AIM： To screen the immunogenic membrane proteins of Shigella Aexneri 2a 2457T. METHODS： The routine two-dimensional polyacrylamide gel electrophoresis （2-DE） and Western blotting were combined to screen immunogeni...AIM： To screen the immunogenic membrane proteins of Shigella Aexneri 2a 2457T. METHODS： The routine two-dimensional polyacrylamide gel electrophoresis （2-DE） and Western blotting were combined to screen immunogenic proteins of S. Aexneri 2a 2457T. Serum was gained from rabbits immunized with the same bacteria. Immunogenic spots were cut out from the polyacrylamide gel and digested by trypsin in-gel. Matrix-assisted laser desorption/ionization time of flight-mass spectrometry （MALDI-TOF-MS） was performed to determine the molecular weight of peptides. Electrospray ionization （ESI-MS/MS） was performed to determine the sequences of the interesting peptides. RESULTS： A total of 20 spots were successfully identified from Coomassie brilliant blue stained gels representing 13 protein entries, 5 known antigens and 8 novel antigens. A hypothetical protein （YaeT） was detected, which might be a candidate target of vaccine. CONCLUSION： Membrane proteins of S. flexneri 2a 2457T were successfully observed by 2-DE. Several known and novel antigens were identified by mass spectrum.展开更多
Comparative genome analysis is performed between Shigella flexneri 2a strain 301 and its close relatives, the nonpathogenic E. Coli K-12 strain MG1655. Result shows that there are 136 DNA segments whose size is larger...Comparative genome analysis is performed between Shigella flexneri 2a strain 301 and its close relatives, the nonpathogenic E. Coli K-12 strain MG1655. Result shows that there are 136 DNA segments whose size is larger than 1000 bp absent from Shigella flexneri 2a strain 301, which is up to 717253 bp in total length. These deleted segments altogether contain 670 open reading frames (ORFs). Prediction of these ORFs indicates that there are 40% genes of unknown function. The other genes of definite functions encode metabolic enzymes, structure proteins, transcription regulatory factors and some elements correlated with horizontal transfer. Here we compare the complete genomic sequences of the two closely related species, which differ in pathogenic phenotype. To our knowledge, this not only reveals the difference of genomic sequence between the two important enteric pathogens for the first time, but also provides valuable clues to further researches in its process of physiological activity, pathogenesis and the evolution of enteric bacteria.展开更多
In order to overcome the defects of difficult gene operations in low-copy suicide plasmid pCVD442,Gateway technology was applied in the construction process of recombinant plasmid for gene knockout in this study.With ...In order to overcome the defects of difficult gene operations in low-copy suicide plasmid pCVD442,Gateway technology was applied in the construction process of recombinant plasmid for gene knockout in this study.With this improved knockout system,we inactivated sitC gene,which is associated with iron transport in Shigella flexneri 2a strain 301,to yield the mutant,MTS.The functional detection of the mutant was performed at the level of culture medium,cell and animal experiment,respectively.The gene expression profiles were compared with DNA microarray between the mutant and the wild type under iron-restricted conditions.The results showed that MTS grew obviously less well than the wild-type strains in L broth containing 150μmol/L iron chelator DIP(2,2′-dipyridyl).Addition of iron or manganese to the cultures stimulated the growth of MTS to wild-type levels in rich culture medium.In either the experiment on the ability of intracellular multiplication and cell-to-cell spread in HeLa and U937 cell lines,or the experiment on keratoconjunctivitis in guinea pigs,MTS showed no obvious changes in virulence compared with the parental strain Sf301.When 65μmol/L DIP was added to the cultured HeLa cells,the ability of intracellular multiplication of MTS reduced about 51.6%as compared with that of Sf301.The analysis of expression profiles under iron-limited condition showed that MTS was more sensitive for the change of iron deficiency than Sf301.There are 106 more up-regulated genes in MTS than in wild-type strains,which are involved in membrane transportation,amino acid metabolism and uncategorized function genes,while down-regulated genes are mainly in-volved in energy and carbohydrate metabolism.Under low iron conditions,the expression levels of known iron-transport associated genes generally increased.Additionally,the number of these genes and their increase amplitude in MTS are more than those in Sf301.Together,these results confirmed that Sit iron-transport system is important for the growth of Shigella.展开更多
基金Supported by the Capital "248" Key Innovation Project, No. H010210360119, State Basic Research Development Program of China No. 973 Program, G1999054103 and 2005CB22904 and National Natural Science Foundation of China No. 30470101
文摘AIM： To screen the immunogenic membrane proteins of Shigella Aexneri 2a 2457T. METHODS： The routine two-dimensional polyacrylamide gel electrophoresis （2-DE） and Western blotting were combined to screen immunogenic proteins of S. Aexneri 2a 2457T. Serum was gained from rabbits immunized with the same bacteria. Immunogenic spots were cut out from the polyacrylamide gel and digested by trypsin in-gel. Matrix-assisted laser desorption/ionization time of flight-mass spectrometry （MALDI-TOF-MS） was performed to determine the molecular weight of peptides. Electrospray ionization （ESI-MS/MS） was performed to determine the sequences of the interesting peptides. RESULTS： A total of 20 spots were successfully identified from Coomassie brilliant blue stained gels representing 13 protein entries, 5 known antigens and 8 novel antigens. A hypothetical protein （YaeT） was detected, which might be a candidate target of vaccine. CONCLUSION： Membrane proteins of S. flexneri 2a 2457T were successfully observed by 2-DE. Several known and novel antigens were identified by mass spectrum.
