Relationship Between The Mutation Of KatG In Mycobacterium Tuberculosis And Drug-resistance Of INH

Background of study and Objectives Tuberculosis (TB) is a Chronic infectious disease caused by mycobacterium tuberculosis ,which were Infected in all over the body of human organ. In recently years, drug resistant strain of TB appearance,merging AIDS and MTB and flowing of high-risk group make TB epidemic in global. Drug resistance of TB is particularly serious in China ,The total drug resistance rate was 29.8%,Multidrug-resistant rate is12.7%,Thus the prevalence and death rates cased by TB is quite high.Therefore, the research on mechanisms of MTB's resistance to INH become the most urgent task The results show that the gene which most associated with INH resistance is KatG , that the decreased or absent peroxidase activity in Mycobacterium tuberculosis caused by the mutation of KatG gene (Missing and Point mutation) can explain more than 90% of isoniazid (INH) resistance. However, studies have found that some strains may be unrelated with that ,may due to other resistance genes or several other mechanisms ,that pending further discussion.In this study, we have manufactured the missing of KatG gene in the Standard strain of Mycobacterium tuberculosis; Repair point mutations of KatG in the INH resistant strains of Mycobacterium tuberculosis; Guided point mutation of KatG in the INH-sensitive strains of Mycobacterium tuberculosis. Then detect drug resistance of the recombinant strains ,observe the relationship between drug resistance and mutation.Matetials and methods 1,Strain and Plasmid origin.Standard strain H37Rv: conserved by ourselves laboratory. Plasmid pKD46 were honored by Dr. Han Yanping who is from the Chinese People's Liberation Army 309 hospital entire armed forces institute of microorganism epidemic.2,Culture of bacterial strains H37Rv were grown in L-J medium modified medium and Middlebrook 7H9 medium. 3.Preparation of DNA fragments used for knockout..The KatG-pre and KatG-post were amplified by PCR from MTB H37Rv. The KatG-pre and KatG-post were connected by overlapping PCR, which compose the fragment used for the knockout of KatG gene .4.preparation of DNA fragments used for repair the resistance gene .The fragments of repair were amplified by PCR from isoniazid resistant strains , using primers with restoration sites. 5.preparation of DNA fragments used for Guiding mutation .The fragments of lead were amplified by PCR from Isoniazid-sensitive strains of MTB DNA,using primers with Guided mutation. 6.Induced mutation.When H37Rv strain were cultured exponential phase of growth, freshly prepared competent cell were electroporated in the presence of 1ug of plasmid pKD46. H37Rv with pKD46 of exponential phase of growth was induced 48 hour with 10mmol/L L-arabopyranose, which was prepared freshly prepared competent cell, and was were electroporated in the presence of 500ng of vector DNA.The fragment of Knockout, Repair, and guide were introduced into the Corresponding cell.7.Identification of the results of the recombinantion of recombinant strain . Extraction DNA with kit, amplified aim gene with primers ,delivery to sequencing.8.Put the recombinant strains into drug susceptibility test ,calculation the percentage of resistance.Results 1. Construction of the recombinant strains. Sequence analysis confirmed the recombinant strains were Successfully constructed. 2.The results of recombinant strains resistant of INH. After the complete knockout of the katG gene in the Standard strain , 80% of the Standard strain Become completely resistant to INH;After the 315 points restored in the Resistant strains ,4 in 5 strains restored sensitivity to INH. After the 315 points guide mutated in the sensitive strains ,6 in 8 strains Produce Resistance to INH.;After the 463 points restored in the Resistant strains ,only 1 strain in 6 restored sensitivity to INH. After the 463 points guide mutated in the sensitive strains ,only 1 in 9 strains Produce Resistance to INH.Conclusions Construction of KatG gene deletion strains of the standard strain, repaired strains of the INH resistant strains ,and mutant-guided strains of the INH sensitive strains. This study shows that 80% of the katG deletion mutant strains resist INH ,which instruct that the absence of KatG of Mycobacterium tuberculosis is an important mechanism resistant to INH. The Differences between before and after katG 315 mutation sites's repairation are significantly, which instruct that there is relationship between KatG codon 315 changes in gene and INH Resistant The Differences between before and after repair katG 463 mutation sites are not significantly, which Instruct that there are no relationship between KatG codon 463 changes in gene and INH Resistant ,codon 463 mutation does not mean that INH Resistant mutations.