Method of identifying a novel target for use in drug development and treatment of tuberculosis

摘要

A method of identifying novel targets for use in the development of therapeutic agents with an effective therapy against tuberculosis: Destroy the devR genes present in EcoRI-HindIII insert of about 3,3kb (1) plasmid pJT53.34, Constructs a pJQ200SkdevR :: kan from devR genes that have been (2) the destruction, Is introduced into the (M. tuberculosis) H37Rv Mycobacterium tuberculosis (3) the plasmid, In 7H10 Middlebrook agar plates containing (4) kanamycin, to select the cross-transformants showing a single integration of the plasmid, Polymerase chain reaction (PCR), and analyzed for the presence of sucrose resistant devR, Km ^R and the (SacB) gene sequences, and (5) the transformed difference intersection, Is subjected to the process of Southern analysis with devR probe, devS probe kanamycin resistant gene probe, and (6) the gene sequence, Mycobacterium tuberculosis, including copies destroyed in the devR locus and a wild-type (M. tuberculosis) Identify the Dup devR, Grown tuberculosis bacteria (M. tuberculosis) Dup devR in 7H9 medium Middlebrook containing sucrose (7) kanamycin, Put the plates each having a 7H10 medium containing sucrose and kanamycin, Mycobacterium tuberculosis (8) the growing (M. tuberculosis) Dup devR strain, to obtain a kanamycin-resistant transformants, Subjected to Southern hybridization, Mycobacterium tuberculosis (9) the proliferation (M. tuberculosis) devR, then, by the polymerase chain reaction method, check the allele exchange, It is subjected to a process of polymerase chain reaction analysis, (10) transformant, analyzes the devR kan gene disruption, It is subjected to a process of immune electron microscopy and Western blotting, the (11) devR kan gene disruption, check the function of the gene disruption, Regard devS and gene expression (12) devR, to evaluate the viability of Mycobacterium tuberculosis (M. tuberculosis) devR mutant strain in oxygen-limited conditions, Regard devS and gene expression (13) devR, to evaluate the viability of said binding Kakukin (M. tuberculosis) strains devR in oxygen-limited conditions aerobic, Is subjected to the process of reverse transcription PCR analysis strains (14) the growth, to obtain a transcript from the Rv3134c-devR-deS operon, The scanned using ultra violet (Ultra-Violet) manufactured by gel recording system, and (15) said transfer Utsushi-tai, further, subjected to the process of the densitometer analysis using computer software, it, By microbiological methods and histopathological analysis of (lung and liver) (16) organization, to quantify the amount of bacteria in the spleen of guinea pigs infected with devR mutant strain, pathogenicity in guinea pig tuberculosis (M . above method comprising testing a tuberculosis) devR mutant strain, that.