Screening And Cloning Diferentially Expressed Genes Of Children Multi-drug Resitance Tuberculosis

Objective： In this study, we aimed to screen for differentiallyexpressed genes in multidrug-resistant tuberculosis (MDR-TB) anddrug-sensitive TB, to identify those genes that have effects on MDR in TBand to discuss the mechanisms of gene regulation in MDR in TB.Methods：In infections and gastroenterology department, We collectedthe culture-positive tuberculosis strains from2003.7～2010.12.In the basisof genotyping and drug identification, the cDNA from an MDR strain ofTB was used as the experimental group (Tester), while the cDNA from asensitive strain of TB was used as the control (Driver). Suppressionsubtractive hybridization (SSH) technology was used in combination withthe T/A family of cloning technology to build a library of cDNAs thatwere differentially expressed in the MDR and sensitive strains. From thiscDNA library, genes that were expressed in the MDR-TB but not in thedrug-sensitive TB were selected for gene sequencing and homologyanalysis.Results：After two subtractive hybridization and PCR amplification,the products were cloned to the PMD-18T vector by the"A/T clone", and transfected to the competent Escherichia coli DH5,among the colonies,about90%are the white colonies.A subtracted cDNA library of genes thatwere differentially expressed in an MDR-TB strain was successfullyconstructed. From this library,113positive clones were obtained, theircDNA fragments were sequenced and homology analysis was performed.Five new sequences were identified. Among the remaining108knowngenes that were identified, only five of them were genes with sequencesthat had been reported to be linked to MDR in TB. Its main functionalcategory were as follows:1.PE and PPE protein family, such as Rv3343c,PPE54, Rv1918c and PPE35;2. Genes for cell wall and cell processesproteins, such as Rv0987, Rv3783and Rv2318;3. Genes involved inintermediary metabolism and respiration, such as Rv3858c, Rv3784andRv3068c;4. virulence, detoxification and adaptability-related genes, suchas Rv2477c, Rv0783c and Rv2303c;5. Bacterial lipid metabolism-relatedgenes, such as Rv2933, Rv2931and Rv3515c;6. Regulatory proteins, suchas Rv3765c, Rv2799c and Rv0984;7. Hypothetical proteins, such asRv1498A, Rv2603and Rv0690c; and certain signaling and regulatoryproteins and insertion sequences.Conclusions： Suppression subtractive hybridization (SSH) is aneffective method for screening new function genes．Many genes bothknown and unknown were found to relate to the MDR in TB．Geneexpression in MDR-TB and drug-sensitive TB clones is significantlydifferent. This difference is closely related to the appearance of the MDR inTB. Studying the expression and function of the differentially expressedgenes will further reveal the molecular mechanisms underlying MDR in TB.This research will hopefully provide new gene targets for the clinicaldiagnosis of MDR-TB. Our study did not found any difference in the mechanism of MDR-TB between adult and children amongmultidrug-resistant genes we have known.