosis can affect dogs, definitive hosts, although they can also act as intermediate hosts, where it causes polymyositis, encephalitis, paralysis and can cause death (Lindsay and Dubey. 1989. J. Parasitol. 75, 163-165 ; Buxton et al . 2002. Trends Parasitol. 18, 546-552).

Like T. gondii , N. caninum has a life cycle comprising three stages (Dubey and Lindsay 1996. Vet. Parasitol. 67, 1-59; Basso et al . 2001. J. Parasitol. 87, 612-618). The sporozoites infect the intermediate host by ingestion of the oocysts removed by the definitive host that sporulate into the environment. In the intermediate host two different intracellular phases develop: tachyzoites, rapid replication and responsible for the acute phase of the infection, and bradyzoites, the slow multiplication of the parasite giving rise to tissue cysts located primarily in the central nervous system . These bradyzoites remain dormant in the tissue cysts until they are reactivated (Antony and Williamson. 2001. New Zeal. Vet. J. 49, 42-47; Buxton et al . 2002. Trends Parasitol. 18, 546-552). Several studies in T. gondii and N. caninum indicate that the differentiation of tachyzoite to bradyzoite in these parasites is a stress-induced phenomenon, although the molecular mechanisms that determine the passage from one phase of the cycle to another are not known exactly. Likewise, an implication of the immune response in latency and reactivation of infection in chronically infected animals has been suggested (Lyons et al ., 2002, Trends Parasitol. 18, 198-201).

N. caninum antigens

Regarding the comparison of the antigenic composition between tachyzoite and bradyzoite of N. caninum , the information available so far is scarce, a fact that contrasts with the studies carried out in T. gondii , where there are several specific antigens of stage identified and characterized.

In N. caninum , specific tachyzoite antigens or shared by both stages have been identified (Fuchs et al . 1998. J. Parasitol. 84, 753-758; Lee et al . 2004. J. Vet. Sci. 5, 139-145 ; Shin et al . 2004. Proteomics 4, 3600-3609; Shin et al . 2005. Vet. Parasitol. 134, 41-52). Among these antigens two surface proteins, NcSAG1 (Hemphill et al . 1997. Parasitology 115, 371-380), specific for tachyzoite, whose gene was cloned by Howe et al . (1998. Infect. Immun. 66, 5322-5328) and NcSRS2 (Hemphill et al . 1996. Parasitol. Res. 82, 497-504), which is expressed jointly in tachyzoites and bradyzoites. Three micronema proteins have been identified, NcMIC3 (Sonda et al . 2000. Mol. Biochem. Parasitol. 108, 39-51), NcMIC1 (Keller et al . 2002. Infect. Immun. 70, 3187-3198), present in both stages, and NcMIC4 (Keller et al . Infect. Immun. 2004. 72, 4791-4800), identified from tachyzoites. Likewise, two Neospora sequences called Nc MIC10 and Nc MIC11 have been included in the gene bank, which could encode two micronema proteins, since their sequences have a high homology with the sequences encoding the T. gondii TgMIC10 proteins . and TgMIC11, respectively. Other genes that have been donated are Nc GRA6 and Nc GRA7 , which encode dense granule proteins of N. caninum tachyzoite (Lally et al . 1996. Clin. Diagn. Lab. Immunol. 3, 275-279). In addition to these dense granule proteins, other so-called NcNTPase-I have been described (Asai et al . 1998. Exp. Parasitol. 90, 277-285), NcGRA2 (Ellis et al . 2000. Parasitology 120 (Pt 4), 383 -390) and NcGRA1 (Lee et al . 2003. Proteomics 3, 2339-2350). The NcGRA1, NcGRA2, and NcGRA7 proteins are located not only in the tachyzoite phase but in the wall of cysts produced in vitro (Vonlaufen et al . 2004. 72, 576-83). Likewise, a 56 kDa antigen present in the apical complex of several stages of parasite development has been recently described (Jenkins et al . 2004. J. Parasitol. 90, 660-663). Based on several of these antigens, several recombinant ELISAs have been developed for the diagnosis of N. caninum infection (reviewed in: Ortega-Mora et al . 2006. Acta Parasitol. 51, 1-14).

Finally, three genes that express enzymes have been cloned in N. caninum : Nc SUB1 that expresses a 65 kDa serine protease, located in the tachyzoite micronems (Louie and Conrad. 1999. Mol. Biochem. Parasitol. 103, 211- 223; Louie et al . 2002. J. Parasitol. 88, 1113-1119); a gene encoding a superoxide dismutase (Fe-SOD) that is expressed in both stages of the parasite, tachyzoites and bradyzoites (Cho et al . 2004. J. Parasitol. 90, 278-85) and the gene encoding an isomerase disulfide ( NcPDI) specific to tachyzoite and involved in the interaction of the parasite with the host cell (Naguleswaran et al . 2005. Int J Parasitol. 35, 1459-1472).

In relation to the specific antigens of N. caninum bradyzoite, only the Nc SAG4 gene has been recently identified, cloned and characterized (WO2005053505). However, in T. gondii , several genes that express specific Bradizoite antigens have been described, including several surface ones such as TgSAG4 (Ödberg-Ferragut et al . 1996. Mol. Biochem. Parasitol. 82, 237-244) and TgBSR4 (Knoll LJ, Boothroyd JC. 1998. Mol. Cell. Biol. 18, 807-814).

Diagnosis of neosporosis

For the diagnosis of bovine neosporosis, epidemiological data and clinical history are very important and can guide, but confirmation is always necessary through laboratory diagnosis (Anderson et al . 2000. Anim Reprod Sci. 60-61,417-431). For this, it is advisable to examine both the aborted fetus and the maternal serology. In this sense, two main problems arise in the diagnosis of bovine neosporosis: the diagnostic criteria and techniques used in the aborted fetus and, on the other hand, the diagnosis of infection and / or disease in the live animal, due to the absence of clinical signs in the chronically infected animal.

The etiological diagnosis of abortion in cattle is complex and laborious. In those cases in which an etiological diagnosis is reached, more than 90% correspond to infectious and parasitic agents among which, currently, N. caninum occupies a prominent place. The fetal diagnosis of abortion by N. caninum is mainly carried out by the observation of compatible lesions by histological techniques, examining their severity and extent, and the detection of the parasite by PCR or immunohistochemistry, mainly in the CNS.

