MXPA98010218A - Desoxirribonucleico acid, which codifies for neosp diethydropholate reductase-timidylate synthetase - Google Patents

Abstract

The present invention provides isolated polynucleotide molecules that contain a nucleotide sequence encoding the DHFR-TS protein of Neospora, these polynucleotide molecules being useful in the preparation of live vaccines modified against neosporosis, and as diagnostic reagents. The present invention further provides for cloning and expression vectors, transformed host cells, substantially purified DHFR-TS proteins and peptide fragments, genetic constructs useful for the targeted deletion of a gene, and

Description

DESOXIRRIBONUCLEICO ACID THAT CODIFIES FOR NEOSPORA DIETHYDROPHOLATE REDUCTASE-TIMIDYLATE SINTETASE 1. - FIELD OF THE INVENTION The present invention is framed in the field of animal health and refers to the compositions of vaccines and the diagnoses of a disease. More especially, the present invention relates to polynucleotide molecules comprising a sequence of nucleotides encoding the protein dihydrofolate reductase-thymidylate synthetase (DHFR-TS) from Neospora, these polynucleotide molecules being useful in the production of vaccines against neosporosis and as diagnostic reagents. 2. BACKGROUND OF THE INVENTION Neospora of a pathogenic protozoan parasite of animals that has been recognized as the main cause of abortion, neonatal death, congenital infection and encephalitic disease in mammals. Dubey and Lindsay, 1996, Vet. Parasitol. 67: 1-59; Dubey and Lindsay, 1993, Paraditology Today, 9: 452-458. N. caninum infects dogs and congenitally infects puppies, often causing paralysis. The tachyzoites of N. caninum have been isolated from naturally infected puppies. Linday and Bubey, 1989, J. Parasitol. 75: 163-165. Neospora is the leading cause of abortion in dairy cows. Cases of Neospora-related disease, ie, neosporosis, have also been reported in goats, sheep and horses. Although N. caninum is superficially similar to the pathogen Toxoplasma gondii, N. canunum and T. gondii has been distinguished from other antigenic and ultrastructural entities. Dubey and Lindsay, 1993, above. Likewise, it was found that the protozoan parasites of the Neospora type isolated from the brains of aborted bovine fetuses and continuously cultured in vitro were antigenically and ultrastructurally distinct from T. gondii and Hammondia hammondi, being more similar to N. caninum. Conrad et al., 1993, Parasitology 106: 239-249. Likewise, analysis of the nuclear genes of the ribosomal RNA of the small subunit did not reveal any difference in nucleotides between the Neospora strains isolated from cows and dogs, but nevertheless they had evident differences between Neospora and T. gondi. Marsh et al., 1995, J. Parasitol. 81: 530-535. The etiological role of a Neospora bovine isolate in bovine abortion and congenital disease has been confirmed. Barr et al., 1994, J. Vet. Diag. Invest. 6: 207-215. A rodent model of neosporosis in the central nervous system has been developed using inbred BALB / c mice infected with N. caninum, Lindsay et al., 1995. J. Parasitol. 81: 313-315. Also, Colé et al., 1995. J. Parasitol. 81: 730-732 and Long et al., 1996, J. Parasitol, 82: 608-611 have described models for studying the transplacental transmission of N. caninum in exogamous and inbred pregnant female mice respectively. Likewise, Lindsay et al., 1995, Am. J. Vet. Res. 56: 1176-1180 has demonstrated an experimental model in pygmy goat with N. caninum that closely resembles abortion in cows induced by Neospora naturally acquired. In protozoa such as T. gondii and Neospora, it is known that the essential metabolic enzymes dihydrofolate reductase (DHFR) and thymidylate synthetase (TS) reside in the same protein molecule in two distinct enzymatic domains, ie, "the DHFR domain" and the "TS domain". It has been described that in T. gondii the DHFR and TS domains are separated by a binding region of -70 amino acids. Roos, 1993, J. Biol. Chem. 268: 6269-6280. This bifunctional protein has served in at least one species of parasite as a deletion target to create an attenuated strain for use in a live vaccine. Thus, Titus et al., 1995, Proc. Nati Acad. Sci. 92: 10267-10271 describes deletion directed by homologous recombination of the DHFR-TS gene from the protozoan parasite Leishmania best to produce null dhfr-ts mutant cells for use in a vaccine against a virulent strain of L.p. 3. SUMMARY OF THE INVENTION The present invention provides a polynucleotide molecule isolate containing a nucleotide sequence encoding a DHFR-TS protein from Neospora. In a preferred embodiment, the DHFR-TS protein from Neospora contains the amino acid sequence of SEQ ID NO: 3 or the amino acid sequence of a DHFR-TS protein as encoded by the DHFR-TS gene as present in phage lambdaNclDHFRTS (ATCC registration number 209512). In a non-limiting embodiment, the isolate of the polynucleotide molecule contains the nucleotide sequence of the DHFR-TS gene of Neospora. In a preferred embodiment, the isolate of the polynucleotide molecule containing the nucleotide sequence of the Neospora DHFR-TS gene contains the nucleotide sequence SEQ ID NO: 1 from about nt 2405 to about nt 8199 or a nucleotide sequence which is the same as the nucleotide sequence which is the same as the nucleotide sequence of the DHFR-TS gene as present in phage lambdaNclDHFRTS (ATCC registration no. 209512). In another non-limiting embodiment, the polynucleotide molecule encoding the DHFR-TS protein comprises the nucleotide sequence of SEQ ID NO: 2. The present invention further provides a polynucleotide molecule isolate that is substantially homologous to a polynucleotide molecule which comprises the nucleotide sequence of the DHFR-TS gene as seen in SEQ ID NO: 1 from about nt 2405 to about nt 0199, or the nucleotide sequence of the DHFR-TS gene as present in lambda phage NclDHFRTS ( ATCC registration no. 209512) or the nucleotide sequence of SEQ ID NO: 2. The present invention further provides an isolate of a polynucleotide molecule comprising a nucleotide sequence encoding a polypeptide that is substantially homologous to a DHFR-protein. Neospora TS having the amino acid sequence of SEQ ID NO: 3, or the amino acid sequence of a DHFR-TS protein as the codi ficated by the DHFR-TS gene as present in phage lambdaNclDHFRTS (ATCC registration no. 209512). The present invention further provides a polynucleotide molecule that consists of a nucleotide sequence that is a substantial part of any of the aforementioned polynucleotide molecules. In a preferred embodiment, the polynucleotide molecule consists of a nucleotide sequence encoding a peptide fragment of any one of the aforementioned Neospora DHFR-TS proteins or substantially homologous polypeptides, such as a polypeptide consisting of the DHFR domain or the TS domain of the DHFR-TS protein. In addition to the nucleotide sequences of any of the polynucleotide molecules related to the aforementioned DHRF-TS proteins, the polynucleotide molecules of the present invention may further comprise, or alternatively may consist of nucleotide sequences which naturally flank the DHFR-TS gene in situ in N. caninum, such as, for example, the flanking nucleotide sequences shown in SEQ ID NO: 1 or parts thereof. The present invention also provides compositions and methods for the cloning and expression of a polynucleotide molecule of the present invention, including cloning vectors, expression vectors and transformed host cells containing said vectors. In a non-limiting embodiment, the present invention provides a cloning vector containing a polynucleotide molecule having the nucleotide sequence of the DHFR-TS gene of N. caninum strain NC-1, such as, for example, a vector of cloning lambda phage designated as lambdaNclDHFRTS (ATCC registration No. 209512). The present invention further provides a partially or substantially purified protein containing the amino acid sequence of the DHFR-TS protein of Neospora. In a non-limiting embodiment, the protein contains the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence of a DHFR-TS protein as encoded by the DHFR-TS gene present in phage lambdaNclDHFRTS (ATCC registration no. 209512). The present invention further provides polypeptides that are substantially homologous to the DHFR-TS protein of Neospora. The present invention further provides fragments of peptides of any of the aforementioned proteins or polypeptides, such as, for example, a polypeptide consisting of an isolate of the DHRF or TS domain of Neospora. The present invention further provides antibodies obtained against a DHFR-TS protein from Neospora or against a peptide fragment of said protein. The present invention further provides a genetic construct that contains any of the aforementioned polynucleotide molecules, such as, for example, a polynucleotide molecule containing the nucleotide sequence of the DHFR-TS gene, as shown in SEQ ID NO: l from about nt 2405 to about nt 8199 or as present in phage lambdaclDHFRTS (ATCC registration No. 209512), or a polynucleotide molecule consisting of a nucleotide sequence, which is a substantial part of either said nucleotide sequences, but modified by having one or more deletions, insertions and / or nucleotide substitutions, or consisting of one or more nucleotide sequences that naturally flank the DHFR-TS gene in situ in N. caninum; so that the polynucleotide molecule when inserted into a wild-type DHFR-TS gene, or used to replace a part of it, produces a modified sequence of the DHFR-TS gene that encodes a partially defective or totally defective protein, or that does not encode or does not produce any protein at all. Said genetic constructs are useful for producing modified Neospora cells in which the DHFR-TS gene or a portion thereof have been partially or completely inactivated, resulting in cells having a dhfr- or ts- phenotype (hereinafter collectively referred to as "phenotype"). dhfr-ts-). The present invention further provides modified live Neospora cells in which the native DHFR-TS gene or a mixture thereof has been partially or completely inactivated. In a preferred embodiment, the Neospora cells have a dhfr-ts- mutant phenotype as a consequence of a disruption of the DHFR-TS gene by homologous recombination with a genetic construct of the invention. Said modified live Neospora cells are useful in vaccine compositions to protect mammals against neosporosis. The present invention further provides a method for preparing modified live Neospora cells. The present invention further provides a vaccine against neosporosis, which contains an immunologically effective amount of the modified live Neospora cells of the present invention and a veterinarily acceptable carrier. The present invention further provides a combination vaccine for the protection of a mammal against neosporosis and, optionally, one or more other diseases or pathological conditions that may affect the mammal, said combination vaccine containing an immunologically effective amount of a first component. containing modified live Neospora cells of the present invention; an immunologically effective amount of a second component capable of inducing a protective response against a pathological disease or disorder which may affect a mammal and a veterinarily acceptable vehicle. The present invention also provides a method for the preparation of the vaccine of the present invention, which comprises combining an immunologically effective amount of the modified live Neospora cells of the present invention with a veterinarily acceptable carrier. The present invention further provides a method for vaccinating a mammal against neosporosis, comprising administering to the mammal the vaccine of the present invention. The present invention further provides a kit for vaccination of a mammal against neosporosis, comprising a first container having a composition containing an immunologically effective amount of modified live Neospora cells of the present invention and a second container having a vehicle. or veterinarily acceptable diluent. 4. DETAILED DESCRIPTION OF THE INVENTION 4. 1 Polynucleotide molecules are encoded for Neospora DHFR-TS The present invention provides: (i) a polynucleotide molecule isolate comprising a nucleotide sequence encoding a DHFR-TS protein from Neospora, including a molecule isolate of polynucleotide containing the nucleotide sequence of the DHFR-TS gene of Neospora; (ii) a polynucleotide molecule isolate that is substantially homologous to any of the aforementioned polynucleotide molecules; (iii) a polynucleotide molecule isolate containing a nucleotide sequence encoding a polypeptide that is substantially homologous to that of the Neospora DHFR-TS protein; and (iv) a polynucleotide molecule consisting of a nucleotide sequence that is a substantial part of any of the aforementioned polynucleotide molecules, including a polynucleotide molecule consisting of a nucleotide sequence encoding a peptide fragment of any of the substantially homologous proteins or polypeptides of Neospora DHFR-TS. As used herein, the term "gene", "polynucleotide molecule", "nucleotide sequence", "coding sequence" and "coding region" is intended to include both DNA and RNA molecules, which can be single or double stranded Also, as used herein, the term "gene", "coding sequence" and "coding region" is intended to refer to polynucleotide molecules that can be transcribed and translated (DNA), or translated (RNA) into a DHFR-TS protein from Neospora, or in a polypeptide that is substantially homologous to a DHFR-TS protein from Neospora, or in a peptide fragment of the aforementioned DHFR-TS protein of Neospora, or in a substantially homologous polypeptide, in a expression system in a host cell when placed in operative association with the appropriate regulatory elements. Polynucleotide molecules can include, but are not limited to, one or more prokaryotic sequences, eukaryotic sequences, cDNA sequences, genomic DNA sequences (exons or introns), and chemically synthesized DNA and RNA sequences, or any combination thereof . A polynucleotide molecule isolate of the present invention can have a nucleotide sequence of any of the Neospora species or strains, but it is preferable that it comes from a pathogenic Neospora species, such as N. caninum. A non-limiting example of a strain of N. caninum from which the polynucleotide molecule of the present invention can be isolated or derived is strain NC-1, which is available in the MARC-145 monkey kidney host cells with the registration number CRL-12231 of the American Type Culture Collection (ATCC), located at 12301 Parklawn Drive, Rockville, MD 20852, USA. Strain NC-1 is also described in Dubey et al., 1988, j. Am. Vet. Med. Assoc. 193: 1259-1263, the publication of which is incorporated herein by reference. Alternatively, Neospora strains or pathogenic species for use in the practice of the present invention can be isolated from organs, tissues or body fluids of infected animals using standard isolation techniques, such as those described in the publications indicated above. . The present invention provides a polynucleotide molecule isolate containing a nucleotide sequence encoding a DHFR-TS protein from Neospora. In a preferred embodiment, the DHFR-TS protein from Neospora contains the amino acid sequence of SEQ ID NO: 3, or the amino acid sequence of a DHFR-TS protein as encoded by the DHFR-TS gene as present in the phage lambdaclDHFRTS (Registration No. 209512). In a non-limiting embodiment, the polynucleotide isolate molecule contains the nucleotide sequence of the DHFR-TS gene of Neospora. In a preferred embodiment, the polynucleotide isolate molecule contains the nucleotide sequence of the DHFR-TS gene of the N. caninum strain NC-1, which contains the nucleotide sequence of SEQ ID NO: 1 from about nt 2405 to about nt 8199, or a nucleotide sequence that is the same as the nucleotide sequence of the DHFR-TS gene as present in phage lambdaclDHFRTS (Registration No. 209512). In SEQ ID NO: 1, the predicted DHFR domain of the DHFR-TS gene is encoded from about nt 2405 to about nt 4664; the predicted TS domain of the DHFR-TS gene is encoded from about nt 4665 to about nt 8199. In another non-limiting embodiment, the polynucleotide molecule encoding the DHFR-TS protein contains the nucleotide sequence of SEQ ID NO: 2, in which the predicted DHFR domain is encoded by about nt 1 to about nt 969 and the predicted domain TS is encoded by about nt 970 to about nt 1836. The present invention further provides an isolated one molecule of polynucleotide that is substantially homologous to a polynucleotide molecule containing the nucleotide sequence of the DHFR-TS gene as shown in SEQ ID NO: 1 from about nt 2405 to about nt 8199, or the nucleotide sequence of the DHFR gene -TS as it is present in phage lambdaclDHFRTS (registration No. 209512) or the nucleotide sequence of SEQ ID NO: 2. When used the term "substantially homologous" to refer to a polynucleotide molecule related to DHFR-TS, indicates a polynucleotide molecule having a nucleotide sequence: (a) that codes for the same protein as the nucleotide sequence of the DHFR gene -TS shown in SEQ ID NO: l from about nt 2405 to about nt 8199, or as it is present in phage lambdaclDHFRTS (registration No. 209512) or the nucleotide sequence of SEQ ID NO: 2, but which includes one or more silent changes in the nucleotide sequence according to the degeneracy of the genetic code; or (b) that hybridizes with the complement of a polynucleotide molecule having a nucleotide sequence encoding the same protein as the nucleotide sequence of the DHFR-TS gene shown in SEQ ID N0: 1 from about nt 2405 to approximately nt 8199, or as is present in phage lambdaclDHFRTS (Registration No. 209512) or the nucleotide sequence of SEQ ID NO: 2, under moderately stringent conditions, i.e., hybridization with DNA bound to the filter in NaHP04 0.5 M, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65 ° C and washing in 0.2xSSC / 0.1% SDS AL, at 42 ° C (see Ausubel et al., (Eds.), 1989, Current Protocols in Molecular Bioloay, Vol. I, Green Pubiishing Associates, Inc., and John Wiley &Sons, Inc., New York, at 2.10.3) and which is useful for the practice of the present invention. In a preferred embodiment, the substantially homologous polynucleotide molecule hybridizes with the complement of a polynucleotide molecule having a nucleotide sequence that codes for the same protein as the nucleotide sequence of the DHFR-TS gene shown in SEQ ID NO: l from about nt 2405 to about nt 8199, or as present in the phage the bdaclDHFRTS (registration No. 209512) or the nucleotide sequence of SEQ ID NO: 2, under slightly stringent conditions, i.e., hybridization with DNA bound to the filter in 0.5 M NaHP04, 7% SDS, 1 mM EDTA at 65 ° C and washed at 0. lxSSC / 0.1% SDS at 68 ° C (Ausubel et al., 1989, above) and is useful for the practice of the present invention. As used herein, a polynucleotide molecule is "useful for the practice of the present invention" when the polynucleotide molecule can be used as a diagnostic reagent to detect the presence of a Neospora-specific polynucleotide in a fluid sample or tissue from an animal infected with Neospora, or when the polynucleotide molecule can be used to prepare a genetic construct useful in the preparation of modified live Neospora cells of the present invention, as described later in section 4.4. The substantially homologous polynucleotide molecules of the present invention do not include polynucleotide molecules having a nucleotide sequence that encodes the T. crondii DHFR-TS protein. The present invention further provides a polynucleotide molecule isolate containing a nucleotide sequence encoding a polypeptide that is substantially homologous to a Neospora DHFR-TS protein having the amino acid sequence of SEQ ID NO: 3 or the sequence of amino acids of a DHFR-TS protein as encoded by the DHFR-TS gene as it is present in phage lambdaclDHFRTS (Acquisition No. 209512). As used herein to refer to polypeptides, the term "substantially homologous" refers to a polypeptide having an amino acid sequence: (a) which is preferably at least about 70%, more preferably at least about 80% and even more preferably at least about 90% like that of the DHFR-TS protein having the amino acid sequence of SEQ ID NO: 3, or the amino acid sequence of a DHFR-TS protein as encoded by the DHFR-TS gene as it is present in the phage lambdaclDHFRTS (Registration No. 209512) and whose substantially homologous polypeptide is useful for the practice of the present invention; and / or (b) wherein one or more amino acid residues present in a DHFR-TS protein of Neospora has the amino acid sequence of SEQ ID NO: 3, or the amino acid sequence of a DHFR-TS protein as the encoded by the DHFR-TS gene as present in phage lambdaclDHFRTS (Registry No. 20915), has been conservatively substituted with a different amino acid residue, the polypeptide being useful in the practice of the present invention. Conservative amino acid substitutions are well known in the art. For example, it can reasonably be expected that one or more amino acid residues of a Neospora DHFR-TS protein can be substituted conservatively with an amino acid residue of similar charge, size or polarity, the resulting polypeptide being useful for the practice of the present invention. invention. The rules for making such substitutions include those described by Dayhof, M.D., 1978, Nat. Biomed. Res. Found. , Washington, D.C., Vol. 5 Sup. 3, among others. More specifically, conservative amino acid substitutions are those that generally take place within a family of amino acids that are related in their side chains. The genetically encoded amino acids are generally divided into four groups: (1) acids = aspartate, glutamate; (2) basic lysine, arginine, histidine; (3) non-polar = alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) polar no charge = glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan and triosine are also classified together as aromatic amino acids. One or more substitutions within any particular group, for example, of a leucine by isoleucine or valine, of an aspartate by glutamate, or of a threonine by serine, or of any other amino acid residue by a structurally related amino acid residue, will generally have a negligible effect on the usefulness of the resultant polypeptide in the practice of the present invention. As used herein, a protein or polypeptide "useful for the practice of the present invention" is considered when the protein or polypeptide can be used for any one or more of a variety of purposes, including, for example, the selection of agents inhibitors directed specifically to either of the two enzymatic domains of the DHFR-TS protein of Neospora, or to obtain antibodies against the entire protein or one of the two enzymatic domains thereof. The present invention further provides a polynucleotide molecule that consists of a nucleotide sequence that is a substantial part of any of the aforementioned polynucleotide molecules. As used herein, a "substantial part" of any of the aforementioned polynucleotide molecules indicates a polynucleotide molecule that consists of less than the complete nucleotide sequence of the particular polynucleotide molecule related to the DHFR-TS. , but comprising at least about 50% of the nucleotide sequence of the polynucleotide molecule related to the DHFR-TS and which is useful for the practice of the present invention, the utility being defined according to the above for the polynucleotide molecules. In a preferred embodiment, the polynucleotide molecule consists of a nucleotide sequence that encodes a peptide fragment of any of the aforementioned substantially homologous proteins or polypeptides of the Neospora DHFT-TS, such as a polypeptide consisting of the DHFR domain or the TS domain of the DHFR-TS protein. As used herein, the "peptide fragment" refers to a polypeptide consisting of one or more sub-sequences of the complete amino acid sequence of the DHFR-TS protein of Neospora, or of the substantially homologous polypeptide, being the shorter sub-sequences in length than the full-length molecule, and in which the resulting peptide fragment is useful in the practice of the present invention, the utility being as defined above for the polypeptides. So, if the full-length molecule is represented by having "n" amino acid residues, a peptide fragment thereof would be any polypeptide smaller than the full length sequence, which includes a polypeptide having n-1 amino acid residues, said polypeptide being useful in the practice of the present invention. Peptide fragments of the present invention preferably have at least about 10 amino acid residues in length. In a non-limiting embodiment, the peptide fragment consists of the DHFR domain or the TS domain of the DHFR-TS protein of Neospora When the peptide fragment consists of more than one sub-sequence of a DHFR-TS protein or substantially homologous polypeptide, the polynucleotide molecule encoding the peptide fragment can be formed such that several sub-sequences are grouped by placing them contiguously in the fragment peptide where the corresponding sub-sequences were not contiguous in the polypeptide or in the full-length protein. Also, a polynucleotide molecule encoding a peptide fragment can be formed in such a way that different sub-sequences comprising the peptide fragment are placed in a relative order different from each other as compared to the full-length protein or polypeptide, or so that the coding peptide fragment comprises multiple copies of a specific sub-sequence. For example, the polynucleotide molecule can encode multiple copies of the DHFR domain or the TS domain, for selected epitope regions thereof or a combination thereof. The present invention also encompasses the polynucleotide colcules encoding a full-length polypeptide (n), in which the sub-sequences of the native protein are located close to one another, in addition to the polynucleotide molecules encoding polypeptides which are longer than the native protein, including polypeptide up to 2n in length. Such polypeptides may contain multiple copies of the entire protein, of the individual domains, of the epitope regions or of some combination thereof. In addition to the nucleotide sequences of any of the aforementioned DHFR-TS related polynucleotide molecules, the polynucleotide molecules of the present invention may also contain, or alternatively may consist of, one or more nucleotide sequences selected from the sequences which naturally flank the DHFR-TS gene in situ in N. caninum, such as, for example, the flanking nucleotide sequences shown in SEQ ID NO: 1 or parts thereof. The sequences of polynucleotide molecules of the present invention also provide the information necessary to construct oligonucleotide molecules that can be used as primers in amplification techniques, or as probes in the differential diagnosis of diseases, and which can be easily designed by those skilled in the art. in the art in light of this description, said oligonucleotides will preferably have a length of at least about 15 nucleotides. The amplification can be carried out using conveniently designed oligonucleotides applying conventional techniques, such as, for example, the polymerase chain reaction (PCR), which is described, inter alia, in Innis et al., (Eds), 1995 , PCR Strategies, Academic Press, Inc., San Diego; and Erlich (ed), 1992, PCR Technology, Oxford Uneversity Press, New York, whose publications are incorporated herein by reference. As for diagnosis, the oligonucleotides of the present invention can be used in PCR amplification to detect the presence of Neospora-specific polynucleotide molecules in a sample of a tissue or fluid from an animal, such as brain tissue. , lung tissue, placental tissue, blood fluid, cerebrospinal fluid, mucus, urine, amniotic fluid, etc. The production of a specific amplification product can be used to support a diagnosis of Neospora infection, while the lack of an amplified product can point to an absence of infection. Generally, for PCR, a mixture is prepared that contains the appropriately designed primers, a template containing the nucleotide sequence to be amplified, and the appropriate enzymes and buffers for PCR and is processed according to conventional protocols to amplify a polynucleotide molecule specific to the Neospora DHFR-TS of the template or a portion thereof. Other amplification techniques known in the art, for example, the ligase chain reaction, can be used as an alternative. It is intended that all subsequent references to a "polynucleotide molecule" include any of the aforementioned polynucleotide molecules of the present invention, including polynucleotide molecules that contain the nucleotide sequence of the DHFR-TS gene as shown in FIG. SEQ ID NO: 1 from about nt 2405 to about nt 8199, or as it is present in phage NclDHFRTS (ATCC registration no. 209512), or the nucleotide sequence of SEQ ID NO: 2; polynucleotide molecules that are substantially homologous to a polynucleotide molecule comprising the nucleotide sequence of the DHFR-TS gene as shown in SEQ ID NO: 1 from about nt 2405 to about nt 8199, or the nucleotide sequence of the DHFR-TS gene as it is present in the phage NclDHFRTS (ATCC registration No. 209512) or the nucleotide sequence of SEQ ID NO: 2; polynucleotide molecules that contain a nucleotide sequence that encodes a polypeptide that is substantially homologous to a Neospora DHFR-TS protein having the amino acid sequence of SEQ ID NO: 3, or the amino acid sequence of a protein DHFR-TS encoding the DHFR-TS gene as present in the phage NclDHFRTS (ATCC registration No. 209512); and polynucleotide molecules that consist of a nucleotide sequence that is a substantial part of any of the aforementioned polynucleotide molecules, including polynucleotide molecules that consist of a nucleotide sequence encoding a peptide fragment of any of the Neospora DHFR-TS proteins mentioned above or substantially homologous polypeptides, unless indicated otherwise. It is intended that all subsequent references to a "DHFR-TS protein", "protein", or "polypeptide" include a protein having the amino acid sequence of SEQ ID NO: 3, or the amino acid sequence of a DHFR protein -TS as encoded by the DHFR-TS gene as it is present in the phage NclDHFRTS (ATCC registration no. 209512); polypeptides that are substantially homologous to any of the aforementioned proteins, and peptide fragments of any of the aforementioned proteins or polypeptides, unless otherwise specified. The production and manipulation of the polynucleotide and oligonucleotide molecules of the invention are within the art and can be performed according to known genetic techniques, described among other sites, in Maniatis et al., 1989, Molecular Cloning, A Laboratorv Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel et al., 1989, supra; Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y .; Innis et al., 1995, above; and Erlich, 1992, supra, which are incorporated herein by reference. 4. 2. Recombinant expression systems 4.2.1. Cloning and Expression Vectors The present invention further provides cloning and expression vectors containing a polynucleotide molecule of the present invention. In a non-limiting embodiment, a cloning vector provided by the present invention is the phage NclDHFRTS (ATCC registration no. 209512), which contains a polynucleotide molecule having a nucleotide sequence of the complete DHFR-TS gene of the strain NC-1 of N. caninum. The expression vectors of the present invention are preferably constructed such that the polynucleotide molecule is in operative association with one or more regulatory elements necessary for transcription and translation. The expression vector is used in an expression system, such as a transformed host cell, to produce a recombinantly expressed DHFR-TS protein. As used herein, the term "regulatory element" includes, but is not limited to, the nucleotide sequences encoding promoters, enhancers, inducible and non-inducible operators, and other elements known in the art that serve to direct and / or regulate the expression of a sequence of coding polynucleotides. As used herein, the DHFR-TS coding sequence is in "operative association" with one or more regulatory elements, the regulatory elements are recorded efficiently and provide the transcription of the DHFR-TS coding sequence or the translation of the DHFR-TS coding sequence. your mRNA, or both. There are methods well known in the art for the construction of expression vectors containing particular coding sequences in operative association with the appropriate regulatory elements and these may be used for the practice of the present invention. Such methods include in vitro recombination techniques, synthetic techniques and in vivo genetic recombination, synthetic techniques and in vivo genetic recombination, as has been described among others in Maniatis et al., 1989, supra; Ausubel et al., 1989, supra; and Sambrook et al., 1989, above. Several expression vectors are known in the art that can be used to express the coding sequence of a polynucleotide molecule of the present invention, including bacteriophage recombinant DNA, plasmid DNA and cosmid DNA expression vectors containing the DHFR coding sequence. -TS for the transformation of a bacterium or a yeast; and expression vectors in recombinant viruses, such as for example, baculoviruses, containing the coding sequence DHFR-TS for the transfection of insect cells, or adenovirus or vaccinia virus containing the DHFR-TS coding sequence for the transfection of cells of mammal, among others. Plasmids, typical prokaryotic expression vectors that can be constructed to contain a polynucleotide molecule of the present invention, include pUC8, pUC9, pBR322 and pBR329 (Biorad Laboratories, Richmond, CA) and pPL and pKK223 (Pharmacia, Piscata ay, NJ), among others. Typical eukaryotic expression vectors that can be constructed to contain a polynucleotide molecule of the present invention include an ecdysone-inducible mammalian expression system (Invitrogen, Carlsbad, CA), promoter-enhancer-based systems in cytomegalovirus (Promega, Madison, Wl; stratagene, La Jolla, CA: Invitrogen) and baculovirus-based expression systems (Promega), among others.

