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Enterohaemorrhagic escherichia coli phage and application thereof

A technology for enterohemorrhagic Escherichia coli, which is applied in the field of enterohemorrhagic Escherichia coli phage and its application, can solve problems such as inability to effectively control transmission, and achieve the effect of preventing survival and reproduction, pollution and overgrowth

Active Publication Date: 2017-05-31
韩鸣
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to provide a new EHEC O157 that can specifically crack EHEC O157 in view of the current prevention and treatment of animal diarrhea and poisoning events caused by EHEC O157:H7. :H7 Phage and Its Application

Method used

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  • Enterohaemorrhagic escherichia coli phage and application thereof
  • Enterohaemorrhagic escherichia coli phage and application thereof
  • Enterohaemorrhagic escherichia coli phage and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0021] Embodiment 1, the separation and preparation of phage

[0022] The sample of the present invention is collected from pig farm cleaning site sewage, filtered through double-layer filter paper, 5000 × g.min -1 Centrifuge for 10 min, and filter the supernatant through a 0.22 μm filter membrane.

[0023] Take 50ml of the filtered supernatant, add 2ml of phage host bacteria EHEC O157:H7 overnight culture, add 20ml of LB medium, incubate at room temperature for 30min, and then place it at 37°C for 12-16h. The next day, take the above-mentioned culture and wash it at 14000×g.min -1 Centrifuge for 30min, take the supernatant; 14000×g.min -1 Centrifuge again for 20 min, take the supernatant and add 0.1% chloroform to form a phage stock solution.

[0024] Take 0.1ml of bacteriophage stock solution, carry out 10-fold dilution, take 10 2 、10 4 and 10 6 Mix 0.1ml each of the dilution solution with 0.1ml of the overnight cultured host bacterial solution. After 15 minutes at roo...

Embodiment 2

[0025] Embodiment 2, purification and transmission electron microscope analysis of phage

[0026] Get 2ml of freshly cultivated host bacteria, centrifuge, resuspend in 0.4ml LB medium, add 0.1ml phage (according to the ratio of 1:1, 1:10 and 1:100 of the single phage culture obtained in Example 1 and the host bacteria respectively) Proportion). Add maltose (0.2%), MgSO 4 (10mM), incubate at 37°C for 20min to make the phage particles adsorb to the host bacteria; add 100ml LB liquid medium containing maltose (0.2%), MgSO 4 (10mM), shake at 37°C for 9-12h; add 0.1ml chloroform, continue shaking at 37°C for 10-20min to obtain a lysate.

[0027] Transfer the lysate to a centrifuge tube and centrifuge at 8000×g.min -1 , to remove bacterial fragments, take the supernatant; add RNase A, DNase I to 1 μg / ml, incubate at 37°C for 30 minutes; add 9.3g PEG 8000, 5.8g NaCl, shake well until dissolved, ice-bath for 1h or overnight at 4°C; 4 Centrifuge at 10000×g.min -1 20min, remove th...

Embodiment 3

[0029] Embodiment 3, the influence of temperature and pH on phage stability

[0030] Take 0.1ml 1×10 respectively 8 pfu / ml of purified phage was reacted in a water bath at 40°C to 90°C for 1 hour, and the titer was measured after the sample was cooled in an ice bath; peptone water with pH 2.0 to 9.0 and 2×10 8 Pfu / ml purified phages were mixed in equal amounts, and the potency was measured after 2 hours in a water bath at 37°C.

[0031] The results show that if figure 2 As shown, after the phage was treated at 20°C to 50°C for 1 hour, its activity did not change significantly; at 60°C for 1 hour, the activity decreased; after acting at 70°C, the titer decreased to less than 50% of the initial titer; at 90°C , almost inactivated.

[0032] The results after the phage were treated with different pH image 3 , the titers of phages were all at 10 after pH6.0~8.0 8 pfu / ml, its activity did not change significantly; when the pH was 6.0 and 9.0, respectively, its titer decreased t...

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Abstract

The invention relates to enterohaemorrhagic escherichia coli phage and an application thereof. The enterohaemorrhagic escherichia coli phage is characterized in that the preservation number of a phage strain is CCTCC NO:M 2016539, and the phage strain is preserved at the China Center for Type Culture Collection at Wuhan university in China on September 29, 2016, and is classified and named asenterohaemorrhagic coliphage vB-ECM-MIE, entero-haemorrhagic-Escherichia-coli-O157:H7 phage vB-ECM-MIE. The enterohaemorrhagic escherichia coli phage has efficient sterilization capacity on EHEC.

Description

technical field [0001] Enterohemorrhagic Escherichia coli (EHEC) is a group of Escherichia coli that can cause hemorrhagic diarrhea and enteritis in humans, and its representative strain is O157:H7 serotype. Background technique [0002] In recent years, food-borne pathogenic bacteria-Escherichia coli 0157:H7 has become a worldwide public health problem, seriously endangering human health, causing large economic losses, and causing a great degree of negative impact on social stability and environmental safety. According to reports, In the United States alone, there are 73,000 cases of food-borne diseases caused by E. coli 0157:H7 every year, causing economic losses of 70 million US dollars per year. Hemorrhagic Escherichia coli mainly includes O157:H7, O26:Hll and 0111:NM, wherein serotype 0157 is the main pathogenic bacteria causing hemorrhagic colitis, and it is reported that 90% of hemolytic uremic syndrome cases are caused by its subtype 0157:H7, so research on Escheric...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00A61K35/76A61P1/12A61P31/04A61L2/18A23L29/00C12R1/92
CPCA61K35/76A61L2/18A61L2202/25C12N7/00C12N2795/00021Y02A50/30
Inventor 韩鸣
Owner 韩鸣
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