METHOD OF SCREENING DRUG-RESISTANCE PROTEIN OF MYCOBACTERIUM TUBERCULOSIS

摘要

The present invention belongs to the field of molecular biology. Specially, the invention relates to the screenings of a new drug target, a vaccine antigen and a test target of anti-tuberculosis treatment. Specially, based on the differential proteome technology and by the techniques of gene sequencing, in vitro protein expression, immunological detection and gene recombination, the invention primarily certifies that the up-regulated expression of Protein Rv2629 and the mutation of Gene 191A/C of Mycobacterium tuberculosis are related to the Rifampicin (RFP) drug-resistance, which provides useful information for the drug-resistance detection of Mycobacterium tuberculosis, the novel drug design and the vaccine development. Firstly, the drug-resistant strains are cultured and the proteins from drug resistance strains and sensitive strains are isolated respectively. Secondly, the proteins from the drug resistance strains and the sensitive strains are compared to determine the differential protein sites, such that the drug-resistant proteins are identified by means of Mass Spectrometry. Next, the up-regulated expression of Protein Rv2629 and the mutation of gene are correlated to the drug-resistance of Mycobacterium tuberculosis by means of gene sequencing technique. Finally, after the gene cloning, the in vitro expression and purification of the protein, the wild-type and mutant-type recombinant protein of Rv2629 are obtained. The polyclonal antibody is prepared, and the protein subcellular localization is carried out. The Minimum Inhibitory Concentrations (MICs) of the strains containing different plasmids obtained by the transformation of recombinant shuttle plasmids are also measured. The gene encoding the protein Rv2629 in the genome of Mycobacterium tuberculosis is isolated and is up-regulation expressed in a Rifampicin (RFP)-resistant strains. And the A/C gene mutation at nucleotide 191 which is related to drug-resistance is detected. The relationship between the mutation and the Rifampicin (RFP)-resistance is primarily certified by molecular biology technique and the in vitro experiments. This invention provides methods used to accurately isolate and identify the drug-resistance protein of Mycobacterium tuberculosis. The A/C mutation of Rv2629 at nucleotide 191 related to the drug-resistance of Mycobacterium tuberculosis is isolated, which shows new approach for the further study of Mycobacterium tuberculosis drug-resistant mechanism, the rapid detection of clinical drug-resistance strains, as well as the developments of new drug targets and vaccines.