US2003017494A1PendingUtilityA1
Genes encoding mycobacterial proteins associated with cell binding and cell entry and uses thereof
Priority: Jul 10, 1996Filed: Jun 24, 2002Published: Jan 23, 2003
Est. expiryJul 10, 2016(expired)· nominal 20-yr term from priority
A61K 39/00A61K 48/00C07K 14/35
57
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Claims
Abstract
A gene from a strain of Mycobacterium encoding a protein of molecular weight between about 45 to about 60 kDa and associated with cell binding and cell entry was cloned. The genes and encoded protein have utility in immunogenic preparations or diagnostic applications.
Claims
exact text as granted — not AI-modifiedWhat we claim is:
1 . An isolated nucleic acid molecule encoding a mycobacterial protein associated with cell binding and cell entry of a Mycobacterium strain and having a molecular weight of about 45 to about 60 kDa.
2 . The nucleic acid molecule of claim 1 wherein said Mycobacterium species is a species of the tuberculosis complex.
3 . The nucleic acid molecule of claim 2 wherein said Mycobacterium strain is a strain of Mycobacterium tuberculosis.
4 . The nucleic acid molecule of claim 2 wherein said Mycobacterium strain is a strain of Mycobacterium bovis.
5 . The nucleic acid molecule of claim 1 wherein said mycobacterial protein has substantially the following amino acid composition:
Number of
Amino Acids
Residues
Leucine
53
Arginine
32
Threonine
45
Alanine
60
Valine
40
Serine
43
Proline
38
Aspartic Acid
29
Isoleucine
24
Asparagine
23
Tyrosine
16
Glycine
45
Phenylalanine
14
Glutamic Acid
15
Lysine
15
Glutamine
12
Tryptophan
7
Methionine
7
Cysteine
6
Histidine
3
6 . The nucleic acid molecule of claim 1 having a restriction map substantially as shown in FIG. 5 for M. bovis strain BCG or the corresponding restriction map from other mycobacteria from the tuberculosis complex.
7 . The nucleic acid molecule of claim 1 having a DNA sequence as shown in FIG. 6 (SEQ ID No: 2) for M. bovis strain BCG or the sequence of the corresponding gene of other mycobacteria from the tuberculosis complex.
8 . The nucleic acid molecule of claim 1 encoding an amino acid sequence as shown in FIG. 7 (SEQ ID No: 3) for M. bovis strain BCG or the corresponding sequence in other mycobacteria from the tuberculosis complex.
9 . An isolated DNA molecule amplifiable by polymerase chain reaction (PCR) by a pair of primers having the sequences of primers 4879 (SEQ ID No: 12) and 4882 (SEQ ID No: 15); or 4879 (SEQ ID No: 12) and 4865 (SEQ ID No: 11); or 4879 (SEQ ID No: 12) and 4812 (SEQ ID No: 10).
10 . The DNA molecule of claim 9 from a Mycobacterium strain of the tuberculosis complex.
11 . The DNA molecule of claim 9 from a Mycobacterium strain of Mycobacterium tuberculosis.
12 . The DNA molecule of claim 9 from a Mycobacterium strain of Mycobacterium bovis.
13 . A vector for transformation of a host comprising the nucleic acid molecule of claim 1 or the DNA molecule of claim 9 .
14 . The vector of claim 13 further comprising DNA sequences for expression of said protein in said host.
15 . A transformed host containing an expression vector as claimed in claim 14 .
16 . A recombinant mycobacterial protein associated with cell binding and cell entry producible by the transformed host of claim 15 .
17 . A plasmid containing the gene encoding a mycobacterial protein associated with cell binding and cell entry of a strain of Mycobacterium having the identifying characteristics of plasmid pBCGcepX having ATCC Deposit No. 97511.
18 . A method of determining the presence, in a sample, of nucleic acid encoding a mycobacterial protein associated with cell binding and cell entry and having a molecular weight between about 45 and about 60 kDa of a strain of Mycobacterium, which comprises the steps of:
(a) contacting the sample with the nucleic acid molecule of claim 1 or the DNA molecule of claim 9 to produce duplexes comprising the nucleic acid molecule or DNA molecule and any nucleic acid molecule encoding the protein present in the sample and specifically hybridizable therewith; and (b) determining production of the duplexes.
19 . A diagnostic kit for determining the presence, in a sample, of nucleic acid encoding a mycobacterial protein associated with cell binding and cell entry and having a molecular weight of between about 45 and about 60 kDa of a strain of Mycobacterium, comprising:
(a) the nucleic acid molecule of claim 1 or the DNA molecule of claim 9 ; (b) contacting means to contact the nucleic acid molecule or DNA molecule with the sample to produce duplexes comprising the nucleic acid molecule or DNA molecule and any nucleic acid molecule encoding the protein present in the sample and hybridizable with the nucleic acid or DNA molecule; and (c) identifying means to determine production of the duplexes.
20 . A method for detection of a strain of Mycobacterium in a sample of tissue or body fluids, which comprises:
providing a pair of primers which are primers 4879 (SEQ ID No: 12) and 4882 (SEQ ID No: 15); or 4879 (SEQ ID No. 12) and 4865 (SEQ ID No: 11); or 4879 (SEQ ID No: 12) and 4812 (SEQ ID No: 10); contacting the sample with the probes; performing a polymerase chain reaction to amplify an amplifiable nucleic acid sequence encoding a mycobacterial protein associated with cell binding or cell entry; and detecting the amplified DNA sequence which indicates the presence of a strain of Mycobacterium in the sample.
21 . An oligonucleotide primer selected from the group consisting of primers 4879 (SEQ ID No: 12), 4882 (SEQ ID No: 15), 4865 (SEQ ID No: 11) and 4812 (SEQ ID No: 10).
22 . A method of producing a substantially pure recombinant mycobacterial protein associated with cell binding and cell entry and having a molecular weight between about 45 kDa and 60 kDa, which comprises:
transforming a host with a vector as claimed in claim 14 ; growing the transformed host to express the protein, and isolating and purifying the protein free from other proteinaceous and cellular material.
23 . An isolated and purified mycobacterial protein associated with cell binding and cell entry of a Mycobacterium strain and having a molecular weight of between about 45 and about 60 kDa.
24 . The protein of claim 23 which is that of a Mycobacterium strain of the tuberculosis complex.
25 . The protein of claim 23 which is that of a Mycobacterium strain of Mycobacterium tuberculosis.
26 . The protein of claim 23 which is that of a strain of Mycobacterium bovis.
27 . The protein of claim 23 produced by the procedure of claim 22 .
28 . The protein of claim 23 having substantively the following amino acid composition:
Number of
Amino Acids
Residues
Leucine
53
Arginine
32
Threonine
45
Alanine
60
Valine
40
Serine
43
Proline
38
Aspartic Acid
29
Isoleucine
24
Asparagine
23
Tyrosine
16
Glycine
45
Phenylalanine
14
Glutamic Acid
15
Lysine
15
Glutamine
12
Tryptophan
7
Methionine
7
Cysteine
6
Histidine
3
29 . An immunogenic composition, comprising at least one component selected from the group consisting of at least one isolated nucleic acid molecule as claimed in claim 1 , at least one isolated DNA molecule as claimed in claim 9 , at least one recombinant mycobacterial protein as claimed in claim 16 and at least one protein as claimed in claim 23 as an active component thereof, and a pharmaceutically acceptable carrier therefor.
30 . A method of generating an immune response in a host, which comprises administering to the host an immunoeffective amount of the immunogenic composition of claim 29 .Join the waitlist — get patent alerts
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