Vibrio alginolyticus bacteriophage and application thereof to prevention of sea cucumber disease

A technology of Vibrio alginolyticus and phage, applied in the field of sea cucumber disease prevention, in the field of phage isolates, can solve problems such as unclear mechanism of action and unresolved key problems of phage, and achieve good environmental tolerance and great potential , the effect of broad application prospects

Active Publication Date: 2014-07-02
DALIAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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 re

Method used

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  • Vibrio alginolyticus bacteriophage and application thereof to prevention of sea cucumber disease
  • Vibrio alginolyticus bacteriophage and application thereof to prevention of sea cucumber disease
  • Vibrio alginolyticus bacteriophage and application thereof to prevention of sea cucumber disease

Examples

Experimental program
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Example Embodiment

[0022] Example 1

[0023] Screening and purification of phage

[0024] (1) Water sample collection and processing

[0025] (1) Preparation of host bacteria

[0026] After bacteriological identification, Vibrio alginolyticus was inoculated into 2216E solid medium and stored in a refrigerator at 4°C. The host bacteria for screening were cultured overnight at 28°C, and a single colony was picked and inoculated in 2216E liquid medium. After shaking culture at 28°C for 8 hours, it was used to separate phage.

[0027] (2) Treatment of water samples

[0028] Take 200 mL of wastewater samples from a sea cucumber farm in Dalian and a sewage outlet in an aquatic product market in Dalian, and pretreat the water samples: add CaCl2, MgCl 2 , Make the final concentration 1mmol / L, and act for about 10min.

[0029] (2) Enrichment of water-like phage

[0030] Centrifuge the above-mentioned pretreated water sample at 10000g for 5 minutes, and collect the supernatant; pass the supernatant through a 0.22μm f...

Example Embodiment

[0037] Example 2

[0038] In vitro antibacterial effect of phage

[0039] (1) In vitro antibacterial experiment of phage

[0040] The experiment is set to 4 groups, and 4 50mL centrifuge tubes are taken and labeled as A, B, C, and D respectively; A is the negative control group, B and C are the positive control group, and D is the experimental group. Add 30mL 2216E liquid medium and 1mL bacterial liquid (concentration of 10 8 CFU / ml), 1mL PBS; Add 30mL 2216E liquid medium, 1mL bacterial solution, 1mL Kanamycin sulfate (Sulfate Kanamycin) concentration to 10mg / L into B; add 30mL 2216E liquid medium, 1mL bacterial solution, 1mL strong Doxycycline (Doxycycline) concentration is 5mg / L; 30mL 2216E liquid medium, 1mL bacterial solution, 1mL PVA1 phage enrichment solution are also added to D, the titer is 10 9 PFU / mL; After shaking for 10 minutes, put it in a microplate reader to measure the OD600nm value and record it; place it in a shaker at 28°C, sample and measure the OD600nm value eve...

Example Embodiment

[0052] Example 3

[0053] Environmental tolerance of phage

[0054] Obtain the high titer enrichment solution of phage PVA1 (titer is 4.2×10 10 PFU / ml). Put 100mL cultured seawater, 1 9g healthy sea cucumber and phage solution into a 500mL beaker, the initial titer is 1.5×10 7 PFU / ml, cultured at room temperature, the titer was measured by the double-layer plate method every 24h, and the degradation of phage was recorded within 14 days.

[0055] The results showed that under the conditions of cultured sea water and cultured room temperature, the phage fluid dropped by two orders of magnitude within 14 days, and the phage environmental tolerance was better, which provided the possibility for the application of phage in environmental water. See the results 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 (Vibrio alginolyticus) is a common marine pathogen, which is one of the main...

Claims

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Application Information

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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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