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Novel Vibrio cholerae Phage and Its Application

A Vibrio cholerae, phage technology, applied in the field of microbiology, can solve problems such as the absence of Vibrio cholerae phages

Active Publication Date: 2021-08-27
QINGDAO PHAGEPHARM BIO TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, there is currently no effective Vibrio cholerae phage that can be used to prevent and treat white stool syndrome in Penaeus vannamei. Therefore, the existing technology needs to be further improved

Method used

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  • Novel Vibrio cholerae Phage and Its Application
  • Novel Vibrio cholerae Phage and Its Application
  • Novel Vibrio cholerae Phage and Its Application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Isolation and cultivation of bacteriophage

[0027] (1) Recovery culture of strains and preparation of bacterial suspension

[0028] Pick the frozen bacteria solution of Vibrio cholerae and line it in three areas on the TCBS plate, separate the single colonies, and culture them in the incubator at 37°C for 16~24h. Pick a single colony, inoculate it into 5ml of 2216E broth, and culture it in an air shaker at 37°C at 170rpm for 16h to obtain a single bacterial suspension.

[0029] (2) Isolation and purification of phage

[0030] Take water samples from a cultured shrimp pond in Binzhou City, Shandong Province and put them into sampling bottles filled with 2216E broth. Add 0.5% of each strain of Vibrio cholerae to each sampling bottle. 170rpm shaking culture overnight, 10000rpm centrifugation 10min, 0.22μm bacterial filter filter sterilization. Mix the filtrate and the host bacteria, incubate at 37°C for 5 minutes, then add the mixture to the melted upper laye...

Embodiment 2

[0031] Example 2 Determination of biological characteristics of phage

[0032] (1) Morphology of bacteriophage observed by transmission electron microscope

[0033] Take higher than 1×10 8 20 μl of PFU / ml phage sample was dropped on the microporous copper grid, settled for 15 minutes, and the excess liquid was absorbed with filter paper. Add 15 μl of 2% phosphotungstic acid (PTA) dropwise on the copper grid, stain for 5 minutes, absorb excess dye solution with filter paper, observe and take pictures with transmission electron microscope after drying.

[0034] Electron microscope observation shows that the head of the phage has a polyhedral structure, the diameter of the head is about 60nm, and the length of the tail is about 180-190nm. According to the definition of the International Committee on Taxonomy of Viruses (ICTV), the shape of the phage conforms to the characteristics of the long-tailed phage family and belongs to the long-tailed family. Phage family, named vB-VchS...

Embodiment 3

[0047] Example 3 Determination of phage lysis profile and in vitro lysis test

[0048] (1) Determination of phage lysis profile

[0049] The lysis spectrum of the phage is measured by the double-layer plate method, and the steps are as follows: obtain the bacterium suspension of the host bacterium according to the method in Example 1, and select 40 strains to separate from the hepatopancreas of Penaeus vannamei in Binzhou, Shandong, and Nantong, Jiangsu, where the disease has occurred. Vibrio cholerae, respectively R01 ~ R40, 40 Vibrio cholerae non-O1, non-O139 serotype. And detect the relevant virulence genes, respectively detect 40 strains of host bacteria enterotoxin gene ctxh, enterotoxin gene ctxB, outer membrane protein gene ompU, zonule junction toxin gene zot, toxin regulatory protein gene toxR, classical biotype (Clas - sical biotype) toxin co-regulated pilus gene tcpA Class, El Tor biotype (El Tor biotype) toxin co-regulated pilus gene tcpA ET, toxin co-regulated pi...

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Abstract

The invention belongs to the field of biotechnology, and discloses a novel Vibrio cholerae phage and its application. The Latin name of the bacteriophage is Vibrio cholerae phage, named vB‑VchS‑PR02, and has broad-spectrum bactericidal ability to Vibrio cholerae , was deposited in the General Microorganism Center of China Microbiological Culture Collection Management Committee on November 04, 2019, and the preservation number is CGMCC NO.18861. The phage has strong cracking effect on Vibrio cholerae, can be used to prepare medicines for preventing and treating Vibrio cholerae infection, biological food therapy additives, and disinfectants, and provides a safe and effective method for the prevention and treatment of Vibrio cholerae infection diseases.

Description

technical field [0001] The invention relates to the technical field of microbes, in particular to novel vibrio cholerae phages and applications thereof. Background technique [0002] Vannamei (Penaeus vannamei) is one of the three most productive varieties in the world's aquaculture industry. It has three characteristics: strong adaptability, fast growth, and strong disease resistance. In the 1980s, the development of my country's shrimp farming industry led to the rapid development of my country's aquaculture industry and promoted the economic development of my country's coastal areas. However, with the rapid increase in the scale and density of Penaeus vannamei aquaculture in recent years, the aquaculture water body has gradually deteriorated, and disease problems have become increasingly serious. [0003] Vibrio cholerae is a common marine pathogen. Vibrio cholerae infection of Penaeus vannamei is an important cause of white stool syndrome. White feces syndrome has bec...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/00A61K35/76A23K10/18A61P31/04C02F3/34C02F1/50C12R1/92
CPCA61K35/76A23K10/18A61P31/04C02F1/50C02F3/34C02F2305/06C12N7/00C12N2795/10321C12N2795/10332Y02A50/30
Inventor 潘强任慧英孙虎芝闫艳新丁同燕
Owner QINGDAO PHAGEPHARM BIO TECH CO LTD
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