Objective To explore the feasibility of detecting the mutations of rpoB gene in rifampin-resistant Mycobacterium tuberculosis strains by gene chip. Methods The DNA chip was designed according to the sequence of Mycobacterium tuberculosis rpoB gene. The DNA fragments which contained hot mutation sites of rpoB gene were labelled with cy3 fluorescence, amplified by PCR technique and hybridized with gene chip. The DNA sequencing was used as the control. Results Eighteen of the Mycobacterium tuberculosis strains were detected for the mutations of rpoB gene by gene chip. Among them, one sensitive strain was identical with the type strain, and all of 17 strains with rifampin-resistant, rpoB mutations were detected. Of the 17 strains with rpoB mutations, 14 were detected as single point mutation, one was detected as double sites point mutations with the sites of 511 and 516, and two were detected as single point mutation with the sites of 518 and 526, respectively. Because the probe of 517 rifampin-resistant did not be dotted on the gene chip, only sites of 518 and 526 mutations were detected, which were consistent with the results of DNA sequencing. Conclusion Using the gene chip could detect mass rpoB gene mutation of rifampin-resistant with higher specialty and sensitivity, which could be used for clinical detection of rifampin-resistant strains and clinical medicine.

Objective To evaluate the protective effectiveness of MPT64 and ESAT6 DNA vaccines against M. tuberculosis. Methods BALB/c mice were randomly divided into 14 groups and subjected to following treatments respectively, i.e. immunized with. ESAT6 (25?g)+MPT64 (25?g)(A), ESAT6(100?g)+IFN-?(100?g) (B), ESAT6 (75?g)+MPT64 (25?g)(C), ESAT6(100?g)+IL-12(100?g) (D), MPT64(100?g)+IL-12(100?g) (E), ESAT6 (25?g)+MPT64 (75?g)(F), MPT64 (100?g)(G), Pvax1 (H),ESAT6 (100?g)(I), ESAT6 (100?g)+MPT64 (100?g)(J), ESAT6 (50?g)+MPT64 (50?g)(K), MPT64(100?g)+IFN-?(100?g)(L), BCG(M ), and saline(N). Then they were infected with M. tuberculosis H37Rv via intravenous route. The pathological changes in the lung, liver, and spleen were observed after the infection. Results Eight weeks after the inoculation, there were only alveolar exudation and capillary hyperemia in the lung lesions in the mice of group N. In the mice of group M and J, main pathological changes included tuberculous granulomas consisting of numerous lymphocytes, macrophages, epithelioid cells and Langhans giant cells, and moderate hyperplasia in alveolar walls. The lung lesions of the other groups were similar, and both hyperplasia and exudate were found (A, B, C, D, E, G, H, I, K, L). No necrosis was found in all the above groups. There were hyperemia, dense lymphocytes infiltration in the portal area, and tuberculous granuloma in the liver in all the groups. No difference was found among all the groups. The pathological changes in spleen induced hyperplasia and fusion of splenic lymph nodule. The reactions in group M and J were stronger than that of the other groups. Conclusions MPT64 and ESAT6 DNA vaccines from M.tuberculosis could enhance immunity against M. tuberculosis, either they were used in combination with different dosages or with IL-12 or IFN-?. The vaccine used in group J showed the strongest effect in enhancing immunity, almost reaching that of combined use of BCG, IFN-? and IL-12.

Objectives To understand the mutations of embB genes in M. tuberculosis isolates, and to evaluate their clinical value. Method 102 clinical isolates were identified for their mycobacterial species, and then analyzed their embB genes with PCR SSCP, PCR RFLP, and PCR direct sequencing. Results Mycobacterium tuberculosis strain H 37 R v was used as a control. 102 clinical isolates all had the same 16S rDNA SSCP profiles as M. tuberculosis . Forty one drug sensitive isolates had normal embB SSCP and RFLP profiles. Of 61 ethambutol resistant isolates, 23 (37.7%) displayed abnormal embB SSCP profiles. Eight isolates had abnormal RFLP profiles. All embB mutations situated at codon 306, whose EMB MICs were more than 20 ?g/ml. Eight isolates had ATG to ATA or ATT mutations at codon 306. Thirty isolates had ATG to GTG or CTG mutations at codon 306, whose EMB MICs were more than 30 ?g/ml. Conclusions Ethabutol resistances in some M. tuberculosis isolates were due to mutations on embB genes. PCR SSCP and PCR RFLP method might become a simple and rapid diagnostic test for genotypes of M. tuberculosis ethabutol resistance.

