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
Conjugate vaccines represent one of the most effective means for controlling the occurrence of bacterial diseases.Although nanotechnology has been greatly applied in the field of vaccines,it is seldom used for conjuga...Conjugate vaccines represent one of the most effective means for controlling the occurrence of bacterial diseases.Although nanotechnology has been greatly applied in the field of vaccines,it is seldom used for conjugate vaccine research because it is very difficult to connect polysaccharides and nanocarriers.In this work,an orthogonal and modular biosynthesis method was used to produce nanoconjugate vaccines using the SpyTag/SpyCatcher system.When SpyTag/SpyCatcher system is combined with protein glycosylation technology,bacterial O-polysaccharide obtained from Shigela flexneri 2a can be conjugated onto the surfaces of different virus-like particles(VLPs)in a biocompatible and controlled manner.After confirming the excellent lymph node targeting and humoral immune activation abilities,these nanoconjugate vaccines further induced efficient prophylactic effects against infection in a mouse model.These results demonstrated that natural polysaccharide antigens can be easily connected to VLPs to prepare highly efficient nanoconjugate vaccines.To the best of the researchers1 knowledge,this is the first time VLP-based nanoconjugate vaccines are produced efficiently,and this strategy could be applied to develop various pathogenic nanoconjugate vaccines.展开更多
An in vivo expression technology (IVET) was applied to screen s.flexneri 2a genes induced after invasion of epithelial cells, and virulence-related genes were further identified by mutational analysis. Thirteen intrac...An in vivo expression technology (IVET) was applied to screen s.flexneri 2a genes induced after invasion of epithelial cells, and virulence-related genes were further identified by mutational analysis. Thirteen intracellular induced genes were identified with a HeLa cell infection model. Of these, two were identified as alkylation-related genes; one was related to metabolism; one encoded a transcriptional regulator; three were identified as insertion elements; three ap- peared to be antisense to genes involved in the transmethylation,biosyntheseis, and phos- photransferase system;and three were predicted to encode polypeptides with unknown functions. Intracellular survival assavs showed that the mutants of alkA,citC and wcaJ genes had lower capability of intracellular replication or survival than the the wild-type strain.The results indicated that alkA, citC and wcaJ genes could take part in the intracellular survival or replication of S. flexneri 2a and the capability of intracellular survival or replication could be one of the major virulence elements. However, the yaiC mutant was able to survive in the murine infection assay but almost not in HeLa cell infection assay. Very possibly, yaiC gene was involved in the other mechanism of S. flexneri virulence. This study might lead to a better understanding of the intra- cellular survival or proliferation process of S. flexneri 2a and perhaps provide insights into the pathogenicity of this pathogen.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
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
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 National Natural Science Foundation of China(Nos.81930122 and U20A20361)the National Key Research and Development Project of China(No.2021YFC2102101).
文摘Conjugate vaccines represent one of the most effective means for controlling the occurrence of bacterial diseases.Although nanotechnology has been greatly applied in the field of vaccines,it is seldom used for conjugate vaccine research because it is very difficult to connect polysaccharides and nanocarriers.In this work,an orthogonal and modular biosynthesis method was used to produce nanoconjugate vaccines using the SpyTag/SpyCatcher system.When SpyTag/SpyCatcher system is combined with protein glycosylation technology,bacterial O-polysaccharide obtained from Shigela flexneri 2a can be conjugated onto the surfaces of different virus-like particles(VLPs)in a biocompatible and controlled manner.After confirming the excellent lymph node targeting and humoral immune activation abilities,these nanoconjugate vaccines further induced efficient prophylactic effects against infection in a mouse model.These results demonstrated that natural polysaccharide antigens can be easily connected to VLPs to prepare highly efficient nanoconjugate vaccines.To the best of the researchers1 knowledge,this is the first time VLP-based nanoconjugate vaccines are produced efficiently,and this strategy could be applied to develop various pathogenic nanoconjugate vaccines.
文摘An in vivo expression technology (IVET) was applied to screen s.flexneri 2a genes induced after invasion of epithelial cells, and virulence-related genes were further identified by mutational analysis. Thirteen intracellular induced genes were identified with a HeLa cell infection model. Of these, two were identified as alkylation-related genes; one was related to metabolism; one encoded a transcriptional regulator; three were identified as insertion elements; three ap- peared to be antisense to genes involved in the transmethylation,biosyntheseis, and phos- photransferase system;and three were predicted to encode polypeptides with unknown functions. Intracellular survival assavs showed that the mutants of alkA,citC and wcaJ genes had lower capability of intracellular replication or survival than the the wild-type strain.The results indicated that alkA, citC and wcaJ genes could take part in the intracellular survival or replication of S. flexneri 2a and the capability of intracellular survival or replication could be one of the major virulence elements. However, the yaiC mutant was able to survive in the murine infection assay but almost not in HeLa cell infection assay. Very possibly, yaiC gene was involved in the other mechanism of S. flexneri virulence. This study might lead to a better understanding of the intra- cellular survival or proliferation process of S. flexneri 2a and perhaps provide insights into the pathogenicity of this pathogen.
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.
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.