The regulatory elements of these and other vectors can vary in their potency and specificity. Depending on the host / vector system used, any of a number of appropriate transcription and translation elements may be used. For example, when cloning into mammalian cell systems, promoters isolated from the genome of mammalian cells can be used., for example, the mouse metallothionein promoter, or viruses that develop in these cells, for example, the 7.5K vaccine virus promoter or the long terminal repeat of Moloney murine sarcoma virus. Promoters obtained by recombinant DNA or synthetic techniques can also be used for the transcription of the inserted sequence. Also, the expression of certain promoters can be increased in the presence of particular inducers, for example, zinc and cadmium ions for metallothionein promoters. Non-limiting examples of the transcriptional regulatory regions or promoters include for the bacteria, the β-gal promoter, the T7 promoter, the TAC promoter, the left and right lambda promoters, the trp and lac promoters, the trp-lac fused promoters, etc; for yeasts, the glycolytic enzyme promoters, such as the ADH-I and II promoters, the GPK promoter, the PGI promoter, the TRP promoter, etc .; for mammalian cells, the SV40 early and late promoters, the adenovirus major late promoters, etc.

Specific initiation signals are also required for sufficient translation of inserted DHFR-TS coding sequences. These signals typically include an ATG start codon and adjacent sequences. In cases where the polynucleotide molecule of the present invention, including its own starter codon and adjacent sequences, are inserted into the appropriate expression vector, no additional translation control signals are needed. However, in cases where only part of a coding sequence is inserted, exogenous signals are required for translation control, including the ATG start codon. These exogenous translation control signals and initiator codons can be obtained from a variety of sources, both natural and synthetic. Also, the initiation codon must be in phase with the reading frame of the DHFR-TS coding region to ensure translation within the framework of the complete insert. The fusion protein expression vectors can be used to express a DHFR-TS fusion protein. The purified fusion protein can be used to obtain an antiserum against the DHFR-TS protein, to study the biochemical properties of the DHFT-TS protein, to construct DHFR-TS fusion proteins with different enzymatic activities, or to facilitate the identification or purification of the DHFR-TS protein expressed. Possible fusion protein expression vectors include, but are not limited to, vectors that incorporate sequences encoding ß-galactosidase and trpE fusions, maltose binding protein fusions, glutathione-S fusions -transferase and fusions of polyhistidine (vehicle regions). The methods known in the art can be used to construct expression vectors encoding said DHFR-TS fusion proteins. As said above, the fusion protein may be useful to facilitate the purification of the expressed protein. For example, fusions of DHFR-TS-maltose binding protein can be purified using amylose resin; DHFR-TS-glutathione-S-transferase fusion proteins can be purified using glutathione-agarose beads; and DHFR-TS-polyhistidine fusions can be purified using divalent nickel resin. Alternatively, antibodies against a protein or peptide carrier can be used for purification by affinity chromatography of the fusion protein. For example, a nucleotide sequence encoding the target epitope of a monoclonal antibody can be constructed in the expression vector in operable association with the regulatory elements, and positioned such that the expressed epitope is fused to the DHFR-TS protein . For example, a nucleotide sequence encoding the FLAG ™ tag epitope (International Biotechnologies Inc.), which is a hydrophilic label peptide, can be inserted by conventional techniques into the expression vector at a corresponding point, for example, at carboxyl terminal of the DHFR-TS protein. The expressed FLAG ™ epitope DHFR-TS-protein fusion product can be detected and purified by affinity using commercially available anti-FLAG T1M antibodies. The expression vector can also be constructed to contain polylinker sequences, which code for the cleavage sites of a specific protease, so that the expressed DHFR-TS protein can be released from the carrier or fusion partner region by treatment with a specific protease. For example, the fusion protein vector may include DNA sequences that code for the thrombin or factor Xa cut sites, among others. A signal sequence upstream and in reading frame can be constructed with the DHFR-TS coding region in the expression vector by known methods to direct the conduction and secretion of the expressed protein. Non-limiting examples of signal sequences include those of the alpha factor, immunoglobulins, outer membrane proteins, penicillinase and T cell receptors, among others. To facilitate the selection of host cells transformed or transfected with an expression vector of the present invention, the expression vector can be constructed so as to further contain a coding sequence for an informative gene product or other selectable marker. A coding sequence of this type is preferably in operative association with the coding sequences of the regulatory element, as described above. Informational genes that are useful in the invention are well known in the art and include those that code for chloramphenicol acetyltransferase (CAT), green fluorescent protein, firefly luciferase, and human growth hormone, among others. Nucleotide sequences that encode selectable markers are well known in the art and include those that code for gene products that confer resistance to antibiotics and anti-metabolites, or that provide an auxotrophic requirement. Examples of such sequences include those that code for thymidine kinase activity or resistance to methotrexate, ampicillin, kenamycin, chloramphenicol, zeocin, pyrimethamine, aminoglycosides, or himicromycin, among others. 4. 2.2. Transformation of host cells The present invention provides transformed host cells containing a polynucleotide molecule or recombinant expression vector of the present invention and cell lines derived therefrom. Useful host cells useful in the practice of the invention can be eukaryotic, although prokaryotic cells are preferred. Said transformed host cells, include but are not limited to microorganisms, such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors; or yeast transformed with a recombinant expression vector; or animal cells, such as insect cells infected with a recombinant expression vector; or animal cells, such as insect cells infected with a recombinant viral expression vector, e.g., baculovirus, or recombinant mammalian cells, e.g., baculovirus, or mammalian cells infected with a recombinant viral expression vector, for example, adenovirus or vaccine virus among others. Bacterial cells can be used host cells. For example, an E. coli strain, such as, for example, the DH5Ó strain, available from the ATCC, may be used.

