Novel enteropathogenic e. coli bacteriophage esc-chp-2 and use thereof for inhibiting proliferation of enteropathogenic e. coli
A technology of Escherichia coli and pathogenicity, applied in the field of bacteriophage, can solve the problems of economic loss, high cost of treatment and prevention, death, etc., and achieve the effect of high specificity, weakened immunity, and less side effects
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Embodiment 1
[0032] Example 1: Isolation of phage capable of killing enteropathogenic E. coli
[0033] Samples were collected from nature to screen for phages capable of killing enteropathogenic E. coli. The enteropathogenic E. coli used for phage isolation herein is one that the inventors have previously isolated and identified as enteropathogenic E. coli.
[0034] The isolation procedure of phage is described in detail below. The collected samples were added to TSB medium (tryptic soy broth) (tryptic digest of casein, 17 g / L; papain digest of soybean, 3 g / L; glucose, 2.5 g / L; sodium chloride , 5g / L; dipotassium hydrogen phosphate, 2.5g / L), inoculate enteropathogenic Escherichia coli at a ratio of 1 / 1000, and then shake and culture at 37°C for 3-4 hours. After the culture was completed, the supernatant was collected by centrifugation at 8000 rpm for 20 minutes. The recovered supernatant was inoculated with enteropathogenic E. coli at a ratio of 1 / 1000, followed by shaking culture at 37...
Embodiment 2
[0039] Example 2: Isolation and sequence analysis of the phage Esc-CHP-2 genome
[0040] The genome of phage Esc-CHP-2 was isolated as follows. The genome was isolated from the phage suspension obtained in Example 1. First, in order to remove DNA and RNA of enteropathogenic Escherichia coli in the suspension, 200 U each of DNase I and RNase A were added to 10 ml of the phage suspension, and incubated at 37°C for 30 minutes. After 30 minutes, in order to remove DNase I and RNase A activities, 500 μl of 0.5 M ethylenediaminetetraacetic acid (EDTA) was added and incubated for 10 minutes. The suspension was further incubated at 65°C for 10 minutes, then 100 μl of proteinase K (20 mg / ml) was added to disrupt the outer wall of the phage, followed by incubation at 37°C for 20 minutes. After that, 500 μl of 10% sodium dodecyl sulfate (SDS) solution was added, followed by incubation at 65°C for 1 hour. Add 10 ml of a 25:24:1 mixture of phenol:chloroform:isoamyl alcohol and mix well....
Embodiment 3
[0044] Example 3: Study on the killing ability of bacteriophage Esc-CHP-2 on enteropathogenic Escherichia coli
[0045] The killing ability of the isolated phage Esc-CHP-2 against enteropathogenic Escherichia coli was investigated. For this purpose, the formation of transparent regions was observed by the spot test in the same manner as described in Example 1. A total of 12 strains of enteropathogenic E. coli were used for this study, which had been previously isolated and identified as enteropathogenic E. coli by the present inventors. Phage Esc-CHP-2 showed the ability to kill 9 enteropathogenic E. coli used in this experiment. The representative results of the lethality test are as follows: figure 2 shown. At the same time, the phage Esc-CHP-2 was also studied to kill Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, Lactobacillus plantarum, Streptococcus uberis uberis) and Pseudomonas aeruginosa. As a result, it was confirmed that phage Esc-CHP-2 di...
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