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Iron is involved in the virulence and pathogenic effects of certain intracellular parasites.In the pathogenic process of Brucella,the uptaking and metabolism of host iron are closely related to intracellular parasitism and immunity escape of Brucella.In this paper,we elucidated the iron transport system,iron response regulators and nutrient immunity of iron based on the latest report and data about Brucella.
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We detected the isoniazid resistance in clinical Mycobacterium tuberculosis isolates by high-resolution melting (HRM) curve analysis and assessed the application value of the assay.The isoniazid resistance of 49 M.tuberculosis isolates preserved in laboratory was analyzed by the drug sensitivity test (traditional proportion method).Further analysis was made on the sequencing of the isoniazid resistance determining region in these test strains,and their mutation sites were screened.Specific primers used in the HRM curve analysis were designed based on the screened mutation sites,DNA mutations were assayed in the isoniazid-resistant gene determining region by the HRM curve analysis,and an assessment was made of the detection efficiency of the assay in isoniazid resistance in M.tuberculosis.Results of the drug sensitivity test (proportion method) showed that,of the 49 test strains,there were 20 isoniazid-resistant strains,29 isoniazid-sensitive strains.Results of the sequencing analysis showed that:1) KatG gene had four mutation patterns,i.e.,point mutations at site 234,at sites 234 and 315,at sites 234 and 463,and at sites 234,315 and 463;2) there were three mutations were detected in inhA gene,i.e.,mutations in inhA-8,-15 and-152.Analysis of gene mutation in drug-resistant strains found that of the 20 isoniazid-resistant strains,11 (55 ％) were mutated at codon 315 of KatG gene;6 (30％) were mutated in inhA-15 (4/20),-8 (1/20) and-153 (1/20) of inhA gene;two (10％) were mutated at codon 315 of KatG gene and in inhA-15;in one strain (5％),no mutation was detected in KatG and inhA genes.Through the gene mutation detection,the sensitivity and specificity of isoniazid resistance in M.tuberculosis were 95 ％ and 100 ％,respectively.Results of HRM curve analysis of drug-resistance gene mutations in test strains showed gene mutations were present in 18 strains and absent in 24 ones;referring to DNA sequencing results,the sensitivity and specificity of the assay were 94.7％ and 80％,respectively.Judged by mutations as drug-resistance via the HRM curve analysis,19 resistant and 24 sensitive strains were tested.With the drug sensitivity test results by the proportion method as controls,the sensitivity and specificity of the assay were 95 ％ and 82.76 ％,respectively.Use of the HRM curve in the detection of resistance of M.tuberculosis to isoniazid is characterized by good sensitivity and short time consuming,and has certain value in the rapid diagnosis of isoniazid-resistant tuberculosis.
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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 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 study the expressions of myeloid cell leukemia-1 (Mcl-1 ) gene in mouse macrophages Raw264.7 and human macrophages THP-1,to screen out the cell lines with high levels of expression as the experimental cells,and based on the screening results to construct the short hairpin RNA(shRNA)eukaryotic expression plasmid targeting mice Mcl-1 gene for transfection and further screen out the shRNA expression plasmid with the most obvious effect in silencing Mcl-1 gene.Methods Semi-quantitative PCR method was used to detect the expression of Mcl-1 mRNA in the two kinds of macrophages.Western blotting was adopted to detect the expressions of Mcl-1 proteins in the two kinds of macrophages.Three different gene loci for Mcl-1 shRNA fragments were designed with small molecules interfering RNA (siRNA)software.Eukaryotic expression plasmid Mcl-1 shRNA 1-3 carrying the shRNA fragments was constructed by a company.And the eukaryotic expression plasmid vector was transfected into scavenger cells Raw264.7 mice via through the liposome.The transfection results 24 h and 48 h after transfection were observed under the inverted fluorescence microscope;Mcl-1 mRNA and protein expression were detected by real time quantitative PCR and Western blot,respectively.Results The relative expression levels of Mcl-1 mRNA and protein in mouse macrophages Raw264.7 were significantly higher than those of human macrophages (P <0.05).The shRNA expression vector constructed within 24 h and 48 h could decrease Mcl-1 mRNA and protein levels in Raw264.7 cells,especially with the most obvious silencing effect at 48 h.The 48-h transfection group differed significantly from normal group,liposome group and negative control group (P <0.05).Compared with Mcl-1shRNA1and Mcl-1shRNA2,Mcl-1shRNA3 had the strongest effect in silencing Mcl-1mRNA and protein.Conclusion We have successfully screened out experimental Raw264.7 cells and Mcl-1 shRNA eukaryotic expression plasmid which has an obvious silencing effect targeting on Mcl-1 in mice macrophages Raw264.7.