As for the in vivo diagnosis of N. caninum infection, the laboratory diagnosis is mainly carried out by detecting specific antibodies in the serum or in the milk, both at the individual and the exploitation level, having developed various techniques throughout the last years (reviewed in: Ortega-Mora et al . 2006. Acta Parasitol. 51, 1-14). These techniques are very valuable tools to study the relationship between N. caninum infection and abortion, the predominant route of transmission on the farm and for the establishment of basic control measures on farms (Ortega-Mora et al . 2006 Parasitol Act 51, 1-14).

In this way, the improvement of the diagnosis is of the utmost importance in order to determine precisely the health status of the animals. In this sense, in the last years a series of studies have been carried out with the aim of validating the serological techniques that are currently used, clarifying some controversial aspects, such as the choice of the cut-off point depending on the age of the animal and the technique used (Alvarez-García et al . 2003. Vet. Res. 34, 341-352; Blumroder et al . 2004. Vet. Parasitol. 120, 11-22). Likewise, techniques such as avidity have been developed (Björkman et al . 1999. J Vet Diagn Invest. 11, 41-44; Aguado-Martínez et al . 2005. J Vet Diagn Invest. 17, 442-450), which they allow to distinguish between a recent and a chronic infection, and it gives us information about the predominant transmission route in the farm. On the other hand, the use of the ELISA technique in milk tank has been applied (Chanlun et al . 2002. Vet. Parasitol. 110, 35-44) that allows to know the state of the exploitation in a first approximation or else Can use for monitoring control programs. Likewise, various ELISAs have been developed based on the use of recombinant antigens (reviewed in: Ortega-Mora et al . 2006. Acta Parasitol. 51, 1-14), although so far none of the diagnostic techniques used against N. caninum allows to distinguish between a chronic infection and a reactivation of the infection or a reinfection.

Prevention of neosporosis

Regarding disease control, due to that the vertical transmission of the disease seems to be the route main disease establishment persistently on farms and that neosporosis is one of the main causes of abortions and neonatal mortality in cattle bovine, with subsequent economic losses, control must go, fundamentally, to reduce the prevalence of farm infection, establishing disposal measures and Selective replacement to reduce infected cash. Further, since it has been shown that postnatal infection by ingestion oocysts can take place on farms, you have to apply  management measures such as control of dogs' access to exploitation, which act as definitive hosts, and the ingestion of placentas and other fetal tissues (Reichel and Ellis. 2002. N Z Vet J. 50, 86-92).

On the other hand, the pharmacological control of neosporosis in cattle is unfeasible at present, it is not has experience in the pharmacological treatment of the disease in cattle and the available data are not very encouraging. Without However, the high costs of a possible treatment, the appearance of possible resistance and residues in meat or milk, limit Chemotherapy as a control measure. In this way at herd management measures should be added the immunoprophylaxis

In this regard, research carried out in recent years for the development of a vaccine against N. caninum has included the evaluation of dead vaccines with variable results, finding some protection or reduction of vertical transmission in a murine model ( Liddell et al ., 1999, J. Parasitol. 85: 1072-1075; Miller et al . 2005. Int J Parasitol. 35, 821-8), but not in a bovine model (Andrianarivo et al ., 1999, Int. J. Parasitol 30: 985-990), although a so-called Bovilis-NeoGuard (Intervet) is currently marketed with a reasonable effect on the abortion rate (Romero et al . 2004. Vet. Parasitol. 123 (3-4 ), 149-59). Immunization with live vaccines has also been used, based on less virulent isolates (Atkinson et al . 1999. Parasitology 118: 363-370) or temperature sensitive mutants (Lindsay et al . 1999. J. Parasitol. 85: 64- 67), in vaccination trials in mice, obtaining promising but not definitive results, presenting the problem of originating persistently infected animals. On the other hand, subunit vaccines have a number of advantages over traditional vaccines, including the safety and relative stability of recombinant proteins, compared to live parasites, the flexibility to incorporate only those antigens that induce an immune response. protective, as well as the ability to establish large-scale production (Jenkins. 2001. Vet. Parasitol. 101, 291-310). The development of subunit vaccines for the prevention of infection, abortion or vertical transmission of the infection, based on the antigens of N. caninum , provides a new instrument for its control (Innes et al . 2002. Trends Parasitol 18, 497-504; Nishikawa et al . 2002. J. vet. Med. Sci. 64, 1-5). At the moment, the work done in this sense is scarce, and only tachyzoite antigens or shared by both stages have been used, inoculated as recombinant proteins, as DNA vaccines or as a combination of both systems. Trials conducted so far have used murine models, some of them pregnant to measure congenital transmission. Thus the following proteins have been used: NcMIC3 (Cannas et al . 2003. J. Parasitol. 89 (1), 44-50), NcMIC1 (Alaeddine et al . 2005. J. Parasitol. 91, 657-65), NcGRA7 (Liddell et al . 2003. J. Parasitol. 89, 496-500; Jenkins et al . 2004. Infect Immun. 72, 1817-9), NcsHSP33 (Liddell et al . 2003. J. Parasitol. 89, 496-500 ), NcSAG1 (Cannas et al . 2003. Parasitology 126, 303-312) and NcSRS2 (Cannas et al . 2003. Parasitology 126, 303-312; Pinitkiatisakul et al . 2005. Vet Parasitol. 129, 25-34), these two last proteins of the surface of the tachyzoite, obtaining good results. In a recent work, a combination of recombinant antigens (NcSAG1, NcSRS2, NcGRA6 and NcGRA7) has been used, obtaining the best results of in vitro and in vivo protection with the combination of NcSRS2 and NcGRA7 (Cho et al . 2005. Korean J Parasitol. 43, 19-25). On the other hand, a recombinant "vaccinia" virus has been tested to express NcSRS2 in the prevention of vertical transmission of N. caninum in a murine model (Nishikawa et al . 2001. Vaccine 19, 1710-1716).

However, so far the development of these vaccines has not been based on specific antigens of the bradyzoite stage, since the first one described in N. caninum is that produced by the Nc SAG4 gene (WO2005053505) and the one set forth in the description of the invention of this specification (Nc BSR4 ) is the second.

As stated in these antecedents, the isolation of genes that are specifically expressed in each stage is of great interest for the study of this disease and of the molecular mechanisms that determine the establishment of chronic infection, as well as the reactivation of the infection, improving knowledge and control of this disease. In this sense, the isolation of the Nc BSR4 gene in N. caninum , ortholog of the Tg BSR4 gene, which encodes a specific protein of the bradizoite stage in T. gondii , its cloning and the expression of the specific antigen of the NcBSR4 stage as a recombinant protein , as described in the description of the invention herein, provides a very useful solution for the diagnosis of neosporosis, as well as for the analysis of the pathogenesis of the disease, and its vaccine use represents an alternative for the control of the same.