Rockville, MD, USA (Registration No. 31343) or from Strategene (La Jolla, CA). Eukaryotic host cells include yeast cells, although mammalian cells, such as mouse, hamster, cow, monkey or a human cell line can also be used efficiently. Examples of fond host cells that can be used to express Chinese hamster ovary protein (OCH) (e.g., ATCC Registry No. CCL-61), NIH NIH / 3T3 Swiss mouse embryonic cells (e.g. , Registration No. CRL-1658), Madin-Darby Bovine Kidney Cells (MDBK) Registered No. CCL-229 and Thymidine Kinase Deficient Cells, for example, LM (TK) (Registration No. CCL-1.3 ) and tk-tsl3 (Registration No. CRL-1632). The recombinant expression vector of the invention is preferably transformed or transfected into one or more host cells of a substantially homogeneous cell culture. The expression vector is generally introduced into host cells according to known techniques, such as, for example, calcium phosphate precipitation, calcium chloride treatment, microinjection, electroporation, liposome-mediated transfection, transfection with DEAE. -dextreme, transduction, conjugation or bombardment with microproyetiles, among others. The selection of the transformants can be carried out by conventional methods, such as the selection of cells expressing a selectable marker, for example, antibiotic resistance associated with the recombinant expression vector. Once the expression vector has been introduced into the host cell, the integration and maintenance of the polynucleotide molecule of the present invention, either in the geoma of the host cell or else episode, can be confirmed by conventional techniques, for example, by Southern hybridization analysis, restriction enzyme analysis, PCR analysis including reverse transcriptase PCR (rt-PCR) or by immunological assay to detect the expected protein product. Host cells that contain and / or express the polynucleotide molecule of the present invention can be identified by any of at least four general procedures, which are known in the art, and which include: (i) DNA-DNA hybridization, DNA -RNA or antisense RNA-RNA; (ii) detection of the presence of "marker" gene functions; (iii) the determination of the level of transcription as measured by the expression, for example, of mRNA transcripts specific to DHFR-TS in the host cell; or (iv) detection of the presence of the mature polypeptide product, for example, by immunoassay or by detection of an enzymatic activity, such as, for example, the conversion of dihydrofolate to tetrahydrofolate catalyzed by the oxidation of NADPH or a TS enzymatic activity, such as, for example, the conversion of dihydrofolate to tetrahydrofolate catalyzed by the oxidation of NADPH or a TS enzymatic activity, such as, for example, the conversion of deoxyuridine monophosphate into deoxythymidine monophosphate, as is known in the art. 4. 2.3. Expression and purification of recombinant polypeptides Once the polynucleotide molecule of the present invention has been stably introduced into an appropriate host cell, the transformed host cell is propagated clonally and the resulting cells grow under conditions that result in maximum production of the encoded DHFR-TS protein. Such conditions typically include the growth of the transformed cells to a high density. When the expression vector contains an inducible promoter, appropriate induction conditions are used, such as, for example, temperature oscillation, nutrient depletion, the addition of unnecessary inducers (eg, carbohydrate analogues, such as isoporopil). -β-D-thiogalactopyranoside (IPTG), the accumulation of excess metabolic byproducts or the like, as needed to induce expression.When the expressed DHFR-TS protein is retained inside the host cells, the cells are harvested and are lysed and the product is purified from the lysate under extraction conditions known in the art to minimize protein degradation, such as, for example, at 4 ° C, or in the presence of protease inhibitors, or both, When the DHFR-protein The expressed TS is secreted out of the host cells, the depleted nutrient medium is simply collected and the protein is isolated from it The DHFR-TS protein The expressed one can be purified from the cellular ones or from the culture medium, as appropriate, using conventional procedures, including, but not limited to one or more of the following procedures: precipitation with ammonium sulfate, fractionation by size, ion exchange chromatography, HPLC, density centrifugation and affinity chromatography. When the expressed DHFR-TS protein exhibits enzymatic activity, for example, the ability to convert or dihydrofolate to tetrahydrofolate, as when catalyzed by the oxidation of NADPH (DHFR), or deoxyuridine monophosphate in deoxythymidine monophosphate (TS), the increase The purity of the preparation can be controlled with each step of the purification process by the use of an appropriate assay, as is known in the art. If the expressed protein has no biological activity, it can be detected based on, for example, the size or reactivity with an antibody that is otherwise specific for the DHFR-TS protein, or the presence of a fusion tag. For use in the practice of the present invention, the recombinantly expressed DHFR-TS protein may be in an unpurified state, such as that produced in the culture or as it is present in a cell lysate, or it may be partially or substantially purified from the same. Thus, for the present invention it provides a method for the preparation of the DHFR-TS protein comprising culturing the host cell transformed with a recombinant expression vector containing said recombinant expression vector a polynucleotide molecule of the present invention in operative association with one or more regulatory elements, under conditions that produce the expression of the DHFR-TS protein and the collection of the DHFR-TS protein from the cell culture. The present invention further provides a DHFR-TS DE Neospora protein isolate containing the amino acid sequence presented as SEQ ID NO: 3, or a protein containing the amino acid sequence of the DHFR-TS protein encoded by the DHFR-TS gene present in phage lambdaNclDHFRTS (ATCC Registration No. 209512); polypeptides substantially homologous thereto and peptide fragments of the aforementioned proteins and polypeptides. For example, the peptide fragments of the invention may consist of the DHFR domain or the TS domain of the DHFR-TS protein of Neospora. An amino acid sequence of the DHFR domain predicted in the DHFR-TS protein of Neospora is shown in SEQ ID NO: 3 from about amino acid residue 323; an amino acid sequence of the predicted TS domain ranges from about this amino acid 612. 4. 3 Use of DHFR-TS proteins Once a DHFR-TS protein of sufficient purity has been obtained, it can be characterized by conventional procedures, including SDS-PAGE, gel permeation chromatography, amino acid sequence analysis, determination of biological activity, etc. The DHFR-TS protein can also be characterized using hydrophilicity analysis (see, for example, Hopp and Woods, 1981, Proc. Nati, Acad. Sci. USA 78: 3824) or analogous software algorithms, to identify hydrophobic regions and hydrophilic. Structural analysis can be carried out to identify the regions of the DHFR-TS protein that assumes specific secondary structures. Biophysical procedures, such as X-ray crystallography (Engstrom, 1974, Biochem. Exp. Biol. 11: 7-13), computer simulation (Fletterick and Zoller (eds), 1986, in Cúrrente Communications in Molecular Biology) can be used. , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) and nuclear magnetic resonance (NMR) to map and study the interaction sites between the DHFR-TS protein and any of its substrates. The information obtained from these studies can be used to design more efficient deletion mutants and compositions for vaccines, or to design or select therapeutic or pharmacological compounds that can specifically block the enzymatic activity of the DHFR or TS domain of the DHFR-protein. Neospora TS in vivo. The DHFR-TS protein isolates of the invention, recombinant or not, are useful for a variety of purposes. For example, the protein can be used to look for inhibitory agents that specifically block the enzymatic activity of the DHFR or TS domain. Such selection procedures are well known in the art. The DHFR-TS proteins of the invention can also be used as antigens to obtain polyclonal or monoclonal antibodies, as described below, that specifically react with the protein. Such antibodies may be useful, for example, as affinity reagents with which to purify the native or recombinant DHFR-TS protein, or as diagnostic reagents for detecting the presence of a Neospora-specific DHFR-TS protein in the cell, tissue sample or fluid from an animal, such as, for example, by ELISA or Western blot. Antibodies can be obtained against a protein DHFR-TS using known procedures. Several host animals, including pigs, cows, horses, rabbits, goats, sheep and mice, can be immunized with the partial or substantially purified antigen. An adjuvant, such as that described below, can be used to enhance the production of antibodies. Polyclonal antibodies can be obtained from the serum of an immunized animal and assayed for the specificity of the anti-DHFR-TS protein using monoclonal techniques against the DHFR-TS protein using any technique that provides the production of antibody molecules by cell lines continuous in cultivation. These include, but are not limited to, the hybridoma technique, originally described by Kohler and Milstein (Nature, 1975, 256: 495-497), the human B-cell hybridoma technique (Kosbor et al., 1983 Immunology Today 4:72 Cote et al., 1983, Proc. Nati, Acad. Sci. USA 80: 2026-2030) and the EBV-hybridoma technique (Colé et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77.96), Alternatively, the techniques described for the production of single chain antibodies (see, for example, US Pat. No. 4,946,778) can be adapted to produce single chain antibodies specific for the DHFR-TS antigen. The fragments of antibody containing specific binding sites for a DHFR-TS antigen are also included within the present invention and can be generalized by known techniques., but are not limited to the F (ab ') 2' 'fragments which can be obtained by digestion with pepsin or an intact antibody molecule, and the Fab fragments, which can be obtained by reduction of the disulphide bridges. F (ab ') 2- fragments. Alternatively, Fab expression libraries can be constructed (Huse et al., 1989, Science 246: 1275-1281) that allow rapid identification of Fab fragments having the desired specificity for the DHFR-TS antigen. Techniques for the production of monoclonal antibodies and antibody fragments are well known in t and are further described, inter alia, in Harlow and Lane, 1988, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory and in JW Goding, 1986, Monoclonal Antibodies: Principles and Practice, Academic Press, London, which are incorporated herein by reference. 4. 4. Directed mutation of the DHFR-TS gene of Neospora 4.4.4. Genetic Constructs Based on the description of the polynucleotide molecules of the present invention, genetic constructs can be prepared for their use of the inactivation of a DHFR-TS gene from Neospora. The gene of DHFR-TS of Neospora can be deactivated using a gene constructs designed in an appropriate way in combination with genetic techniques known at present or to be developed in the future. For example, a DHFR-TS gene from Neospora can be inactivated using a genetic construct of the present invention that functions to: (a) suppress all or a portion of the DHFR-TS gene; or (b) replacing a part of the DHFR-TS gene with a different nucleotide sequence; or (c) an oligonucleotide molecule, or a polynucleotide molecule, which may contain a nucleotide sequence from Neospora or from another source. Neospora cells in which a DHFR-TS gene has been deactivated are useful in the practice of the present invention when deactivation of the DHFR-TS gene reduces the pathogenicity of Neospora cells carrying the DHFR-TS gene inactivated in comparison with the cells of the same Neospora strain of which the DHFR-TS gene has not yet been deactivated, and wherein said Neospora cells carrying the DHFR-TS gene inactivated can be used in a composition for a vaccine, especially in a modified live vaccine, to induce the protective response in a mammal against neosporosis. In a non-limiting embodiment, the genetic construct of the present invention is used to inactivate a wild-type Neospora DHFR-TS gene by replacing the nucleotide sequence of the wild-type DHFR-TS gene or a parasite thereof, with a mutated Neospora DHFR-TS gene or portion thereof. The sequences of the DHFR-TS genes of Neospora mutated for use in such a genetic construct can be produced by several known methods, including the use of error-prone PCT or by module mutagenesis. For example, oligonucleotide-directed mutagenesis can be used to alter the ORF sequence of the wild type Neospora DHFR-TS gene in a defined manner, for example, to introduce a frame shift or termination codon into specific regions within sequence. Alternatively, or in addition, a mutated nucleotide sequence for use in the genetic construct of the present invention can be prepared by insertion into the Neospora DHFR-TS gene of one or more nucleotides, oligonucleotide molecules or polynucleotide molecules, or by replacing a part of the DHFR-TS d Neospora gene with one or more nucleotides, oligonucleotide molecules or polynucleotide molecules. Said oligonucleotide molecules or polynucleotide molecules can be obtained from any source that exists naturally or can be synthetic. The inserted sequence may simply serve to interrupt the reading frame of the Neospora DHFR-TS gene, or it may also encode a heterologous gene product, such as a selectable marker. Random mutagenesis can also be used to produce a mutated sequence of the Neospora DHFR-TS gene for use in a gene construct of the present invention. Random mutagenesis can be carried out by any technique currently known or to be developed in the future, such as, for example, by exposing the cells carrying the DHFR-TS gene from Neospora or ultraviolet radiation or x-rays to chemical mutagens, such as N-methyl-N '-nitrosoguanidine, ethyl methanesulfonate, nitrous acid or nitrogen mustard and then selecting the cells carrying a mutation in the DHFR-TS gene. See, for example, Ausubel, 1989, supra, for a study of mutagenesis techniques. Mutations for the production of modified Neospora cells that are useful in the practice of the present invention, as defined above, can be produced anywhere in the Neospora DHFR-TS gene, including in the ORF region or in the promoter region, or in any other sequence flanking the gel or ORF. Said Neospora cells may be mutants in which a modified form of the protein encoded by the DHFR-TS gene of Neospora is produced, or in which no such protein is produced. In a preferred embodiment, said Neospora cells have a mutant phenotype dhfr or ts or dhfr-ts (hereinafter collectively referred to as the dhfr-ts phenotype). Likewise, said Neospora cells can be null, conditional or deficient mutants. Alternatively, a genetic construct of the present invention may contain nucleotide sequences that naturally flank the Neospora DHFR-TS gene or the ORF in situ, as selected from the flanking sequences shown in SEQ ID N0: 1 , presenting few or no nucleotide sequences of the coding region of the gene itself. A genetic construct of this type would be useful, for example, to suppress the entire gene or the ORF. In a preferred embodiment, a generic construct of the present invention contains a polynucleotide molecule that can be used to inactivate a Neospora DHFR-TS gene, which contains: (a) a polynucleotide molecule having a nucleotide sequence that codes for the DHFR-TS protein of N. caninum, but whose nucleotide sequence also contains one or more inactivating mutations; or (b) a polynucleotide polynucleotide protein polynucleotide olefin polymerization that naturally flanks the ORF of a DHFR-TS gene from Neospora in situ. Once transformed into cells of a Neospora strain, the polynucleotide molecule the genetic construct specifically targets the DHFR-TS gene of Neospora, for example, by homologous recombination and thereby replaces the gene or part thereof, or is inserted in the gene. As a consequence of this recombination event, the DHFR-TS gene, otherwise native to that particular Neospora strain, is deactivated. Methods for performing homologous genetic substitution on parasitic protozoa are known in the art and are described, inter alia, in Cruz and Beverley, 1990, Nature 348: 171-173; Cruz and others, 1991. Proc. Nati Acad.d Scie. USA 88: 7170-7174; Donald and Roos, 1994, Mol. Biochem. Parasitol. 