AIM:To evaluate how widely Helicobacter pylori (H. pylori ) HopE and HopV porins are expressed among Chilean isolates and how seroprevalent they are among infected patients in Chile.METHODS: H. pylori hopE and hopV ge...AIM:To evaluate how widely Helicobacter pylori (H. pylori ) HopE and HopV porins are expressed among Chilean isolates and how seroprevalent they are among infected patients in Chile.METHODS: H. pylori hopE and hopV genes derived from strain CHCTX-1 were cloned by polymerase chain reaction (PCR), sequenced and expressed in Escherichia coli AD494 (DE3). Gel-purified porins were used to prepare polyclonal antibodies. The presence of both genes was tested by PCR in a collection of H. pylori clinical isolates and their expression was detected in lysates by immunoblotting. Immune responses against HopE, HopV and other H. pylori antigens in sera from infected and non-infected patients were tested by Western blotting using these sera as f irst antibody on recombinant H. pylori antigens.RESULTS: PCR and Western blotting assays revealed that 60 and 82 out of 130 Chilean isolates carried hopE and hopV genes, respectively, but only 16 and 9, respectively, expressed these porins. IgG serum immunoreactivity evaluation of 69 H. pylori-infected patients revealed that HopE and HopV were infrequently recognized (8.7% and 10.1% respectively) compared to H. pylori VacA (68.1%) and CagA (59.5%) antigens. Similar values were detected for IgA serum immunoreactivity against HopE (11.6%) and HopV (10.5%) although lower values for VacA (42%) and CagA (17.4%) were obtained when compared to the IgG response.CONCLUSION: A scarce expression of HopE and HopV among Chilean isolates was found, in agreement with the infrequent seroconversion against these antigens when tested in infected Chilean patients.展开更多
Helicobacterpylori （1f. pylori） infection causes peptic and duodenal diseases in humans. Among a 32-protein family of outer membrane proteins, a porin-like protein, HopE, has been a subject of note, mainly for its c...Helicobacterpylori （1f. pylori） infection causes peptic and duodenal diseases in humans. Among a 32-protein family of outer membrane proteins, a porin-like protein, HopE, has been a subject of note, mainly for its conservative nature among 11. pylori, and for its potential as a vaccine candidate. To achieve stable surface expression of this host cell-toxic protein, hopE gene was introduced into pBAD expression system. After induction with arabinose, all 15 randomly-chosen E. coli LMG 194 colonies from 3 successive passages could express Hope protein, while only 1 from 5 E. coli colonies that contained lac operon-regulated plasmid encoding hopE gene could express HopE. Indirect immunofluorescence confirmed the expression of HopE on E. coli cell surface展开更多
In order to provide a rational research basis for clinical detection and genetic engineering vaccine, plasmid pET-28a (+) encoding both Porin gene PIA and PIB of Neisseria gonorrhoeae was constructed and a fusion prot...In order to provide a rational research basis for clinical detection and genetic engineering vaccine, plasmid pET-28a (+) encoding both Porin gene PIA and PIB of Neisseria gonorrhoeae was constructed and a fusion protein in E.coli DE3 expressed. The fragments of PIA and PIB gene of Neisseria gonorrhoeae were amplified and cloned into prokaryotic expression plasmid pET-28a(+) with double restriction endonuclease cut to construct recombinant pET-PIB-PIA. The recombinant was verified with restriction endonuclease and sequenced and transformed into E.coli DE3 to express the fusion protein PIB-PIA after induced with IPTG. The results showed PIA-PIB fusion DNA fragment was proved correct through sequencing. A 67 kD (1 kD=0 992 1 ku) fusion protein had been detected by SDS-PAGE. It was concluded that the fusion protein was successively expressed.展开更多