Description of the invention

Use of the Nc BSR4 gene for the diagnosis and prevention of neosporosis, and as a marker for the analysis of pathogenesis.

The invention provides a reagent useful for use for diagnostic and vaccination purposes against N. caninum . In addition, it provides a marker for the analysis of the pathogenesis of neosporosis, mainly for the study of the establishment of the chronic phase of the infection, as well as the reactivation of the infection.

A molecular technology is described that has allowed the identification, isolation and characterization of the Nc BSR4 gene of N. caninum (described in SEQ ID NO: 13), this being the second one specific to the bradyzoite stage described in this parasite. For this, the chromosomal walk technique has been used to obtain the complete sequence of the gene, designing four oligonucleotides based on a sequence of N. caninum (NcEST3c28hO2.y1) that shares an identity of 44% with the previously existing Tg BSR4 gene in the databases. Thus, oligonucleotides R1BSR4 (SEQ ID NO: 1) and R2BSR4 (SEQ ID NO: 2) were used to complete the gene in the 5 'sense. Oligonucleotides F1BSR4 (SEQ ID NO: 3) and F2BSR4 (SEQ ID NO: 4) were used to complete the gene in the 3 'direction. To complete and confirm the internal sequence, two more oligonucleotide pairs were used: NcESTF (SEQ ID NO: 5), NcESTR (SEQ ID NO: 6), FNCEST5 (SEQ ID NO: 7) and RNCEST3 (SEQ ID NO: 8) .

One aspect of the present invention relates to a method of donation of the Nc BSR4 gene, by means of its PCR amplification, using oligonucleotides specially designed for it and its subsequent digestion with restriction enzymes. This cloning is performed in a plasmid for expression in a prokaryotic system in which the Nc BSR4 gene or a part thereof is inserted. Preferably, the plasmid pRSET (Invitrogen) is used and, also preferably, the fragment of the Nc BSR4 gene encoding from amino acid 40 to 408 included, corresponding to the sequence of the NcBSR4 protein is inserted, excluding the region of the end signal peptide amino thereof. For this, the Nc BSR4 gene, or the corresponding region to be cloned, is amplified by PCR, from N. caninum genomic DNA, using the oligonucleotides designed for this purpose called FBamHNcBSR4 (SEQ ID NO: 9) and RHindIIINcBSR4 (SEQ ID NO: 10). Systems are used in which the expression of the rNcBSR4 recombinant protein itself facilitates its purification by usual methods. To produce the recombinant rNcBSR4 protein, cells that allow the expression of the recombinant proteins and, preferably, cells that induce such expression are used. Preferably, the rosetta strain (DE3) pLysS of E. coli (Novagen), which induces phage RNA T7 RNA polymerase, and induces the expression of the rNcBSR4 recombinant protein by adding IPTG to the culture medium is used. The use of this recombinant protein for serological diagnosis of neosporosis, provides a very valuable novel tool for the differential diagnosis of the different phases, acute and chronic, of the infection, by ELISA, radioimmunoassay (RIA), or any other method based on the antigenic capacity of said polypeptides. For this purpose, chemically or enzymatically modified sequences derived from homologs to the NcBSR4 protein, NcBSR4 derived polypeptides or recombinant proteins that include NcBSR4 or include NcBSR4 derived polypeptides can also be used, provided they retain the antigenic characteristics of this protein. For example, by means of the "western blot" technique, antibodies against the rNcBSR4 protein are detected, separated by electrophoresis under denaturing conditions in 15% acrylamide-DATD gels, in sera of cattle with chronic natural infection by N. caninum . This demonstrates the immunogenicity of this protein and therefore its usefulness for the diagnosis and prevention of the disease through its use as a vaccine.

Similarly, the development of monoclonal antibodies and monospecific polyclonal sera against the recombinant rNcBSR4 protein represents an alternative for diagnosis by developing techniques based on these, such as the competition ELISA. Likewise, these monoclonal antibodies or specific polyclonal sera against the polypeptides described in this invention are used for the diagnosis of chronic N. caninum infection in animal tissues by immunohistochemistry, immunofluorescence or any other method based on the detection of parasite by the said serum.

Another aspect of the present invention is that of provide vaccines and vaccination kits against neosporosis, vertical transmission of infection establishment of the chronic infection The heterologous gene expression system in prokaryotes, has the advantage of being able to produce on a large scale the recombinant antigen, using it as a subunit vaccine. These vaccines have another advantage and is to be used as marked vaccines, being the immune response they produce easily distinguishable from that produced by the parasite in the infected animals, which is of vital importance in campaigns of eradication of diseases. In addition these vaccines are safe, which represents a benefit from vaccines based on other recombinant vectors such as viral.

This type of recombinant proteins used as a subunit vaccine, they induce an immune response mainly of humoral type. So, to direct the answer Adjuvants that are also immune to a cell-based base are used. Orient it in this regard, including various cytokines in alone or with other adjuvants, preferably particulate. Likewise, to guide the cellular base response and ensure the proper folding of proteins, vaccines are used DNA

On the other hand, the present invention includes the use of the Nc BSR4 gene as a marker of the bradyzoite phase of N. caninum associated with chronic infection. This gene is used as a marker by using the nucleotide molecules described in this invention for the diagnosis of chronic infection by N. caninum , from tissues or fluids of infected animals, by PCR or RT-PCR, in situ hybridization. with DNA probes, or any other detection method based on the nucleic acids of the zarásito. A method of detecting the expression of the Nc BSR4 gene in the parasite is detailed by means of a real-time quantitative RT-PCR, using two oligonucleotides designed for this purpose, called NcBSR4F3 (SEQ ID NO: 11) and NcBSR4R3 (SEQ ID NO : 12). This RT-PCR allows the use of the NcBSR4 gene as a marker of the change of stage of N. caninum , in the intermediate host. It is a necessary tool for the analysis of the mechanisms that determine the establishment of chronic infection, of the mechanisms involved in the reactivation of the infection in persistently infected animals, and of the factors that influence these processes. This tool also serves to determine the efficiency of vaccine products against N. caninum in terms of protection against the establishment of chronic infection and reactivation. Similarly, the use of the Nc BSR4 gene promoter to express heterologous genes in transfected N. caninum cells thanks to genetic constructs made with the said promoter, allows the analysis of the molecular mechanisms that determine the conversion of tachyzoite to bradyzoite stage and conversely, both in vitro and in vivo expression systems, in cell cultures or in experimental animals.

