Use of NcSAG4 for diagnosis and prevention of Neospora disease and use as a marker for analysis of pathogenesis

摘要

A polynucleotide molecule (P1) which comprises 601 nucleotides isolated from Neospora caninum and characterised by SEQ ID NO. 9 corresponding to the gene NcSAG4, comprising an Open Reading Frame (ORF) of 522 nucleotides encoding the antigenic protein NcSAG4 made up of 173 amino acids and characterised by SEQ ID NO. 10, is new. Independent claims are also included for the following: (1) polynucleotide molecule (P2) comprising the ORF sequence for the NcSAG4 gene as described for (P1), included in an expression vector, preferably the plasmid pcDNA3.1-His-C (Invitrogen), by insertion via polymerase chain reaction (PCR) amplification using the oligonucleotides FNcSAG4 and ReNcSAG4 characterised by SEQ ID NOS. 11 and 12; (2) polynucleotide molecule (P3) comprising the sequence between nucleotides 83 and 444 in the ORF for the NcSAG4 gene as described for (P2), included in an expression vector, preferably the plasmid pRSET-C, by insertion via PCR amplification using the oligonucleotides F85NcSAG4 and Re444NcSAG4 characterised by SEQ ID NOS. 13 and 14; (3) polynucleotide molecule (P4) comprising a sequence including any fragment of the gene NcSAG4 described for (P1) or sequences obtained by any chemical, physical, enzymatic or other natural modification resulting in a change in this sequence and included in any recombinant vector; (4) use of the oligonucleotides SAG4-2, SAG4-3, SAG4-4, 1R5SAG4, 2R5SAG4, 1F3SAG4, 2F3SAG4, FNcSAG4, ReNcSAG4, F85NcSAG4 and Re444NcSAG4 characterised by SEQ ID NOS. 2-14 respectively for detecting N. caninum by PCR or a reverse transcriptase-polymerase chain reaction (RT-PCR), as DNA probes or for amplifying any fragment of SEQ ID NOS. 9 or 10 by PCR; (5) recombinant vector (V) comprising the SEQ ID NO. 9; (6) eucaryotic host cells (C1) transfected with (V); (7) prokaryotic host cells (C2) transfected with (V); (8) substantially pure or isolated polypeptide (PP) selected from (a) the protein NcSAG4 isolated from N. caninum and characterised by SEQ ID NO. 9, (b) chemically or enzymatically modified derivatives of this protein, (c) a polypeptide comprising a major portion of this protein or a chemically or enzymatically modified derivative thereof or (d) a recombinant protein comprising one of the proteins (a)-(c); (9) use of the promoter for the gene NcSAG4 for expressing heterologous genes in N. caninum cells transfected with genetic constructions obtained using this promoter; (10) use of monoclonal antibodies or specific polyclonal serum against (PP) for the serological diagnosis of chronic infections caused by N. caninum, by means of an ELISA technique; (11) use of monoclonal antibodies or specific polyclonal serum against (PP) for the diagnosis of chronic infections caused by N. caninum in animal tissue, by means of an immunohistochemical or immunofluorescence technique or any other method based on the detection of the parasite by this serum; (12) immunogenic composition (IC) comprising (PP), any one of (P1)-(P4), (V), (C1) or (C2), used as a vaccine against neosporosis; (13) preparation of (IC) by formulating one or more of (PP), (P1)-(P4), (V), (C1) or (C2); and (14) kit for vaccinating mammals against neosporosis, which includes (IC). ACTIVITY : Antiparasitic. MECHANISM OF ACTION : Vaccine.