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Kinetic analysis of phage infection towered phage therapy

噬菌体感染塔式噬菌体疗法的动力学分析

基本信息

批准号：
13450340
负责人：
TANJI Yasunori
金额：
$ 10.3万
依托单位：
Tokyo Institute of Technology
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2003
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13450340/
关键词：
Bacteriophage E.colt O157: H7 Receptor Phage-therapy GFP Pathogenic E.coli Host cell recognition Fluorcent detection バクテリオファージ外膜タンパク質 LPS 病原性大腸菌O157 大腸菌

项目摘要

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.

T-Even型噬菌体PP01利用外膜蛋白OMPC作为受体，特异性感染大肠杆菌O157：H7。PPO01抗性细胞由于OMPC上游14kbp的缺失而失去了OMPC的表达。分离到两个感染OmpG缺失突变体的寄主范围突变体，并从这两个寄主范围突变体中对编码受体识别蛋白Gp38的基因38进行了测序。根据推导的氨基酸序列，发现Gp38基因发生了突变。最常见的突变将第161位的谷氨酰胺和第208位的甘氨酸改变为碱性氨基酸。PP01也用于检测其宿主细胞E.coil 0157：H7。以噬菌体衣壳蛋白(SOC)为平台，在噬菌体衣壳上呈现标志蛋白绿色荧光蛋白(GFP)。用聚合酶链式反应扩增SoC周围的DNA片段，并进行测序。SoC与其上游区域的基因序列为g56-so2-so1-soc，与T2噬菌体的基因序列一致。GFP被引入到C-末端和N-末端区域。分别生产重组噬菌体PP01-GFP/SOC和PP01-SOC/GFP。GFP与SOC的融合没有改变PP01的寄主范围。另一方面，重组噬菌体与宿主细胞的结合亲和力增加。但是，重组噬菌体在碱性溶液中的稳定性降低。将GFP标记的噬菌体吸附到大肠杆菌细胞表面，荧光显微镜观察到，GFP标记的PP01不仅可以吸附在可培养的E.COIL细胞上，而且还可以吸附在活的但不可培养的(VBNC)和巴氏杀菌的细胞上。不敏感细胞K12(W3110)的共存不影响GFP标记的PPO01在大肠杆菌O157：H7上吸附的特异性和亲和力。GFP标记的PP01噬菌体可快速、灵敏地检测O157：H7大肠杆菌。