Description of the drawings

To complement the description and in order to help better compression of the characteristics of the invention is accompanied by the present specification, as integral part of it, a game of drawings where with illustrative and non-limiting nature, what has been represented next:

Figure one

Panel A: Represents the expression plasmid that carries the corresponding coding region of the Nc BSR4 gene, as explained in example 2 of this specification. Panel B: the rNcBSR4 recombinant protein scheme, as explained in example 2 herein.

Figure 2

The figure shows the selection of plasmids recombinant by digestion with restriction enzymes, as explained in example 2 of this report. Recombinant plasmids pBSR4.5 (lane 1) and pBSR4.7 (lane 3) released a fragment of the expected size (1122 bp) and undigested plasmids were visualized on the same gel (lanes 2 and 4, respectively). In addition, the vector pRSET-C was digested as a control of digestion (lane 5). As molecular marker (L) was used "1Kb Plus DNA ladder (Invitrogen)".

Figure 3

Panel A: Expression of the recombinant rNcBSR4 protein in E. coli , as explained in example 2 of this specification, where PP is "Precision Plus Protein Standards (Bio-rad)". Panel B: rNcBSR4 purified by affinity chromatography (IMAC), MW being "Molecular weight standards, low range (Bio-rad)".

Figure 4

Characterization of the immunogenicity of the recombinant rNcBSR4 protein, as explained in example 4 of this specification. The figure shows the reaction of a specific rabbit polyclonal serum produced against the rNcBSR4 protein, with a mixed extract of N. caninum bradyzoites and tachyzoites produced in vitro (B) by "western blot" (lane 3). As a control, an extract of tachyzoites produced in vitro (T) was used in which no protein was recognized (lane 4). As a marker of bradyzoites, another rabbit polyclonal serum directed against the rNcSAG4 protein was used, which recognized the NcSAG4 protein, specific to the bradyzoite phase, in the bradyzoite extract (B) and not in the tachyzoite (T) extract (Figure 4, lanes 1 and 2, respectively).

Figure 5

Regulation of the transcription of the Nc BSR4 gene in the bradyzoite stage of N. caninum measured by real-time RT-PCR, as explained in example 5 of this report. The figure shows the variation in messenger RNA levels (expressed as n-fold induction and represented in the ordinate axis) of the various genes analyzed: NcSAG1 (SAG1), NcSAG4 (SAG4) and NcBSR4 (BSR4), throughout the conversion test on days 1, 2, 3, 5 and 7 (lanes 1,2,3, 5 and 7, respectively).

Embodiment of the invention

The present invention is further illustrated. through the following examples, which are not limiting of its scope, which is defined exclusively by the note attached claim.

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Example one

Isolation and characterization of the Nc BSR4 gene

For the isolation of the Nc BSR4 gene from N. caninum , the method called chromosomal walk or "genome-walking" was used, which allows amplifying by PCR DNA fragments whose sequence is unknown but flanking regions of known DNA.

The NcEST3c28h02.y1 sequence of N. caninum present in the databases (NcGI: TIGR Nespora caninum gene index, www.tigr.org/tdb/tgi) was used to design the necessary oligonucleotides for the chromosomal path . 44% with the TgBSR4 gene that expresses a specific antigen of the bradyzoite phase of T. gondii . To perform the chromosomal walk in the 5 'and 3' direction from this sequence the genomic DNA of N. caninum was used, using a commercial kit (Universal Genome WalkerTM kit, BD Biosciences Clontech). The first step to carry out this technique was the digestion of genomic DNA with four different restriction enzymes that produce blunt ends: Eco RV, Dra I, Pvu II and Stu I, and subsequent ligation to adapters, following the instructions from the commercial house. Then, with each of the libraries thus obtained, a double PCR was performed using oligonucleotides that bind to the adapter (AP1 and AP2) and the gene-specific oligonucleotides, designed according to the known genomic sequence. The oligonucleotides designed to perform the chromosomal walk in the 5 'direction were designated R1BSR4 (SEQ ID NO: 1) and R2BSR4 (SEQ ID NO: 2) and those designed to perform the chromosomal walk in the 3' direction were designated F1BSR4 (SEQ ID NO : 3) and F2BSR4 (SEQ ID NO: 4). The amplification was performed by a nested PCR, using a long-distance thermostable DNA polymerase (Ecotaq Plus, Ecogen), in the corresponding buffer, in the presence of Cl2 Mg (2 mM), 200 µM of each dNTP and 10 µM of each oligonucleotide, in a reaction volume of 50 µl. The first PCR was performed with each of the "genomic DNA libraries" using oligonucleotides AP1 and R1BSR4 or F1BSR4, to perform the chromosomal walk in the 5 'or 3' direction, respectively, following the instructions of the commercial house. The conditions under which the PCR was performed were 7 cycles of 25 seconds at 94 ° C and 3 minutes at 72 ° C followed by 32 cycles of 25 seconds at 94 ° C and 3 minutes at 67 ° C, with a final elongation at 67 ° C for 7 minutes. The second PCR was carried out from 1 µl of a 1:50 dilution of the products of the first PCR and was performed with oligonucleotides AP2 and R2BSR4 or F2BSR4, to perform the chromosomal walk in the 5 'or 3' direction respectively. The conditions under which the second PCR was performed were: 5 cycles of 25 seconds at 94 ° C and 3 minutes at 72 ° C, followed by 20 cycles of 25 seconds at 94 ° C and 3 minutes at 67 ° C with a final elongation at 67 ° C for 7 minutes. Finally, to confirm the internal sequence by PCR and sequencing, two pairs of oligonucleotides called: NcESTF (SEQ ID NO: 5) and NcESTR (SEQ ID NO: 6) were designed; FNCEST5 (SEQ ID NO: 7) and RNCEST3 (SEQ ID NO: 8). The PCR was performed using a high fidelity enzyme (Expand High Fidelity Plus PCR System, Roche), following the indications of the commercial house, using the following conditions: an initial denaturation of 2 minutes at 94 ° C followed by 10 cycles of 15 seconds at 94 ° C, 30 seconds 60 ° C and 1 minute at 72 ° C, followed by 20 cycles of 15 seconds at 94 ° C and 30 seconds 60 ° C and 1 minute at 72 ° C increasing 5 seconds in each cycle, with a final elongation at 72 ° C for 7 minutes. The DNA fragments obtained by the chromosomal walk were purified using a commercial kit (GENECLEAN Turbo nucleic acid purification kit, Q-BIOgene) and sequenced.

