Escherichia coli bacteriophage esc-cop-9 and its use in inhibiting proliferation of pathogenic Escherichia coli
A technology of esc-cop-9 and Escherichia coli is applied in the direction of phage, virus/phage, medical raw materials derived from virus/phage, etc., to achieve the effect of small side effects and high specificity
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Embodiment 1
[0034] Example 1: Isolation of phages that can kill Escherichia coli
[0035] When isolating bacteriophages capable of killing Escherichia coli, samples secured from natural environments were used. In addition, Escherichia coli used for phage isolation has been previously isolated by the present inventors and identified as pathogenic Escherichia coli.
[0036] Next, the isolation procedure of the phage will be described in detail. Inoculate the TSB (Tryptic Soy Broth) medium (casein digest, 17g / L; soybean digest, 3g / L; glucose, 2.5g / L; NaCl, 5g / L; Dipotassium hydrogen phosphate, 2.5g / L) was added together with the collected sample (sample), and then cultured with shaking at 37°C for 3-4 hours. After culturing, centrifugation was performed at 8,000 rpm for 20 minutes, and the supernatant was recovered. Escherichia coli was inoculated into the recovered supernatant at a ratio of 1 / 1000, and then cultured again with shaking at 37° C. for 3-4 hours. In the case that the phag...
Embodiment 2
[0041] Embodiment 2: Genome isolation and sequence analysis of phage Esc-COP-9
[0042] The genome of phage Esc-COP-9 was isolated as follows. The genome was isolated using the phage suspension obtained in the same manner as in Example 1. First, in order to remove DNA and RNA of Escherichia coli that may be contained in the suspension, 200 U of DNase I and RNase A were added to 10 ml of phage suspension, and then left at 37°C for 30 minutes. After standing for 30 minutes, in order to eliminate the activity of DNase I and RNase A, 500 μl of 0.5M ethylenediaminetetraacetic acid (EDTA) was added, and then left to stand for 10 minutes. Then, it was left still at 65° C. for 10 minutes. Next, in order to disrupt the outer wall of the phage, 100 μl of proteinase K (20 ㎎ / ml) was added and reacted at 37° C. for 20 minutes. Then, 500 μl of 10% sodium dodecylsulfate (SDS) was added, and then allowed to react at 65° C. for 1 hour. After reacting for 1 hour, 10 ml of a mixture compos...
Embodiment 3
[0046] Embodiment 3: the investigation of the ability of phage Esc-COP-9 to kill pathogenic escherichia coli
[0047] The ability of the isolated phage Esc-COP-9 to kill pathogenic Escherichia coli was investigated. The killing ability investigation was carried out by investigating whether or not a transparent ring was formed by the spot test proposed in Example 1. The pathogenic Escherichia coli used for the investigation of killing ability was isolated and identified as pathogenic Escherichia coli by the present inventors, and a total of 10 strains were used. The phage Esc-COP-9 has the ability to kill a total of 9 strains of the 10 pathogenic Escherichia coli strains used as the experimental object. Representative experimental results are as follows: figure 2 shown. In addition, the effects of phage Esc-COP-9 on Bordetella bronchiseptica, Enterococcus faecalis, Enterococcus faecium, Streptococcus mitis, Streptococcus uberis were also investigated. (Streptococcus uberi...
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