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Bacteriophage vB_Yen_X1 and application thereof in prevention and treatment of Yersinia pestis infestation

A phage, Yersinia technology, applied in the field of bioengineering, can solve problems such as narrow bactericidal spectrum

Active Publication Date: 2021-04-23
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At the same time, due to the strong specificity of phage, its bactericidal spectrum is narrow, which limits its practical application to a certain extent. The key to realize its application is to separate and obtain phage with broad-spectrum and high-efficiency bactericidal ability.

Method used

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  • Bacteriophage vB_Yen_X1 and application thereof in prevention and treatment of Yersinia pestis infestation
  • Bacteriophage vB_Yen_X1 and application thereof in prevention and treatment of Yersinia pestis infestation
  • Bacteriophage vB_Yen_X1 and application thereof in prevention and treatment of Yersinia pestis infestation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Phage isolation and preparation

[0019] The fecal liquid sewage sample in the present invention is collected from Huoshao Li Sewage Ditch in Changchun City; the host bacteria is Yersinia enterocolitica 23715. Collect sewage, filter it with gauze, or centrifuge at 6000 g / min for 10 min, and take the supernatant; replace ddH with treated sewage 2 O Prepare Yersinia enterocolitica expansion medium (sodium chloride 0.5 g, tryptone 0.5 g, beef extract powder 0.4 g) (100 mL); take 1 mL of the overnight cultured bacterial solution, mix it, and shake In bed (37°C, 165 r / min overnight culture); take 1 mL of overnight culture, centrifuge at 12,000 g / min for 5 min, filter the supernatant with a 0.22 µm filter to form a phage stock solution and save it, and use the obtained filtrate for the spot test , to check whether the phage stock contains phages capable of killing Yersinia enterocolitica.

[0020] The plaque test was performed as follows: Yersinia enterocolitica 23715 was i...

Embodiment 2

[0024] Phage amplification and purification

[0025] On the double-layer plate forming plaques, use a sterile pipette tip to pick up a single plaque with a large diameter, round and translucent, and inoculate it in 5 mL of Yersinia enterocolitica expansion culture Add 200 µL of phage host bacterial solution to the base, mix well, incubate at room temperature for 15 min, incubate at 37°C for 10-14 h, centrifuge at 12,000 g, 4°C for 10 min, and take the supernatant; repeat the double-layer plate experiment 4-5 times, In this way, single phage plaques were picked repeatedly 4-5 times, and the phages were purified into phage plaques of the same size.

[0026] Take 1 mL of freshly cultured host bacteria and add 300 µL of phage lysate (the ratio of a single phage culture to host bacteria is 1:1, 1:10 and 1:100, respectively). Incubate at 37°C for 20 min to make the phage particles adsorb to the host bacteria; add 800 mL Yersinia enterocolitica amplification medium, and then add CaC...

Embodiment 3

[0035] Transmission electron microscope observation of bacteriophage vB_Yen_X1

[0036] Take the PEG-purified phage in Example 2 for electron microscope observation. The specific operation steps are: add 10 µL of sample and drop it on the copper grid, wait for it to settle for 15 min, absorb excess liquid with filter paper, and use 2% phosphotungstic acid (PTA ) stained for 1-2 min, and observed with a transmission electron microscope (Hitachi H-7650) after drying; the observation results are as follows figure 2 As shown, the head is in the shape of an icosahedron, the diameter of the head is about 64 nm, and the length of the tail is about 94 nm. According to the "Classification of Viruses—The Eighth Report of the International Committee on Taxonomy of Viruses" published by the International Committee on Taxonomy of Viruses (ICTV) in 2005, vB_Yen_X1 belongs to the family Myoviridae.

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Abstract

The invention discloses a bacteriophage vB_Yen_X1 and application thereof in prevention and treatment of Yersinia pestis infection, and provides a novel Yersinia enterocolitica bacteriophage vB_Yen_X1. The bacteriophage vB_Yen_X1 has a wide bactericidal spectrum, can infect and kill Yersinia enterocolitica, and more importantly, shows an extremely wide infection and bactericidal spectrum on Yersinia enterocolitica, can be used alone or in combination with other substances, and can effectively purify pestis infection focus, urgently prevent and control Yersinia pestis infection and pestis caused by the Yersinia pestis infection.

Description

technical field [0001] The invention discloses a new Yersinia enterocolitica phage vB_Yen_X1, which can effectively purify plague foci, urgently prevent and control Yersinia pestis infection and cause plague, and belongs to the technical field of bioengineering. Background technique [0002] Yersinia enterocolitica (Yersinia enterocolitica) is a Gram-negative bacillus or cocci, belonging to Enterobacteriaceae, Yersinia genus. Yersinia enterocolitica is a type of zoonotic pathogen and a very important food-borne pathogen. It has cryophagocytosis and can grow and proliferate in a cold storage environment. Human infection can cause respiratory system, gastrointestinal system, cardiovascular system and other diseases, and may also cause acute appendicitis and sepsis. [0003] Yersinia pestis (Yersinia pestis), which belongs to Enterobacteriaceae and Yersinia genus, commonly known as Yersinia pestis, is the pathogenic bacteria that causes plague infection. Plague is a zoonotic ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00A61K35/76A61P31/04C12R1/92
CPCC12N7/00A61K35/76A61P31/04C12N2795/10321C12N2795/10332Y02A50/30
Inventor 顾敬敏韩文瑜薛一冰王子晶冀亚路
Owner JILIN UNIV
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