A prokaryotic expression recombinant plasmid pET-PIB to express porin B (PIB) of Neisseria gonorrhoeae in E.coli DE_3 was constructed in order to provide a basis of research in detection, prophylactic and therapeutic ...A prokaryotic expression recombinant plasmid pET-PIB to express porin B (PIB) of Neisseria gonorrhoeae in E.coli DE_3 was constructed in order to provide a basis of research in detection, prophylactic and therapeutic vaccine against the pathogen infection. The gene encoding PIB was amplified by PCR from Neisseria gonorrhoeae and cloned into prokaryotic expression plasmid pET-28a(+) to construct a pET-PIB recombinant, which was verified by restriction endonuclease and DNA sequencing. Protein PIB was expressed in E.coli DE_3 induced with IPTG. The antigenicity of the expressed protein was evaluated by indirect ELISA. Rabbits were immunized with the protein and serum was collected after immunization. To assess the immunogenicity of the protein, the titer of serum to protein PIB was determined by ELISA. DNA sequence analysis showed that the nucleic acid sequence of PIB gene was 99.28 % of homology compared with that (NGPIB18) published in GenBank. A 41 kD fused protein was detected by SDS-PAGE and was proven to have reactivity with anti-PIB polyclonal antibody from mouse. A polyclonal antibody to PIB of 1:4000 titer determined by indirect ELISA was obtained from rabbit immunized with the purified product. Recombinant plasmid encoding PIB of Neisseria gonorrhoeae was constructed. Protein PIB with antigenicity and immunogenicity was successfully expressed.展开更多
目的分析浙江地区淋病奈瑟菌株外膜孔蛋白PⅠ基因序列及其点突变与耐药性关系,了解本地区淋病奈瑟菌株PⅠA与PⅠB基因分布及其优势基因型。方法采用高保真PCR扩增34株淋病奈瑟菌全长PⅠ基因序列,T-A克隆后测序。根据测序结果,建立多重PC...目的分析浙江地区淋病奈瑟菌株外膜孔蛋白PⅠ基因序列及其点突变与耐药性关系,了解本地区淋病奈瑟菌株PⅠA与PⅠB基因分布及其优势基因型。方法采用高保真PCR扩增34株淋病奈瑟菌全长PⅠ基因序列,T-A克隆后测序。根据测序结果,建立多重PCR同时检测113株淋病奈瑟菌PⅠA和PⅠB基因。分析测序菌株PⅠB基因中与耐药性密切相关的G120和A121变异情况,采用二倍琼脂稀释法确定PⅠB菌株的耐药性。结果11株PⅠA+淋病奈瑟菌均为ⅠA6血清型。23株PⅠB+淋病奈瑟菌中,6株(26．1％)为ⅠB3血清型、5株(21．7％)为ⅠB3／6血清型、2株(8．7％)为ⅠB4血清型、9株(39．1％)为ⅠB3／6-ⅠB2嵌合血清型、1株(4．3％)为ⅠB2-ⅠB4嵌合血清型。所建立的多重PCR可特异和准确地对PⅠA和PⅠB基因分型,检测灵敏度为10 ng DNA模板。113株淋病奈瑟菌中,26株(23．0％)和87株(77．0％)分别携带PⅠA和PⅠB基因。经测序的23株PⅠB+淋病奈瑟菌中,1株(4．3％)G120和A121均未突变,3株(13．0％)G120或A121突变,2株8．7％)G120突变但A121缺失,17株(73．9％)双位点突变。22株G120和／或A121突变的PⅠB+菌株均对青霉素和四环素耐药。结论所建立的多重PCR可用于淋病奈瑟菌PⅠA和PⅠB基因的分型检测。本地区流行的淋病奈瑟菌主要携带PⅠB基因。ⅠA6为PⅠA菌株优势血清型。PⅠB菌株以ⅠB3／6-ⅠB2嵌合、ⅠB3和ⅠB3／6血清型常见,但主要为耐药性G120和,或A121突变株。展开更多
Background Carbapenems are an important class of drugs for the treatment of Pseudomonas aeruginosa(P. aeruginosa) infections. However, carbapenem resistance has been commonly observed in nonfermenter species of bacter...Background Carbapenems are an important class of drugs for the treatment of Pseudomonas aeruginosa(P. aeruginosa) infections. However, carbapenem resistance has been commonly observed in nonfermenter species of bacteria. The purpose of this study was to investigate the molecular epidemiology and carbapenem resistant mechanisms of P. aeruginosa isolated from a surgical intensive care unit(SICU) in China. Methods The molecular typing was analyzed by REP-PCR. Enzyme activity was measured with a 260 nm wavelength spectrophotometer. The levels of outer membrane proteins OprD and OprN were measured by Western blotting. The levels of mexA gene transcriptional expression were measured by quantitative real-time PCR. The metallo-beta-lactamase genes IMP, VIM, SPM, GES, and GIM were amplified by PCR. DNA fragments were sequenced by an automated ABI PRISM 3700.Results Forty-two strains resistant to carbapenems isolated from a SICU were analyzed. REP-PCR revealed 34 belonging to type A, a predominant strain in this SICU. But we did not find metallo-beta-lactamases IMP, VIM, SPM, GES, or GIM genes by PCR. With a three-dimensional extract test, we found 34 strains producing high levels of AmpC enzymes. We also observed the activity of beta-lactamases enzymes in the imipenem resistant group, which was statistically different from the sensitive group. Western blotting revealed that 23 strains showed loss of OprD, 18 strains had decreased OprD expression, and 14 strains expressed OprN. We discovered 27 strains that overexpressed mexA by quantitative real-time PCR, and the resistance rate to meropenem was statistically different between the overexpressing group and the lowexpressing group. Nucleotide sequences and deduced amino acid sequence