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Vibrio alginolyticus bacteriophage preparation and preparation method and application thereof

A technology of vibrio alginolyticus and bacteriophage, which is applied in botanical equipment and methods, applications, and medical raw materials derived from viruses/phages, etc. Easy to obtain, low price, small usage effect

Inactive Publication Date: 2018-10-16
广州诺晶生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In the previous work, we have obtained bacteriophage Vp670, also known as ΦPE333, but we have not carried out infection research and preparation development, nor pointed out its application path

Method used

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  • Vibrio alginolyticus bacteriophage preparation and preparation method and application thereof
  • Vibrio alginolyticus bacteriophage preparation and preparation method and application thereof
  • Vibrio alginolyticus bacteriophage preparation and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] The separation and purification of embodiment 1 phage prepares phage

[0049] (1) Seawater treatment and phage enrichment

[0050] Vibrio alginolyticus E06333 was cultured overnight as the host bacteria for phage isolation. Seawater samples were centrifuged at 10,000 rpm for 10 min, and the supernatant was filtered through a 0.45 μm filter membrane to retain the filtrate. Add the filtrate to 2×LB medium at a volume ratio of 1:1, then add the overnight cultured E06333 bacterial solution at a volume ratio of 1%, culture at 30°C for 12 hours, and after centrifugation, filter the supernatant through 0.22 μm After membrane filtration, the phage enrichment solution was obtained.

[0051] (2) Phage purification

[0052] Using the double-layer plate method, the phage enrichment solution was serially diluted with sterilized SM buffer (0.05M Tris-HCl, pH 7.5), and 100 μL of Vibrio alginolyticus E06333 in the exponential phase was mixed with 100 μL of the phage serial dilution ...

Embodiment 2

[0055] Example 2 Electron Microscopic Detection of Vibrio alginolyticus Phage Vp670

[0056] The Vibrio alginolyticus E06333 culture solution and the bacteriophage Vp670 preservation solution in the above-mentioned Example 1 were respectively dropped on the copper grid, and naturally precipitated for 2-3 minutes. Absorb excess liquid sideways with filter paper. Then stain with 2% phosphotungstic acid for 10 min. Then use filter paper to absorb excess dye solution from the side and air dry at room temperature. The morphology of Vibrio alginolyticus and phage Vp670 was observed and recorded by Hitachi transmission electron microscope.

[0057] Electron microscope observation results such as figure 2 As shown, it can be seen that the normal host bacteria cell structure is complete, and the texture inside the cell is uniform. The outer membrane layer of the Vibrio alginolyticus host bacteria infected by phage Vp670 disappeared, and a large light-colored area appeared inside, a...

Embodiment 3

[0058] Example 3 Molecular identification of Vibrio alginolyticus phage Vp670

[0059] According to the genome sequence of phage Vp670 (GenBank: KY290756.1), its DNA polymerase (DNAP) sequence was found, and the phylogenetic tree was constructed using DNAP and DNAP sequences of similar phages, using the tree building tool MEGA 6.0. Phylogenetic Tree See image 3 . Depend on image 3 It can be seen that Vp670 is clustered with multiple phages of the Brachyphage family from different sources, and this branch is obviously different from the branches formed by other bacteriophage families. Therefore, combined with the electron microscope morphology observation, it is determined that the phage Vp670 belongs to the Brachyphage family.

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Abstract

The invention discloses a vibrio alginolyticus bacteriophage preparation and a preparation method and application thereof. Auxiliary agents such as vibrio alginolyticus bacteriophage Vp670 and trehalose are adopted for developing the corresponding bacteriophage preparation. After application, the bacteriophage preparation continuously exists in a water environment for a long time to keep quantitybalance of pathogenic host bacteria and bacteriophage, so that effect durability and low consumption are achieved as compared with those of antibiotics. The bacteriophage Vp670 preparation is free oftoxic and side effects, is a novel green biological sterilization preparation and can be used for prevention and control of vibrio alginolyticus infection of animals in marine aquaculture to promote healthy development of the aquaculture industry.

Description

technical field [0001] The invention relates to the technical field of aquaculture, in particular to a Vibrio alginolyticus phage preparation, a preparation method and an application thereof. Background technique [0002] Bacterial diseases have plagued the development of the aquaculture industry, among which epidemic diseases caused by outbreaks of pathogenic Vibrio alginolyticus have caused huge economic losses to the aquaculture industry. The common method to treat Vibrio alginolyticus and other pathogenic bacteria is to use various antibiotics, but the use of antibiotics in aquaculture not only causes food safety problems, but also causes a series of environmental ecological problems, including a large number of drug-resistant bacteria in the marine environment. Emergence and spread of resistance genes. It is estimated that approximately 90% of bacteria isolated from seawater are resistant to at least one antibiotic, and approximately 20% of bacteria are resistant to at...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01N63/00A01N1/00A61K35/76A61P31/04A61P31/02
CPCA01N63/00A61K35/76A61P31/02A61P31/04
Inventor 罗鹏卢泽禹田雨顺云龙朱婉君
Owner 广州诺晶生物技术有限公司
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