63: 243-253; and Titus et al., 1995, Proc. Nati Acad. Scie. USA 92: 10267-10271, all of which are incorporated herein by reference. For genetic mutation directed by homologous recombination, the genetic construct is preferably a plasmid, circular or linearized, containing a mutated nucleotide sequence, as described above. In a non-limiting embodiment, at least about 200 nucleotides of the mutated sequence are used to specifically target the genetic construct of the present invention for homologous recombination of the Neospora DHFR-TS gene, although shorter nucleotide lengths may also be effective. Also, the plasmid preferably contains an additional nucleotide sequence encoding a product of an informative gene or other selectable marker that is constructed so as to be inserted into the Neospora genome in operative association with the regulatory element coding for the sequences of a native DHFR-TS gene from Neospora. Informational genes that can be used in the practice of the invention are well known in the art and include those that code for CAT, green fluorescent protein and β-galactosidase, among others. Nucleotide sequences that encode selectable markers are well known in the art and include those that code for gene products that confer resistance to antibiotics and anti-metabolites, or that provide an auxotrophic requirement. Examples of such sequences include those that code for resistance to pyrimethamine or neomycin phosphotransferase (which confers resistance to aminoglycosides) or hygromycin phosphotransferase (which confers hygromycin resistance). The methods that can be used to create a genetic construct of the present invention are well known in the art and include genetic recombination techniques in vivo, as has been described, among others, in Maniatis et al., 1989, supra; Ausubel, et al., 1989 above; and Sambrook et al., 1989, above. The Neospora cells can be transformed or transfected with a genetic construct of the present invention according to known techniques, such as, for example, electroporation. The selection of the transformants can be carried out using conventional techniques, such as by selection of the cells that express a selectable marker associated with the construct. The identification of the transoformers in which a successful recombination event has occurred and in which the particular target gene has been deactivated, can be carried out by genetic analysis, such as detecting a lack of mRNA transcripts encoding for DHFR-TS protein or by appearance of a new phenotype, such as reduced pathogenicity or cells that do not have DHFR-TS protein, as determined, for example, by immunological analysis or some combination thereof. The Neospora cells that can be modified according to the present invention are preferably tachyzoites, but as an alternative they can be bradyzoites or oosistos. Although the cells at certain stages of the Neospora life cycle are diploid, the tachyzoites are haploid. Thus, the use of tachyzoites in the production of modified Neospora cells expressing the appropriate mutant phenotype is preferred because the tachyzoites require only a single successful recombination event to break the particular Neospora gene. Alternatively, in diploid Neospora cells, two alleles must be broken for each gene. This can be carried out by sequentially selecting the first allele and then the second allele with genetic constructs carrying two different selectable markers. In another non-limiting embodiment, the genetic construct of the present invention may additionally contain a different gene or a Neospora coding region or a different pathogen that can infect an animal, this gene encoding to the coding region an antigen useful for inducing a separate and distinct protective immune response in the animal when vaccinated with the modified live Neospora cells of the present invention. This additional gene or coding region can be further constructed to contain a signal sequence that produces the secretion of the coded antigen from the modified live Neospora cell, thereby allowing the antigen to be displayed to the immune system of the vaccinated animal. The present invention thus provides modified live Neospora cells in which the DHFR-TS gene has been destroyed. In addition, the present invention provides a method for preparing modified live Neospora cells comprising; (a) the transformation of Neospora cells with a genetic construct of the invention; (b) the selection of transformed cells in which the DHFR-TS gene has been destroyed by the genetic construct; and (c) selection among the cells of step (b) of those cells that can be used in a vaccine to protect a mammal from neosporosis. 4. 4.2. Culturing Neospora cells Neospora cells for use in the present invention can be cultured and maintained in vitro by infection of any recipient cell line, preferably in a mammalian cell line, with tachyzoites according to the known techniques described in technique. Mammalian cell lines in which the Neospora tachyzoites can be grown include, for example, human foreskin fibroblasts (Lindsay et al., 1993, Am. J. Vet. Res. 54: 103-106), cardiopulmonary aortic endothelial cells. bovine (Marsh et al., 1995, above), bovine monocytes (Lindsay and Dubey, 1989, above) and monkey kidney cells, among others. For example, N. caninum tachyzoites can be cultured in monolayers of human foreskin fibroblast cells Hs68 (ATCC Registration No. CRL-1635) (Lindsay et al., 1993, supra); and MARC145 monkey kidney cells infected with tachyzoites of N. caninum strain NC-1 for use in the present invention are deposited with the ATCC (Registry No. 12231). Bradyzoites can be grown and manipulated in a similar way. Cultures of mammalian cells can be cultured and cell cultures that have been infected with Neospora can be maintained, in any of the many types of culture media described in the art. For example, stationary monolayer cultures of bovine cardiopulmonary aortic endothelial cells infected with N. caninum tachyzoites can grow in Dulbeco Minimum Essential Medium (DMEM: Gibco Laboratories, NY) supplemented with 10% heat-inactivated fetal bovine serum (FBS). % (v / v) or adult equine serum (ES), 2mM L-glutamine, 50 U / ml penicillin and 50 μg / ml streptomycin (Conrad et al., 1993, supra). Monolayers of human foreskin fibroblast Hs68 cells can be maintained in RPMI 1640 containing 2% FBS (v / v), 1.0 mM sodium pyruvate, 1 x 104 U / ml penicillin, 1 x 10 μg / ml streptomycin, 5 x 10"mM 2-mercaptoethanol and 0.3 mg / ml L-glutamine (maintenance medium) Monolayer cultures of human foreskin fibroblast cells Hs68 infected with Neospora can be maintained in identical media, but in which FBS has been increased to 10% (v / v) (growth medium). Monolayer cultures of Neospora-infected mammalian cells are typically maintained under conventional tissue culture conditions, such as, for example, conventional tissue cultures. , such as, for example, at 37 ° C and 5% CO.The tachyzoites are typically passed to uninfected monolayer cultures when 70-90% of the mammalian cells in the culture have become infected, which can be determined microscopically using techniques conventional The tachyzoites can be harvested from the cultures of the infected mammalian cells using the host cells using any conventional technique and collecting the tachyzoites, for example, by filtration or by centrifugation. Neospora cells that have been modified according to the present invention and which have a dhfr-ts phenotype can be cultured in mammalian cells, as described above, in medium containing thymidine. 4. 5. Anti-Neospora Vaccines The present invention provides a vaccine against neosporis, which contains an immunologically effective amount of living modified Neospora cells, such as for example, the above-mentioned null mutants dhfr ts and a veterinarily acceptable carrier. The present invention further provides a method for preparing a vaccine that protects a mammal against neosporosis, comprising the preparation of modified living cells, such as, for example, those having a dhfr ts phenotype prepared as described above and the combination of an immunologically effective amount of the living cells modified with a veterinarily acceptable carrier in a form appropriate for its acid to the mammal. As used in this, the term "immunologically effective amount" refers to that amount of modified live Neospora cells of the present invention capable of inducing a protective response against neosporosis when administered to a member of an animal species after a single administration or after multiple administrations. The phrase "capable of inducing a protective response" is widely used herein to include the induction or enhancement of any immune-based response in the animal in response to vaccination, including either a cell-mediated immunity antibody or both, which serves to protect the vaccinated animal against neosporosis. The terms "protective response" and "protects", as used herein, refer not only to the absolute prevention of neosporosis or to the absolute prevention of infection by a pathogen that causes neosporosis, but also to any detectable reduction in the degree or rate of infection by said pathogen, or any detectable reduction in the severity of the disease or any symptom or disorder resulting from infection with the pathogen, include, for example, any detectable reduction in the rate of formation or in the absolute number of lesions formed in one or more tissues, or any detectable reduction in the rate of formation or in the absolute number of lesions formed in one or more tissues, or any detectable reduction in the production of abortions or the transmission of infection from a pregnant mammal to its fetus, or from a parental mammal to its offspring in the vaccinated animal in com paration with an unvaccinated infected animal of the same species. The vaccine may simply contain an aliquot of the culture fluid containing modified live Neospora cells, free in the medium or resident in mammalian host cells or a combination of both, which is administered directly to the mammal, or may instead be administered to the mammal. containing modified live Neospora cells combined with a veterinarily acceptable carrier selected from those known in the art depending on the route of administration. It is preferred that at least some degree of viability of the modified live Neospora cells be maintained in the composition of the vaccine. For example, a composition of the vaccine of the present invention can be formulated following accepted conventional procedures using conventional buffers, carriers, stabilizers, diluents, preservatives and / or solubilizers and can also be formulated to facilitate sustained release. The diluents may also include water, saline, dextrose, ethanol, glycerol, and the like. Additives for isotonicity may include sodium chloride, dextrose, mannitol, sorbitol and lactose, among others. Other suitable vehicles and additives are known, which are particularly useful in modified live vaccines or will be apparent to those skilled in the art. See, for example, Remington's Pharmaceutical Science, 18th ed., 1990, Mack Pubiishing, which is incorporated herein by reference. The modified live Neospora cells that can be used in the vaccine of the present invention are preferably tachyzoites, but may alternatively be bradyzoites or oocysts, or some combination thereof. The vaccine of the present invention may further contain one or more immunomodulatory components, such as, for example, an adjuvant or a cytokine, among others, as long as a certain degree of viability of the modified live Neospora cells is maintained. Non-limiting examples of adjuvants that can be used in the vaccine of the present invention include the RIBI adjuvant system (Ribi Inc., Hamilton, MT), alum, mineral gels, such as aluminum hydroxide gel, oil emulsions in water, water-in-oil emulsions, such as for example, complete and incomplete Freund's adjuvants, block copolymers (CytRx, Atlanta GA), adjuvant QS-21 (Cambridge Biotech Inc., Cambridge MA) and SAF-M (Chiron, Emeryville CA), AMPHIGENR, saponin, Quil A or other fraction of saponin, monophosphoryl lipid A and lipid-amino adjuvant Avridine. Specific non-limiting examples of water oil emulsions useful for the vaccine of the invention include modified SEAM62 and SEAM 1/2 formulations. SEAM62 modified in a water oil emulsion containing 5% squalene (v / v) (Sigma), detergent SPANR 85 (ICI Surfactants) at 1% (v / v), TWEENR 80 0.7% detergent (v / v) (ICI Surfactants), ethanol 2.5% (v / v), 200 μg / ml Quil A, 100 μg / ml cholesterol and 0.5% lecithin (v / v). The SEAM 1/2 modified is an oil-in-water emulsion containing 5% squalene (v / v), SPANR 85 1% v / v detergent, 0.7% TWEENR 80 (v / v) detergent, 2.5% ethanol ( v / v), 100 μg / ml of Quil A and 50 μg / ml of cholesterol. Other immunomodulatory agents that can be included in the vaccine include, for example, one or more interleukins, interferons or other known cytokines. The vaccine can be stored frozen, thawing it before administration. The vaccine of the present invention can optionally be formulated for the sustained release of modified live Neospora cells, as long as at least some degree of viability of the modified live Neospora cells is maintained in the vaccine composition. Examples of sustained release formulations of this type include modified live INeospora cells in combination with biocompatible polymer composite materials, such as, for example, poly (lactic acid), poly (lactic-co-glycolic acid), hypromellose, acid hyaluronic, collagen and the like. The structure, selection and use of degradable polymers in drug delivery vehicles has been indicated in several publications including A. Domb et al., 1992. Polymers for Advances Technologies 3: 279-292, which is incorporated herein by reference. In the text by M. Chasin and R. Langer (eds), 1990, "Biodegradable Polymers as Drug Delivery Systems" in: Drugs and the Pharmaceutical Sciences. Vol. 45, Dekker, NY, an additional guide can be found for the selection and use of polymers in pharmaceutical formulations, which is also incorporated herein by reference. Alternatively, or in addition, modified live Neospora cells can be microencapsulated to improve administration and efficacy, as long as at least some degree of viability of the modified live Neospora cells is maintained in the vaccine composition. Methods for microencapsulation of antigens are well known in the art and include techniques described, for example, in U.S. Patent 3,137,631; U.S. Patent 3,959,457; U.S. Patent 4,205,060; U.S. Patent 4,606,940; U.S. Patent 4,744,933; U.S. Patent 5,132,117 and International Publication WO 95/28227, all of which are incorporated herein by reference. Liposomes can also be used to provide sustained release of the modified live Neospora cells of the invention, as long as a certain degree of viability of the modified live Neospora cells is maintained in the vaccine composition. Details concerning how to prepare and use liposome formulations can be found among others, in U.S. Patent 4,016,100; U.S. Patent 4,452,747; U.S. Patent 4,921,706; U.S. Patent 4,927,637; U.S. Patent 5,008,050 and U.S. Patent 5,009,956, all of which are incorporated herein by reference. The present invention further provides a combination vaccine for the protection of a mammal against neosporosis and, optionally, one or more diseases or pathological disorders that may affect the mammal, this combination vaccine comprising an immunologically effective amount in a first component containing modified live Neospora cells; an immunologically effective amount of a second component capable of inducing a protective response against a pathological disease or disorder affecting a mammal, and a veterinarily acceptable carrier. The second component of the combination vaccine is selected on the basis of its ability to induce a protective response against neosporosis or another disease or pathological disorder that may affect members of the species of mammals, as is known in the art. Any known immunogenic composition that may be useful in a vaccine composition in the particular mammalian species may be used in the second component of the combination vaccine, as long as a certain degree of viability of the modified live Neospora cells is maintained. in the resulting vaccine composition. Such immunogenic compositions include, but are not limited to those that provide protection against pathogens selected from the group consisting of bovine herpes virus (without: infectious bovine rhinotracheitis), bovine respiratory syncytial virus, bovine viral diarrhea virus, bovine viral parainfluenza type I, II or III, Leptosispira spp., Campylobacter spp., Staphylococcus aureus, Streptococcus agalactiae, Mvcoplasma spp., Klebsiella spp., Salmonella spp., rotavirus, coronavirus, rabies virus, Pasteurella hemolytica. Pasteurella multocida, Clostridia spp., Tetanus toxoid, E. Coli, Cyptosporidium spp., Eimeria spp. , Trichomonas spp., And other eukaryotic parasites, among many others. The combination vaccine of the present invention may further contain one or more additional immunomodulatory components, including for example, an adjuvant or a cytokine, as described above, as long as the viability of the cells of vaccine composition is maintained. . The antigens of the combination vaccine can be stored frozen and thawed before administration. The present invention further provides a method for the protection of a mammal against neosporosis, comprising administering to the mammal a vaccine containing an immunologically effective amount of modified Neospora living cells of the present invention and a veterinarily acceptable carrier. The vaccine is preferably administered parenterally, for example, by include subcutaneous or intramuscular. However, the vaccine can be administered by intraperitoneal or intravenous injection by other routes, including, for example, the oral, intranasal, rectal, vaginal, intraocular or via combination routes, and also by delayed release devices as are known. in the technique. The person skilled in the art will know how to formulate the composition of the vaccine according to the chosen route. An effective dosage can be determined by conventional means, starting with a low dose of modified live Neospora cells and then increasing the dose while controlling the effects. Numerous factors can be taken into consideration when determining an optimal dose per animal. Among the main ones are the species, size, age and general condition of the animal, the presence of other drugs in the animal, the virulence of a particular species of the Neospora strain against which it is going to be vaccinated and the like. The actual dosage is preferably chosen after considering the results from other studies in animals. Vaccine regimens can also be selected based on the factors described above. The vaccine of the invention can also be administered at any time during the life of a particular animal depending on several factors including, for example, the appearance of an outbreak of neosporosis in other animals, etc. The vaccine can be administered to animals at weaning age or younger or more mature animals, etc., as a vaccine before crossing to protect them against congenital disease or abortion related to Neospora. Effective protection may only require a primary vaccination or one or more booster vaccinations may be needed. A method for detecting whether adequate immune protection has been obtained is to determine seroconversion and antibody titration in the animal after vaccination. The vaccination guideline and the number of booster shots, as the case may be, will preferably be determined by a veterinarian based on the analysis of all relevant factors, some of which have been described above. The amount of live Neospora cells modified in the vaccine ranges preferably between about 1 x 10 3 and about 1 x 10 8 / ml and more preferably between about 1 x 10 5 and about 1 x 10 / ml. An appropriate dosage size ranges from about 0.5 ml to about 10 ml and more preferably between about 1 ml and about 5 ml. The vaccine of the present invention is useful to protect mammals against neosporosis. As used herein, the "mammalian" tea refers to any animal species that can be protected against neosporosis using the vaccine of the invention, including dogs, cows, goats, sheep and horses among others. The vaccine is useful to protect both pregnant and nonpregnant mammals. The present invention further provides a kit for the vaccination of an animal against neosporosis, which contains a first package having an immunologically effective amount of modified live Neospora cells of the present invention and a second package having a veterinarily acceptable carrier or diluent. . The modified living cells of the kit can be stored frozen, thawing them before administration. The following example is illustrative only and is not intended to limit the scope of the present invention.

