| Summary: | 碩士 === 國立臺灣大學 === 植物學研究所 === 91 === P56, a protein with a molecular weight of 56 kDa, was previously isolated and purified from the cell-free supernatant of Salmonella choleraesuis CH12440 culture in this laboratory. It causes the degeneration and necrosis of mouse macrophage cell lines, and was regarded as a cytotoxin. Comparison of amino acids sequence with other protein, P56 was found to be a flagellin, the product of fliC gene. It has a 99.2% similarity of nucleotide sequence and a 98.6% similarity of amino acid sequence with the fliC gene of other S. choleraesuis. Furthermore, P56 contains a sequence of 12 amino acids that is absent in other fliC genes. This region has been suspected to be the toxic region. The subject of this study was to testify this hypothesis using the deletion mutants. The analysis of the toxicity of normal and mutant proteins were performed on mouse macrophage cell lines by MTT assay. The results indicated that the deletion of the first 4 amino acids of the region and retain the 4 amino acids of the middle region had a decrease survival rate of cells. The deletions of the first 8 amino acids and all 12 amino acids in the region had no effect on the survival of cells. These results suggested that the sequence of the later 8 amino acids could be responsible for the toxicity of the protein P56. The three-dimensional structure of P56 suggested 7 amino acids of the toxic region could be the active sites, especially the following ones: Gly250, Val251, Ala252, Asp253, Ala254, Glu255, and Ala256.
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