1.Expression Purification And Primary Identification Of Decaprenylphosphoryl-β-d-ribose-2’-epimerase Sub-unit From Mycobacterium Tuberculosis H₃₇Rv2.Study On Susceptibility For Rifampin Of Mycobacterium Tuberculosis From Sputum Detection Wit

Objective: Expression purification and primary identification ofDecaprenylphosphoryl-β-d-ribose-2’-epimerase sub-unit from Mycobacteriumtuberculosis H37Rv. Methods: The H37Rv DprE1coding gene DprE1was amplifiedand cloned into pET16b expression vector. The recombinant DprE1protein wasexpressed in Escherichia coli BL21(DE3)pLysS induced by IPTG and purifiedthrough immobilized metal affinity chromatography with Ni2+-NTA agarose andconfirmed by SDS-PAGE. Basis on DprE1catalyzing decaprenylphosphorylribose(DPR) to form decaprenylphosphoryl-X(DPX) with oxidation of FAD and adecrease in absorbance signal at450nm wavelength, we primary identificated thisenzyme activity. Results: We finally purified Decaprenylphosphoryl-2’-epimerasesub-unit DprE1with activity. Conclusion: We finally purifiedDecaprenylphosphoryl-β-D-2’-epimerase sub-unit DprE1with activity, and primaryidentificated this enzyme activity, providing help for building a screening modeltargeting DprE1in the future. Objective: To evaluate the detection capability of direct and rapid of RollingCircle Amplification(RCA) for rifampin(RIF) susceptibility of mycobacteriumtuberculosis. Methods: By use the selected sputum specimens of32initial treatmentand re-treatment sputum smear(SS+)pulmonary TB cases hospitalized in BeijingChest Hospital, utilizing the RCA, direct DNA sequencing and conventional drugsusceptibility testing absolute concentration method, detect the MycobacteriumTuberculosis sensitivity to RIF, furthermore compare the results from above threemeasurements. Results: Of32Mycobacterium tuberculosis isolates, by use ofRCA,24had mutations in the amplified region of rpoB.23strains of mono pointmutations, there were2occur at codon516,12occur at codon526and9occur atcodon531,1strains516and526-bit double locus mutation. The results are consistentwith the results of sequencing. Compared the results of three methods of32sputumspecimens,28strains were consistent. There were4cases inconsistent, among them2strains of RCA and sequencing without mutations, drug susceptibility results showedresistance;2strains of RCA and sequencing showed mutations, drug susceptibilityresults showed that for sensitive. Conclusion: In the sputum specimens RCA methodused for the direct detection of Mycobacterium tuberculosis sensitivity to RIF has thesame equal effects as sequencing. Compared with the conventional drug susceptibilitytesting, the RCA method can greatly save time, moreover it possess good clinical application and prospects.