EXAMPLE 5 ISOLATION OF THE DHFR-TS GENE OF NEOSPORA . 1 Amplification of exon 1 of the DHFR Domain of N. caninum Oligonucleotide primers of specific base pairs of length were designed for the 5 'and 3' ends of the DNA sequence of exon 1 of the DHFR-TS gene of T. gondii and were synthesized based on the published sequence of the DHFR-TS gene from T. gondii (Roos, 1993, J. Biol. Chem. 268: 6269-6280). The genomic DNA of N. caninum strain NC-1 was prepared using the GNOME ™ kit (Bio 101, La Jolla, CA) using the reagents and protocols provided by the manufacturer. The PCR was carried out with 0.5 μg of genomic DNA; 120 pmol of each of the primers Tgdhfrexon 1-5 '(5'-ATGCAGAAACCGGTGTGTCT) (SEQ ID NO: 4) and Tgdhfrexonl-3' (51-AGGAAGAGGAAACGACGAT) (SEQ ID NO: 5); 2.5 mM dATP, dGTP, dCTP and dTTP (Perkin-Elmer, Norwalk, CT); PCR buffer (Perkin-Elmer); and 1.5 U of DNA polymerase AMPLITAQ ™ (Perkin-Elmer). The PCR cycle was 1 cycle of 94 ° C for 1 min and 29 cycles of 94 ° C for 1 minute, 55 ° C for 1 minute, 72 ° C for 1 minute. An approximately 0.3 kb PCR product was obtained and cloned into a pCRII vector (Invitrogen, Carlsbad, CA), without further processing using T4 DNA ligase. The cloned fragment of approximately 0.3 kb was sequenced by the dideoxy chain termination sequencing procedure and the sequence was determined to be > 85% homologous with the sequence of the corresponding exon 1 DHFR-TS of T. gondii. . 2. Cloning and sequencing of the Neospora DHFR-TS gene A genomic DNA bank of the NC-1 strain of N. caninum was made in the bacteriophage lambda vector DASHII (Stratagene, La Jolla, CA). The above ~ 0.3 kb fragment was radioactively labeled with O -P-dCTP and the reactivity of the labeled fragment was searched for in about 5 x 105 phage clones by plaque hybridization. After three rounds of selection to enrich the reactive clones, 12 phage clones reactive with the sequence of ~ 0.3 kb were identified. The lambda bacteriophage clone DNA was prepared from 3 of the 12 positive clones using a lambda DNA isolation system (Qiagen, Chatsworth, CA) according to the manufacturer's protocols. The DNA of one of the lambda phage clones, which has been designated as lambdaNclDHFRTS (ATCC registration no. 209512) (also referred to as 4C13 or lambdaCY50) was digested with Notl releasing an 11 kg DNA fragment, which was subcloned according to the procedures described in Sambrook et al., 1989, supra. PCR was performed on the DNA of a plasmid subclone containing the 11 kb DNA fragment using the following oligonucleotides, which were designed based on the sequence DHFR-TS: 5 '-CCCCTCGTGGACCGGCTGAATA (SEQ ID NO: 6). 5 '-TCCGTGCGTGCCAAGA-GACTG (SEQ ID NO: 7); 5-ATGGAGATGGCGATGGGAGGAC (SEQ ID NO: 8); And 5 ' AGTATGTACACGAAGCCTCAAT (SEQ ID NO: 9). Oligonucleotides were used for PCR in the following combinations: SEQ ID NOS: 6 & 8; SEQ ID NOS: 6 & 9; SEQ ID NOS: 7 & 8; and SEQ ID NOS: 7 & 9. PCR yielded specific products suggesting the presence of the Neospora DHFR-TS gene within the 11 kb fragment. Additional restriction analysis of the plasmid subclone containing the 11 kb fragment indicated the presence of an asymmetric HindIII site within approximately 1.5 kb of one of the Notl sites. Next, the DNA sequence of the NotI site at one end of the HindIII site and at the other end (~ 9.6 kb) was determined by primer walking using the dideoxy chain termination sequencing procedure. The DNA sequences thus obtained were analyzed using the DNASTAR software program (DNASTAR Inc., Madison, Wl) The length of the fragment previously sequenced is 9603 bp (SEQ ID NO: 1) and contains the sequence of the DHFR-gene. Whole TS of the NC-1 strain of N caninum The nucleotide sequence of the DHFR-TS gene from Neospora was compared to that of the published sequence of the DHFR-TS gene for T. gondii to predict the locations of exons and introns, as well as as the boundaries of the DHFR-TS and TS domains. It is believed that the DHFR-TS gene from Neospora contains 10 exons and 9 introns in the following way: Exon 1 -from <; 2405-2724; Exon 2 -from 3212-3348; Exon 3 -from 3925-4262; Exon 4 -from 4491-4737; Exon 5 -from 5214-5307; Exon 6 -from 5678-5750; Exon 7 -from 6129-6270; Exon 8 -from 6685-6777; Exon 9 -from 7264-7574; and Exon 10 -from 8116- > 8199). The consensus splicing signals are present in the intron-exon junctions. The predicted DHFR domain ranges from about nt 2405 to about nt 4664; and the predicted TS domain ranges from about nt 4665 to about nt 1899, based on the structural analogy with the DHFR-TS sequence published for T. gondii. The predicted open reading frame (ORF) coding for the DHFR-TS protein of N. caninum strain NC-1 is 1,839 bp in length (SEQ ID NO: 2). The ORF frame coding for the predicted domain ranges from about nt 1 to about nucleotide 969. The ORF encoding the predicted TS domain ranges from about nt 870 to about nt 1836. The deduced amino acid sequence of the DHFR protein -TS of N. caninum strain NC-1 is 612 amino acids in length (SEQ ID NO: 3). The deduced amino acid sequence comprising the predicted DHFR-TS domain ranges from about amino acid residue 1 to about amino acid 323; the deduced amino acid sequence comprising the predicted TS domain ranges from approximately amino acid residue 324 to approximately amino acid residue 612. Sequence analysis indicates that there are 73 differences between amino acids in the DHFR domain and 15 difference amino acids in the TS domain between the deduced amino acid sequence of N. caninum and the predicted DHFR-TS sequence of T. gondii.

