Objective:In this study,we examined the HLA-DPB1 alleles in patients with tuberculosis,and health individuals attempt to investigate the association between the polymorphism of HLA-DPB1 gene and pulmonary tuberculosis in Han population in Shihezi area in Xinjiang uygur autonomous region of China.Methods:High-resolution typing of DPB1 was performed by the sequence-based typing ( SBT) method using the SBT-HLA-DPB1 generic DNA typing kit.Results:In the controls ,17 HLA-DPB1 alleles were ob-served,the HLA DPB1*0501 (28.1%) and HLA DPB1*0201(27.6%) frequency was significantly higher than other sites ,the first and second respectively.The frequency of HLA-DPB1* 0201 was observed significantly increased in patient group compared with control group ( P<0.05 );the frequency of HLA-DPB1* 0501 was observed significantly decreased in patient group compared with control group ( P<0.05 ).Conclusion: The gene frequencies of HLA-DPB1 in Xinjiang Shihezi Han population are roughly in consistence with other northern Chinese Han population;the HLA-DPB1*0201 may be the protective factor to pulmonary tuberculosis , and HLA-DPB1*0501 may be susceptible to pulmonary tuberculosis in Han population from the Xinjiang uygur autonomous region of China.

OBJECTIVE To investigate the antimicrobial susceptibility of Streptococcus pneumoniae isolated in Yantai and their mechanisms of resistance to macrolides.METHODS Antimicrobial susceptibility of S.pneumoniae was determined by agar dilution method.Phenotypes of macrolide-resistant S.pneumoniae were determined using double disk test with erythromycin and clindamycin disks.ermB And mefE genes were amplified by PCR.RESULTS Among 42 strains of S.pneumoniae,65.0% were intermediate to and no strain was resistant to penicillin.The resistance rates to erythromycin and clindamycin were 93.0%,respectively.Of 41 erythromycin resistantstrains,93.0% were constitutive resistant.ermB Was detected in 40 strains and mefE in 1 strain,both ermB and mefE genes were found in 9 strains.CONCLUSIONS The resistance rate of S.pneumoniae to penicillin is high in Yantai area,the resistance rates to erythromycin and clindamycin are very high.Target modification by ermB methylase is the predominant mechanism in macrolide-resistant S.pneumoniae in Yantai.

Objective To determine whether 3'UTR polymorphisms of the NRAMP1 gene are as-sociated with tuberculosis in Uighurs. Methods 3'UTR polymorphisms of NRAMP1 gene were typed by PCR-RFLP among 224 patients with active tuberculosis and 225 healthy individuals. The relationship be-tween 3'UTR polymorphisms and susceptibility to tuberculosis was studied, and cases were grouped accord-ing to genotypes. Results In the tuberculosis patients, genotype TGTG/TGTG,TGTG/TGTG deleted, and TGTG deleted/TGTG deleted were observed in 159,56 and 9 cases respectively, while the genotypes of the healthy controls were TGTG/TGTG in 185, TGTG/TGTG deleted in 36 and TGTG deleted/TGTG deleted in 4 case. The frequency of the genotype TGTG/TGTG was found more often among controls than that in pa-tients (X2=7.94 ,P <0.01). The frequency of allele TGTG and the frequency of variant allele were 0.87 and 0.13 respectively. Conclusion 3'UTR polymorphisms of NRAMP1 gene are associated with suscepti-bility to tuberculosis in Uighurs.

We studied the effect of the Mycobacterium tuberculosis prokaryotic ubiquitin-like protein-proteasome system on mono-resistant to rifampin resistance to M.tuberculosis.A resazurin-based assay was employed to evaluate minimum inhibitory concentration (MIC) and comparative research on mono-resistant to rifampin MTB with Pup,Dop,PafA,Mpa genes expression and deletion of the difference.Above testing strains,respectively,carbonyl cyanide chlorobenzene hydrazone (CCCP),reserpine (RP),verapamil (VP)and chlorpromazine (CPZ) were tested.We compared and analyzed the change of rifampicin MICs on the various strains.Compared with rifampin resistant MTB,overexpression of Pup,Dop,PafA and Mpa genes were able to make monorifampicin of M.tuberculosis to enhance resistance to rifampin.Deletion of Pup gene,Mpa gene,Dop gene,PafA gene significantly decreased the resistance to rifampicin alone MTB,and the P value was ＜0.05.Results indicated that 4 kinds of efflux pump inhibitors can reduce the degree of rifampin MIC in different strains.Through the factorial analysis,there were some interactions between MTB and PPS efflux pump inhibitors,and the P value was ＜0.05.MTB PPS has influence on mono-rifampin resistance to MTB and it may regulate the efflux pathway related protein to influence its resistance.

