Arcanobacterium pyogenes phage and medical application thereof
A technology of Cryptobacterium pyogenes and phages, which is applied in the field of bioengineering, can solve the problem of not finding Cryptobacterium pyogenes phages, etc., and achieve the effect of strong lytic activity
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Embodiment 1
[0019] Phage isolation and preparation
[0020] The isolation procedure of phage is described in detail below. The manure sewage sample in the present invention is collected from Huoshao Li in Changchun City, and the host bacteria is Cryptobacterium pyogenes BS. Collect sewage, filter it with gauze, centrifuge at 6000r / min for 10min, take the supernatant, and use treated sewage instead of ddH2O to prepare LB medium (100mL); add 1mL of overnight cultured host bacteria BS to the medium, and incubate at 37°C for 10-12h Take 1 mL of the culture, centrifuge at 12000r / min for 5min, filter the supernatant with a 0.22 μm filter to form a phage stock solution and save it, and use the obtained filtrate for a plaque test to check whether it includes phage capable of lysing Cryptobacter pyogenes.
[0021] Plaque test: Inoculate Cryptobacterium pyogenes BS in 5ml LB liquid medium at a ratio of 2%, and cultivate overnight at 37°C with shaking. Take 0.1ml of the bacterial culture solution ...
Embodiment 2
[0024] Phage amplification and purification
[0025] On the double-layer plate forming phage plaques, use the tip of a sterile pipette to pick up a single phage plaque with a large diameter, relatively round and translucent, inoculate it in 5 ml LB liquid medium, and add phage 200 μL of host bacterial solution, mix well, act at room temperature for 15 minutes, incubate at 37°C for 10-14 hours, centrifuge at 12,000 rpm, 4°C for 10 minutes, and take the supernatant; repeat the double-layer plate experiment, and pick a single phage plaque repeatedly in this way for 4-5 times. Phage were purified into plaques of equal size.
[0026] Take 1 mL of freshly cultured host bacteria and add 300 μL of phage lysate (the ratio of a single phage culture to host bacteria is 1:1, 1:10 and 1:100, respectively). Incubate at 37°C for 20 minutes to make the phage particles adsorb to the host bacteria; add 800mL LB liquid medium, then add CaCl 2 The mother solution was grown to a final concentra...
Embodiment 3
[0032] Transmission electron microscope observation of bacteriophage vB_ApyS_QF1
[0033]Take the PEG-purified phage in Example 2 for electron microscope observation. The specific operation steps are: add 10 μL of the sample and drop it on the copper grid, wait for it to settle for 15 minutes, absorb the excess liquid with filter paper, and use 2% phosphotungstic acid (PTA) Stain for 1-2min, observe with a transmission electron microscope (Hitachi H-7650) after drying; the observation results are as follows figure 2 As shown, the head is in the shape of an icosahedron, the diameter of the head is about 100 nm, and the length of the tail is about 320 nm. According to the "Classification of Viruses—The Eighth Report of the International Committee on Taxonomy of Viruses" published by the International Committee on Taxonomy of Viruses (ICTV) in 2005, vB_ApyS_QF1 belongs to the family Siphoviridae.
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