AIM: Bacillary dysentery caused by Shigella flexneriis still a threat to human health. Of four invasion plasmid antigen proteins (IpaA, B,C and D), IpaC plays an important role in the pathogenicity of this pathogen. T...AIM: Bacillary dysentery caused by Shigella flexneriis still a threat to human health. Of four invasion plasmid antigen proteins (IpaA, B,C and D), IpaC plays an important role in the pathogenicity of this pathogen. The purpose of this study was to investigate the proteins interacting with IpaC in the host cell during the pathogenic process of this disease.
METHODS: By applying two-hybrid system, the bait plasmid containing ipaC gene was constructed and designated pGBKr-ipaC. The bait plasmid was transformed AH109, and proved to express IpaC and then HeLa cDNA library plasmids were introduced into the above transformed AHL09. The transformation mixture was plated on medium lacking Trp, Leu, and His in the initial screen, then restreaked on medium lacking Trp, Leu, His and Ade. Colonies growing on the selection medium were further assayed for β-galactosidase activity. BLAST was carried out in the database after sequencing the inserted cDNA of the positive library plasmid.
RESULTS: Among the 2x106 transformants, 64 positive clones were obtained as determined by activation of His,Ade and LacZ reporter genes. Sequence analysis revealed that cDNA inserts of two colonies were highly homologous to a known human protein, RanBPM.
CONCLUSION: These results provide evidence that IpaC may be involved in the invasion process of S. flexneri by interacting with RanBPM, and RanBPM is most likely to be the downstream target of IpaC in the cascade events of S.flexneri infection.展开更多
AIM: In vivo induced genes are thought to play an important role during infection of host. ,AlkA was identified as an in vivo-induced gene by in vivo expression technology (IVET),but its virulence in Shigella flexneri...AIM: In vivo induced genes are thought to play an important role during infection of host. ,AlkA was identified as an in vivo-induced gene by in vivo expression technology (IVET),but its virulence in Shigella flexneri was not reported. The purpose of this study was to identify the role of alkA gene in the pathogenesis of S. flexneriMETHODS: PCR was used to amplify alkA gene of S. flexneri 2a and fragment 028pKm. The fragment was then transformed into 2457T05 strain, a S flexneri 2a strain containing Red recombination system, which was constructed with a recombinant suicide plasmid pXLkd46. By in vivo homologous recombination, alkA mutants were obtained and verified by PCR and sequencing. Intracellular survival assay and virulence assay were used to test the intracellular survival ability in HeLa cell model and the virulence in mice lung infection model respectively.RESULTS: Deletion mutant of S. flexneri 2a alkA was successfully constructed by λ Red recombination system.The mutant exhibited significant survival defects and much significant virulence defects in mice infection assay.CONCLUSION: AlkA gene plays an important role in the infection of epithelial cells and is a virulent gene of Shigella spp.展开更多
基金Capital"248"Key Innovation Project(H010210360119)State key Research and Development Project(G1999054103)
文摘AIM: Bacillary dysentery caused by Shigella flexneriis still a threat to human health. Of four invasion plasmid antigen proteins (IpaA, B,C and D), IpaC plays an important role in the pathogenicity of this pathogen. The purpose of this study was to investigate the proteins interacting with IpaC in the host cell during the pathogenic process of this disease.
METHODS: By applying two-hybrid system, the bait plasmid containing ipaC gene was constructed and designated pGBKr-ipaC. The bait plasmid was transformed AH109, and proved to express IpaC and then HeLa cDNA library plasmids were introduced into the above transformed AHL09. The transformation mixture was plated on medium lacking Trp, Leu, and His in the initial screen, then restreaked on medium lacking Trp, Leu, His and Ade. Colonies growing on the selection medium were further assayed for β-galactosidase activity. BLAST was carried out in the database after sequencing the inserted cDNA of the positive library plasmid.
RESULTS: Among the 2x106 transformants, 64 positive clones were obtained as determined by activation of His,Ade and LacZ reporter genes. Sequence analysis revealed that cDNA inserts of two colonies were highly homologous to a known human protein, RanBPM.
CONCLUSION: These results provide evidence that IpaC may be involved in the invasion process of S. flexneri by interacting with RanBPM, and RanBPM is most likely to be the downstream target of IpaC in the cascade events of S.flexneri infection.
基金the Major State Basic Research Development Program of China(973 Program),No.G1999054103National Hightechnology Research and Development Program of China(863 Program),No.2001AA215211Capital"248"Key Innovation Program of China,No.H010210360119
文摘AIM: In vivo induced genes are thought to play an important role during infection of host. ,AlkA was identified as an in vivo-induced gene by in vivo expression technology (IVET),but its virulence in Shigella flexneri was not reported. The purpose of this study was to identify the role of alkA gene in the pathogenesis of S. flexneriMETHODS: PCR was used to amplify alkA gene of S. flexneri 2a and fragment 028pKm. The fragment was then transformed into 2457T05 strain, a S flexneri 2a strain containing Red recombination system, which was constructed with a recombinant suicide plasmid pXLkd46. By in vivo homologous recombination, alkA mutants were obtained and verified by PCR and sequencing. Intracellular survival assay and virulence assay were used to test the intracellular survival ability in HeLa cell model and the virulence in mice lung infection model respectively.RESULTS: Deletion mutant of S. flexneri 2a alkA was successfully constructed by λ Red recombination system.The mutant exhibited significant survival defects and much significant virulence defects in mice infection assay.CONCLUSION: AlkA gene plays an important role in the infection of epithelial cells and is a virulent gene of Shigella spp.
