Reporter gene based method for the screening of anti-tuberculosis drugs by using essential and regulatory genes of mycobacteria as drug target
AssigneeCouncil of Scientific and Industrial Research
InventorsVishal Soni, Lakshami Pathi Khandrika, Pushpa Agrawal
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Abstract
The present invention relates to an in vivo drug screening method taking advantage of the yeast two-hybrid system provides a method of using the whiB genes which are regulatory genes of mycobacteria, whose functions have not been reported so far, as drug target by using the yeast Saccharomyces cerevisiae as a surrogate host.
Claims27
A reporter gene based method for the screening of anti-tuberculosis drugs comprising using whiB like genes (whiB1, whiB2 whiB3 whiB4) present in Mycobacterium tuberculosis H37Rv, Mycobacterium bovis BCG and Mycobacterium leprae having DNA and protein sequence as shown in sequence ID 1 to 4 as drug target for the screening of anti-tuberculosis and anti-leprosis drugs.
The presence of functional whiB1 gene of Mycobacterium tuberculosis H37Rv and Mycobacterium bovis BCG is essential for their survival.
The whiB1 gene of Mycobacterium tuberculosis H37Rv and Mycobactrium bovis BCG code for a DNA binding protein.
The whiB1 gene of Mycobacterium tuberculosis H37Rv and Mycobacterium bovis BCG can be used for the screening of anti-tuberculosis drugs.
The presence of functional whiB2 gene is important for the normal growth of Mycobacterium tuberculosis H37Rv and Mycobacterium bovis BCG.
The whiB2 gene of Mycobacterium tuberculosis H37Rv and Mycobacterium bovis BCG code for a DNA binding protein.
The presence of functional whiB3 gene is essential for the normal cell division of Mycobacterium tuberculosis H37Rv and Mycobacterium bovis BCG.
The whiB3 gene of Mycobacterium tuberculosis H37Rv and Mycobacterium bovis BCG code for a DNA binding protein.
The whiB3 gene of Mycobacterium tuberculosis H37Rv and Mycobacterium bovis BCG can be used for the screening of anti-tuberculosis drugs.
The whiB3 gene of Mycobacterium tuberculosis H37Rv and Mycobacterium bovis BCG can be used for the screening of anti-tuberculosis drugs.
The presence of functional whiB4 gene of Mycobacterium tuberculosis H37Rv and Mycobacterium bovis BCG is important for their normal growth.
The whiB4 gene of Mycobacterium tuberculosis H37Rv and Mycobcaterium bovis BCG code for a DNA binding protein.
The drugs against whiB2 and the whiB4 genes of Mycobacterium tuberculosis H37Rv and Mycobacterium bovis BCG will be particularly useful where drug resistance has developed against the whiB1 and whiB3 genes or where the anti-whiB1 and anti-whiB3 drugs are allergic or toxic.
The source of whiB like genes can be Mycobacterium avium, Mycobacterium fortuitum, Mycobacterium gastri, Mycobacterium kansasii, Mycobacterium leprae, Mycobacterium marinum, Mycobacterium microti, Mycobacterium phlei, Mycobacterium scrofulaceum, Mycobacterium smegmatis and Mycobacterium xenopi and related organisms.
A reporter gene based method for the screening of anti-tuberculosis drugs comprising using whiB like genes (whiB1, whiB2 whiB3 whiB4) present in Mycobacterium tuberculosis H37Rv, Mycobacterium bovis BCG and Mycobactereium leprae having DNA and protein sequence as shown in sequence ID 1 to 4 as drug target for the screening of anti-tuberculosis and anti-leprosis drugs, said method comprising the steps of:
- (a) amplifying the whiB genes of Mycobacterium tuberculosis H37Rv,
- (b) cloning of the products obtained in step (a) into plasmid vectors such as pUC 19 or pBluescript SK+ or SK− and transforming into E. coli;
- (c) amplifying the whiB genes of Mycobacterium bovis BCG by using the oligonucleotide primers of Mycobacterium tuberculosis H37Rv;
- (d) cloning the product as obtained in step (c) into a plasmid vector pUC19 or pBluescript SK+ or SK−;
- (e) constructing a gene disruption integrative vector by inserting a kanamycin cassette in between the whiB gene sequences of Mycobacterium tuberculosis H37Rv identified as sequence ID1 for whiB1, ID 2 for whiB2, ID3 for whiB3 and ID4 for whiB4;
- (f) constructing a gene disruption integrative vector by inserting a kanamycin cassette in between the whiB gene sequences of Mycobacterium bovis BCG;
- (g) amplifying whiB genes of Mycobacterium tuberculosis H37Rv by using oligonucleotide primers carrying either EcoRI, or BamHI restriction enzyme sites at the 5′ end and XhoI restriction enzyme site at the 3′ end;
- (h) digesting the fragment as obtained in step (f) with either EcoRI or BamHI or XhoI restriction enzymes and then cloning of the fragment into a E. coli/Saccharomyces cerevisiae shuttle vector pEG202 to produce the cloned gene as a LexA fusion protein and transforming the recombinant plasmid into a E. coli;
- (i) preparation of large amount of the desired recombinant plasmid and then transforming the recombinant whiB1, whiB2, and whiB3/pEG202 into the Saccharomyces cerevisiae strains EGY48 and EGY191,
- (j) co-transforming pJK101, pSH 18-34, pSH17-4, pJG4-5 and pRFHM vectors into the Saccharomyces cervisiae strains EGY48 and EGY191 already harboring whiB genes in the pEG202 vector and then selecting for the clones, which complements for uracil and histidine+uracil or histidine+uracil+tryptophan auxotrophy;
- (k) checking the clones for the activation of both lacZ and leu2 reporter genes by inoculating the recombinant clones in to a yeast-nitrogen base medium;
- (l) the whiB1, whiB2 and whiB3 genes were co-transformed separately with pJK101 plasmid and selected for histidine and uracil auxotrophy;
- and
- (m) using the DNA binding property of the whiB1, whiB2 and whiB3 to screen for anti-tuberculosis drugs.
Dependencies are inferred from the claim text.