Objective To investigate the mutation of embB and pncA genes in mycobacterium tuberculosis isolates, and assess its clinical value. Methods The drug-sensitivity of 106 clinical isolates of mycobacterial species was identified by the tranditional method, and then analyzed the mutation of their embB and pncA genes with PCR-SSCP. Mycobacterium tuberculosis strain H 37 RV was used as a control. Results The mutational frequency of pncA gene in 94 PZA-resistant mycobacterium tuberculosis was 44 6%(42/94). The mutational frequency of embB gene in 83 EMB-resistant isolates was 54 2%. The frequency of both genes mutation was 11 8%(11/94). Conclusion EMB and PZA resistances in some mycobacterium tuberculosis isolates were due to mutations of embB and pncA genes. PCR- SSCP method might become a simple and rapid diagnostic test for genotypes of EMB and PZA resistant mycobacterium tuberculosis.

Objective To study the rapid detection of mycobacterium tuberculosis resistance to streptomycin by reverse dot blot hybridization technique. Methods The oligonucleotide probes of streptomycin-resistant genes (rpsL and rrs) were prepared and dropped on nitrocellulose membrane. The target DNA fragments of M. tuberculosis clinical isolates were labeled with biotin by PCR amplification, and then hybridized with the oligonucleotide probes on the membranes. PCR-SSCP and PCR-direct sequencing (PCR-DS) techniques were used to detect the target fragment of M.tuberculosis as control. Results In 53 M. tuberculosis clinical isolates, the consistent rate of three detection methods was 100%. Both the SSCP mapping of rpsL and rrs genes and the results of membrane hybridization in 9 drug-sensitive strains were identical to those in M. tuberculosis standard strain H37Rv. Of 44 streptomycin-resistant strains, 33 strains had AAG→AGG mutation at the codon 43 of rpsL gene, 6 strains had A→C mutation at the 513 site of rrs gene, 1 strain had A→T mutation at the 513 site of rrs gene, and the detection rate of the target genes mutation was 90 9%. In 53 M.tuberculosis clinical isolates, 40 resistant strains and 9 sensitive strains to streptomycin could be detected using dot blot hybridization and the consistent rate with the in vitro susceptibility test was 92 6%(49/53). Conclusion The reverse dot blot hybridization technique showed high sensitivity and specificity to detect Mycobacterium tuberculosis resistance to streptomycin. It possessed the simple and rapid characteristics, and could be used to detecte streptomycin-resistant M.tuberculosis clinical strains.

M.tuberculosis strains isolated strains from sputum specimens of 134 patients were analyzed by PCR SSCP and traditional drug susceptibility tests. The results sbowed that the gene mutation rate of PZA, SM, RFP,INH and EMB resistance in those clinically isolated strains was 42 7%,71 7%,78 9%,68 6% and 43 9%, respectively. The gene mutation was in relation with drug resistance level of M. tuberculosis. The gene mutation rate was higher in high concentration resistance strains than in low concentration resistance strains.

To study the therapeutic action of tuberculosis Ag85A DNA vaccines. Methods:BALB/c mice were infected by in-traperitoneal injection with M. tuberculosis H37Rv for 8 weeks, and then treated with the saline (A), plasmid vector (B), M. bovis BCG (C), M. vaccae (D), and Ag85A DNA vaccine (E). Ag85A-specific antibodies were determined by ELISA. The lungs, livers and spleens were taken and observed their pathological changes, weighted and performed mycobacterial cultures 2 or 5 monthes after treatment. Spleen cells were tested for proliferative responses.Results:Ag85A-specific antibodies increased in the mice of group E. At 2 months after immunothera-pies, the stimulation rates of spleen cells had no significant difference among each group. The numbers of viable bacteria in the lungs and spleens of therapeutic group were lower than those in the control group. The group C and B could be observed slight .lesion of lung. At 5 months after immunotherapies, the stimulation rates of spleen cells all increased significantly in the immunotherapeutic groups. The numbers of viable bacteria in the lung and spleen had no significant difference among each group. No lesions of lung could be observed in group E and D. Conclusion:The tuberculosis DNA vaccines seem to have some immunotherapeutic actions.

Objective To evaluate the clinic application value of detection of rPOB,katG and rPSL resistance genes to M tuberculosis by polymerase china reaction-single stranded conformation polymorphism (PCR-SSCP).Method The drug-resistance tests of M tuberculosis clinical isolates from 141 patients with the tuberculosis was analyzed by Bactec-960 rapid culture,drug-sensitive and PCR-SSCP techniques.Results As nearly As one of three patients with priliminary treatment had resistance to one kind of the drugs tested at least.The mutation rates of RFP,INH and SM drug-sensitive isolates were 6 5%,11 4% and 27 2% respectively.That of the drug-resistant isolates were 60 2%,45 7% and 62 7%.As drug-resistant concentration heightened,so mutation rate of genes increased.Conclusions Detecting the resistance genes was a new explore in guiding treatment,and multiple drug-sensitive test combinative use have cooperativity each other.It might become good test for clinical management.