analysis revealed that eight strains carried mutations in the mexR gene operon down regulating MexAB-OprM. The nucleotide sequences of mexR genes from PA36, PA41 and PA48 were submitted to the Genebank with accession numbers of AY899299, AY899300, and AY899301. Conclusions There was a predominant strain in the SICU of our hospital. Imipenem resistance is mainly mediated by OprD deficiency or loss, and high activity AmpC enzymes. Overexpression of MexAB-OprM is one of the mechanisms of meropenem resistance, which are partly upregulated by mutations in the mexR gene. The expression of MexEF-OprN also plays an important role in the carbapenem resistance.展开更多
Shwachman-Diamond syndrome （SDS） is a multi-system disorder characterized by bone marrow failure, pancreatic insufficiency, skeletal abnormalities, and increased risk of leukemic transformation. Most patients with S...Shwachman-Diamond syndrome （SDS） is a multi-system disorder characterized by bone marrow failure, pancreatic insufficiency, skeletal abnormalities, and increased risk of leukemic transformation. Most patients with SDS contain mutations in the Shwachman- Bodian-Diamond syndrome gene （SBDS）, encoding a highly conserved protein that has been implicated in ribosome biogenesis. Emerging evidence also suggests a distinct role of SBDS beyond protein translation. Using the yeast model of SDS, we examined the underlying mechanisms that cause cells lacking Sdolp, the yeast SBDS ortholog, to exhibit reduced tolerance to various stress conditions. Our analysis indicates that the environmental stress response （ESR）, heat shock response （HSR）, and endoplasmic reticulum unfolded protein response （UPR） of sdolA cells are functional and that defects in these pathways do not produce the phenotypes observed in sdolh yeast. Depletion of mitochondlial DNA （mtDNA） was observed in sdolh cells, and this is a probable cause of the mitochondrial insufficiency in SDS. Prior disruption of POR1, encoding the mitochondrial voltage dependent anion channel （VDAC）, abrogated the effects of SD01 deletion and substantially restored resistance to environmental stressors and protected against damage to mtDNA. Conversely, wild-type cells over-expressing POR1 exhibited growth impairment and increased stress sensitivity similar to that seen in sdolA cells. Overall, our results suggest that specific VDAC inhibitors may have therapeutic benefits for SDS patients.展开更多
基金Supported by Grants FONDECYT #1030894 and 1085232 and FONDEF DO2I-1067 from CONICYT,Chile
文摘AIM:To evaluate how widely Helicobacter pylori (H. pylori ) HopE and HopV porins are expressed among Chilean isolates and how seroprevalent they are among infected patients in Chile.METHODS: H. pylori hopE and hopV genes derived from strain CHCTX-1 were cloned by polymerase chain reaction (PCR), sequenced and expressed in Escherichia coli AD494 (DE3). Gel-purified porins were used to prepare polyclonal antibodies. The presence of both genes was tested by PCR in a collection of H. pylori clinical isolates and their expression was detected in lysates by immunoblotting. Immune responses against HopE, HopV and other H. pylori antigens in sera from infected and non-infected patients were tested by Western blotting using these sera as f irst antibody on recombinant H. pylori antigens.RESULTS: PCR and Western blotting assays revealed that 60 and 82 out of 130 Chilean isolates carried hopE and hopV genes, respectively, but only 16 and 9, respectively, expressed these porins. IgG serum immunoreactivity evaluation of 69 H. pylori-infected patients revealed that HopE and HopV were infrequently recognized (8.7% and 10.1% respectively) compared to H. pylori VacA (68.1%) and CagA (59.5%) antigens. Similar values were detected for IgA serum immunoreactivity against HopE (11.6%) and HopV (10.5%) although lower values for VacA (42%) and CagA (17.4%) were obtained when compared to the IgG response.CONCLUSION: A scarce expression of HopE and HopV among Chilean isolates was found, in agreement with the infrequent seroconversion against these antigens when tested in infected Chilean patients.
