RU99104772A - GNRH CHIMERS (GIVG) -LEYKOTOKSIN - Google Patents

Claims (24)

1. A chimeric protein containing a leukotoxin polypeptide fused to the first and second multimers, wherein the C-terminus of the first multimer is fused to the N-terminus of the leucotoxin polypeptide, and the N-terminus of the second multimer is fused to the C-terminus of the leucotoxin polypeptide and, in addition , in that each of these multimers contains more than one selected GnRH polypeptide. 2. Chimeric protein according to claim 1, characterized in that the first and second multimers of GnRH are different and are part of the molecules corresponding to the general formula
[GnRH-X-GnRH] n,
where GnRH is a GnRH polypeptide;
X is selected from the group comprising a peptide bond, an amino acid spacer group and a leukotoxin polypeptide;
n means an integer greater than or equal to 1. 3. The chimeric protein according to claim 1, characterized in that the first and second GnRH multimers are the same and are part of the molecules corresponding to the general formula
[GnRH-X-GnRH] n,
where GnRH is a GnRH polypeptide;
X is selected from the group comprising a peptide bond, an amino acid spacer group and a leukotoxin polypeptide;
n means an integer greater than or equal to 1.  4. The chimeric protein according to claim 2 or 3, wherein X is an amino acid spacer group having at least one antigenic determinant of a T-helper.  5. Chimeric protein according to claim 2 or 3, characterized in that n = 4.  6. The chimeric protein according to claim 1, wherein the leukotoxin polypeptide has no cytotoxic activity.  7. The chimeric protein according to claim 6, wherein the leukotoxin polypeptide is LKT 352.  8. Chimeric protein according to any one of claims 1 to 7, characterized in that the first multimer further comprises an amino acid sequence fused at its N-terminus.  9. The chimeric protein according to claim 1, containing the amino acid sequence depicted in Figures 9-1 through 9-6, or an amino acid sequence that is almost homologous and functionally equivalent to it.  10. A vaccine formulation containing a chimeric block according to any one of claims 1 to 9 and a pharmaceutically acceptable carrier.  11. The method of providing selected GnRH multimers to an individual, comprising prescribing an effective amount of a vaccine formulation according to claim 10 to said individual.  12. A DNA construct encoding a chimeric protein, characterized in that said protein is a leukotoxin polypeptide fused to the first and second multimers, so that the C-terminus of the first multimer is fused to the N-terminus of the leucotoxin polypeptide and the N-terminus of the second multimer is fused The C-terminus of a leukotoxin polypeptide; and, in that each of said multimers contains more than one GnRH polypeptide, wherein said DNA construct comprises: a first nucleotide sequence encoding a GnRH multimer; and the second nucleotide sequence encoding the second multimer GnRH; moreover, these first and second nucleotide sequences are functionally linked using a third nucleotide sequence that encodes a leukotoxin polypeptide. 13. DNA construction according to item 12, characterized in that the first and second GnRH multimers are different and are part of the molecules corresponding to the general formula
[GnRH-X-GnRH] n,
in which GnRH is a GnRH polypeptide;
X is selected from the group comprising a peptide bond, an amino acid spacer group and a leukotoxin polypeptide;
n means an integer greater than or equal to 1. 14. The DNA construct according to claim 12, characterized in that the first and second GnRH multimers are the same and are part of the molecules corresponding to the general formula
[GnRH-X-GnRH] n,
in which GnRH is a GnRH polypeptide;
X is selected from the group comprising a peptide bond, an amino acid spacer group and a leukotoxin polypeptide;
n means an integer greater than or equal to 1.  15. The DNA construct of claim 13 or 14, wherein X is an amino acid spacer group having at least one antigenic determinant of a T-helper.  16. The DNA construct according to item 13 or 14, characterized in that n = 4.  17. The DNA construct according to claim 12, wherein the leukotoxin polypeptide has no cytotoxic activity.  18. The DNA construct of claim 17, wherein the leukotoxin polypeptide is LKT 352.  19. A DNA construct according to any one of claims 12-18, wherein the first multimer further comprises an amino acid sequence (Met-Ala-Thr-Val-Ile-Asp-Arg-Ser) fused at its N-terminus.  20. The DNA construct of claim 12, wherein the chimeric protein contains the amino acid sequence shown in FIG. 9-1 to 9-6, or the amino acid sequence that is practically homologous to it and functionally equivalent to it.  21. An expression cassette consisting of: (a) a DNA construct according to any one of claims 12 to 20; and (b) control sequences that direct the transcription of the indicated construct, on the basis of which the said construct can be transcribed and translated in the host cell.  22. A host cell transformed by the expression cassette according to claim 21.  23. A method of producing a recombinant polypeptide, which consists in: using the host cell population according to claim 22, which is cultured under conditions in which it expresses a polypeptide encoded by said expression cassette.  24. The way to reduce the number of cases of breast tumors in mammals individuals, which consists in the introduction of a therapeutically effective amount of the vaccine formulation according to claim 10 to the indicated individual.