Vibrio parahaemolyticus phage and application thereof in prevention of stichopus japonicus disease

A technology for hemolytic Vibrio and disease prevention, applied in the direction of phage, virus/phage, medical raw materials derived from virus/phage, etc., can solve problems such as unsatisfactory treatment effect, drug-resistant strains and drug residues, and achieve animal-derived food The effects of safety, abundance reduction, and economic loss reduction

Active Publication Date: 2019-11-19
DALIAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the prevention and treatment measures for Vibrio parahaemolyticus infection are mainly prevention and supplemented by treatment. According to reports, the treatment effect of antibiotics after the onset of the disease is not ideal, and it is easy to produce a large number of drug-resistant strains and drug residues.

Method used

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  • Vibrio parahaemolyticus phage and application thereof in prevention of stichopus japonicus disease
  • Vibrio parahaemolyticus phage and application thereof in prevention of stichopus japonicus disease
  • Vibrio parahaemolyticus phage and application thereof in prevention of stichopus japonicus disease

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Experimental program
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Effect test

Embodiment 1

[0024] Phage Screening and Purification

[0025] (1) Water sample collection and treatment

[0026] (1) Preparation of host bacteria

[0027] Samples of diseased sea cucumbers from sea cucumber farms were collected, and TCBS medium was used to isolate suspected colonies of Vibrio parahaemolyticus. The genomic DNA of the strain of interest was extracted, and the 16s rDNA of the pathogenic bacteria was amplified by PCR. After sequencing, NCBI comparison (MG589511), identified as Vibrio parahaemolyticus, named Vibrio parahaemolyticus VP-ABTNL.

[0028] Vibrio parahaemolyticus VP-ABTNL was inoculated in 2216E solid medium and stored in a refrigerator at 4°C. The host bacteria used for screening were cultivated overnight at 28°C, and a single colony was picked, inoculated in 2216E liquid medium, shaken at 28°C for 8 hours, and then used to isolate phage, attached figure 1 .

[0029] (2) Treatment of water samples

[0030] Take 200mL each of wastewater samples from a sea cucum...

Embodiment 2

[0040] Phage one-step growth curve assay

[0041] The multiplicity of infection (MOI) refers to the ratio of the number of phage added to the host bacteria at the initial infection. Add phage with a multiplicity of infection of 0.1 to the host bacterial solution, mix well, let stand at 28°C, and absorb for 15 minutes; centrifuge at 11,000 rpm at 4°C for 10 minutes, discard the supernatant, resuspend the precipitate with 2216E liquid medium, repeat the above centrifugation, and remove Unadsorbed phage. The pellet was resuspended in 100mL 2216E liquid medium, and the time was T 0 =0; culture at 120 rpm at 28°C, sample every 10 minutes to determine phage titer.

[0042] The results showed that the incubation period of the phage infecting the host bacteria was about 30 minutes, and the lysing amount was about 125 PFU / cell, and the lysing ability was strong. Figure 4 .

Embodiment 3

[0044] Safety experiment

[0045] Select 26 8-week-old female SPF grade BALB / c mice, 30g ± 2g, and then divide them into two groups with 13 mice in each group. 9 pfu / kg of phage liquid, the control group was given the same amount of normal saline, and after 20 days of continuous administration, the mice were killed by dislocation of the neck. After autopsy, no symptoms of pathological changes were found in the lungs, liver, kidneys, and abdominal cavity. Preliminary verification of this measurement of phage liquid It has no effect on mice and is safe.

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Abstract

The invention belongs to the technical field of biology, and relates to a phage isolate capable of specifically cracking Vibrio parahaemolyticus and application of the phage in the field of sea cucumber disease prevention, so that the economic benefit of sea cucumber breeding enterprises is improved, and food safety is guaranteed from the source. The vibrio parahaemolyticus phage vB_VpaP_VP-ABTNL-1 has a preservation number of CGMCC No.17991. The vibrio parahaemolyticus phage provided by the invention is applied to the prevention of the stichopus japonicus disease and can effectively prevent the infection of the vibrio parahaemolyticus in the breeding process.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a phage isolate capable of specifically lysing Vibrio parahaemolyticus (Vibrioparahaemolyticus) strains and its application in the field of sea cucumber disease prevention. Background technique [0002] Sea cucumber (Apostichopus japonicus) is the largest economical mariculture animal in the northern coastal area of ​​my country, and has extremely high medicinal and commercial value. In 2017, the total output of sea cucumbers in my country reached 220,000 tons, and the breeding area reached 219,000 hectares, of which Liaoning, Shandong, and Fujian accounted for the vast majority. At present, the output value of my country's sea cucumber industry has exceeded 50 billion yuan. However, with the rapid increase in the scale and density of sea cucumber farming in recent years, the water body of sea cucumbers has gradually deteriorated, and the problem of diseases has become increasingly se...

Claims

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Application Information

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IPC IPC(8): C12N7/00A61K35/76A61P31/04C12R1/91
CPCC12N7/00A61K35/76A61P31/04C12N2795/10221Y02A50/30
Inventor 徐永平任宏宇李振徐乐李晓宇王丽丽
Owner DALIAN UNIV OF TECH
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