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Objective:To study the different gene expressions of Pup-proteasome system between the original drug resistance Mtb and the cultured Mtb( INH-Mtb,RFP-Mtb,SM-Mtb,EB-Mtb,MDR) which were at the different concentrations of the Mtb drug,and to explore whether the different Pup-proteasome system gene expression was relevent to the clinical isolates of Mtb-resistant which was widely spreaded in Xinjiang region. Methods:Culturing INH-Mtb,RFP-Mtb,SM-Mtb,EB-Mtb,MDR strains at Original drug resistance Medium,low concentrations of drug Medium and high concentrations of drug Medium to the logarithmic phase,and extract total RNA from each group of Mtb. Using SYBR GreenⅠreal-time PCR to detect the Pup-proteasome system expression level in different concen-trations of drug Mtb in each group of Mtb. Results: Compared with the original state of drug-resistant strains,Pup gene in INH-Mtb, RFP-Mtb,SM-Mtb and MDR strains group were down-regulated 0. 74,0. 23,0. 28,0. 57 times;Dop gene were up-regulated 1. 33,1. 63, 1. 14,2. 88 times;PafA gene were up-regulated 1. 69,1. 30,1. 58,1. 32 times;Mpa gene were up-regulated 3. 05,1. 79,1. 31,2. 27 times in low concentrations of Anti-Mtb drugs condition, the difference was statistically significant ( P<0. 05 ) . Compared with the original state of drug-resistant strains,Pup gene in INH-Mtb,RFP-Mtb,SM-Mtb,MDR strains group were down-regulate 0. 58,0. 37, 0. 43,0. 78 times;Dop gene were up-regulated 2. 62,2. 49,1. 69,2. 95 times;PafA gene were up-regulated 2. 16,1. 48,2. 02,2. 21 times;Mpa gene were up-regulated 1. 63,3. 22,1. 13,3. 94 times in the high concentrations of Anti-Mtb drugs condition,the difference was statistically significant(P<0. 05). Conclusion:Through the different concentrations of antibiotic selection pressure,these groups of Mtb strains expressions in the Pup-proteasome system of Pup gene,Dop gene,Mpa gene and PafA gene were different,The results reveal that Pup-proteasome system is associated with the drug resistance in Mtb which was spreaded in Xinjiang region.
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Objective:To explore the regulation of prokaryotic ubiquitin-like protein ( Pup )-proteasome system on the persistence of Mycobacterium tuberculosis by inducing Mycobacterium tuberculosis to being persistence state under hypoxia conditions and then analyzing the difference on the expression levels of Pup,Dop,PafA and Mpa gene at various time and different conditions. Methods:The total mRNA of the international standard virulent strains of Mycobacterium tuberculosis(H37Rv),which were cultured under hypoxia and aerobic conditions, were extracted from each group at various time and purity of the mRNA were identified.The expression of Pup,Dop,PafA and Mpa genes of M.tuberculosis strains in each group were quantified by SYBR Green I qRT-PCR,which aimed at finding the difference among the expression of Pup,Dop,PafA and Mpa genes at various time and different conditions.Results:The expression levels of Pup, Dop, PafA and Mpa genes in Mycobacterium tuberculosis under hypoxia conditions were measured at various times.The expression levels of Pup,Dop,PafA and Mpa genes:compared with the 0 d,the expression of the Pup gene was up-regulated 1.66,2.43 and 2.76-fold at 4 d,7 d,10 d respectively(P<0.05);the expression of the Dop gene was up-regulated 1.38, 1.91,2.54 and 3.28-fold at 2 d,4 d,7 d,10 d respectively(P<0.05);the expression of the PafA gene was up-regulated 1.22,1.75, 2.37 and 2.67-fold at 2 d,4 d,7 d,10 d respectively( P<0.05);the expression of the Mpa gene was up-regulated 1.66,2.21 and 2.63-fold at 4 d,7 d,10 d respectively(P<0.05).Take the aerobic conditions as control,under hypoxic conditions with the same culture time,the expression of the Pup gene was up-regulated 1.85,2.81 and 2.93-fold in 4 d,7 d,10 d respectively(P<0.05);the ex-pression of the Dop gene was up-regulated 1.20,1.76,2.01 and 3.01-fold in 2 d,4 d,7 d,10 d,respectively( P<0.05);the expression of the PafA gene was up-regulated 1.22,1.57,2.29 and 2.29-fold in 2 d,4 d,7 d,10 d,respectively(P<0.05);the expression of the Mpa gene was up-regulated 1.16,1.58,2.16 and 2.69-fold in 2 d,4 d,7 d,10 d,respectively(P<0.05).Conclusion:Under hypoxic conditions,there were significant differences on the expressions of Pup, Dop, PafA and Mpa genes at various times;what′s more, significant differences on the expressions of Pup,Dop,PafA and Mpa genes exist between hypoxic and aerobic conditions at the same time,Prokaryotic ubiquitin-like protein( Pup)-proteasome system plays a regulatory role on M.tuberculosis′s persistence.
