A strain of Proteus mirabilis phage rdp-sa-16033 and its industrial production process

A technology of RDP-SA-16033, Proteus mirabilis, applied in the field of microorganisms, can solve the problems of antibiotic abuse and bacterial resistance, and achieve the effects of high titer, good product stability, and high temperature tolerance

Active Publication Date: 2020-05-22
RECOM QINGDAO BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The invention is based on the order of the corresponding country to comprehensively ban antibiotics, solves the problems of bacterial drug resistance and antibiotic abuse, and provides a safe and reliable lytic proteus mirabilis phage for treating poultry proteus mirabilis disease

Method used

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  • A strain of Proteus mirabilis phage rdp-sa-16033 and its industrial production process
  • A strain of Proteus mirabilis phage rdp-sa-16033 and its industrial production process
  • A strain of Proteus mirabilis phage rdp-sa-16033 and its industrial production process

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Isolation and identification of pathogenic Proteus mirabilis S5

[0038] Sampling from the diseased farm, taking the liver of the diseased poultry through aseptic operation, streaking on the selective medium, and culturing at 37°C for 18-24 hours, a red colony with round, flat, neat edges, smooth and moist surface formed on the medium , pick a typical colony and continue to streak and purify 3 times, then pick a single colony and inoculate it in 5 mL of LB broth medium, shake it at 200 rpm for 8 hours at 37°C, and obtain a uniform turbid bacterial suspension. Then through 16sRNA molecular identification and serotype identification, it was determined to be pathogenic Proteus mirabilis, and one of them was named S5, which was stored in a -80°C refrigerator by glycerol preservation.

Embodiment 2

[0039] Example 2 Isolation and Identification of Phage RDP-SA-16033

[0040] Sample processing: collect diseased material from the broiler farm, dissect the liver, grind it with a sterilized grinder, then add 2 times the volume of normal saline, shake for 10 minutes, then centrifuge at 12000 rpm for 10 minutes, and take the supernatant to pass through a 0.22 μm filter , collect the filtrate for later use.

[0041] Concentration of phage: Add 10% GEG8000 to the collected supernatant, let it stand at 4°C for 4-6 hours, centrifuge at 12000rpm for 10min, discard the supernatant, dissolve the precipitate with a small amount of normal saline, and collect the solution for later use.

[0042]Phage proliferation: Add 0.2mL of bacterial suspension and 0.5mL of lysate to 5mL of LB broth, culture overnight at 37°C with shaking at 200rpm, then centrifuge at 12000rpm for 5min, take the supernatant and pass it through a 0.22μm filter, and collect the filtrate for later use.

[0043] Phage i...

Embodiment 3

[0046] Embodiment 3: Electron microscope observation of phage

[0047] 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 filter paper to absorb the staining solution from the side, and observe the phage morphology with an electron microscope after the sample is dry.

[0048] The bacteriophage RDP-SA-16033 has a polyhedral three-dimensional symmetrical head wrapped around nucleic acid, with a diameter of about 70nm, a tail of about 150nm in length, a tail sheath, and a neck connecting the head and tail. According to the Ninth Report of the International Virus Taxonomy Organization Virus Classification, the phage can be classified as Myoviridae of the order Myoviridae. refer to figure 1 shown.

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Abstract

The present invention discloses a Proteus mirabilis phage RDP-SA-16033. The host of the Proteus mirabilis phage is Proteus mirabilis S5. The phage may form a plaque having a diameter of 4-6 mm on a double-layer plate. When observed through an electron microscope, the phage has a head in polyhedral cubic symmetry, is coated by nucleic acid, has a diameter of about 70 nm, has a tail of about 150 nm long, has a tail sheath, has a neck connected to the head and the tail, and belongs to tailed virales myovirus division. The present invention further provides a production process of the phage. After centrifugation of value-added liquid, a titer of the phage is increased by membrane concentration, and then residual host and other infectious microbes in value-added are effectively removed by a ceramic membrane and a 0.22 μm polyethersulfone filter membrane. Meanwhile, the phage is reserved to the utmost extent.

Description

technical field [0001] The invention relates to the field of microorganisms, in particular to a Proteus mirabilis phage RDP-SA-16033 and its industrial production process. Background technique [0002] Proteus mirabilis is a Gram-negative bacterium with no spores, no capsule, flagella all over the body, and obvious pleomorphism. Its growth and reproduction do not require high nutrition, and it can reproduce at 4-7°C. The bacterium widely exists in human and animal feces, sewage, soil and clinical specimens, and the toxin it produces can cause poisoning, so it is a common opportunistic pathogen. Proteus mirabilis not only infects common domestic animals such as chickens, pigeons, goats, cows and pigs, but also animals such as monkeys, foxes, pandas and minks. It can cause surgical infections, urinary system infections, diarrhea and bacterial blood disease etc. In recent years, Proteus mirabilis disease outbreaks have continued to occur in chicken flocks in Henan, Hebei, Sha...

Claims

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Application Information

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IPC IPC(8): C12N7/00C12R1/92
CPCC12N7/00C12N2795/10121C12N2795/10151C12N2795/00051
Inventor 李先胜杜新永刘玉庆张庆罗成盛马如霞赵丹丹
Owner RECOM QINGDAO BIOTECH CO LTD
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