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
Of ICE protease family, CPP32 (apopain or Yama) plays a central role in different apoptotic pathways. To study the molecules regulating CPP32, yeast two_hybrid system was used to identify proteins (peptides) that inte...Of ICE protease family, CPP32 (apopain or Yama) plays a central role in different apoptotic pathways. To study the molecules regulating CPP32, yeast two_hybrid system was used to identify proteins (peptides) that interact with CPP32. First, the CPP32 gene was cloned into plasmid vector pGBT9. The resulting recombinant plasmid was designated as pGBT9/CPP. The pGBT9 /CPP plasmid was transformed into the yeast strain HF7C, then the leukemia library was introduced. The transformation mixture was plated on medium lacking Trp, Leu and His in the initial screen. Colonies growing on the selection medium were further assayed for β galactosidase activity. Within 42 Trp +Leu +His + colonies only 5 turned blue in the presence of X_Gal. Plasmid DNA from 5 positive yeast colonies was prepared respectively and used to transform HB101 by electroporation. The transformation mixture was plated on medium lacking Leu to be selected for the library plasmid. Finally, only one library plasmid, designated as pY1, was determined to be truly positive by retransformation of pGBT9/CPP and the library plasmid into HF7C. The inserted cDNA of pY1 encodes a peptide of 15 amino acids, suggesting that it may be the domain interacting with CPP32.展开更多
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.
文摘Of ICE protease family, CPP32 (apopain or Yama) plays a central role in different apoptotic pathways. To study the molecules regulating CPP32, yeast two_hybrid system was used to identify proteins (peptides) that interact with CPP32. First, the CPP32 gene was cloned into plasmid vector pGBT9. The resulting recombinant plasmid was designated as pGBT9/CPP. The pGBT9 /CPP plasmid was transformed into the yeast strain HF7C, then the leukemia library was introduced. The transformation mixture was plated on medium lacking Trp, Leu and His in the initial screen. Colonies growing on the selection medium were further assayed for β galactosidase activity. Within 42 Trp +Leu +His + colonies only 5 turned blue in the presence of X_Gal. Plasmid DNA from 5 positive yeast colonies was prepared respectively and used to transform HB101 by electroporation. The transformation mixture was plated on medium lacking Leu to be selected for the library plasmid. Finally, only one library plasmid, designated as pY1, was determined to be truly positive by retransformation of pGBT9/CPP and the library plasmid into HF7C. The inserted cDNA of pY1 encodes a peptide of 15 amino acids, suggesting that it may be the domain interacting with CPP32.
