Claims
- 1. A method for identification and the selection of essential genes for the survival or the virulence of mycobacterium species which comprises:
a. Aligning the genomic sequence of a first mycobacterium species on a genomic sequence of the genomic sequence of a second mycobacterium species, b. Selecting a polypolynucleotide sequence highly conserved in both genomes with no counterparts in other bacterial genomic sequences and which corresponds to an essential gene for the survival or the virulence of mycobacterium species, and c. Optionally, testing the polypolynucleotide selected in step b) for its capacity of virulence or involved in the survival of a mycobacterium species said testing being based on the activation or inactivation of said polypolynucleotide in a bacterial host or said testing being based on the activity of the product of expression of said polynucleotide in vivo or in vitro.
- 2. A method according to claim 1, wherein the first genomic sequence of mycobacterium belongs to Mycobacterium tuberculosis.
- 3. A method according to claim 1, wherein the second genomic sequence of mycobacterium belongs to Mycobacterium leprae.
- 4. A method according to any one of claims 1 to 3, wherein the complete genomic sequence of said mycobacterium species is analysed.
- 5. A method for the identification and the selection in silico of essential genes for the survival or the virulence of mycobacterium species according to any one of claims 1 to 4.
- 6. Purified polynucleotide molecule obtained by the method according to any one of claims 1 to 5.
- 7. A purified polynucleotide molecule of claim 6 which encodes essential proteins or fragments of proteins of Mycobacteium species.
- 8. A purified polynucleotide molecule of a formula selected from the group consisting of polynucleotidic sequences, which encode for polypeptides and regulatory sequences essential for the virulence and/or the survival of mycobacterium which are, in one hand, specific to Mycobacterium tuberculosis and, in the other hand, specific to Mycobacterium leprae, that is to say, said polynucleotidic sequences are not found in publicly accessible banks of non-Mycobacterium tuberculosis and non-Mycobacterium leprae genome.
- 9. A purified polynucleotide molecule according to claim 8 obtained by the method according to any one of claims 1 to 5.
- 10. A purified polynucleotide molecule that hybridizes to either strand of a denratured, double-stranded DNA comprising the purified polynucleotide sequence according to claims 6 to 9 under conditions of moderate stringency in 50% formamide and 6× SSC at 42° C. with washing conditions of 60° C., 0.5×SSC, 0.1% of SDS.
- 11. The purified polynucleotide molecule as claimed in claim 10, wherein said purified polynucleotide molecule is derived by mutagenesis.
- 12. A purified polynucleotide molecule degenerate from the purified polynucleotide molecule according to any one of claims 6 to 9 as a result of the genetic code.
- 13. A purified polynucleotide according to any one of claims 6 to 9 which encodes M. tuberculosis or M. leprae marker polypeptide.
- 14. A purified polynucleotide molecule according to any one of claims 6 to 9 which encodes an allelic variant of M. tuberculosis or M. leprae marker polypeptide according to claim 13.
- 15. A purified polynucleotide molecule according to any one of claims 6 to 9 which encodes a similar sequence of M. tuberculosis or M. leprae marker polypeptide DNA according to claim 13.
- 16. A purified polypeptide encoded by a polynucleotide molecule according to claim 8.
- 17. A purified polypeptide of a formula selected from the group consisting of SEQ ID NO: X to SEQ ID NO: Y.
- 18. A purified polypeptide according to claim 17 encoded by a polynucleotide molecule according to claims 6 or 9.
- 19. A purified polypeptide according to claim 16 in non-glycosylated form.
- 20. A purified polypeptide according to claim 16 in glycosylated form.
- 21. A purified polypeptide according to claim 17 in non-glycosylated form.
- 22. A purified polypeptide according to claim 17 in glycosylated form.
- 23. A purified polypeptide according to claim 18 in non-glycosylated form.
- 24. A purified polypeptide according to claim 18 in non-glycosylated form.
- 25. Process of screening of active molecules comprising:
a. Preparation of at least one purified polynucleotidic molecule or fragment thereof according to claims 6 to 9 in an acceptable medium, b. Contacting the purified polynucleotide sequence or a fragment thereof corresponding to said essential gene of interest with an active molecule to be tested, and c. Selecting the active molecule by inhibition or activation of the activity of the purified polynucleotide compared to the standard activity of the essential gene in absence of said active molecule.
- 26. Process of screening of an active molecule comprising:
a. Preparation of at least one purified polypeptide according to claims 16 to 24 or fragment thereof to be used as a target in an acceptable medium, b. Contacting said purified polypeptide or fragment thereof obtained in step a) with an active molecule to be tested, and c. Selecting the active molecule by inhibition or activation of the activity of the purified polypeptide obtainable after expression of the essential gene selected according to claim 1 and compared to the standard activity of said polypeptide.
