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A phage of Vibrio alginolyticus and its application in disease prevention of sea cucumbers

A technology of Vibrio alginolyticus and bacteriophage, applied in the biological field, can solve problems such as unclear mechanism of action and unsolved key problems of bacteriophage, and achieve good environmental tolerance, great potential and broad application prospects

Active Publication Date: 2016-04-06
DALIAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the effect of phages on the prevention and treatment of bacterial diseases in terrestrial animals and aquatic animals (yellowfish, sweetfish, prawns, etc.) has been confirmed in the laboratory, there are no research reports on the use of phages in the prevention and control of sea cucumber diseases. Some key issues in the process of phage therapy have not been resolved, and the specific mechanism of action is still unclear

Method used

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  • A phage of Vibrio alginolyticus and its application in disease prevention of sea cucumbers
  • A phage of Vibrio alginolyticus and its application in disease prevention of sea cucumbers
  • A phage of Vibrio alginolyticus and its application in disease prevention of sea cucumbers

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Experimental program
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Effect test

Embodiment 1

[0022] Phage Screening and Purification

[0023] (1) Water sample collection and treatment

[0024] (1) Preparation of host bacteria

[0025] Vibrio alginolyticus was inoculated in 2216E solid medium after bacteriological identification, 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.

[0026] (2) Treatment of water samples

[0027] Take 200mL of wastewater samples from a sea cucumber farm in Dalian and a sewage outfall of an aquatic product market in Dalian, and pretreat the water samples: add CaCl2, MgCl 2 , so that the final concentration is 1mmol / L, and the effect is about 10min. (2) Enrichment of bacteriophage in water samples

[0028] Centrifuge the above-mentioned pretreated water sample at 10,000g for 5min, and collect the supernatant; pass the supernatant through a 0.22μm filter...

Embodiment 2

[0036] Antibacterial effect of phage in vitro

[0037] (1) In vitro antibacterial test of phage

[0038] The experiment was set up as 4 groups. Take 4 50mL centrifuge tubes and mark them as A, B, C, and D respectively; where A is the negative control group, B and C are the positive control groups, and D is the experimental group. Add 30mL2216E liquid culture medium and 1mL bacterial solution (concentration is 10 8CFU / ml), 1mL LPBS; add 30mL 2216E liquid medium, 1mL bacterial liquid, 1mL kanamycin (SulfateKanamycin) concentration to 10mg / L in B; add 30mL 2216E liquid medium, 1mL bacterial liquid, 1mL doxycycline in C (Doxycycline) at a concentration of 5 mg / L; D also added 30 mL of 2216E liquid medium, 1 mL of bacterial liquid, and 1 mL of PVA1 phage enrichment solution, and the titer was 10 9 PFU / mL; After shaking for 10 minutes, put it into a microplate reader to measure the value of OD600nm and record it; put it into a shaker at 28°C for cultivation, take a sample every ha...

Embodiment 3

[0051] Environmental Tolerance of Phages

[0052] Obtain the high-titer enrichment solution of phage PVA1 (titer is 4.2×10 10 PFU / ml). Put 100mL cultured seawater, 1 9g head of healthy sea cucumber and phage liquid into a 500mL beaker, the initial titer is 1.5×10 7 PFU / ml, placed at room temperature for cultivation, and the titer was measured every 24 hours using the double-layer plate method, and the degradation of the phage within 14 days was recorded.

[0053] The results show that: under the conditions of seawater culture and culture room temperature, the phage liquid decreased by two orders of magnitude within 14 days, and the environmental tolerance of the phage is better, which provides the possibility for the application of the phage in the environmental water body. The results are shown in the appendix Figure 4 .

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Abstract

The invention discloses a vibrio alginolyticus bacteriophage and application thereof to prevention of sea cucumber disease, belonging to the technical field of biology. The bacteriophage is collected in the China General Microbiological Culture Collection Center on March 11, 2014 and has a collection number of CGMCC No.8792 and a classification name of Vibrio alginolyticus bacteriophage; the vibrio alginolyticus bacteriophage is a short-tailed phage, has a head diameter being 50 nanometers and tail length being 15 nanometers, and has no shrinkage tail sheath and six short tail fibers. The bacteriophage is used for preventing infection and pollution caused by corresponding vibrio alginolyticus in the culturing process of sea cucumbers, and the potency is more than or equal to 10<5>PFU / mL. The bacteriophage has an inhibiting function on vibrio alginolyticus, and no vibrio alginolyticus-resistant strain is produced within 12 hours; the incubation period lasts for 28 minutes, the burse size is 79, and two orders of magnitude are lowered within 14 days in a seawater culturing environment.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a phage isolate capable of specifically lysing Vibrio alginolyticus strains and its application in the field of sea cucumber disease prevention. Background technique [0002] Sea cucumber (Holothurioidea) is one of the marine animals with the largest production value in the northern coastal areas of China. According to statistics, my country's current sea cucumber cultivation area is nearly 90,000 hectares, the output is nearly 100,000 tons, and the annual output value is more than 10 billion yuan (China Sea Cucumber Academic Research and Industry Development Forum, 2009). 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 serious. Vibrio alginolyticus (Vibrioalginolyticus) is a common marine pathogen, which is one of the main ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20A61P31/04C12R1/63
Inventor 徐永平张建城曹振辉王丽丽李振徐乐
Owner DALIAN UNIV OF TECH
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