Light green balloon bacteriophage AVP and use thereof
A light green, phage technology, applied in the field of microorganisms, to achieve the effect of strong killing activity and wide cracking spectrum
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Embodiment 1
[0026] The phage of the present invention is a novel phage isolated from sewage, and the phage has an icosahedral head and a retractable tail; the phage can form translucent plaques on 1.5% BHI agar medium without halos around the edges, Clear and regular, with a diameter of 0.1-1mm; the restriction map of genomic nucleic acid shows that the phage nucleic acid is double-stranded DNA (dsDNA); it has been preserved in the China Center for Type Culture Collection on March 25, 2018, and the preservation number is CCTCC NO: M 2018155, Address: Wuhan University, Wuhan, China.
Embodiment 2
[0028] Phage isolation and preparation
[0029] The isolation procedure of phage is described in detail below. The fecal liquid sewage sample in the present invention is collected from Huoshao Li Sewage Ditch in Changchun City; Collect sewage, filter it with gauze, or centrifuge at 6000r / min for 10min, and take the supernatant; use the treated sewage instead of ddH2O to prepare BHI medium (100mL); add 1mL of overnight cultured bacteria to the medium, and incubate at 37°C for 10-12h; Take 1 mL of the culture, centrifuge at 12,000 r / min for 5 min, filter the supernatant with a 0.22 μm filter and save it, and use the obtained filtrate for a spot test to check whether it includes phages capable of killing Coccus aureus.
[0030] Plaque test was carried out as follows: inoculate Coccus aureus AV-XHY in BHI liquid medium at a ratio of 2%, and cultivate overnight at 37°C with shaking. Take 100 μL (OD 600 =1.5) of the bacterial culture solution prepared above and spread it on the BH...
Embodiment 3
[0033] Phage amplification and purification
[0034]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 BHI 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.
[0035] 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 BHI liquid medium, then add CaCl 2 The mother solution was grown to a final concentr...
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