文摘Helicobacterpylori （1f. pylori） infection causes peptic and duodenal diseases in humans. Among a 32-protein family of outer membrane proteins, a porin-like protein, HopE, has been a subject of note, mainly for its conservative nature among 11. pylori, and for its potential as a vaccine candidate. To achieve stable surface expression of this host cell-toxic protein, hopE gene was introduced into pBAD expression system. After induction with arabinose, all 15 randomly-chosen E. coli LMG 194 colonies from 3 successive passages could express Hope protein, while only 1 from 5 E. coli colonies that contained lac operon-regulated plasmid encoding hopE gene could express HopE. Indirect immunofluorescence confirmed the expression of HopE on E. coli cell surface
文摘In order to provide a rational research basis for clinical detection and genetic engineering vaccine, plasmid pET-28a (+) encoding both Porin gene PIA and PIB of Neisseria gonorrhoeae was constructed and a fusion protein in E.coli DE3 expressed. The fragments of PIA and PIB gene of Neisseria gonorrhoeae were amplified and cloned into prokaryotic expression plasmid pET-28a(+) with double restriction endonuclease cut to construct recombinant pET-PIB-PIA. The recombinant was verified with restriction endonuclease and sequenced and transformed into E.coli DE3 to express the fusion protein PIB-PIA after induced with IPTG. The results showed PIA-PIB fusion DNA fragment was proved correct through sequencing. A 67 kD (1 kD=0 992 1 ku) fusion protein had been detected by SDS-PAGE. It was concluded that the fusion protein was successively expressed.
文摘A prokaryotic expression recombinant plasmid pET-PIB to express porin B (PIB) of Neisseria gonorrhoeae in E.coli DE_3 was constructed in order to provide a basis of research in detection, prophylactic and therapeutic vaccine against the pathogen infection. The gene encoding PIB was amplified by PCR from Neisseria gonorrhoeae and cloned into prokaryotic expression plasmid pET-28a(+) to construct a pET-PIB recombinant, which was verified by restriction endonuclease and DNA sequencing. Protein PIB was expressed in E.coli DE_3 induced with IPTG. The antigenicity of the expressed protein was evaluated by indirect ELISA. Rabbits were immunized with the protein and serum was collected after immunization. To assess the immunogenicity of the protein, the titer of serum to protein PIB was determined by ELISA. DNA sequence analysis showed that the nucleic acid sequence of PIB gene was 99.28 % of homology compared with that (NGPIB18) published in GenBank. A 41 kD fused protein was detected by SDS-PAGE and was proven to have reactivity with anti-PIB polyclonal antibody from mouse. A polyclonal antibody to PIB of 1:4000 titer determined by indirect ELISA was obtained from rabbit immunized with the purified product. Recombinant plasmid encoding PIB of Neisseria gonorrhoeae was constructed. Protein PIB with antigenicity and immunogenicity was successfully expressed.
文摘目的分析浙江地区淋病奈瑟菌株外膜孔蛋白PⅠ基因序列及其点突变与耐药性关系,了解本地区淋病奈瑟菌株PⅠA与PⅠB基因分布及其优势基因型。方法采用高保真PCR扩增34株淋病奈瑟菌全长PⅠ基因序列,T-A克隆后测序。根据测序结果,建立多重PCR同时检测113株淋病奈瑟菌PⅠA和PⅠB基因。分析测序菌株PⅠB基因中与耐药性密切相关的G120和A121变异情况,采用二倍琼脂稀释法确定PⅠB菌株的耐药性。结果11株PⅠA+淋病奈瑟菌均为ⅠA6血清型。23株PⅠB+淋病奈瑟菌中,6株(26．1％)为ⅠB3血清型、5株(21．7％)为ⅠB3／6血清型、2株(8．7％)为ⅠB4血清型、9株(39．1％)为ⅠB3／6-ⅠB2嵌合血清型、1株(4．3％)为ⅠB2-ⅠB4嵌合血清型。所建立的多重PCR可特异和准确地对PⅠA和PⅠB基因分型,检测灵敏度为10 ng DNA模板。113株淋病奈瑟菌中,26株(23．0％)和87株(77．0％)分别携带PⅠA和PⅠB基因。经测序的23株PⅠB+淋病奈瑟菌中,1株(4．3％)G120和A121均未突变,3株(13．0％)G120或A121突变,2株8．7％)G120突变但A121缺失,17株(73．9％)双位点突变。22株G120和／或A121突变的PⅠB+菌株均对青霉素和四环素耐药。结论所建立的多重PCR可用于淋病奈瑟菌PⅠA和PⅠB基因的分型检测。本地区流行的淋病奈瑟菌主要携带PⅠB基因。ⅠA6为PⅠA菌株优势血清型。PⅠB菌株以ⅠB3／6-ⅠB2嵌合、ⅠB3和ⅠB3／6血清型常见,但主要为耐药性G120和,或A121突变株。
基金This work was supported by grants from the National ＂863＂ High Technology Research and Development Project of China （No. 2002AA2Z341D）, National Natural Science Foundation of China （No. 30940080）, and Foundation of China-Japan Friendship Hospital, Ministry of Health （No. 2010-ms-54）.