基金supported by the State“973”Key Basic Research Program(Grant No.G1999054103)the State“863”High-Tech Project(Grant No.Z19-02-05-01)+1 种基金Beijing Innovation Engineering(Grant No.955020700)the North China Pharmaceutical Corporation(NCPC)
文摘Comparative genome analysis is performed between Shigella flexneri 2a strain 301 and its close relatives, the nonpathogenic E. Coli K-12 strain MG1655. Result shows that there are 136 DNA segments whose size is larger than 1000 bp absent from Shigella flexneri 2a strain 301, which is up to 717253 bp in total length. These deleted segments altogether contain 670 open reading frames (ORFs). Prediction of these ORFs indicates that there are 40% genes of unknown function. The other genes of definite functions encode metabolic enzymes, structure proteins, transcription regulatory factors and some elements correlated with horizontal transfer. Here we compare the complete genomic sequences of the two closely related species, which differ in pathogenic phenotype. To our knowledge, this not only reveals the difference of genomic sequence between the two important enteric pathogens for the first time, but also provides valuable clues to further researches in its process of physiological activity, pathogenesis and the evolution of enteric bacteria.
基金This work was supported by the High Technology Project(Grant No.2001AA223011)the State Key Basic Research Program(Grant No.G1999054105).
文摘In order to overcome the defects of difficult gene operations in low-copy suicide plasmid pCVD442,Gateway technology was applied in the construction process of recombinant plasmid for gene knockout in this study.With this improved knockout system,we inactivated sitC gene,which is associated with iron transport in Shigella flexneri 2a strain 301,to yield the mutant,MTS.The functional detection of the mutant was performed at the level of culture medium,cell and animal experiment,respectively.The gene expression profiles were compared with DNA microarray between the mutant and the wild type under iron-restricted conditions.The results showed that MTS grew obviously less well than the wild-type strains in L broth containing 150μmol/L iron chelator DIP(2,2′-dipyridyl).Addition of iron or manganese to the cultures stimulated the growth of MTS to wild-type levels in rich culture medium.In either the experiment on the ability of intracellular multiplication and cell-to-cell spread in HeLa and U937 cell lines,or the experiment on keratoconjunctivitis in guinea pigs,MTS showed no obvious changes in virulence compared with the parental strain Sf301.When 65μmol/L DIP was added to the cultured HeLa cells,the ability of intracellular multiplication of MTS reduced about 51.6%as compared with that of Sf301.The analysis of expression profiles under iron-limited condition showed that MTS was more sensitive for the change of iron deficiency than Sf301.There are 106 more up-regulated genes in MTS than in wild-type strains,which are involved in membrane transportation,amino acid metabolism and uncategorized function genes,while down-regulated genes are mainly in-volved in energy and carbohydrate metabolism.Under low iron conditions,the expression levels of known iron-transport associated genes generally increased.Additionally,the number of these genes and their increase amplitude in MTS are more than those in Sf301.Together,these results confirmed that Sit iron-transport system is important for the growth of Shigella.