Deposit of biological material The following biological material was deposited at the American Type Culture Collection (ATCC) at 12301 Parklawn Drive, Rockville, MD, 20852, USA. on December 3, 1997 and was awarded the following registration number: phage lambdaNclDHFRTS (ATCC registration No. 209512). All patents, patent applications and publications cited above are incorporated herein by reference in their entirety. The present invention is not limited in scope by the specific embodiments described, which are only intended to be concrete illustrations of the individual aspects of the invention. The functionally equivalent compositions and methods are within the scope of the invention. Thus, various modifications of the invention, in addition to those shown and described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

LIST OF FREQUENCIES < 110 > Kn siman, B. Will Yodar retain? . Chx? atine Durtschi. Beclty A. < 120 > DNA THAT CODIFIES FOR DIHYDROFOLATE REDUCTASE TIMIDILATE SINTETA8A FROM NEOSPORA < 130 > DHGR-TS < 140 > < 141 > < 150 > 60 / 067,501 < 151 > 1997-12-04 < 160 > 9 < 170 > Paceptln Ver. 2.0 - scholarship < 210 > 1 < 211 > 9603 < 212 > DNA < 213 > Neospora caninum < 220 > < 221 > gene < 222 > (2405) ..18199) < 400 > 1 gcggccgccg gcacaatgec gagggcagcg cagggaaaaa ccggccgaag ctgcttcacg 60 tgcctgaaaa tagagccagc gctactrteg cttcaaagaa cgagacttcc gcgccacaaa 120 tcggtagtga cggcggccgg aagcgaaaat tttacgacga gcagctgcca gtcggcgtgt 180 accgtcacca gcagaagtat gtcgcgaact gggtagaccc gaaaacccgg agacaaatca 240 aggtctgttt ccccatcgac gtgtg GGAG acrctcaagc tcgcaacatg gctgccgttg 300 cgaggcgtga gcgctgcgtg gatgtcgacg AGGT gctge tattttcaat cgtgaagagc 360 gcaccaagac atcaggacgt catccgagtc cttcgaggga tgacagcaaa cagacagcgt 420 ctttcaatag tgctgttagg atgccagcag tccagggtgt ggattcaaaa acggagacgc 480 actcggctcc ggcgctcgaa agcgcgtaga gcctgaggcc acaccacttc tggttttcag 540 gcactcacaa agagggcgtc ttttcttcga cttctttccg cacactgggg ccgtgtgctc 600 gaaeactttt tatccgtgtg accatgtgcc cgaacacttt ttatccgtgt gaccatgtgc 660 ccacgaacgc ggcgaggett ccctgtgctc gtggatttta ttgagtgatr ttettgtata 720 cagaaaacct gtttcgtttt gctgccagtc ttcacatacg cttgcaagca gagcgtgagt 780 agggggggtt ccttgggaga gagcgcgaaa ggcgactttt tttacgacca gcgagagcgc 640 cgcagcagca caacgat cag aaaaeaettg ggacaaattg gctctcgcca gggagaagtt 900 ctgcagtgga gataactaaa gaatccaaga tgcgaacgca gccegcaaaa acacggcttc 960 agagctgcat gttcctttga tttaacgtgc acaccttgtc gtgcaaggtg ggcat cactg 1020 agggacttcg ccatcccgcg tactcgtcgc agcagtgcca cgcttctccg ggcgtccact 1080 gggccgaagt ttaatcgggc ggatczcaag etaccgtcaa gtcccaaatc ttcagctgtt 1140 tgcaggaagg ttgctccgca cttgggtact gccgtgagaa gcagcggttc actgtgctgc 1200 gtcacctcgt ccacaggaac agacacactg gagctatgtc ggtggaccgg ctgcgtattc 1260 gatgaggttc agctgcggtc gttgcagagc tgcttttccc tctt? ccaag acgatacgaa 1320 tagagtgtag cggaaggcac tgtacccaga agcattcagt tgcgctgttt cggatttcag 1380 tcgacacaga tctgttcgcg aaegacggcc ccgcagatct tgtgcccctg ßetcgggact 1440 cgtacagaag gcggttcgac gacgtttaga tgcgggcgac aagaattcgt gtcttaactt 1S0O gctttcagcc tggtgtctac gtgccaaagt tcgctgaccg aaaagtcacc acggaagtgg 1560 attgctacgc ßgtcatatge ccgatgccca ctccactgtg ctctttccgc agtgcttcgc 1620 cgccgaaaaa acgaggcgac tccgg TTGC tccgtcctgc tccacaaaac tgaaggttcc 1680 tcagaccctt tagagactca a aacgcagcc ccgacgcgca tccaggacga gaagaaacca 1740 gcacgccact gctctctccg cctccagcgt gectttgtgt ctcgtggcac ggcggtcggt 1800 ggttccgcct gcgtctcgcc tttecgcgtt ccctgtctca gcgtgaacac catcactaca 1860 cccaccgcca gtgctagaac tggtccgaca ctctttcttc ctccaacgag cgcttcatta 1920 ggtcctttgt ttttcgacac tgcaaacttg cgtggttttt cacttcgcac gcggaagacc 1980 cgaaeetgga ctgccgecgc ttgcacacta tagcggcctc gtccgccgtc tccgtcggag 2040 cctcccccct cgtcaacttt acggtccggt gccgcgaaag cgcatttggc gaatatatct 2100 ccggaaactc cacgcttttt aaagcegtca ggctctcttc gttcttctcg tcgagctcgt 2160 tttctctgct tccttgactt gcttcgatc- gcgcatcttc ctactcctcc ctggcccgtt 2220 aaaettegag acaagggacg aggtgtgcac atctttcgct tgatacccaa ctctccccgg 2280 cttcgtgctg accgtttccc acttttacct cacggcaatg ccgagcgcgc ctctgagtgc 2340 ggctgggacg ggtgaagttg gaccgtctgt tgccgttcca actcgtggag ttgtgcagcg 2400 aaacatgcag aaaccagtgt ctcttattgc cgcgatgacc cccaggaggg gcatcggcgt 2460 caacaacggc ctgccatggc cccacttggc cacagatttc aaacactttt ctcgcgtgac 2520 gaaaacgacg gccgacgaag tctctcgcct gaacgcatgg cttccgaaaa aaattgccaa 2580 sacgggcgat tcgggacttc cctctcccgc cttcggtgtc aacagattca acgctgttgt 2640 catgggacga aaaacctggg agagcetgcc gctaaaattt cstcccctcg tggaccggct 2700 gaatatcgcg gtttcctcct ccctgtaagc acacggcgca ggcgatgtgt ctgtgcgctc 2760 gcgacgtctc cgtgcgtgcc aagagactgg acgattccct ctgaaaagcc ttcgccggct 2820 ccattcctcg tagacgaacc acaaaccgcg agtgcgctt cctcggagag aaaggecgca 2880 cgtctggatg caacacactg gggggactgc gatcgtatgt cctgataacc gaaatccgga 2940 aggctga ctg aagacgcact ggcgttcgca aacatttctc catcgcttcg cgcggaccgg 3000 tgaacctgct ctctgcacaa ccagaetcgt ggttcctacg ttgctgagca cggctgeata 3060 tatatatata tatatatata tacatacgca tatgcgtata gaggttcccg cccgaagcgt 3120 gcttctgtcc gtcttccgtc cctgctgtcc tcccatcgcc atctccattt cctacgtgtc 3180 cccttgttca tttcgtectt gcaaagaaga acggttgtca agacatcgcg gcggagaagc 3240 aggccagcaa ctctagtcga cgcgtgcgag tetgcgattc actceccgca gcectgcgee 3300 agagtacaga ttgtggacga gagtctgttg accagattta tgttgtgggt aggtgtgcca 3360 agagcggcac agattcctct ctcgctcaga agtgcctggc aactgatctg cggcacgcgc 3420 cgcggatccc gatgcacatt gaggcttcgt gtacatactc cctgcgagct accagagggg 3480 ctgagaatct ccagataagc gtatatttct atgtgtttat gcatagatat ttagaacagg 3540 ctgaggagat gcggggaagg ctgacactga cggaatgcct tgccgcccac cagcagagga 3600 aggcggatgg ggacaacggc atgcagcctg ggacagcggc tgattcacgc cgacagcgca 3660 gcccccgacg tcggtgaagc gtctcgaccg cctettttgg cgcgcetctc cgttccttct 3720 ttcgcccacg gcttcgtttc tctccattcc cactgcgtgt tgsagcccgt cegtccagtc 3780 tctctcagtt ctcttctttc ccttcagttg ctgeactctt ttcgccgtgc tccagttcct 3840 tgcgttgtgg ccttttccgc tcttcctatt ggcgtctcgc atccattgcg ttttctcggg 3900 tggtatccct ccgcctgcct tcaggaggag cggggctcta tgaggaagec ctgtctctgg 3960 gcgtggtgtc tcacctctac atcacccgcg tggcgcgtga ctteccatgc gacgttttct 4020 tteeegctt t ccccggagac tccattcctt caaacaagca ggcggcctcc gcgagtcagc 4080 cttcggctgc tgcggaaccg gtgtttgetc cgtettgcee ccagctcggg agagagaaga 4140 gcaatgaagc gtcgtaccga CCCat cttta tttcaaagac ctactcggac aacggagtgc 4200 cctacgact t? eggttctt ggaaggctga gaaaaaggga cgcttgcagc gccacggaat 4260 cggtaagtgg ctacggaggg gaaaagacga gagaaagagc gggcatccgg aacgagtgtc 4320 gcgaggcgac gttccatgcg tatccaagag agagggaaga gggaacacgc agcttgaggg 4380 gtaagctacg cgctcttttc etttctccgt gacgccgtaa ggccgcggga agacggtgca 4440 aggac gcgca CGG? cgtttc gccgtzttcg gtttctgtcc actctcccag tgcgagcttc 4500 gcggcccttg gacctccacc ggagagacgt cgccagagac gaggcttccg tcttcctccg 4560 cctcagccgt tgcccaggtg ttggcttgga tggccgacga agaccggaaa aaatgcgaga 4620 agaaagaaat cattcgggca gtgcctcacg tccactttcg gggccacgaa gaattccagt 4680 acctcgacct cattgccgat attatcaaca aeggagcgac aatggacgac cgaacgggta 4740 cgggaaggca aacgcgacca tcccgtccat gcggggctgt ctgctgagcc tcggttcctt 4800 cttccgcgct gtgcggcttt cccc ggggt ctttgcttct ctctgttcgc cgccttgcct 4860 gccacatttt cccttcattt tttttctctg tctcccttgc tcttttgtcc taacgttcgt 4920 actcgccttc gtctcgcagt ccacgcttca aaacagacgg gctaccgaaa cgtgttttcc 4980 cectgcacgg cct ttttcac acgccgttgt cgcttgactc tcgctgacgc ggggtttgcc 5040 gcttcctgga agaaagaagg ecttt cctca ectgttegcc cttttcgctg tttcacagaa 5100 agacgagaga gattccgcct ccattttctc aattege? tt .c; tgccc = a agcagatgtg 5160 ccctgatctg gaggctcttc gccgctcccc ttctcccct? tcgccgcttt caggcgttgg 5220 agtcatctcc aagttcggct geaccatgcg gttctcgct; ? Gataaggcct tccctcecct 5280 caccacaaag cgtgtgttct ggaaagg ta cggcgcccta caaaaatctg tatatattta 5340 acaggcacac gtgtgcgtgt ctgacctgca cacgtgttca taaacgtgca cgcaattgta 5400 p: gtggctg sc gtggagtcgt ccacgaacag gaaatattcs catgcatgca ctctacagac 5460 tgcttctcta gtgcctggac ccttcgtttg tctgtttatt tgctttaatt tcgcccggtg 5520 accgtcgcgc ctcegtctga ccgtgcattt gcttgcgect catcgtagtg tgcgtatcga 5580 gcatgtgacg agacgagaga egccgagta ctgttgtcta: gagaagtecg cacgacggtc 5640 cc gctgaacgat gttctttt gtgtgggttg ctctcaga i cctcgaggag ctgttgtggt 5700 tcatccgcgg tgacacgaac gcgaatcacc tctctgaaaa gggcgtaaag gcaagtcttc 5760 aagcaccgct gctctcgttc aggctcctcc gcagacttgg cgctttcctt cgcggcgtca 5820 cccctccgag gcttcacact eacattgagt gtacecgtf. gtcttctaga ccgtctgctg 5880 cgtttgcagg cccccgcgtg agegcaggcc cttcatcgee gagtgtggcc gtagctttgc 5940 gcgacgaaga cagtcgatag gccttccaga gcacgttcct tctgtctccc gttttccccg 6000 tttttttccg cgtcttcctc cgacagcctc gcacggctea catcccctct gagccgggac 6060 agggctcgcc taaggcagag taccacgctc cgtaacttcc ggcatgcgtt tctgggtttt 6120 cgttttagat ctgggacaag aatgtgacaa gagagttcct tgattcacgc aatctttccc 6180 accgagaggt cggagacatc ggeccgggtt acggcttcca gtggagacac ttcggcgcga 6240 cctacaagga catgcacacg gactacactg gtatgtcccg gcgttctttg aggggggaag 6300 ggaaagcagc cgaacccgcg aaacggcgct gatgccegtt ctcgcttcgt gtgctggacc 6360 gaccgttcca gccatatcgc aggttecaat agccgccaca aacggagatg aaaatgcaag 6420 gcgacgactc tgcgctcgcg cacaactggt gacagacgcc actccgtgtg gacgaattcg 6480 gttgcaaact gccaagcgat gaaagggccg tcgggtggta actccgtgcc cgggcgcetg 6540 cacacaaata cccgtgtgtg tgtgtgtgtg cgtatgttga tgcaaagata cccctacgtg 6600 tgcatgtgta catacacgtg agaaattggt gcccgtcgtt saaaaatgct cactcccatc 6660 ectcctggcg ttgcccattt gcaggtcagg gcgtagacca accgaagaag gtgatcaaca 6720 gaatccaaca tgctgagaac gaccggcgca tgctcaegac cgcttggaac cctgcgggtg 67B0 agatctccgt cttcaatctc tcctttccag ataatacgtg catttcaact ggaagctcte 6840 acacagccgt gtgcacaacg ggaagacgct gacacatacg tgttggtccc cccaacgtta 6900 tcteccgtgc cgtatctgtg tggctgctec tctaaggeta ttgcgccgtg gtgatgtctc 696C tcgtatctcg tctgtgcttt tcgctggatg cctctgtgcc tagacatctc aaagtgtccc 7020 tcccgtgcgg ggtcccgcga aacacttgcc actggccttt tcgcctcttg ccgctgtcgt 7080 ctgttcctcg ggattcccea ctcggggacc tgccggtttc agtgcetttc ctccgcgggt 7140 gcttcttccc ccgtcctcgc gccttgtgtt tctttgccgt ggcgactgcg ccgccgtgca 7200 tgetgetcac ttccccccgt gcgcgtgtgt tttgtcctcg ccgtctctct ctttcccgtt 7260 cagcgctgga cgaaatggcg ttgccgcctt gccacttgce gtgtcagttc tacgtggaga 7320 acgacagaga cttgtcttgc gtcatgtatc agcggtcctg cgacgttggc ctcggggtgc 7380 cgttcaacat tgcgtcctat tcccttctga cgctcatggt tgcgcacgtc tgcaacctga 7440 agccga Agga gttcattcac ttcatgggca acacgcacgt ctactcgaac cacgtcgagg 7500 ccctgaagga gcagctgcgc agagaaccga gacctttccc catcgtgaac atcctgaaca 7560 aggaacgcat ccaggtgcga agcaactggg aaggaaacgg cacaacggac acgcaaacaa 7620 gcagaagagg cgaaacggac GACG? CAGCC gaggccccgg ccactgcgag ccgagcgcag 7680 acacgctgct tccagccggc ctatattcgg