文摘Background Carbapenems are an important class of drugs for the treatment of Pseudomonas aeruginosa(P. aeruginosa) infections. However, carbapenem resistance has been commonly observed in nonfermenter species of bacteria. The purpose of this study was to investigate the molecular epidemiology and carbapenem resistant mechanisms of P. aeruginosa isolated from a surgical intensive care unit(SICU) in China. Methods The molecular typing was analyzed by REP-PCR. Enzyme activity was measured with a 260 nm wavelength spectrophotometer. The levels of outer membrane proteins OprD and OprN were measured by Western blotting. The levels of mexA gene transcriptional expression were measured by quantitative real-time PCR. The metallo-beta-lactamase genes IMP, VIM, SPM, GES, and GIM were amplified by PCR. DNA fragments were sequenced by an automated ABI PRISM 3700.Results Forty-two strains resistant to carbapenems isolated from a SICU were analyzed. REP-PCR revealed 34 belonging to type A, a predominant strain in this SICU. But we did not find metallo-beta-lactamases IMP, VIM, SPM, GES, or GIM genes by PCR. With a three-dimensional extract test, we found 34 strains producing high levels of AmpC enzymes. We also observed the activity of beta-lactamases enzymes in the imipenem resistant group, which was statistically different from the sensitive group. Western blotting revealed that 23 strains showed loss of OprD, 18 strains had decreased OprD expression, and 14 strains expressed OprN. We discovered 27 strains that overexpressed mexA by quantitative real-time PCR, and the resistance rate to meropenem was statistically different between the overexpressing group and the lowexpressing group. Nucleotide sequences and deduced amino acid sequence analysis revealed that eight strains carried mutations in the mexR gene operon down regulating MexAB-OprM. The nucleotide sequences of mexR genes from PA36, PA41 and PA48 were submitted to the Genebank with accession numbers of AY899299, AY899300, and AY899301. Conclusions There was a predominant strain in the SICU of our hospital. Imipenem resistance is mainly mediated by OprD deficiency or loss, and high activity AmpC enzymes. Overexpression of MexAB-OprM is one of the mechanisms of meropenem resistance, which are partly upregulated by mutations in the mexR gene. The expression of MexEF-OprN also plays an important role in the carbapenem resistance.
基金supported by the Faculty of Science,Mahidol University(ANJ)
文摘Shwachman-Diamond syndrome （SDS） is a multi-system disorder characterized by bone marrow failure, pancreatic insufficiency, skeletal abnormalities, and increased risk of leukemic transformation. Most patients with SDS contain mutations in the Shwachman- Bodian-Diamond syndrome gene （SBDS）, encoding a highly conserved protein that has been implicated in ribosome biogenesis. Emerging evidence also suggests a distinct role of SBDS beyond protein translation. Using the yeast model of SDS, we examined the underlying mechanisms that cause cells lacking Sdolp, the yeast SBDS ortholog, to exhibit reduced tolerance to various stress conditions. Our analysis indicates that the environmental stress response （ESR）, heat shock response （HSR）, and endoplasmic reticulum unfolded protein response （UPR） of sdolA cells are functional and that defects in these pathways do not produce the phenotypes observed in sdolh yeast. Depletion of mitochondlial DNA （mtDNA） was observed in sdolh cells, and this is a probable cause of the mitochondrial insufficiency in SDS. Prior disruption of POR1, encoding the mitochondrial voltage dependent anion channel （VDAC）, abrogated the effects of SD01 deletion and substantially restored resistance to environmental stressors and protected against damage to mtDNA. Conversely, wild-type cells over-expressing POR1 exhibited growth impairment and increased stress sensitivity similar to that seen in sdolA cells. Overall, our results suggest that specific VDAC inhibitors may have therapeutic benefits for SDS patients.