- 27. A recombinant BAC containing a fragment of M. tuberculosis genome deposited on Feb. 20, 2001 at the C.N.C.M. under the accession number I-2625.
- 28. A recombinant BAC containing a fragment of M. tuberculosis genome deposited on Feb. 20, 2001 at the C.N.C.M. under the accession number I-2626.
- 29. A recombinant BAC containing a fragment of M. tuberculosis genome deposited on Feb. 20, 2001 at the C.N.C.M. under the accession number I-2627.
- 30. A recombinant BAC containing a fragment of M. tuberculosis genome deposited on Feb. 20, 2001 at the C.N.C.M. under the accession number I-2628.
- 31. A recombinant BAC containing a fragment of M. tuberculosis genome deposited on Feb. 20, 2001 at the C.N.C.M. under the accession number I-2629.
- 32. A recombinant cosmid containing a fragment of M. leprae genome deposited on Feb. 21, 2001 at the C.N.C.M. under the accession number I-2632.
- 33. A recombinant cosmid containing a fragment of M. leprae genome deposited on Feb. 21, 2001 at the C.N.C.M. under the accession number I-2633.
- 34. A recombinant purified vector that directs the expression of a polynucleotide molecule selected from the group consisting of purified polynucleotide molecule selected according to claims 1 or 6, 7, 8, 9, 13, 14 and 15.
- 35. A recombinant purified vector that directs the expression of a purified polynucleotide molecule according to claims 10 or 11.
- 36. A recombinant purified vector that directs the expression of a purified polynucleotide molecule according to claim 12.
- 37. A recombinant purified vector containing a part of the polynucleotide insert of claims 27 to 32 or claims 33 to 35 which is a plasmid.
- 38. A host cell transfected or transduced with the vector of claim 34.
- 39. A host cell transfected or transduced with the vector of claim 35.
- 40. A host cell transfected or transduced with the vector of claim 36.
- 41. A host cell transfected or transduced with the plasmid of claim 37.
- 42. A method for the production of mycobacterium purified marker polypeptide comprising culturing a host cell of claim 38 under conditions promoting expression, and recovering the polypeptide from the culture medium.
- 43. A method for the production of mycobacterium purified marker polypeptide comprising culturing a host cell of claim 39 under conditions promoting expression, and recovering the polypeptide from the culture medium.
- 44. A method for the production of mycobacterium purified marker polypeptide comprising culturing a host cell of claim 40 under conditions promoting expression, and recovering the polypeptide from the culture medium.
- 45. A method for the production of mycobacterium purified marker polypeptide comprising culturing a host cell of claim 41 under conditions promoting expression, and recovering the polypeptide from the culture medium.
- 46. The method according to any one of claims 42 to 45, wherein the host cell is selected from the group consisting of bacterial cells, yeast cells, plant cells, and mammalian cells.
- 47. An immunological complex comprising a Mycobacterium purified marker polypeptide produced by a method according to any one of claims 42 to 45 and an antibody that specifically recognizes said polypeptide.
- 48. A composition comprising at least a mycobacterium purified polypeptide marker produced by a method according to any one of claims 42 to 45.
- 49. A method for detecting infection by mycobacteria, said method comprises:
a. Providing a composition according to claim 48 with a biological sample suspected to be infected with a mycobacterium, b. Assaying for the presence of said mycobacterium, and c. Optionally, detecting the presence of mycobacteria in said biological sample if infected.
- 50. The method of claim 49, which in step b) the assay is performed by electrophoresis or by immunoassay with antibodies that are immunologically reactive with M. tuberculosis and/or M. leprae.
- 51. An in vitro diagnostic method for the detection of the presence or the absence of antibodies which bind to an antigen or fragment of antigen comprising a mycobacterium purified polypeptide molecule according to any one of claims 16 to 24, wherein the method comprises contacting the antigen or fragment of antigen with a biological fluid for a time and under conditions sufficient for the antigen and antibodies in the biological fluid to form an immunological complex, detecting the formation of the complex, and optionally measuring the formation of the immunologicalcomplex.
- 52. The method as claimed in claim 51, wherein the formation of the immunological complex is detected by immunoassay method based on western blot technique, ELISA, indirect immuno-fluorescense assay, or immunoprecipitation assay.
- 53. An in vitro diagnostic method for the detection of the presence or the absence of antibodies which bind to an antigen or fragment of antigen comprising a mycobacterium purified polypeptide molecule obtained by a method according to any one of claims 42 to 45, wherein the method comprises contacting the antigen or fragment of antigen with a biological fluid for a time and under conditions sufficient for the antigen and antibodies in the biological fluid to form an immunological complex, detecting the formation of the complex, and optionally measuring the formation of the immunological complex.