aaagaaaggg agggagcaca acagtgtcga 7740 cgagacgcaa caacgaaaag gaaacgcgat gcgtcgcaga tcggctcacc tatgtggtgc 7800 gcggtgcggc gcccagacgg cgcggctgca gcgctcgaag gaagactgtc tttgggtgcg 7860 tgtgaacgtt tgtccctgac gcgcggctga cagaacatga gagggctctt ttctgtgttg 7920 cacgctctcc ggatgcatct ctttctgtgc cacgggaagc aaagacgtgt gtgttccccg 7980 aaagacccga ggaattcgga gcatccgctg ccggcgatgg ggggggaggg gccgggcatt 8040 ggatgcctcc cgcctcgtct tgtcgacggc cccaaaaccc gtcagtgcca cgttgtttgt 8100 gagtgtgttc gccaggaaat cgacgacttc accgccgagg tie ecgaggt cgtgggctac 8160 gacgaatcca gtgccgcatg gatggagatg gctget 8220 TAG-ggaaaaatce gaaatatata tatatatata tatatatata tatatatagg t tcctggttt tgcaccgtte tttcttctcc 82B0 ctccgaaggc attggtgaga gagcggtgga tgcgagggcg ctgaggccaa ttcagcggct 8340 gtttggtccc tcggggaagc aagaaacggg ttecgettcg cctcgttgct ttccgaaaca accttaccgc 8400 gtttcaaagt cttttctctt tgtcaatgag cgccactact ttgtgggagt 8460 cacgaatgtg cgcgtatccg gccttgtaeg gaggtgcggc tgccgcgctc gtcgccggaa 8520 cgctggactg tctgttgctt tcggggccte tggcgtgctc cggcgegggc gggggagtgg 8580 caggcgctac ccccccgccg gecctcggeg tgctcttgac tcetggcgag tgcgtgaaac 8640 ceaaaececg acttgtt tcg ctcgataagc tgcacaccga gteccccgca gcacegaaga B700 cattatgagg ccgcgcgctc tetcgegcct ggcacacacg cctaacacag ggtagctgcc 8760 cctatcgcca egataaactg ccaagggagc tgcccagcca agttgtcggt ggacaaaagt 8820 cctcacacgc cccgcagacc cggaagcaca cgtttcagag accaccggaa acgcatagtt 8880 cttcgtgccc tcgtcgttaa aeacgtttcc tgtctcgtct gctggttttt gcttcgtgcg 8940 tt cgctcgcg ctaagatgtc tagaaacggt ggacgccgtt tgcggctetc tctgccgccc 9000 ttccgctgtg actettccga cctgacctca cgcgcgtgtt ccacgtgaga caaccggtcg 9060 gcggccgagg caggagactt gcgagaaagg aaacaagtcg gacgcacgaa cgtacgtaac 9120 cccgcctcca aagttccccg ctccaaagag gaacgcggcg aggcgactcc eccggcgttg 9180 cctaccecgc ctccacgcct aaagaggact gcagtgggtc gacgctcccg tccgcattgt 9240 aaaaaagggg aaacaagacg agattcacgt ggaagaacca gaaacaacac cgaagcgacg 9300 ctgtcaaecc ccacgggcgc gcagccttce ctccgccgca cccgaccgca tgcaagagcc 9360 ctcgagtgct cccccgcagc tggcctttcc ctgtcgcctt ggaagcagag tgaacacaaa 9420 gagcagagac tagccagggc gaggaagcct cagaaaaact gacggcgagg aacggcagtc 9480 cgccggaaac aggggaaggc agacggcgaa tccgccgccg aagagaggac aaaaagagaa 9540 gaaaaaaggc aaccgcgtgc cgaaaactgg gtaacgggac gacaggcagg aggeataaag 9600 9603 < 210 > 2 < 2U > 1839 < 212 > DNA < 2i3 > Neospora canimim < 220 > < 221 > CDS < 222 > ( 1 ) . . (1B39) < 400 > 2 atg cag aaa cea gtg tet ctt aet gee gcg atg aec eec agg agg ggc 48 Met Gln Lys Pro Val Ser Leu He Ala Ala Thr Pro Arg Arg Gly 1 5 10 15 ate ggc gtc aac aac ggc ctg cea tgg ecc cae tzz gcc here gat ttc 96 He Gly Val Asn Asn Gly Leu Pro Trp Pro Has Le. Aia Thr Asp Phe 20 25 30 aaa falls ttt tet cgc gtg acg aaa acg acg gcc gac gaa gtc tet cgc 144 Lys His Phe Ser Arg Val Thr Lys Thr Thr Wing Asp Glu Val Ser Arg 35 «0 45 ctg aac gca tgg ctt ecg aaa aaa att gcc aag acg ggc gat teg gga 192 Leu? sn Wing Trp Leu Pro Lys Lys He Wing Lys Thr Gly Asp Ser Gly 50 55 6C ctt ecc tet ecc gcc ttc ggt gtc aac aga ttc aac gct gtt gtc atg 240 Leu Pro Ser Pro Wing Phe Gly val Asn Arg Phe Asn Wing Val Val Het 65 70 75 80 gga cga aaa acc tgg gag age ttg ceg cta aaa ttt cgt ecc etc gtg 288 Gly Arg Lys Thr Trp Glu Ser Leu Pro Leu Lys Phe Arg Pro Leu Val 85 90 95 gac cgg ctg aat ate gtg tec tech tec etc aaa gaa gaa gac ate 336 Asp Arg Leu Asn He Val Val Ser Ser Leu Lys Glu Glu Asp He 100 105 110 gcg gcg gag aag ect cta gtc gaa ggc cag caa cgc gtg cga gtc tgc 384 Wing Wing Glu Lys Pro Leu Val Glu Gly Gln Gln Aro Val Arg Val cys 115 120 125 gat tea etc ecc gca gcc ctg cgc ctt gtg gac gas gag tac aga gag 432 Aap Ser Leu Pío Ala Ala Leu Arg Leu Val Asp Glu Glu Tyr Arg Glu 130 135 1 40 tet gtt gae cag att tae gtt gtg gga gga gcg ggg etc tat gag gaa 480 Ser Val Asp Gln Ue Tyr Val Val Gly Gly Wing Gly Leu Tyr Glu Glu 145 150 155 160 gcc ctg ter ctg ggc gtg gtg tet drops etc tac ate acc cgc gtg gcg 528 Wing Leu Ser Leu Gly Val Val be Hls Leu Tyr He Thr Arg Val Wing 165 170 175 cgt gac ttt cea tgc gac gtt ttc ttt ecc gct tcc ecc gga gac tec 576 Arg Asp Phe Pro Cys Asp Val Phe Phe Pro Wing Phe Pro Gly Asp Ser 180 185 190 att ctt tea aac aag cag gc gc gc gc g ag g ect g gct gct 62 « Ue Leu Being Asn Lys Gln Wing Wing Being Wing Being Gln Pro Being Wing Wing 195 200 205 gcg gaa ceg gtg tet gtt ceg ttt tac ecc cag etc ggg aga gag aag 672 Wing Glu Pro to Phe Val Pro Phe Cys Pro Gln Leu Gly Arg Glu Lys 210 215 220 age aat gaa gcg teg tac cga ecc ate ttt att tea aag acc tac teg 720 Ser Asn Glu Wing Ser Tyr Arg Pro He Phe He Ser Lya Thr Tyr Ser 225 230 235 240 gac aac gga gtg ecc tac gac ttt gtg gtt ctt gaa aaa ggg agg aag 768 Asp Asn Gly Val Pro Tyr Asp Phe Val Val Leu Glu Lys Gly Arg Lys 245 250 235 gct gac gct tgc age gcc acg gaa teg tgc gag ctt cgc ggc ect tgg 816 Wing Asp Wing Cys Ser Wing Thr Glu Ser Cys Glu Leu Arg Gly Pro Trp 260 265 270 acc tec acc gga gag acg tec cea gag acg agg ctt ceg tet tec tec B64 Thr Ser Thr Gly Glu Thr Ser Pro Glu Thr Arg Leu Pro Ser Ser Ser 275 280 285 gcc gcc tea gtt gcc cag gt? ttg gct tgg atg gcc gac gaa gac cgg 912 Wing Wing Wing Val Wing Gln Val Leu Wing Trp Met Wing Asp Glu Asp? Rg 290 295 300 aaa aaa tgc gag aag aaa gae ate att cgg gca gtg ect cae gtc falls 960 Lys Lys Cys Glu Lys Lys Glu He He Arg Ala Val Pro Hls Val His 305 310 315 320 ttt cgg ggc falls gaa gaa ttc cag tac etc gac etc att gcc gat att 1008 Phe Arg Gly Hia Glu Glu Phe Gln Tyr Leu Asp Leu He Wing Asp He 325 330 335 ate aac aac gga gcg here atg gac gac cga acg ggc gtt gga gtc ate 1056 He Asn Asn Gly Ala Thr Met Asp Asp Arg Thr Gly Val Gly Val He 340 345 350 tec aag tec ggc tgt acc atg cgg ttc teg ctg gat aag gcc ttc ect 1104 Be Lys Phe Gly Cys Thr Met Arg Phe Ser Leu Asp Lys Wing Phe Pro 355 360 365 etc etc acc here aag cgt gtg ttc tgg aaa gga gtc etc gag gag ctg 1152 Leu Leu Thr Thr Lys Arg Val Pne Trp Lys Gly Val Leu Glu Glu Leu 370 375 380 teg tgg tec ate cgc ggt gac acg aac gcg aat cae cte tet gaa aag 1200 Leu Trp Phß He Arg Gly Asp Thr Asn Wing Asn His Leu Ser Glu Lys 385 390 395 400 ggc gta aag ate tgg gac aag aat gtg here aga gag etc ctt gat tea 1248 Gly Val Lys He Trp Asp Lys Asn Val Thr Arg Glu Phe Leu Asp Ser 405 410 415 cgc aat ctt tec drops cga gag gtc gga gac ate ggc ceg gge eac ggc 1296 Arg Asn Leu Ser Hxs Arg Glu Val Gly Asp He Gly Pro Gly Tyr Gly 420 425 430 etc cag tgg aga falls ttc ggc gcg ace tac aag gac atg falls acg gac 1344 Phe Gln Trp Arg His Phe Gly Wing Thr Tyr Lys Asp Met His Thr Asp 435 440 445 tac ace ggt cag ggc gta gac caa ctg aag aag gtg ate aac atg ctg 1392 Tyr Thr Gly Gln Gly Val Asp Gln Leu Lys Lys Val He Asn Het Leu 450 455 460 aga acg aat cea ac gac cgg cgc atg cte atg aec gct tgg aac ect 1440 Arg Thr Asn Pro Thr Asp Arg Axg Met Leu Met Thr Wing Trp Asn Pro 465 470 475 480 gcg gcg ctg gac gaa atg gcg ttg ceg cet tgc falls ttg ctg tgt cag 1488 Ala Ala Leu Asp Glu Met Ala Leu Pro Pro Cys His Leu Leu Cys Gln 485 490 495 ttc tac gtg gag aac gac aga gac ttg tet tgc gtc atg tat cag cgg 1536 Phe Tyr Val Glu Asn Asp Arg Asp Leu Ser Cys Val Met Tyr Gln Arg 500 505 510 tec tgc gac gtt ggc etc ggg gtg ceg ttc aac att gcg tec tat tec 1584 Ser Cys Asp Val Gly Leu Gly Val Pro Phe Asn lie Wing Ser Tyr Ser 515 520 525 ctt ctg acg etc atg gtt gcg drops gtc tgc aac ctg aag ceg aag gag 1632 Leu Leu Thr Leu Met Val Wing His Val Cys Asn Leu Lys Pro Lys Glu 530 535 540 ttc att falls ttc atg ggc aac acg drops gte tac teg aac drops gtc gag 1680 phe he his phe met gly asn thr his val tyr ser asn his val ßlu 545 550 555 560 gcc ctg aag gag cag ctg cgc aga gaa ceg aga ect ttc ecc ate gtg 1728 Wing Leu Lys Glu Gln Leu Arg Arg Glu Pro Arg Pro Phe Pro He Val 565 S70 575 aac ate ctg aac aag gaa cgc ate cag gaa ate gac gac tte acc gcc 1776 Asn Ue Leu Asn Lys Glu Arg He Gln Glu Ue Asp Asp Phe Thr Wing 580 585 590 gag gar etc gag gec geg ggc tac gtg ceg cat gga cga ate cag atg 1B24 Glu Asp Phe Glu Val Val Gly Tyr Val Pro His Gly Arg Ue Gln Met 595 600 605 gag atg get gtt tag 133g Glu Met Wing Val 610 < 210 > 3 < 211 > 612 < 212 > PRT < 213 > Neospora caninum < 400 > 3 Met Gln Lys Pro Val be Leu Ue Ala Wing Het Thr Pro? Rg Arg Gly 1 5 10 15 He Gly Val Asn Asn Gly Leu Pro Trp Pro His Leu Wing Thr Asp Phe 20 25 30 Lys His Phe Ser Arg Val Thr Lys Thr Thr Wing Asp Glu Val Ser Arg 35 40 45 Leu Asn Wing Trp Leu Pro Lys Lys Ue Wing Lys Thr Gly Asp Ser Gly 50 55 60 Leu Pro Ser Pro Wing Phe Gly Val Asn Arg Phe Asn Wing Val Val Met 65 70 75 80 Gly Arg Lys Thr Trp Glu Ser Leu Pro Leu Lys Phe Arg Pro Leu Val 85 90 95 Asp? Rg Leu Asn Ue Val Val Ser Ser Leu Lys Glu Glu Asp Ue 100 105 110 Wing Wing Glu Lys Pro Leu Val Glu Gly Gln Gln ? rg Val? rg Val Cys 115 120 125 Asp Ser Leu Pro Ala Ala Leu Arg Leu Val Asp Glu Glu Tyr Arg Glu 130 135 140 Ser Val Asp Gln Ue Tyr Val Val Gly Gly Ala Giy Leu Tyr Glu Glu 145 150 155 160 Ala Leu Ser Leu Gly Val Val Ser His Leu Tyr He Thr Arg Val Ala 165 170 175 Arg Asp Phe Pro ys Asp Val Phe Phe Pro Wing Phe Pro Gly Asp Ser 180 185 190 Tle Leu Ser Asn Lys G n Wing Wing Ser Wing Ser G n Pro Wing Wing 195 200 205 Wing Glu Pro Val Phe Val Pro Phe Cys Pro Gln Leu Gly Arg Glu Lys 210 215 220 Ser Asn Glu Wing Ser Tyr Arg Pro Ue Phe He Ser Lys Thr Tyr Ser 225 230 235 240 Aßp Aan Gly Val Pro Tyr Asp Phe Val Val Leu Glu Lys Gly Arg Lys 245 250 255 Ala Asp Ala Cys Ser Ala Thr Glu Ser Cys Glu Leu? Xg Gly Pro Trp 260 265 270 Thr Ser Thr Gly Glu Thr Ser Pro Glu Thr Arg Leu Pro Ser Ser Ser 275 280 285 Ala Ala Ala Ala Ala Gln Val Leu Ala Trp Met Ala? Sp Gl? Asp Arg 290 295 300 Lys Lys Cys Glu Lys Lys Glu He He Arg Ala Val Pro His Val His 305 310 315 320 Phe Arg Gly H s Glu Glu Phe Gln Tyr Leu Asp Leu Ue Wing Asp He 325 330 335 Ue Asn Asn Gly Ala Thr Met? Sp? Sp Arg Thr Gly Val Gly Val Ue 340 345 350 Be Lys Phe Gly Cys Thr Met Arg Phß Ser Leu Asp Lys Wing Phe Pro 355 360 365 Leu Leu Thr Thr Lys Arg Val Phe Trp Lys Gly Val Leu Glu Glu Leu 370 375 380 Leu Trp Phe Ue Arg Gly? Sp Thr Asn Ala Asn His Leu Ser Glu Lys < 400 > 4 atgcagaaac cggtgtgtct < 210 > 5 < 211 > 20 < 212 > DNA < 213 > Neospora caninum < 400 > 5 agggaagagg aaacgacgat 20 < 210 > 6 < 211 > 22 < 212 > DNA < 213 > Neospora caninum < 400 > 6 cccctcgtgg accggctgaa ca < 210 > 7 < 211 > 21 < 212 > DNA < 213 > Neospora caninum < 400 > 7 tccgtgcgtg ccaagagaec g 21 < 210 > 8 < 211 > 22 < 212 > DNA < 213 > Neospora caninuro < 400 > 8 atggagatgg egatgggagg ac 22 < 210 > 9 < 211? 22 < 212 > DNA < 213 > Neospora caninum < 400 > 9 agtatgtaca cgaagcctca at 22