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Objective:To transfect Mcl-1shRNA into Raw264.7 cells,and screen out specific shRNA eukaryotic expression plasmids with the most significant effect of silent Mcl-1 gene to figure out the effect of shRNA on Mcl-1 expression in murine macrophage cell line Raw 264.7.Methods: Specific shRNA was transfected into murine macrophage cell line Raw 264.7 via lipofectamine.Semi-quantitative RT-PCR and Western blot were respectively employed to test the changes in Mcl-1 mRNA level and Mcl-1 protein expressions 24 h and 48 h after the transfection ,and the silencing effects of the three pairs of specific shRNA fragments corresponding to different sites were analyzed.Results: Specific shRNA fragments at 24 h and 48 h could effectively reduce Mcl-1 mRNA and protein level ,with higher silencing effects than those of the normal group ,the lipofectamine group and the negative control group.There were statistically significant differences among them ( P<0.05 ).Among the three pairs of specific shRNA fragments corre-sponding to different sites ,Mcl-1 shRNA3 showed the most significant inhibiting effect on Mcl-1 mRNA and proteins.Conclusion:RNA interference can downregulate the level of Mcl-1 mRNA in murine macrophage cell line Raw 264.7 and greatly downregulate the expression of Mcl-1protein.Specific shRNA eukaryotic expression plasmids with the most significant effect of silent Mcl-1 gene have been screened out successfully.
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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 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 evaluate the possibilities of using circulating miR-155-5p, miR-21-5p, miR-29a and miR-142-5p as biomarkers for the diagnosis of active pulmonary tuberculosis (TB).Methods Plasma samples were collected from 60 healthy subjects, 40 patients with active pulmonary TB and 20 sub-jects with latent tuberculosis infection ( LTBI) to extract miRNAs.The levels of miR-155-5p, miR-21-5p, miR-29a and miR-142-5p in plasma samples were detected by using reverse transcription-quantitative PCR. Results The levels of miR-155-5p, miR-21-5p and miR-29a in patients with active pulmonary TB were re-spectively 10.13, 7.34 and 2.74 times as much as those in healthy subjects(P<0.05).No significant differences with the level of miR-142-5p were observed between the two groups.The receiver operating char-acteristic (ROC) curve analysis of miR-155-5p, miR-21-5p and miR-29a in patients with active pulmonary TB and healthy subjects showed that the areas under the curve (AUC) were respectively 0.905, 0.830 and 0.687.The level of miR-155-5p in patients with LTBI was 3.1 times than that in healthy subjects ( P<0. 05).No differences with miR-21-5p, miR-29a and miR-142-5p were found between patients with LTBI and healthy subjects.The levels of miR-155-5p, miR-21-5p and miR-29a in patients with active pulmonary TB were respectively 3.26, 6.69 and 1.98 times than those in patients with LTBI (P<0.05).The rate of LTBI was 40.58%in people who were in close contact with patients with active pulmonary TB.Conclusion Sig-nificant differences with the levels of miR-155-5p, miR-21-5p and miR-29a were observed among healthy subjects, patients with active pulmonary TB and patients with LTBI, but no difference with the level of miR-142-5p was observed among them.miR-155-5p, miR-21-5p and miR-29a could be used as the potential bio-markers for the diagnosis of active pulmonary tuberculosis and latent tuberculosis infection.People who were in close contact with patients with active pulmonary TB could have a higher LTBI rate.