- 54. The method as claimed in claim 53, wherein the formation of the immunological complex is detected by immunoassay method based on western blot technique, ELISA, indirect immuno-fluorescence assay, or immunoprecipitation assay.
- 55. A kit for the in vitro diagnostics of mycobacterium infections comprising:
a. A mycobacterium purified polypeptide molecule according to any one of claims 16 to 24 or mixture thereof, b. Antibodies capable of forming an immunological complex with said polypeptides, and c. Acceptable medium to permit the detection of the formation of the complex thereof.
- 56. A kit as claimed in claim 55 useful for the detection of M. tuberculosis infections.
- 57. A kit as claimed in claim 55 useful for the detection of M. leprae infections.
- 58. An immunogenic composition comprising at least a purified polypeptide according to any one of claims 16 to 24 in an amount sufficient to induce an immunogenic or protective response in vivo, and a pharmaceutically acceptable carrier therefor.
- 59. A polynucleotidic probe comprising a purified polynucleotide molecule or fragment thereof according to any one of claims 6 to 15.
- 60. A polynucleotidic probe which is complementary to the full length sequence of a purified nucleic acid that hybridizes under conditions of moderate stringency in 50% formamide and 6× SSC at 42° C. with washing conditions of 60° C., 0.5×SSC, 0.1% SDS with a nucleic acid encoding a purified polypeptide according to any one of claims 6 to 24.
- 61. A method for the detection of the presence or the absence of mycobacteria in a sample comprising:
a. contacting a sample suspected to contain genetic material of mycobacteria with at least one probe according to claims 59 or 60, b. Detecting the hybridization under conditions of moderate stringency in 50% formamide and 6× SSC at 42° C. with washing conditions of 60° C., 0.5×SSC, 0.1% SDS.
- 62. A method for the detection of the presence or the absence of mycobacteria according to claim 61, wherein said method is specific for the detection of M. tuberculosis infections.
- 63. A method for the detection of the presence or the absence of mycobacteria according to claim 61, wherein said method is specific for the detection of M. leprae infections.
- 64. A method for the detection of the presence or the absence of mycobacteria according to any one of claims 61 to 63, wherein the sample contains nucleic acids of at least one microorganism other than the mycobacteria.
- 65. A method of selection according to claim 1, wherein the comparison of the genetic informations of different types of organisms, wherein the method comprises:
a. Providing a database including sequence libraries for a plurality of types of organism, said libraries having multiple genomic sequences, b. Providing one or more probe sequences according to claims 59 or 60, c. Determining homologous matches between one or more of said probe sequences and one or more sequences of said sequences in said genomic libraries; and d. Displaying the results of said determination.
- 66. A method according to claims 1 to 4, wherein the genomic sequence of a first mycobacterium species is the recombinant BAC deposited at the C.N.C.M. according to claim 27.
- 67. A method according to claims 1 to 4, wherein the genomic sequence of a first mycobacterium species is the recombinant BAC deposited at the C.N.C.M. according to claim 28.
- 68. A method according to claims 1 to 4, wherein the genomic sequence of a first mycobaterium species is the recombinant BAC deposited at the C.N.C.M. according to claim 29.
- 69. A method according to claims 1 to 4, wherein the genomic sequence of a first mycobacterium species is the recombinant BAC deposited at the C.N.C.M. according to claim 30.
- 70. A method according to claims 1 to 4, wherein the genomic sequence of a first mycobacterium species is the recombinant BAC deposited at the C.N.C.M. according to claim 31.
- 71. A method according to claims 1 to 4, wherein the genomic sequence of the second mycobacterium species is the recombinant BAC deposited at the C.N.C.M. according to claim 32.
- 72. A method according to claims 1 to 4, wherein the genomic sequence of the second mycobacterium species is the recombinant BAC deposited at the C.N.C.M. according to claim 33.
- 73. An in vitro diagnostic method for the detection of the presence or the absence of essential nucleotidic sequences for the survival or the virulence in mycobacterium by hybridization or amplification of said specific sequence comprising:
a. Providing a composition comprising a probe according to claims 59 or 60 with a sequence library of interest to be tested in an acceptable medium and in sufficient time to obtain an hybridization and/or an amplification of said sequence, b. Purifying the sequence which hybridizes with said probe; and c. Optionally, quantifying said sequence.
- 74. A method according to claim 1, wherein the first genomic sequence of mycobacterium belongs to Mycobacterium microti.
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is based on and claims the benefit of U.S. Provisional application Ser. No. 60/270,123, filed Feb. 22, 2001 (attorney docket no. 03495.6061) The entire disclosure of this application is relied upon and incorporated by reference herein.
Provisional Applications (1)
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Number |
Date |
Country |
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60270123 |
Feb 2001 |
US |