A Phage of Aeromonas hydrophila and Its Application

A technology of Aeromonas hydrophila and bacteriophage, applied in the direction of bacteriophage, virus/bacteriophage, application, etc., can solve the problems of high concentration and low virulence of bacteriophage, and achieve short incubation period, good stability and activity, good adaptive effect

Active Publication Date: 2021-06-15
山东宝来利来生物工程股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the phages reported above generally have problems such as low toxicity and high concentration. Therefore, it is necessary to further develop new potent phages for the treatment of Aeromonas hydrophila

Method used

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  • A Phage of Aeromonas hydrophila and Its Application
  • A Phage of Aeromonas hydrophila and Its Application
  • A Phage of Aeromonas hydrophila and Its Application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1: Isolation and preparation of phage

[0054] The freshwater fish viscera sample among the present invention is collected from Wuma Market, Tai'an City, Shandong Province;

[0055] The host bacterium was Aeromonas hydrophila CVCC4002, which was purchased from China Veterinary Microbiology Culture Collection Management Center.

[0056] Fill 20mL Tris-HCl buffer into a 50mL centrifuge tube, then add a small amount of freshwater fish viscera samples, and let it stand overnight at 4°C. The sample was centrifuged at 8000r / min for 10min to remove impurities, and the supernatant was centrifuged at 4000r / min for 10min and filtered with a 0.22μm microporous filter to obtain the filtrate and save it.

[0057] Take the above 20mL filtrate and add it to 20mL TSB medium, then add 1mL suspension of host bacteria (Aeromonas hydrophila CVCC4002) in the logarithmic growth phase, mix well and culture overnight at 30°C with shaking. The next day, centrifuge at 8000r / min for 30m...

Embodiment 2

[0058] Embodiment 2: Amplification culture and purification of phage

[0059] Use a pipette tip to pick out larger diameter plaques, place them in buffer (Tris-HCl buffer), place at 4°C for 3 hours, make 10-fold gradient dilution with buffer, double-layer plate (bottom TSA solid, upper layer TSB +0.75% agar semi-solid) method for single-spot culture. Pick a single phage plaque with a larger diameter and place it in the host bacterial solution (the amount of bacteria is about 10 8 CFU / mL) in liquid TSB medium for 6 hours for a small amount of proliferation, and then observe the morphology of the plaques by the double-layer plate method. After repeating the operation 3 to 5 times, the plaques with the same shape and size can be obtained. Pick a single phage plaque and place it in Tris-HCl buffer, transfer it into 3-5mL TSB medium, add 0.1mL of phage host bacterial solution, mix well, act at room temperature for 15min, and incubate at 30°C for 10-14h , 12000rpm, 4°C, centrifuge...

Embodiment 3

[0068] Example 3: Effects of temperature and pH on the stability of bacteriophage BLCC16-001

[0069] With reference to the method recorded in the patent CN107099511A, the temperature and pH are investigated for the stability of the phage, as follows:

[0070] Take 0.5mL10 each 9 The phage liquid of PFU / mL was placed in a sterile EP tube, and reacted in a water bath at 30°C, 40°C, 50°C, 60°C, 70°C, 80°C, and 90°C for 60 minutes respectively. The sample was cooled in a water bath, and the titer of the phage was determined after dilution. The results are shown in image 3 . Depend on image 3 It can be seen that the titer of Aeromonas hydrophila phage BLCC16-001 was basically unchanged after being treated at 30°C, 40°C, 50°C, and 60°C for 1 hour, and the titer decreased by an order of magnitude after being treated at 70°C for 1 hour, and after being treated at 80°C for 1 hour Afterwards, the titer decreased by 2 orders of magnitude, and all of them were inactivated after be...

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Abstract

The invention discloses a phage of Aeromonas hydrophila and its application. The strain has been preserved in the China Center for Type Culture Collection on August 28, 2020, and its preservation number is: CCTCC NO: M 2020456. The optimal multiplicity of infection of the Aeromonas hydrophila phage provided by the present invention is 0.0001, the incubation period of the phage infection host bacteria is about 20min, the outbreak time is about 120min, the burst amount is 1000PFU / cell, and the dosage is 10 4 PFU / mL can achieve significant antibacterial effect. It is a potent phage that can be used to treat Aeromonas hydrophila infection in freshwater fish in my country, and has broad application prospects in aquaculture.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an Aeromonas hydrophila phage and its application. Background technique [0002] Aeromonas hydrophila is a Gram-negative bacterium belonging to the Aeromonas family and the genus Aeromonas. It is widely distributed in the water environment and is the main pathogen that causes fish motile Aeromonas sepsis. The bacterium is considered to be the main pathogenic bacterium of fulminant infectious diseases of freshwater cultured fish in my country. Two hemolytic toxins, hemolysin and aerolysin, have been found in Aeromonas hydrophila. Hemolytic toxins in Aeromonas hydrophila cause leakage of cell contents, ultimately triggering host cell death. In addition to fish, Aeromonas hydrophila can also cause systemic sepsis or local infection in molluscs, amphibians, reptiles, birds and mammals, often leading to a large number of animal deaths, and can also cause acute acute Gastroenteritis, foo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/00A01N63/40A01P1/00A61K35/76A61P31/04A23K50/80A23K10/16C02F3/34C02F103/20C12R1/92
CPCA61K35/76A23K10/16A23K50/80A61P31/04A01N63/40C02F3/34C02F2103/20C12N1/00C12N7/00C12N2795/10121C12R2001/91
Inventor 王春凤单宝龙高绪娜李金敏桑瑞新陈雷谷巍徐海燕王红赵倩
Owner 山东宝来利来生物工程股份有限公司
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