Effect And Mechanism Of MTB Resistant With The MTB PPS Deletion Mutants

Objecve : Comparative analysis the missing mutations effect on resistance of the Mycobacterium tuberculosis ubiquitin-like protein(Pup)- proteasome system( including Pup gene, Mpa gene, Dop gene, Paf A gene) in Xinjiang widely popular region of drug sensitive strains( intolerance drug strains), a simple first-line anti-TB drugs( rifampicin, isoniazid, streptomycin, ethambutol) of clinical isolates of strains,the clinical isolates of multidrug-resistant Mycobacterium tuberculosis. Methods: Using SOE-PCR technique and T vector method to constructe and identify the missing mutant gene replacement vectors T-Pup-NK-Pup-C, T-Mpa-NK-Mpa-C, T-Dop-NK-Dop-C and T-Paf ANK-Paf A-C;Useing the technique of electroporation to electrical transform and identiy each mutant vectors into different resistant strains;Using Resazurin chromogenic method for detecting strains minimum inhibitory concentration(MIC) in each group rapidly. Comparative analysis Pup- proteasome system gene delete mutation effects on mycobacterium tuberculosis resistance. Results: 1. This study has constructed Pup-proteasome system( including Pup gene,Mpa gene,Dop gene, Paf A gene) replacement vectors deletion mutant T-Pup-NK-Pup-C, T-Mpa-NK-Mpa- C, T-Dop-NK-Dop-C and T-Paf A-NK-Paf A-C of Mycobacterium tuberculosis. 2. Successfully. Constructed Pup gene deletion mutant INH-MTB △ Pup strains, RFP-MTB △ Pup strains, SM-MTB △ Pup strains, EB-MTB △ Pup strains, MDR-MTB △ Pup strains, and lack of Drug-sensitive-MTB △ Pup strains, Mpa gene mutant INH-MTB △ Mpa strains, RFP-MTB △ Mpa strains, SM-MTB △ Mpa strains, EB-MTB △ Mpa strains, MDR-MTB △ Mpa strains, and Drug-sensitive-MTB △ Mpa strains, Dop gene deletion mutant INH-MTB △ Dop strains, RFP-MTB △ Dop strains, SM-MTB △ Dop strains, EB-MTB △ Dop strains, MDR-MTB △ Dop strains, and Drug-sensitive-MTB △ Dop and Paf A gene deletion mutants INH-MTB △ Paf A strains, RFP-MTB △ Paf A strains, SM-MTB △ Paf A strains, EB-MTB △ Paf A strains, MDR-MTB △ Paf A strains and Drug-sensitive-MTB △ Paf A strains. 3. successfully.Resazurin chromogenic method for detecting strains minimum inhibitory concentration(MIC) in each group shows that,Compared with the original drug-resistant strains,the delete mutant Pup gene, Mpa gene, Dop gene of a simple first-line anti-TB drug resistance in clinical isolates of Mycobacterium tuberculosis and multidrug-resistant Mycobacterium tuberculosis clinical isolates MIC are all declining to varying degrees, with Pup gene deletion strains obviously.Simple first-line anti-TB drug resistance in clinical isolates of Mycobacterium tuberculosis and multidrug-resistant Mycobacterium tuberculosis clinical isolates Paf A gene deletion mutants MIC basically unchanged.Drug-sensitive strains of M. Tuberculosis in missing Pup gene,Mpa gene,Dop gene,Paf A gene MIC are fundamental change. Conclusions: The Pup-proteasome system of deletion mutant Pup gene,Mpa gene,Dop gene in Xinjiang region of widespread resistance to first-line anti-TB drugs alone( isoniazid, rifampicin, streptomycin, ethambutol) clinical isolates of Mycobacterium tuberculosis and multidrug-resistant Mycobacterium tuberculosis clinical isolates of drug resistance are reduced in varying degrees, the deletion strains of Pup gene is decrease obviously, almost lost resistance;The deletion strains of Paf A gene resistant basically unchanged, indicating that there is correlated with Pup-proteasome system of Mpa gene,Dopgene and drug resistant in Mycobacterium tuberculosis,and related Pup gene closely,which is good for further study in regulate mechanism of Pup- proteasome system of drug resistance in Mycobacterium tuberculosis.