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Objective:To study the correlation different drug-resistant mycobacterium tuberculosis clinical isolates of prokaryotic ubiquitin-like protein ( Pup )-proteasome of Pup, Dop, PafA, Mpa gene expression level and mycobacterium tuberculosis Pup-proteasome system with Xinjiang region widely popular drug-resistant mycobacterium tuberculosis in clinical isolates resistance.Methods:Total RNA of Mtb was extracted from cultured Mtb during the logarithmic phase in drug-susceptible strains in Xinjiang region,the clinical strains drug sensitive to INH,RFP,SM and EB respectively,and multidrug-resistant(MDR) strains.And then the purity of total RNA was identified.The expressing of Pup-proteasome relevant gene( Pup,Dop,Mpa,PafA) were quantified using SYBR GreenⅠqRT-PCR which aimed at finding the correlation between Mtb Pup-proteasome system and drug resistance of Mtb clinical isolates widespread in Xingjiang region by analyzing the expression of Pup, Dop, PafA, Mpa gene among different isolates.Results:Compared with the drug-senstive clinical isolates, mRNA expression level of Pup, Mpa gene was down-regulated in resistant M.tuberculosis clinical isolates INH ( INH-MTB) ,RFP ( RFP-MTB) ,SM ( SM-MTB) and EB ( EB-MTB) ,mRNA expression levels of genes in Dop and PafA was higher in resistant M.tuberculosis clinical isolates,the difference was statistically significant(P<0.05).Compared with MDR strain, the expression of Pup, Dop, Mpa gene were up-regulated different in the resistant M.tuberculosis clinical isolates isolates ,the expression of PafA gene was down-regulated different,the difference was statistically significant( P<0.05).Conclusion:The differentially expressed gene of Pup、Dop、PafA、Mpa gene in sensitive strains,INH-MTB,RFP-MTB,SM-MTB,EB-MTB and MDR strains.The Mycobacterium tuberculosis Pup-proteasome system.Therefore,the Pup-proteasome system have association with the drug resistance of Mtb strains widespread in Xinjiang region.
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Objective:To explore the correlation between Mycobacterium tuberculosis PhoPR two-component system and the pathogenicity of different virulent MTB by analysing the expression levels difference of PhoP gene and PhoR gene in BCG ,H37Ra, H37Rv and XJ-MTB respectively.Methods:Total RNA extracted from four different virulent MTB strains and the integrity of total RNA were identified by using agarose gel electrophoresis.The expression of PhoP gene and PhoR gene were quantified by using SYBR Green I FQ-PCR.The expression levels difference of these genes were compared in different virulent MTB strains .Results: The relative expression levels of PhoP gene in between four different virulent MTB strains from high to low were XJ -MTB(9.05),H37Rv(1.00), H37Ra(0.25),BCG(0.08) respectively ,and the expression levels difference of PhoP gene were statistically significant in different virulent MTB strains ( P＜0.05 );the relative expression levels of PhoR gene in four different virulent MTB strains from high to low were XJ-MTB(5.72),H37Rv(1.00),H37Ra(0.18),BCG(0.07) respectively,and the expression levels difference of PhoR gene were sta-tistically significant in different virulent MTB strains ( P＜0.05 ).The expression levels of PhoP gene and PhoR gene at XJ-MTB were statistically significant difference compared with BCG ,H37Rv,H37Ra respectively (P＜0.05);the expression levels of PhoP gene and PhoR gene at H37Rv were statistically significant difference compared with BCG ,H37Ra respectively (P＜0.05);the expression levels of PhoP and PhoR gene at BCG were not statistically significant difference compared with H 37Ra (P>0.05).Conclusion:Significant expression levels difference of PhoP gene and PhoR gene are confirmed in different virulent MTB strains .Therefore,the Mycobacterium tuberculosis PhoPR two-component system is correlated with the pathogenicity in different virulent MTB strains.
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Objective To determine the polymorphism in CXC chemokine SDF-1α transcript and its effects on HIV infection.Methods Three groups of study subjects lived in Hang Kong were recruited:278 HIV-heahhy donors of Chinese origin.49 HIV+Caucasians and 13 Chinese with high risk behavior to HIV but kept uninfected.Genomic DNA and RNA were extracted from eripheral blood mononucleal cell. The PCR and RT-PCR reaction were set up accordingly.Sequence of the SDF-1α promoter,the open reading frame(ORF)and the 3'untranslate region(3'UTR)were analyzed.Two steps PCR reaction using two reverseprimers with mismatched nucleic acid were employed to screen the frequency of a novel mutation. Results equencing analysis from 100 subjects indicated that non mutation happened in tlle promoter and ORF of SDF-1α.A novel mutation Was detected from 3'UTR of SDF-1α.It is a "GA" insertion in "G" rich region near the stop code of SDF-1α.The mutation Was named as SDF-1-3’GA+and submitted to GenBank (AY874118).The mutation happened in three roups.with allele frequency of 15.1% in the healthy Chinese.of 30.7% in the high risk Chinese group.Conclusions our results confirm that SDF-1 genes arerelatively conserved.None noteworthy mutation is identified in the promoter and ORF regions of SDF-1α However.a novel mutation is identified from the 3'UTR of SDF-1α. It would be worthwhile to etermine effect of the novel mutation on HIV infection.