A broad-spectrum Escherichia coli phage capable of simultaneously lysing four bacteria and its composition, kit and application
A technology of Escherichia coli and phage, applied in the direction of virus/phage, medical raw materials derived from virus/phage, application, etc., to achieve good stability
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Embodiment 1
[0067] Example 1 Separation, preparation and purification of Escherichia coli phage EC35P1
[0068] The source sample of Escherichia coliphage EC35P1 in this application was collected from domestic sewage in Jiangning District, Nanjing City, Jiangsu Province, filtered through double-layer filter paper, centrifuged at low speed at room temperature, and then filtered with a 0.22 μm filter membrane.
[0069] Phage isolation: Take 10 mL of the filtered supernatant, add 10 mL of 2-fold TSB medium, and add 1 mL of phage host bacteria EC35 log-phase bacterial solution at the same time. Centrifuge for 10 min under conditions, filter the supernatant with a 0.22 μm filter, and set aside. Take 0.5 mL of the phage host bacteria log-phase bacterial solution, add it to 5 mL of semi-solid TSB medium at 48°C, mix well, and pour it onto a TSA plate to prepare a double-layer plate containing the host bacteria. Take 10 μl of the filtered supernatant, drop it on the solidified double-layer plate...
Embodiment 2
[0071] Example 2 Electron microscope observation of Escherichia coli phage EC35P1
[0072] Take the supernatant of the phage EC35P1 culture prepared in Example 1 for electron microscope observation: take 20 μL of the sample and drop it on the copper grid, let it settle naturally for 15 minutes, use filter paper to absorb excess liquid from the side, add 1 drop of 2% phosphotungstic acid (PTA) On the copper mesh, dyed for 10min, sucked the dye solution from the side with filter paper, and observed by electron microscope after drying. The results are shown in Figure 2. The morphology of Escherichia coli phage EC35P1 was observed under an electron microscope, and it was found that it was a tailed phage, without folding, with a polyhedral symmetrical head and a retractable tail. The transverse diameter is about 50-60 nm; the length of the tail is about 95-110 nm, and the width of the tail is about 15-25 nm. Based on its unique size and morphology, E. coli phage EC35P1 is systemat...
Embodiment 3
[0074] Example 3 Preparation of Escherichia coli phage EC35P1 particles and extraction and sequencing of genome
[0075] Take 100 mL of phage EC35P1 prepared in Example 1, add DNaseI and RNaseA with a final concentration of 1 μg / mL, incubate at 37°C for 60 min, add 5.84 g NaCl (final concentration 1 mol / L), dissolve and place in an ice bath for 1 h. Centrifuge at 11,000 rpm for 10 min at 4°C, and transfer the supernatant to a new centrifuge tube. Solid polyethylene glycol (PEG8000) was added to a final concentration of 10% (w / v), and after it was completely dissolved, ice bath for at least 1 h. Centrifuge at 11,000 rpm for 20 min at 4°C, and resuspend the pellet with a small amount of SM solution to obtain a phage particle concentrate, which is stored at 4°C for later use.
[0076] Phage nucleic acid was extracted and sequenced using λ phage genomic DNA kit. After nucleotide sequencing, the Escherichia coliphage EC35P1 (Escherichia coliphage EC35P1) has the nucleotide sequen...
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