Full-text?

The full document isn't yet available to us from the patent office.

Abstract

The following are claimed: (1) a recombinant screening, cloning and/or expression vector that replicates in mycobacteria, comprising: a replicon functional in mycobacteria; a selectable marker; and a reporter cassette comprising (a) a multiple cloning site (polylinker), (b) optionally a mycobacterial transcription terminator upstream of the polylinker, (c) a coding sequence from a gene coding for a protein expression, export and/or secretion marker, the coding sequence lacking its start codon and regulatory sequences, and (d) a coding sequence from a gene coding for a cis-acting promoter activity marker, the coding sequence having its start codon; (2) a recombinant vector as above containing a mycobacterial nucleic acid sequence in one of the cloning sites of the polylinker, where the mycobacterial nucleic acid sequence is one "in which one detects a polypeptide capable of being exported and/or secreted, and/or of being induced or repressed during infection by said mycobacterium or constitutively expressed or produced, as well as associated promoter and/or regulatory sequences capable of permitting or enhancing the export and/or secretion of said polypeptide, or all or part of [a] gene coding for said polypeptide"; (3) a method for screening mycobacterial nucleotide sequences to detect sequences coding for exported and/or secreted polypeptides that may be induced or repressed during infection, promoter and/or (other) regulatory sequences associated with such coding sequences, especially sequences that permit or enhance the export and/or secretion of such polypeptides, or all or part of genes coding for such polypeptides, using a vector as above; (4) a method as in (3) comprising: (a) physically or enzymatically fragmenting mycobacterial DNA sequences and recovering the resulting fragments; (b) inserting the fragments into a compatible cloning site in the vector's polylinker; (c) if necessary, amplifying the fragments contained in the vector, e.g. by replicating the vector in a cell, preferably of E. coli; (d) transforming host cells with the vector; (e) culturing the transformed cells in a medium permitting detection of the export and/or secretion marker and/or the promoter activity marker; (f) detecting positive colonies that express the export and/or secretion marker and/or the promoter activity marker; (g) isolating DNA from the positive colonies and inserting this DNA into a cell identical to that of step (c); (h) "the selection of insertions contained in the vector, permitting clones positive for the export and/or secretion marker, and/or for the promoter activity marker to be obtained"; and (i) isolating and characterising mycobacterial DNA sequences contained in the inserts and optionally sequencing selected inserts; (5) a mycobacterial genomic DNA or cDNA library obtained by the method of (3) and/or by a method comprising steps (a) and (b) or steps (a), (b) and (c) of the method of (4); (6) a mycobacterial nucleotide sequence selectable by the method of (3) or (4); (7) a polynucleotide which has a sequence complementary to the sequence of (6), or has a sequence that is at least 50% identical to the sequence of (6), or hybridises with the sequence of (6) under high-stringency conditions, or consists of a fragment of at least 8 consecutive nucleotides of the sequence of (6); (8) a polypeptide, "their fragments or biologically active fragments or their homologous polypeptides", that is encoded by a mycobacterial nucleotide sequence as in (6) or (7) and is capable of being exported and/or secreted and/or induced and/or repressed or constitutively expressed during infection; (9) a recombinant mycobacterium transformed with a recombinant vector as above; (10) a polypeptide encoded by a mycobacterial nucleotide sequence as in (6) or (7); (11) a polypeptide selected from: (a) a polypeptide having a sequence selected from SEQ ID NO:1 to SEQ ID NO:24C, SEQ ID NO:27A to SEQ ID NO:28 and SEQ ID NO:30 to SEQ ID NO:50F [all defined sequences given in the specification], (b) a polypeptide homologous to the polypeptide of (a), (c) a fragment comprising at least 5 amino acids of the polypeptide of (a) or (b), and (d) a biologically active fragment of the polypeptide of (a), (b) or (c); (12) a recombinant cloning, expression and/or insertion vector containing a nucleotide sequence as in (6) or (7); (13) a host cell transformed with the vector of (12); (14) a process for preparing a polypeptide using the vector of (12); (15) a recombinant polypeptide obtainable by the process of (14); (16) a hybrid polypeptide comprising at least one polypeptide sequence as in (8), (11) or (15) and a sequence of a polypeptide capable of inducing an immune response in humans or other animals; (17) a polynucleotide encoding the hybrid polypeptide of (16); (18) mono- or polyclonal antibodies, antibody fragments or chimeric antibodies capable of specifically recognising the polypeptide of (8), (11) or (15); (19) a method of screening for molecules capable of inhibiting the growth or survival of mycobacteria in a host, characterised in that the molecules block the synthesis or function of polypeptides encoded by the nucleotide sequence of (6) or the polynucleotide of (7); (20) molecules capable of inhibiting the growth or survival of mycobacteria in a host, where the molecules are synthesised according to the structure of polypeptides encoded by the nucleotide sequence of (6) or the polynucleotide of (7).


Claims

Information currently unavailable.

IPC Classifications

Download Citation


Sign in to the Lens

Feedback