Thus, when comparing the sequences of the different amplified fragments, a sequence of 2676 nucleotides could be determined (SEQ ID NO: 13). This sequence was called Nc BSR4 , for its homology with that of the Tg BSR4 gene of T. gondii , which encodes a specific protein of the bradyzoite phase. Likewise, the existence of an open reading phase (ORF) of 1227 bp was verified, which encodes a 408 amino acid protein (SEQ ID NO: 14). When comparing the amino acid sequence encoding this ORF, a similarity of up to 66% was found, with a total of 273 similar residues, with the sequences of the TgBSR4 protein of the different strains of T. gondii existing in the databases (BLASTP, www.toxodb.org).

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Example 2

Expression of the BSR4 Nc antigen as a recombinant protein and its purification

Once the Nc BSR4 gene was identified, for its expression in prokaryotes a part of the gene (from nucleotide 1084 to 2195) that encodes a truncated form of the protein, from amino acid 40 to 408, was inserted into the Bam HI and Hind III of plasmid pRSET-C (Invitrogen) (Figure 1). For this, a PCR was performed from the genomic DNA of N. caninum , using oligonucleotides designed for this purpose: FBamHNcBSR4 (SEQ ID NO: 9), the direct oligonucleotide, at whose 5 'end a specific Bam recognition site was included HI followed by a sequence identical to that of the Nc BSR4 gene from nucleotide 1084 to 1106, and RHindIIINcBSR4 (SEQ ID NO: 10), the reverse oligonucleotide, at whose 5 'end a specific recognition site for Hind III was included followed by the complementary reverse sequence of the 3 'end of the ORF of the Nc BSR4 gene, from nucleotide 2174 to e12195 of the gene.

To perform the PCR, 0.1 µg of genomic DNA was used per reaction, isolated from N. caninum tachyzoites, using a high fidelity enzyme (Expand High Fidelity Plus PCR System, Roche), following the indications of the commercial house, under the conditions previously described in example 1 of this specification. The amplified DNA was purified (GENECLEAN Turbo nucleic acid purification kit, Q-BIOgene) and digested with the enzymes Bam HI (5U) and Hind III (5U) in the appropriate buffer overnight. At the same time and under identical conditions the plasmid vector pRSET-C was digested. Finally, the digestion products were purified (GENECLEAN Turbo nucleic acid purification kit, Q-BlOgene). Once the digested DNA fragment was purified, it was ligated to 50 ng of the linearized vector plasmid in a 1: 3 vector: insert, in the presence of 1U of the enzyme T4DNA ligase, in the appropriate buffer and in a final volume of 15 µl. Ligation was performed in 0.5 ml tubes (M? Multi, Sorenson BioScience), in a thermal cycler for 18 hours at 12 ° C, being subsequently stored at -20 ° C until use.

For the manipulation of plasmids, the methodology described by Sambrook et al. (1989). Once the ligation was carried out, it was used to transform chemically competent NovaBlue (Novagen) strain cells frozen, by thermal shock under the conditions indicated by the commercial house. The competent bacteria were thawed and kept on ice, until 1 µl of the ligation was added. After 5 minutes on ice they were heated at 42 ° C in a water bath for 30 seconds and immediately placed on ice for 2 minutes. The transformed bacteria were immediately resuspended in 250 µl of SOC medium (10 mM MgSO 4, 10 mM MgCl 2 and 20 mM glucose added to SOB medium: 2% bactotriptone, 0.5% yeast extract, NaCl 10 mM and 2.5 mM KC1), were incubated for one hour at 37 ° C and, finally, 50 µl of the cell suspension was plated in LB-agar medium (1.5% noble agar) in the presence of 100 µg / ml of Ampicillin, antibiotic against which the plasmids used are resistant. Bacteria transformed with the plasmids were selected after growth in the plates with the appropriate medium, for 18 hours at 37 ° C. 10 clones of each were selected and incubated overnight at 37 ° C in 3 ml of liquid LB medium with 100 µg / Ampicillin, for subsequent isolation of plasmid DNA. This DNA was obtained by the alkaline lysis method and subsequently visualized on a 0.8% agarose gel in 1x TAE buffer, to select those clones with the plasmid of the expected size. The plasmids of interest were named pBSR4, numbered from 1 to 10 according to the clone. According to the size observed, two clones (pBSR4.5 and pBSR4.7) were selected for characterization by restriction enzymes. Thus, the plasmids obtained with the restriction enzymes Bam HI (5U) and Hind III (5U) were digested in the appropriate buffer, in a final volume of 20 µl, for two hours at 37 ° C in a water bath. The digested plasmid DNA was separated on a 0.8% agarose gel in 1% TAE buffer, observing the release of a fragment of the appropriate size (1122 bp) in each of the plasmids constructed pBSR4.5 and pBSR4.7 (Figure 2, lanes 1 and 3 respectively). Undigested plasmids were visualized on the same gel (Figure 2, lanes 2 and 4). In addition, the vector pRSET-C (Figure 2, lane 5) was digested and used as a control of digestion. Plasmid pBSR4.5 was sequenced using commercial oligonucleotide T7, confirming the correct insertion of the Nc BSR4 gene fragment into the expression vector. Sequencing showed a silent mutation in nucleotide 1824 of the gene (SEQ ID NO: 13) by replacing an adenine (A) of the original sequence of the Nc BSR4 gene, with a guanine (G), which translates into amino acid 282 into a glutamine (E) as in the original sequence of NcBSR4 (SEQ ID NO: 14).

The sequence cloned in plasmid pRSETC was expressed as a recombinant protein in Rosetta (DE3) pLysS cells of Escherichia coli (Novagen) and was designated rNcBSR4 (SEQ ID NO: 15). The recombinant rNCBSR4 protein comprises from amino acid 40 to 408 of the NcBSR4 protein, and corresponds to the amino acid sequence thereof in which the amino acid signal peptide would be missing. RNcBSR4 is a fusion protein attached at its amino terminus to a block of amino acids from the vector that include: 6 histidines, which allows its purification by affinity chromatography, the T7-tag peptide as well as an enterokinase recognition zone . In Figure 1, panel B shows a scheme of the recombinant protein rNcBSR4.

