A Thermostable Escherichia coli Phage rdp-ec-20031 and Its Application

A RDP-EC-20031, Escherichia coli technology, applied in the direction of virus/bacteriophage, medical raw materials derived from virus/phage, application, etc., can solve problems such as untimely treatment, ineffective medication, and drug resistance of pathogenic bacteria

Active Publication Date: 2021-11-26
RECOM QINGDAO BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to provide a high-temperature-resistant coliphage RDP-EC-20031, which aims to solve the problem of untimely treatment of poultry in farms due to acute colibacillosis infection and ineffective medication due to drug resistance of pathogenic bacteria and other difficulties

Method used

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  • A Thermostable Escherichia coli Phage rdp-ec-20031 and Its Application
  • A Thermostable Escherichia coli Phage rdp-ec-20031 and Its Application
  • A Thermostable Escherichia coli Phage rdp-ec-20031 and Its Application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Isolation and identification of pathogenic Escherichia coli BE-20036

[0035] Sampling from the diseased chicken farm, aseptically take the liver of the diseased chicken, streak it on the selective medium (MacConkey agar medium), cultivate it at 37°C for 18-24 hours, and form a round, flat, edge on the medium For red colonies with neat, smooth and moist surface, pick typical colonies and continue to streak and purify for 3 times, then pick a single colony and inoculate in 5mL LB broth, shake and culture at 37°C and 200rpm for 8h to obtain a uniform turbid bacterial suspension. Then it was identified as Escherichia coli through 16sRNA molecular identification, and its serotype was further determined to be O78 through serum identification. O78 has strong pathogenicity, and can be transmitted to humans through food chains and other methods, and has strong pathogenic infectivity; Name it BE-20036 and store it in a -80°C refrigerator.

Embodiment 2

[0036] Example 2 Isolation and identification of coliphage RDP-EC-20031

[0037] (1) Sewage treatment: Take sewage from the farm, centrifuge at 10,000rpm for 5min, take the supernatant and pass it through a 0.22μm filter, and collect the filtered supernatant for later use.

[0038] (2) Preparation of mixed bacterial suspension: Take 0.2mL of bacterial suspension and 0.3mL of filtrate and add it to 5mL of LB broth, shake and culture overnight at 37°C at 200rpm, then centrifuge at 10,000rpm for 5min, take the supernatant and pass it through a 0.22μm filter. Set aside the filtrate.

[0039] (3) Separation of phages: Separation of phages was carried out using the double-plate method. Take 0.2 mL of the filtrate of the mixed bacterial suspension and 0.2 mL of the Escherichia coli suspension and mix evenly, bathe in water at 37°C for 10 minutes, then spread double plates, and incubate at 37°C for 8 After -12h, observe the result. If there are phages, there will be clear plaques on...

Embodiment 3

[0042] Example 3 Morphological observation of bacteriophage

[0043] Take 20 μL of the liquid containing crude phage particles and drop it on the copper grid, let it settle naturally for 15 min, absorb the excess liquid from the side with filter paper, add a drop of 2% phosphotungstic acid (PTA) on the copper grid to stain the phage for 10 min, and then use The staining solution was sucked from the side of the filter paper, and the morphology of the phage was observed with an electron microscope after the sample was dried. The result is as figure 1 shown.

[0044] Depend on figure 1 It can be seen that the bacteriophage RDP-EC-20031 has a polyhedral stereosymmetric head, wrapped in nucleic acid, with a diameter of about 70nm, a tail with a length of about 90nm, a tail sheath, and a neck connecting the head and the tail. According to the Ninth Report of the International Virus Taxonomy Organization Virus Classification, the phage can be classified as Myoviridae of the order ...

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Abstract

The invention discloses a high-temperature-resistant coliphage RDP‑EC‑20031, the preservation number is CGMCCNo.21406, and the gene length is 135812bp. After sequencing the whole gene, it is found that it does not have virulence genes and lysogenic genes. The gene contains multiple lyase genes. The coliphage RDP-EC-20031 provided by the present invention has higher temperature tolerance and wider acid-base tolerance range, which is conducive to industrial production and preparation; it is found by lysis rate experiment and RTD experiment that the lysis of the bacteriophage Rate 98% at dilution 10 7 times, it still has a good cracking effect on pathogenic bacteria. The phage has the characteristics of excellent performance and safety. The phage can be used in the preparation of medicines for preventing and or treating diseases caused by Escherichia coli.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a strain of high-temperature-resistant coliphage RDP-EC-20031 and its application in pathogenic coliform infection of poultry animals. Background technique [0002] Avian colibacillosis refers to the general term for acute or chronic infectious diseases caused by pathogenic Escherichia coli, often leading to extraintestinal diseases in poultry. The incidence rate in chicken flocks is about 11%-69%, and the mortality rate is about 3.8%-72.9%. With the development of intensive breeding industry, poultry colibacillosis has become an important infectious disease threatening the poultry industry, which poses a serious threat to the poultry industry and causes huge economic losses. At present, antibiotics are widely used to prevent and treat avian E. coli disease, which has led to a rapid increase in the resistance of E. coli. Antimicrobial resistance of Escherichia coli from animal sour...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/00A61K35/76A61K45/06A61P31/04A23K10/18A23K20/195A23K50/75C11D3/38C11D3/48A01N63/40A01P1/00C12R1/92
Inventor 杜新永李先胜王立坤赵丹丹张庆刘玉庆罗成盛盖春云马如霞刘爽
Owner RECOM QINGDAO BIOTECH CO LTD
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