Patent FR-2767337-A1 (FR)
Title: Mycobacterial DNA vectors containing reporter constructs
Inventor: GICQUEL BRIGITTE
IPC: C12N1/21
Filed: 1997-09-11
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).