Kinetic analysis of phage infection towered phage therapy

T-even型噬菌体PP01以外膜蛋白OmpC为受体，特异性感染大肠杆菌O157: H7。由于ompC上游14 kbp区域的缺失，ppo01耐药细胞失去了ompC的表达。分离了两个感染OmpG零突变体的宿主范围突变体，并从两个宿主范围突变体中测序了编码受体识别蛋白Gp38的基因38。根据推导出的氨基酸序列，在Gp38中发现了突变。最常见的突变将161位的谷氨酰胺和208位的甘氨酸转变为碱性氨基酸。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，在噬菌体衣壳上呈递标记蛋白绿色荧光蛋白（GFP）。用PCR扩增出soc周围的DNA片段并测序。soc及其上游区基因序列为g56-soc.2-soc。1-soc与T2噬菌体相同。GFP被引入到c端和n端。重组噬菌体分别为PP01-GFP/SOC和PP01-SOC/GFP。GFP与SOC的融合没有改变PP01的宿主范围。另一方面，重组噬菌体对宿主细胞的结合亲和力增强。然而，重组噬菌体在碱性溶液中的稳定性降低。将GFP标记的噬菌体吸附在大肠杆菌细胞表面，通过荧光显微镜观察细胞。GFP标记的PP01不仅吸附在可培养的e.c coil细胞上，而且吸附在活细胞和非培养细胞（VBNC）和巴氏灭菌细胞上。不敏感细胞e.c coil 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噬菌体能够快速、灵敏地检测大肠杆菌O157: H7。

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.