| Co-Investigator(Kenkyū-buntansha) |
MIYANAGA Kazuhiko Tokyo Institute of Technology, Graduate School of Bioscience and Biotechnology, Assistant Prof., 大学院・生命理工学研究科, 助手 (40323810)
HORI Kastutoshi Tokyo Institute of Technology, Graduate School of Bioscience and Biotechnology, Assistant Prof., 大学院・生命理工学研究科, 助手 (50302956)
UNNO Hajime Tokyo Institute of Technology, Graduate School of Bioscience and Biotechnology, Professor, 大学院・生命理工学研究科, 教授 (10087471)
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| Research Abstract |
T-even type coliphage PP01, which specifically infects to Escherichia coil O157: H7, uses the outer membrane protein OmpC as a receptor. The PPO01-resistant cells had lost ompC expression due to the deletion of a 14 kbp region upstream of ompC. Two host range mutants, infective to the OmpG null mutant, were isolated and gene 38, which codes for the receptor recognition protein Gp38, was sequenced from both host range mutant. According to the deduced amino acid sequence, the mutational alterations were found in Gp38. The most common mutations changed glutamine at position 161 and glycine at Dosition 208 into basic amino acids.
PP01 was also used to detect its host cell, E.coil 0157: H7. Phage capsid protein, SOC, was used as a platform to present marker protein, green fluorescent protein (GFP), on the phage capsid. DNA fragment around soc was amplified by PCR and sequenced. The gene alignment of soc and its upstream region was g56-soc.2-soc.1-soc which was the same as that of T2 phage. GFP was introduced into the C-and N-terminal regions. of SOC to produce, recombinant phages, PP01-GFP/SOC and PP01-SOC/GFP, respectively. Fusion of GFP to the SOC did not change host range of PP01. On the other hand, binding affinity of the recombinant phages to the host cell increased. However, stability of the recombinant phages in alkaline solution was reduced. Adsorption of the GFP labeled phages to the E.coli cell surface visualized the cells through fluorescent microscopy., GFP labeled PP01 adsorbed not only on the culturable E.coil cell but also viable but nonculturable (VBNC) and pasteurized cells. Coexistence of insensitive cell, E.coil K12(W3110), did not influence specificity and affinity of GFP labeled PPO01 adsorption on E coli O157: H7. GFP labeled PP01 phage could be rapid and sensitive toll of E.coil O157: H7 detection.
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