To express the recombinant protein in the prokaryotic system, cells of the Rosetta strain (DE3) pLysS of E. coli (Novagen) were transformed with the plasmid pBSR4.5, following the indications of the commercial house. The cells transformed with the plasmid were plated in LB-agar medium with 1% glucose, ampicillin (100 µg / ml) and chloramphenicol (34 µg / ml) and grown overnight at 37 ° C. The next day 10 colonies of the plates were selected and grown overnight at 250 rpm and 37 ° C in 3 ml of the same liquid selection medium. The next day, the 1:10 starter culture was diluted in the same growth medium and maintained in identical conditions until an A 600 of 0.8-0.9 OD was reached. Next, the expression of the recombinant protein was induced in the presence of 1 mM IPTG, for 4 hours under stirring at 250 rpm and 37 ° C. Finally the cells were collected by centrifugation at 3,500 xg for 15 minutes. Once the supernatant was removed, the sediments were stored at -80 until use. Finally, the proteins, solubilized in 1x Laemmli loading buffer (Laemmli. 1970. Nature 227, 680-685), were separated in a proportion of 50 µl / ml of original culture, by electrophoresis under denaturing conditions in acrylamide gels. DADT at 15% and stained with Commassie blue staining. The proteins were separated from both a total extract of the bacteria collected before induction (figure 3, panel A, lane 1), and after induction (figure 3, panel A, lane 2) of each clone, choosing the one of them for induction on a larger scale (pBSR4.5.9) whose expression is shown in Figure 3.

To characterize the expression of the recombinant proteins, the cell sediments, collected above, were lysed in a commercial break solution called 1x BugBuster (Novagen) in 20 mM Tris pH 7.98 at a concentration of 5 ml / g of cell pellet , in the presence of the enzyme benzonasa (1U / ml of sample, Novagen) and lysozyme (1 KU / ml of sample), incubating under gentle agitation and room temperature for 1 hour. The soluble fraction of the sediment, where the inclusion bodies are located, was then separated by centrifugation at 16,000 xg for 20 minutes to determine the location of the protein of interest. Subsequently, the inclusion bodies in the BugBuster and lysozyme solution were washed four times and resuspended in 0.1x BugBuster solution, as indicated by the commercial house. Finally the proteins were separated under the conditions described above. In this way the expression of the recombinant rNcBSR4 protein in Rosetta (DE3) pLysS cells of E. coli (Novagen) and its location in inclusion bodies was confirmed.

For purification of the rNcBSR4 protein by Affinity chromatography (IMAC) clone pBSR4.5.9 was grown in the conditions described above in larger quantities (volume induction end of 1 liter). For the solubilization of the protein, washed inclusion bodies were solubilized in denaturing conditions in 40 ml of a buffer containing salts of sodium phosphate (20 mM), sodium chloride (500 mM), imidazole (40 mM) and urea (8M), in gentle agitation and room temperature for 2 hours They were finally centrifuged at 12,000xg for 15 minutes at 4 ° C and the supernatant was stored at -80 ° C until use.

For purification by IMAC the 1 ml HisTrap HP columns (GE Healthcare) in the Akta system Prime (GE Healthcare), following the directions of the house commercial. For this, the 40 ml described above were punctured. in the column, after an initial equilibration, and the protein was eluted by an increasing gradient of imidazole maintaining the denaturing conditions (8M urea) to reach 500 mM, concentration at which the protein was eluted. The protein obtained separated by electrophoresis under the conditions described above (figure 3, panel B, lane 2), and its concentration is determined by densitometry, using a pattern of different BSA concentrations in the same gel.

In order to confirm the amino acid sequence of the rNcBSR4 protein once purified, it was analyzed by mass spectrophotometry. For the MALDI-TOF analysis and de novo sequencing , the 4700 Proteomic Analyzer (Applied Biosystems) of the proteomics service of the Complutense University of Madrid was used.

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Example 3

Production of polyclonal sera

To obtain polyclonal sera, rabbits of the female New Zealand breed of 2.5 kg live weight were used. The rabbits were kept in the animal shelter of the Complutense University of Madrid, with food and water ad libitum in individual cages. The first immunization was performed intradermally, inoculating a total of 1 ml in 20 points (0.05 ml / point of immunization) per rabbit. The inoculum was prepared in a final volume of 1 m (v / v), with 200 µg of the purified rNcBSR4 protein as described in example 2 herein, in the complete Freund's adjuvant (Difco Laboratories, USA), emulsifying the mixture by sonication and following the usual methodology. Subsequently, 5 immunizations were performed at intervals of 3 weeks, intramuscularly with 0.5 ml of inoculum. In this case, 100 µg of protein was used in incomplete adjuvant (v / v) of Freund (Difco Laboratories, USA). The serum of the animals was collected at 18 weeks after the first immunization and was named
anti-NcBSR4.

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Example 4

Determination of the immunogenicity of the rNcBSR4 protein

The immunogenicity of the rNcSAG4 recombinant protein was determined by "western blot", using sera of bovine origin from animals naturally infected with N. caninum . Infection of these sera had been previously diagnosed by the ELISA and indirect immunofluorescence (IFI) technique, presenting high specific antibody titres. To this end, the rNcBSR4 protein purified by IMAC was used at a concentration of 10 µg in minigeles of 15% acrylamide-DATD, by electrophoresis under denaturing conditions, and finally electrotransfer to a 0.2 µm nitrocellulose membrane was performed (Bio.rad), at 400 mA for one hour immersed in previously cooled transfer buffer (25 mM Tris, 192 mM glycine pH 8.5 and 20% methanol). To perform the "westen blot" basically followed the protocol described above (Álvarez et al . 2002. Vet. Parasitol. 107, 17-27). Thus, the membranes were blocked with 3% BSA in TBS with 0.05% Tween-20 (TBS-T) for one hour under stirring at room temperature. The membranes were then incubated with the bovine sera, at a 1:20 dilution in TBS-T with 0.3% BSA, for 1 hour with stirring at 37 ° C. After three rapid washes with TBS-T, followed by one of 15 minutes and two of 5 minutes, the membranes were incubated with a mouse anti-mouse IgG monoclonal antibody conjugated to peroxidase (Hipra), at a dilution of 1: 200 in TBS-T with 0.3% BSA, for one hour under stirring at 37 ° C. After another series of washes under the above conditions, it was revealed with a substrate solution prepared immediately before use (60 mg of 4-Chloro-1-Naphtol dissolved in 20 ml of methanol, 100 ml of TBS and 0.060 ml of peroxide hydrogen), for 15 minutes at room temperature in darkness and stirring. The sera of bovine origin that were used were from animals in which a chronic infection had been established, which recognized the rNcBSR4 protein. In addition, the negative serum used as a control showed no reaction as expected.