Claims (33)

NOVELTY OF THE INVENTION CLAIMS

1. - An isolated polynucleotide molecule that contains a nucleotide sequence that codes for the DHFR-TS protein of Neospora.

2. The isolated polynucleotide molecule of claim 1, wherein the DHFR-TS protein contains the amino acid sequence of SEQ ID NO: 3 or the amino acid sequence of a DHFR-TS protein as encoded by the gene DHFR-TS present in phage lambdaNclDHFRTS (atcc registration no. 209512).

3. The isolated polynucleotide molecule of claim 2, which contains the nucleotide sequence of SEQ ID NO: 1 from about nt 2405 to about nt 8199, or a nucleotide sequence which is the same as the sequence of nucleotides of the DHFR-TS gene present in lambdaNclDHFRTS phage (ATCC registration no. 209512), or the nucleotide sequence of SEQ ID NO: 2.

4.- An isolated polynucleotide molecule that is substantially homologous to a polynucleotide molecule containing: (a) the nucleotide sequence of SEQ ID NO: l from about nt 2405 to about nt 8199; or (b) the nucleotide sequence of the DHFR-TS gene present in phage lambdaNclDHFRTS (ATCC registration no. 209512), or (c) the nucleotide sequence of SEQ ID NO: 2.

5. - A polynucleotide molecule isolated containing a nucleotide sequence encoding a polypeptide that is substantially homologous to a Neospora DHFR-TS protein having the amino acid sequence of SEQ ID NO: 3, or the amino acid sequence of a DHFR-TS protein as that encoded by the DHFR-TS gene present in phage lambdaNclDHFRTS (ATCC registration no. 209512).

6. A polynucleotide molecule consisting of a nucleotide sequence that is substantially a part of: (a) a polynucleotide molecule containing the nucleotide sequence of SEQ ID NO: 1 from about nt 2405 to about nt 8199; (b) a polynucleotide molecule containing the nucleotide sequence of the DHFR-TS gene present in the phage lambdaNclDHFRTS (ATCC registration number 209512); (c) a polynucleotide molecule containing the nucleotide sequence of the SEQ ID NO: 2; (d) a polynucleotide molecule that is substantially homologous to any of the polynucleotide molecules of (a), (b) or (c); or (e) a polynucleotide molecule containing a nucleotide sequence encoding a polypeptide that is substantially homologous to a neospore DHFR-TS protein having the amino acid sequence of SEQ ID NO: 3, or the amino acid sequence of a DHFR-TS protein as encoded by the DHFR-TS gene present in the phage lambdaNclDHFRTS (ATCC registration no. 209512).

7. The polynucleotide molecule of claim 6, which consists of a nucleotide sequence encoding a peptide fragment of (a) a DHFR-TS protein consisting of the amino acid sequence of SEQ ID NO: 3, or the amino acid sequence of a DHFR-TS protein as encoded by the DHFR-TS gene present in the phage lambdaNclDHFRTS (ATCC registration no. 209512); or (b) a polypeptide that is substantially homologous to a Neospora DHFR-TS protein consisting of the amino acid sequence of SEQ ID NO: 3, or the amino acid sequence of a DHFR-TS protein as encoded by the gene DHFR-TS present in phage lambdaNclDHFRTS (ATCC registration no. 209512).

8. The polynucleotide molecule of claim 7, which consists of a nucleotide sequence encoding the DHFR domain or the TS domain of the DHFR-TS protein.

9. An isolated polynucleotide molecule containing a nucleotide sequence that weakens the DHFR-TS gene in situ in N. caninum, as selected from the flanking nucleotide sequences shown in SEQ ID NO: l.

10. A recombinant vector comprising: (a) a polynucleotide molecule containing the nucleotide sequence of SEQ ID NO: 1 from about nt 2405 to about nt 8199; (b) a polynucleotide molecule containing a nucleotide sequence that is the same as the nucleotide sequence of the DHFR-TS gene present in phage lambdaNclDHFRTS (ATCC registration no. 209512); (c) a polynucleotide molecule containing the nucleotide sequence of SEQ ID NO: 2; (d) a polynucleotide molecule that is substantially homologous to any of the polynucleotide molecules of (a), (b) or (c); or (e) a polynucleotide molecule that contains a nucleotide sequence encoding a polypeptide that is substantially homologous to a Neospora DHFR-TS protein having the amino acid sequence of SEQ ID NO: 3, or the amino acid sequence of a DHFR-TS protein as encoded by the DHFR-TS gene present in the phage lambdaNclDHFRTS (ATCC registration no. 209512); or (f) a polynucleotide molecule consisting of a nucleotide sequence that is a substantial part of any of the polynucleotide molecules of (a), (b), (c), (d) or (e).

11. The recombinant vector of claim 10, wherein the polynucleotide molecule contains the nucleotide sequence of SEQ ID NO: 1 from about nt 2405 to about nt 8199.

12. The recombining vector of the claim 11, which is phage lambdaNclDHFRTS (ATCC registration no. 209512).

13. A transformed host cell, containing the recombinant vector of claim 10.

14. - A substantially purified protein containing the amino acid sequence of SEQ ID NO: 3 or the amino acid sequence of a DFR-TS protein as encoded by the DHFR-TS gene present in phage lambdaNclDHFRTS (ATCC registration no. 209512 ).

15. A substantially purified polypeptide that is substantially homologous to the protein of claim 14.

16. A peptide fragment of the protein of claim 14 or 15.

17. The peptide fragment of claim 16, which consists of a isolated from the DHFR domain or Neospora TS.

18. An antibody that specifically reacts against a DHFR-TS protein from Neospora that contains the amino acid sequence of SEQ ID NO: 3 or the amino acid sequence of a DHFR-TS protein as encoded by the DHFR-TS gene present in phage lambdaNclDHFRTS (ATCC registration No. 209512).

19. A genetic construct that can be used to inactivate a Neospora DHFR-TS gene, said genetic construct containing: (a) a polynucleotide molecule containing the nucleotide sequence of SEQ ID NO: 1 from about nt 2405 to approximately nt 8199, or a nucleotide sequence which is the same as the nucleotide sequence of the DHFR-TS gene present in phage lambdaclDHFRTS (ATCC registration no. 209512); (b) a polynucleotide molecule containing the nucleotide sequence of the nucleotide sequence of SEQ ID NO: 2; (c) a polynucleotide molecule that is substantially homologous to any of the polynucleotide molecules of (a) or (b); or (d) a polynucleotide molecule consisting of a nucleotide sequence that is a substantial part of any of the polynucleotide molecules of (a), (b) or (c); but further containing this nucleotide sequence of the polynucleotide molecule of (a), (b), (c) or (d) one or more mutations capable of deactivating the DHFR-TS gene of Neospora or a part of said gene; or a polynucleotide molecule consisting of one or more nucleotide sequences that flank in situ the open reading frame of a DHFR-TS gene of Neospora; so that said transformation of a cell of Neospora with the genetic construct produces the deactivation of the DHFR-TS gene of Neospora or a part of it.

20. The genetic construct of claim 19 further comprising a selectable marker.

21. The genetic construct of claim 19, which is useful for separating the DHFR domain or the TS domain of the DHFR-TS gene.

22. A Neospora cell transformed with the genetic construct of claim 19.

23. The Neospora cell transformed with the genetic construct of claim 22, which has a phenotype selected from the group consisting of dhfr, ts and dhfr-ts.

24. - A method for preparing the modified live Neospora cells, comprising transforming the Neospora cells with the genetic construct of claim 19 and selecting the transformed cells having a mutant phenotype selected from the group consisting of dhfr, ts and dhfr-ts as a result of this transformation.

25. A vaccine for the protection of a mammal against neosporosis, which contains an immunologically effective amount of modified Neospora living cells that have a mutant phenotype selected from the group consisting of dhfr, ts and dhfr-ts and a veterinarily vehicle acceptable.

26. The vaccine of claim 25, wherein the modified Neospora living cells have a mutant phenotype selected from the group consisting of dhfr, ts and dhfr-ts as a consequence of the transformation with the genetic construct of claim 19

27. The vaccine of claim 25, which also contains an adjuvant or a cytokine.

28. The vaccine of claim 27, wherein the adjuvant is selected from the group consisting of the RIBI adjuvant system, alum, mineral gels, an oil-in-water emulsion, a water-in-oil emulsion, block copolymer, QS-21, SAF-M, adjuvant AMPHIEGENR, saponin, Quil A, monophosphoryl lipid A and lipid-amino-adjuvant Avridine, SEAM62 and SEAM 1/2.

29. A process for the preparation of a vaccine against neosporosis, comprising the combination of an immunologically effective amount of modified Neospora living cells having a mutant phenotype selected from the group consisting of dhfr, ts and dhfr-ts and a veterinarily acceptable vehicle.

30. The use of an immunologically effective amount of Neospora modified living cells that have a mutant phenotype selected from the group consisting of dhfr-ts and dhfr-ts, in the manufacture of a vaccine against neosporosis in a mammal. 31.- A combination vaccine for the protection of a mammal against neosporosis and, optionally, one or more diseases or pathological disorders that may affect the mammal, the combination vaccine containing an immunologically effective amount of a first compound containing cells Modified living Neospora that have a mutant phenotype selected from the group consisting of dhfr-ts and dhfr-ts; an immunologically effective amount of a second component capable of inducing a protective response against the other disease or pathological disorder that may affect the mammal; and a veterinarily acceptable vehicle. 32. - The combination vaccine of claim 31, wherein the second component is capable of inducing a protective response against a pathogen selected from the group consisting of bovine herpesvirus, bovine respiratory syncytial virus, diarrhea virus bovine viral, parainfluenza virus type I, II or III, Leptospira spp., Campylobacter spp., Staphylococcus aureus, Streptococcus aqalactiae, Mycoplasma spp., Klebsiella spp., Salmonella spp., rotavirus, coronavirus, rabvirus, Pasteurella hemolytica , Pasteurella multocida, Clostridia spp., Tetanus toxoid, E. Coli, Cryptosporidium spp., Eimeria spp., And Trichomonas spp. 33.- A kit for the vaccination of a mammal against neosporosis containing a first container having an immunologically effective amount of live modified Neospora cells that have a mutant phenotype selected from the group consisting of dhfr-ts and dhfr-ts; and a second container having a veterinarily acceptable vehicle or diluent.