Efficient lytic phage vB-AsoP-yong of Aeromonas sobria and application of efficient lytic phage vB-AsoP-yong

An Aeromonas and phage technology, applied in the field of Aeromonas Mildew efficient and potent phage, can solve problems such as edema of internal organs, loss of aquaculture, tissue hemorrhage, etc., and achieve high infection rate, low cost and high replication rate. Effect

Inactive Publication Date: 2021-02-23
NINGBO UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Aeromonas sobria infects fish, which can cause internal organ edema, tissue hemorrhage, ulceration and other symptoms, often causing heavy losses to the aquaculture industry

Method used

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  • Efficient lytic phage vB-AsoP-yong of Aeromonas sobria and application of efficient lytic phage vB-AsoP-yong
  • Efficient lytic phage vB-AsoP-yong of Aeromonas sobria and application of efficient lytic phage vB-AsoP-yong
  • Efficient lytic phage vB-AsoP-yong of Aeromonas sobria and application of efficient lytic phage vB-AsoP-yong

Examples

Experimental program
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Effect test

Embodiment 1

[0030] Isolation and Purification of Phage vB_AsoP-Yong

[0031] The water samples were collected from Guanshan River, Cicheng Town, Ningbo City, Zhejiang Province (North latitude: 29.9699719; Eastlongitude: 121.4543285); the target host bacteria was Aeromonas sobria (Aeromonas sobria) LY-23, and the specific preparation method was as follows:

[0032] (1) Activation and cultivation of Aeromonas sobria LY-23

[0033] Aeromonas sobria LY-23 was isolated and identified from the diseased turbot fish body by Professor Bai Fengling's research group from the School of Food Science and Engineering, Bohai University, Liaoning Provincial Key Laboratory of Food Safety, and donated as a gift. In this patent, the LY-23 bacterial classification is streaked and inoculated on an LB solid medium plate containing 1.5% (W / V) agar, and cultured upside down at 29° C. overnight. A single colony was picked from the plate, inoculated into a test tube containing 5 mL of LB liquid medium, and culture...

Embodiment 2

[0046] Morphological Observation of Phage vB_AsoP-Yong

[0047] Take the phage-bacteria culture lysate isolated and purified in Example 1 and centrifuge at low speed (4°C, 12000g, 15min) to discard the precipitate, take 1mL of the supernatant and centrifuge at high speed (4°C, 58000g, 1h) to discard the supernatant, add 200 μL of Suspended in 0.01M PBS to obtain phage suspension. Use a pipette gun to take a drop of phage suspension onto the copper grid, let it stand for 10 minutes, and then use neutral filter paper to absorb excess water from the side. Drop a drop of 3% uranyl acetate on the copper grid, and after staining for 20 seconds, use neutral filter paper to absorb the staining agent from the side. After standing for 10min to dry, the morphology of phage was observed with a transmission electron microscope (Hitachi H-7650).

[0048] The result is as figure 2 As shown, the phage vB_AsoP-Yong has an icosahedral approximately spherical head with a diameter of about 55...

Embodiment 3

[0050] Host range assay of phage vB_AsoP-Yong

[0051] Cultivate Aeromonas sobria LY-23 strain, Aeromonas sobria ATCC43979 strain and other strains to be tested (see Table 1 for details) to logarithmic phase (OD 600 ≈0.6). These logarithmic phase bacterial liquids and phage vB_AsoP-Yong suspension were mixed at a volume ratio of 100:1 as the experimental group. The control group used LB instead of phage, and each group was cultured on a shaker (29°C, 180rpm) overnight. There are two parallels for each group. Group OD was measured with a microplate reader 600 Value, take the average value of the parallel group, calculate the ratio of the average value of the control group and the experimental group, if the ratio is greater than 1.2, it is considered that the phage can infect the bacteria, and it is a positive result; otherwise, it is considered that the phage cannot infect the bacteria, and it is a negative result; naked eye, microscope Observe the infection and lysis, and c...

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Abstract

The invention discloses efficient lytic phage vB-AsoP-yong of Aeromonas sobria and an application of the efficient lytic phage vB-AsoP-yong, and relates to the biological treatment of Aeromonas sobriapollution and infection. The vB-AsoP-yong is preserved in the China General Microbiological Culture Collection Center, and the preservation number of the vB-AsoP-yong is CGMCC No.17097. The vB-AsoP-yong is the first phage obtained by taking a pathogenic Aeromonas sobria LY-23 strain as a target for separation, and can specifically lyse Aeromonas sobria, the optimum MOI is 0.001, the incubation period is about 50 min, the lytic period is 50 min-150 min, and the burst amount is 7665 PFU/Cell; the vB-AsoP-yong has relatively good tolerance to factors such as temperature, pH and chloroform; and the vB-AsoP-yong has the protective effect on animals.

Description

technical field [0001] The invention relates to a phage of pathogenic bacteria, in particular to a highly efficient and virulent phage vB_AsoP-Yong of Aeromonas temperatus and its application. Background technique [0002] In order to prevent and control diseases, antibiotics and disinfectants have been used for a long time and widely. However, the abuse of antibiotics makes bacteria resistant, and super bacteria continue to emerge, which seriously threatens human and animal health; antibiotics and disinfectants are not specific, destroy the normal flora we rely on, damage health and endanger the micro-ecological balance of the environment; Once ingested by the human body, antibiotics and other residual drugs in farmed products will affect the intestinal bacterial community, suppress the immune system, and endanger human health. [0003] Phages are viruses that infect bacteria and fungi. According to different life cycles, phages can be divided into lytic phages and temper...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00C12N7/02A01P1/00
CPCC12N7/00C12N2795/10321C12N2795/10331C12N2795/10332C12N2795/10351
Inventor 秦伟南李登峰孙智同童贻刚许丽华林威
Owner NINGBO UNIV
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