Likewise, a "western blot" was carried out with the rabbit polyclonal serum produced against the rNcBSR4 protein, using as an antigen an extract of bradyzoites (B) and tachyzoites (T) produced " in vitro " (Figure 4, lanes 3 and 4), to confirm the stage specificity of the NcBSR4 protein as well as its antigenicity. To produce and purify tachyzoites and bradyzoites, the protocol previously described by Fernández-García et al . (2006. Mol. Biochem. Parasitol. 146, 89-97). To induce stage conversion, cultures infected with sodium nitroprusside (SNP) were treated at a concentration of 70 µM in the culture medium for 7 days after infection, being replaced every two days. As a negative control, infected cultures were maintained without SNP treatment, for the production of tachyzoites. A mixture of tachyzoites and bradyzoites was collected by scraping off the infected cell tapestry and treated with SNP, centrifuging at 1350 xg for 15 min at 4 ° C. The tachyzoites produced in infected cultures untreated with SNPs were collected in the same way. After two washes in PBS under the same conditions, the zoites were resuspended using a 25G needle and purified using sephadex? G25 columns (PD-10, Amersham Biosciences), to remove cell debris. To do this, after balancing the column with 5 ml of PBS, the suspension of zoites in 5 ml of PBS was allowed to flow by gravity. After two washes with the same volume of PBS, the zoites were collected by centrifugation at 1350 xg for 15 min at 4 ° C. The zoites collected were kept frozen at -80 ° C until use.

To carry out the "western blot", 15% acrylamide-DATD gels were used, under denaturing conditions, using a total extract of zoites at a concentration of 10 7 per lane in the extract of tachyzoites (T) and 5x10 6 in the mixed extract of tachyzoites and bradyzoites (B), solubilized in 50 µl of a 1 x Laemmli rupture solution (Laemmli UK 1970. Nature 227, 680-685), by vigorous stirring, boiled and sonication in bath for 15 min at 15 ° C. The western blot was then performed as described above using a 1: 200 dilution of the rabbit polyclonal anti-rNcBSR4 serum and as a conjugate a peroxidase-bound rabbit anti-IgG (Sigma) at a dilution 1: 1000 The development was carried out by chemiluminescence, using the ECL system (Amersham Biosciences). As a control, a rabbit polyclonal serum directed against N. caninum (anti-TAQUI), specific to the tachyzoite phase, was used. The polyclonal anti-rNcBSR4 serum recognized the NcBSR4 protein in the bradyzoite extract (B) (figure 4, lane 3) and did not recognize any protein in the tachyzoite extract (T) (figure 4, lane 4), confirming the specificity of stage of this protein and its antigenic capacity. As a marker of bradyzoites, another rabbit polyclonal serum directed against the rNcSAG4 protein was used, which recognized the NcSAG4 protein, specific to the bradyzoite phase, in the bradyzoite extract (B) and not in the tachyzoite (T) extract (Figure 4, lanes 1 and 2, respectively).

These results confirm the existence of a response against the NcBSR4 protein in the chronically infected animal in a natural way. Likewise, the specificity of the NcBSR4 protein of the bradyzoite phase is confirmed, as with its homologue in T. gondii (TgBSR4), since there is recognition of the native protein in bradyzoites produced in vitro by a polyclonal serum raised against the recombinant rNcBSR4 protein.

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Example 5

Determination of the transcription of the Nc BSR4 gene in the bradyzoite phase of N. caninum by RT-PCR

To study the regulation of the expression of the Nc BSR4 gene, an analysis of the evolution of the gene transcription was carried out, by measuring the production of its messenger RNA by quantitative RT-PCR, in a conversion assay over several days. For this, the protocol described by Fernández-García et al . (2006. Mol. Biochem. Parasitol. 146, 89-97), designing specific oligonucleotides for the Nc BSR4 gene which were named NcBSR4F3 (SEQ ID NO: 11) and NcBSR4R3 (SEQ ID NO: 12). As a control, the previously described oligonucleotides were used to amplify part of the Nc SAG4 gene, specific to the bradizoite phase and to the Nc SAG1 gene, specific to the tachyzoite phase. As normalizer the ribosomal gene 18S of N. caninum (Nc 18sR ) was used, using previously described oligonucleotides, Nc18sF (SEQ ID NO: 16) and Ncl8sR (SEQ ID NO: 17). For the Nc SAG4 gene, oligonucleotides NcSAG4F (SEQ ID NO: 18) and NcSAG4R (SEQ ID NO: 19) were used. For the NcSAG1 gene the oligonucleotides NcSAG1F (SEQ ID NO: 20) and NcSAG1R (SEQ ID NO: 21) were used.

For the conversion test the protocol previously described in example 4 of this report descriptive Zoites were collected on days 0 (before stress) 3, 5 and 7 post-stress and were purified as they were previously described, but using all reagents prepared to handle RNA, that is to say free of RNAsas, and following the appropriate management measures for this. For the extraction of Total RNA sediments of 10 7 tachyzoites or a mixture of tachyzoites and bradyzoites. The kit was used for this. commercial NucleoSpin RNA II (BD Biosciences Clontech), following the indications of the commercial house. The total RNA obtained is separated by electrophoresis on a 0.8% agarose gel in TAE buffer 1x, to check its quality. RNA concentration Total obtained was measured on a spectrophotometer.

The cDNA was synthesized using 50 ng of total RNA using 2 U of an enzyme called Superscript II RNAse H minus Reverse Transcriptase (Invitrogen), 12.5 nM of "random hexamers" (Applied Biosystems) and 40 U of an RNAse inhibitor ( RNAse inhibitor, Ambion) per reaction, in 20 µl of final volume, following the indications of the commercial house (Invitrogen). Next, to quantify the amount of messenger RNA of each gene with respect to the normalizer at each test point, a 1:10 dilution obtained from the cDNA previously synthesized from each sample was used. Quantitative PCR was carried out by performing triplicates for each gene to be amplified, with 2 µl in each well of each sample dilution, using a commercial kit that uses a Taq polymerase and SYBR Green (Platinum SYBR Green qPCR SuperMix UDG, Invitrogen ), following the indications of the commercial house, in the ABI Prism 7000 Sequence Detector (Applied Biosystems). The PCR mixture contained a final concentration of 1.5 U of enzyme (Platinum Taq DNA polymerase, Invitrogen), 3 mM of MgCl2, 200 µM of each dNTP, 1 U of UDG and 200 nM of each oligonucleotides in 25 µl final volume. The amplification conditions were: one cycle of 50 ° C for 2 minutes followed by 10 minutes at 95 ° C and then 40 cycles of 95 ° C for 15 seconds and 60 ° C for 1 minute. The "Sequence Setection System Software v.1.6." Program was used for amplification, data acquisition and analysis. (Applied Biosystems). The results were expressed as n-times of induction for each gene in each sample, calculated using the formula of 2 Δ Δ Ct. For this purpose, the average of the threshold cycle values ( C t) was initially obtained. ) of the amplification products of each triplicate in each sample (of each day of the test). Then the difference (\ Delta C {t}) between the average value of C {t} of the amplification products of genes under study (Nc BSR4, Nc SAG4 or Nc SAG1) and the mean value was calculated of C t of the amplification product of the Nc 18sR gene, used as a normalizer, for each sample. It was obtained in the same way the \ Delta C {T} of the sample used as a baseline, which are unstressed parasites collected at day 0 before the stress, for each gene. Subsequently, to calculate the Δ Δ C t, the Δ C t of the sample used as the baseline of the Δ C t value of each sample was subtracted for each gene. The absence of contaminating DNA was confirmed by PCR from the total RNA of each sample, using specific oligonucleotides to amplify the Nc 18sR gene.

