| TB is a malignant epidemic disease which seriously threats public health all over the world. China, as one of the countries suffering from the heaviest load of tuberculosis infection, is facing to the problem of serious drug-resistance. Thus, it is very necessary to make research on resistance mechanisms of tuberculosis. Our former studies using LC/MS/MS found that the expression of transcription factors Dev R, which is part of two-component signal transduction system of TB, was increased in the MDR-TB(multiple drug resistant tuberculosis). Dev R is an important dormancy regulation factor of TB and dormancy could lead drug resistance. It implies that Dev R may be closed associated with drug resistance. Based on this, we plan to explore the relationship between differential expression of Dev R and drug resistance.Objective 1. Detect different expression levels of Dev R and study the relationship between the dev R level and drug susceptibility phenotype. 2. Clarify the effect of genetic background on the expression of dev R. 3. Verify the relationship between differential expression of dev R and drug resistance.Method 1. Total RNA was extracted from 32 susceptible strains, 18 INH-resistant strains, 2 RIF-resistant strains and 45 MDR-TB. Then real-time PCR was used to detect the expression level of dev R in different groups; Add INH,RIF,SM or EMB at the half of minimum inhibitory concentration(MIC) to 6 susceptible strains in the logarithmic phase. After 2 hours, detect the expression of dev R gene. 2. Genomic DNA were extracted from 97 clinical isolates of mycobacterium tuberculosis, and 15 MIRU-VNTR locus of genomic DNA were amplified by PCR. Then the data of nucleic acid amplification was upload to MIRU-VNTRplus Database, and the genotypes of clinical isolates was identified by using phylogenetic trees. 3. To obtain DNA fingerprints of 97 clinical isolates, Genomic DNA of strains were amplified by Devsilab rep-PCR. Then cluster analysis of DNA fingerprints was carried out using online software of Deversilab(Version 3.4). 4. The promoter region of dev R was predicted by online software, and the dev R promoter of 97 clinical isolates was amplified by PCR. Then products of nucleic acid amplification were sequenced and performed sequence alignment. 5. The p JV53 plasmid which can express mycobacreriophage-derived recombinase Che C 60-61 recombinase, was transformated into mycoabterium bovis BCG strain for constructing the recombinant BCG strain. 6. Upstream and down homologous arm of dev R were orderly ligated with zeo R cassette. Then the fragment of dev R homologous arm-zeo R cassette was transformated into the above-mentioned recombinant BCG strain. Finally, the deletion mutants of dev R was identified by PCR and sequencing. 7. The dev R was cloned into plasmid p MN437 to construct dev R expressed vector, named as p MN437 dev R. Then the p MN437 dev R was transformated into mycobacterium bovis BCG. Finally, the dev R overexpressed BCG strain was identified by PCR, and fluorescence microscope, etc. 8. Use Resazurin Microtiter Assay Plate Testing to identify the MIC of the wild type strains of mycobactrerium bovis BCG, dev R-deletion and dev R-overexpression strain.Results 1. quantified PCR testing result showed that the expression of dev R was significantly increased to 1.42 times and 7.36 times of the drug-susceptible strain(p<0.05) in INH resistant strain and MDR strain. The expression of dev R in drug-susceptible strains of clinical isolates was significantly could be induced by INH, RIF and EMB, respectively. 2. 15 MIRU-VNTR of loci analysis showed that, 97 strains of Mycobacterium tuberculosis of clinical isolates belong to Beijing genotype. But the laboratory standard strains H37 Rv was found having the copy number variation of repetitive sequence on 3 loci(MIRU-26, Mtub39 and QUB4156), compared to the original reference strains H37 Rv. 3. DNA fingerprints cluster analysis obtained by Devsilab rep-PCR showed obvious polymorphism on genotypes of Mycobacterium tuberculosis strains preserved in our laboratory. Every sample has a unique DNA fingerprint. The overall similarity of all strains is 55%. In 70% cut-off value, all of the strains could be divided into 8 major genotype. But there is no statistical significance in the correlationship between resistant phenotype and Rep-PCR genotypes(p>0.05). Besides, the results of correlation analysis between genotype and the dev R expression showed that the difference of dev R expression in drug-resistant strains and sensitive strains is independent with the genotype similarity(p>0.05). 5. The sequencing of 792 bp gene fragment upstream of dev R showed that 22 strains(6 sensitive strains, 4 INH resistance strains and 12 MDR strains) occurred A base deletion mutation in the dev R-370 bp upstream, which was new found. But there is no expression difference of dev R between the mutant and the wild type strain(p>0.05). 6. Our research successfully constructed the dev R-deletion and dev R-overexpression mycobaterium bovis BCG strain, which identified by PCR, sequencing and fluorescence microscope, etc. 7. The drug sensitivity results showed that the MIC of wild type mycobaterium bovis BCG strain to INH, RIF, SM and EMB was 0.06, 0.17, 0.5 and 2.6 μg/ml, repectively. The MIC of dev R-deletion strain to INH, RIF, SM and EMB was 0.06, 0.20, 0.33 and 3.3 μg/ml, repectively. The MIC of Dev R-overexpression strain to INH, RIF, SM and EMB was 0.08, 0.20, 0.33 and 3.3 μg/ml, repectively.Conclusion The expression of dev R in various strains of phenotype was investigeated systematically. It was identified that the expression of dev R could be induced by INH, SM, and EMB in drug-resistant strains significantly. The differential expression of dev R in drug-resistant and-susceptible strains was not affected by the genotype of strains, suggesting that its expression is closely associated with drug resistance olny. Then, we successfully constructed the dev R-deletion and-overexpression M.bovis BCG strains. It laid the foundation for further research of the relationship between the expression of dev R and the drug-resistance gene mutation of M.tuberculosis. |
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