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.

The aim of this study is to investigate the immune effects of BCG primary immunization and IL-12 combined with Mycobacterium tuberculosis Ag85A DNA vaccine booster immunization on mice .We randomly divided the mice into 7 groups ,namely PBS negative controls ,BCG controls ,pcAg85A controls ,BCG primary immunization combined with Ag85A booster immunization controls ,BCG primary immunization combined with Ag85A and IL-12 booster immunization controls , BCG primary immunization combined with IL-12 booster immunization controls ,and BCG primary immunization combined with pcDNA3 .1 booster immunization controls .Implementing the immune in procedure of BCG primary immunization and cytokine IL-12 booster immunization combined with mycobacterium tuberculosis Ag85A DNA ,we observed the immune effect on mice by detecting the mice serum total IgG , specific lymphocyte proliferation and cytokine levels in 4 ,6 ,8 weeks after the last immunization .Comparing the mice immunized in the strategy of BCG primary immunization and cytokine IL-12 combined with Mycobacterium tuberculosis Ag85A DNA vaccine strengthening immunization to the mice in other groups by other immune ways ,we found that ,in BCG/Ag85A+ IL-12 groups ,IgG in-creased significantly (P＜ 0 .05) ,specific lymphocyte prolifera-ted significantly ,and after strengthening immunization IFN-γlevels ,IL-2 levels and IL-4 levels in the three periods were 128 .2 ±20.4,190.2±16.51,244.2±39.14 ;146.2±17.29,271.6±16.36and16.36±28.12 ;68.6±6.62,96.6±5.5and5.5± 10 .71 ,respectively ,which were higher than those in other groups (P＜0 .05) .It’s suggested that the immunization way of BCG primary immunization and cytokine IL-12 combined with Mycobacterium tuberculosis Ag85A DNA vaccine booster immu-nization could significantly enhance the humoral and cellular immunity of the bodies ,and provide the basis for further study on protective effect test in animals .

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.

AIM:To investigate the effect of inhibiting Mcl-1 gene expression on apoptosis of mouse peritoneal macrophages infected with different virulence of Mycobacterium tuberculosis using a technique of RNA interference .METH-ODS:The BALB/c mice were infected with prepared bacterium of the virulence strains of Xinjiang , H37Rv, H37Ra and BCG.Mcl-1-shRNA was applied to the mouse model of infection , and the control groups were set up .On 1 d, 3 d, 5 d and 7 d, the mouse peritoneal macrophages were collected .The expression of Mcl-1 at mRNA and protein levels was deter-mined by real-time PCR and Western blot .The apoptotic rate of peritoneal macrophages was analyzed by flow cytometry . RESULTS:The expression of Mcl-1 at mRNA and protein levels was up-regulated in the peritoneal macrophages from the mice infected with different virulence of Mycobacterium tuberculosis, and the cells from the mice infected with virulence strains of Xinjiang and H37Rv expressed higher level of Mcl-1 than the uninfected control cells (P<0.05).The expres-sion of Mcl-1 at mRNA and protein levels was reduced by RNA interference as compared with control group ( P<0.05 ) . Inhibition of Mcl-1 expression induced apoptosis of peritoneal macrophages in the mice .CONCLUSION: The Mcl-1 ex-pression at mRNA and protein levels in mouse peritoneal macrophages infected with different virulence of Mycobacterium tu-berculosis was effectively suppressed by Mcl-1-shRNA, which can induce macrophage apoptosis .

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.

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.