Objective To construct recombinant plasmid containing CFP-10 gene of Mycobacterium tuberculosis(MTB).Methods The gene fragment of CFP-10 was amplified by PCR from Mycobacterium tuberculosis H37Rv genomic DNA and cloned to pET-32a(+) vector.The recombinant plasmid pET-32a-CFP-10 was transformed into E.coli BL21 (DE3) and induced by IPTG.Results CFP-10 gene fragment was amplified from genomic DNA of Mycobacterium tuberculosis H37Rv strain,and thepET-32a(+) prokaryotic recombinant plasmid was constructed successfully.The recombinant protein was expressed with the induction of IPTG.Conclusions The prokaryotic expression vector for CFP-10 was successfully constructed and the recombinant protein was highly expressed in E.coli BL21 (DE3),which lays a foundation for its subsequent immunological function study.

Objective To investigate the expression of aquaporins-3 (AQP3) in amniotic epithelial cells regulated by cyclic adenosine monophosphate-protein kinase A (cAMP-PKA) signal pathway and to explore the mechanisms of its expression.Methods The amniotic epithelial cells were collected from 30 patients who underwent elective caesarean sections at term with normal amniotic fluid volume and primarily cultured.The cultured cells were treated with (1) forskolin groups: different concentration (0,2.5,5,50 or 100 μmol/L) of forskolin treated cells for 2 hours,and the optimal concentration of forskolin treated cells with different time (0,1,2,10 or 20 hours) ; (2)SP-cAMP groups: different concentration (0,2.5,5,50 or 100 μmol/L) of SP-cAMP treated cells for 2 hours,and the optimal concentration of SP-cAMP treated cells with different time (0,1,2,10 or 20 hours); (3)H-89 groups: different concentration (0,5,10,50 or 100 μmol/L) of H-89 treated cells for 2 hours,and the optimal concentration of H-89 treated cells with different time (0,1,2,10 or 20 hours).The level of intracellular cAMP and activity of PKA were detected by using ELISA,and immunohistochemistry was used to detect the localization of AQP3,the protein expression of total cAMP-response element binding protein (CREB) and phospho-CREB (p-CREB) and AQP3 were assessed by western blot analysis.Cell proliferation was assessed by cell counting kit-8 (CCK-8)assay.Results (1) The brown staining of AQP3 was detected in both cell membrane and cytoplasm in each group.(2) There was no significant change of the cell proliferation rate among groups with different concentration of forskolin,SP-cAMP and H-89 treatment (P ＞ 0.05).(3) After different concentration of forskolin treated 2 hours,the expression of total CREB had no significant difference among them(P ＞ 0.05).While the expression of cAMP level,PKA activity,p-CREB and AQP3 protein were significantly changed,which were higher in 2.5 μmol/L,5 μmol/L,50 μmol/L forskolin group when compared with 0 μmol/L (P ＜ 0.05).Their expressions in 5 μmol/L forskolin group were higher than that in 2.5 μmol/L and 50 μmol/L (P ＜ 0.05).The optimal forskolin concentration was 5 μmol/L.(4) After different concentration of SP-cAMP treated 2 hours,the expression of total CREB and cAMP level had no significant difference among them (P ＞ 0.05),while the expression of PKA activity,p-CREB and AQP3 protein were significantly changed,which were higher in 5 μμmol/L,50 μmol/L SP-cAMP group when compared with 0 μmol/L (P ＜ 0.05).Their expressions in 50 μmol/L SP-cAMP group were higher than that in 5 μmol/L (P ＜0.05).The optimal SP-cAMP concentration was 50 μmol/L (5) After different concentration of H-89 treated 2 hours,the expression of total CREB and cAMP level had no significant difference among them (P ＞ 0.05),while the expression of PKA activity,p-CREB and AQP3 protein were significantly changed,which were lower in 10 μmol/L,50 μmol/L and 100 μmol/L H-89 group when compared with 0 μmol/L (P ＜ 0.05).Their expressions in 10 μmol/L H-89 group were lower than that in 50 μmol/L,100 μmol/L (P ＜ 0.05).The optimal H-89 concentration was 10 μmol/L.(6) p-CREB and AQP3 protein expression were significantly lower in 5 μmol/L forskolin combined 10 μmol/L H-89 incubating 2 hours group when compared with 5 μmol/L forskolin,but higher than that in 10 μmol/L H-89 treated group (P ＜ 0.05).Total CREB was no significant difference among the three groups (P ＞ 0.05).Conclusion cAMP-PKA signal transduction pathway may regulate AQP3 protein expression in human amniotic epithelial cells.