Thus, the levels of messenger RNA of the Nc BSR4 , Nc SAG4 and Nc SAG1 genes in the N. caninum zoites obtained during the conversion test were analyzed on days 1, 2, 3, 5 and 7, with respect to the Basal levels obtained on day 0, before stress (Figure 5). In this way it was possible to observe an increase in the transcription of the Nc BSR4 gene throughout the conversion test, as the transformation to bradyzoites occurs, reaching a level 15.1 times higher than the baseline level on the 7th day post- stress. Likewise, the expected increase in the Nc SAG4 gene was observed , which was used as a control for conversion to bradizoite, up to 381.8 times higher than the baseline level on day 7 post-stress, and a slight decrease in the levels of Nc SAG1 , which was used as a tachyzoite control, as previously described (Fernández-García et al . 2006. Mol. Biochem. Parasitol. 146, 89-97).

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<400> 18

2. 3

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<210> 19

         \ vskip0.400000 \ baselineskip
      

<211> 23

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<212> DNA

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<213> Neospora caninum

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 19

24

         \ vskip0.400000 \ baselineskip
      

<210> 20

         \ vskip0.400000 \ baselineskip
      

<211> 20

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Neospora caninum

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 20

25

         \ vskip0.400000 \ baselineskip
      

<210> 21

         \ vskip0.400000 \ baselineskip
      

<211> 20

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Neospora caninum

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 21

26

Claims (18)

1. Polynucleotide molecule characterized by the nucleotide sequence SEQ ID NO: 13 containing the Nc BSR4 gene of Neospora caninum . 2. Polynucleotide molecule according to claim 1 characterized in that it encodes the NcBSR4 antigenic protein of Neospora caninum (SEQ ID NO: 14). 3. Polynucleotide molecule according to claim 1 characterized by the sequence between nucleotide 1084 and 2195 of the sequence SEQ ID NO: 13. 4. Polynucleotide molecule selected from the sequence set consisting of (a) the Nc BSR4 gene of Neospora caninum , characterized by SEQ ID NO: 13, (b) sequences homologous to the Nc BSR4 gene encoding the NcBSR4 antigen and (c) fragments of the Nc BSR4 gene encoding polypeptides that retain the antigenic characteristics of Nc BSR4 . 5. Procedure for the in vitro diagnosis of chronic N. caninum infection from infected tissues or fluids characterized by the detection of polynucleotide molecules according to claims 1 to 4 by their enzymatic amplification based on the PCT or RT reaction -PCR, its in situ hybridization with DNA probes or any other method of detecting nucleic acids. 6. Method for the diagnosis of chronic infection by N. caninum according to claim 5 characterized by the use of oligonucleotide-primers and / or probes chosen from the set consisting of the sequences SEQ ID NO: 1 (R1BSR4), SEQ ID NO: 2 (R2BSR4), SEQ ID NO: 3 (F1BSR4), SEQ ID NO: 4 (F2BSR4), SEQ ID NO: 5 (NcESTF), SEQ ID NO: 6 (NcESTR), SEQ ID NO: 7 (FNCEST5), SEQ ID NO: 8 (RNCEST3), SEQ ID NO: 9 (FBamHNcBSR4), SEQ ID NO: 10 (RHindIIINcBSR4), SEQ ID NO: 11 (NcBSR4F3) and SEQ ID NO: 12 (NcBSR4R3). 7. Recombinant vector characterized in that it comprises a nucleotide sequence defined according to claims 1 to 4. 8. Recombinant vector characterized in that it comprises the polynucleotide molecule according to claim 3 and the plasmid pRSET-C. 9. Transfected host eukaryotic cells with the recombinant vectors of claim 7. 10. Prokaryotic host cells transformed with the recombinant vectors of claim 7 or 8. 11. A purified or isolated polypeptide selected from (a) the N. caninum NcBSR4 antigenic protein, characterized by the amino acid sequence SEQ ID NO: 14; (b) chemically or enzymatically modified sequences derived from sequences homologous to that of the NcBSR4 protein (SEQ ID NO: 14) that retain their antigenic characteristics; (c) NcBSR4 derived polypeptides (SEQ ID NO: 14) that retain their antigenic characteristics; or (d) recombinant proteins comprising the NcBSR4 protein (SEQ ID NO: 14) or NcBSR4 derived polypeptides (SEQ ID NO: 14) that retain their antigenic characteristics. 12. Procedure for the serological diagnosis of chronic N. caninum infection characterized by the use of monoclonal antibodies or specific polyclonal serum against proteins and / or polypeptides according to claim 11 by immunohistochemistry, immunofluorescence or any other method based on the detection of said proteins and / or polypeptides by said serum. 13. Procedure for the serological diagnosis of chronic N. caninum infection characterized by the detection of the proteins and / or polypeptides according to claim 11 by enzyme immunoassay (ELISA), radioimmunoassay (RIA), immunoblot or any other capacity based method antigen of said polypeptides. 14. Method for the serological diagnosis of chronic infection by N. caninum according to claim 13 characterized by the detection of the proteins and / or polypeptides according to claim 11 by means of a competition ELISA. 15. An immunogenic composition comprising: (a) a polypeptide according to claim 11, (b) a molecule polynucleotide according to claims 1 to 4; (c) a vector recombinant according to claim 7; (d) some cells transfected hosts according to claims 9; (e) some transformed host cells according to claims 10, formulated as a vaccine against neosporosis. 16. An immunogenic composition according to claim 15, comprising an adjuvant or one or more cytokines

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17. A method of preparing a composition immunogenic comprising a combination of (a) a polypeptide according to claim 11; (b) a polynucleotide molecule that contain a sequence encoding a polypeptide according to the claim 11; (c) a recombinant vector according to the claim 7; (d) transfected host cells according to claims 9; (e) transformed host cells according to claims 10, formulated as a vaccine against neosporosis 18. A vaccination kit for mammals against neosporosis comprising a container that includes a immunogenic composition formulated according to claims 15 and 16.