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Alkali-resistant vibrio alginolyticus phage and composition thereof and kit, and application thereof

A technology of Vibrio alginolyticus and bacteriophage, which is applied in the direction of bacteriophage, virus/phage, medical raw materials derived from virus/phage, etc. good stability

Active Publication Date: 2019-09-06
PHAGELUX (NANJING) BIO-TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Invention patent CN107904213A discloses a phage of Vibrio alginolyticus and a bactericidal composition containing the phage. The phage can quickly inhibit the growth of Vibrio in a short time, and the inhibitory effect lasts for a long time
[0007] At present, the research on Vibrio alginolyticus phages mainly focuses on the bactericidal and antibacterial properties, and the research on Vibrio alginolyticus phages targeting more pathogens and their alkali resistance has rarely been reported.

Method used

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  • Alkali-resistant vibrio alginolyticus phage and composition thereof and kit, and application thereof
  • Alkali-resistant vibrio alginolyticus phage and composition thereof and kit, and application thereof
  • Alkali-resistant vibrio alginolyticus phage and composition thereof and kit, and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Separation and purification of Vibrio alginolyticus phage VAP21

[0044] Collect 50ml of Fujian seafood samples and Hainan seawater samples respectively, centrifuge at 5000rpm for 10min, take 20ml of supernatant and sterilize it, mix it with 20ml of 2 times TSB liquid medium and 2ml of Vibrio alginolyticus VAHN1 bacterial solution in logarithmic phase (10 8 cfu / ml) were evenly mixed, and incubated at 150 rpm overnight at 30°C to enrich the phage. The sample enrichment solution was centrifuged at 5000 rpm for 10 min, and the supernatant was sterilized through a 0.22 μm microporous membrane to obtain a filtrate containing phage. Take 50 μl of the filtrate and mix it evenly with 300 μl of the host Vibrio alginolyticus bacteria liquid, and let it stand for 15 minutes to fully bind with the receptors on the surface of the bacteria. Add the above mixed solution to 4ml of semi-solid agar medium cooled to 50°C, spread it on the solidified TSA plate immediately after...

Embodiment 2

[0046] Example 2 Determination of Vibrio alginolyticus phage VAP21 titer

[0047] Make diluent with SM liquid, the stoste of Vibrio alginolyticus bacteriophage VAP21 (made by embodiment 1) 10 times gradients are diluted step by step to 10 8 times. Take l0 respectively 5 , l0 6 , l0 7 and l0 8 1000 μl of diluted phage culture solution was evenly mixed with 300 μl of its host bacterium Vibrio alginolyticus VAHN1, and left to stand for 15 minutes to allow it to fully bind to the receptors on the surface of the bacteria. Add the above mixed solution to 4ml of semi-solid agar medium cooled to 50°C, mix well and immediately spread on the solidified solid agar plate, and incubate upside down at 30°C for 6-8h after the agar solidifies. Three parallel samples are required for each dilution, and the average of the three parallel samples of this dilution is used for counting. Among them, phage titer (PFU / ml) = average number of plaques × dilution factor

[0048] It can be drawn fr...

Embodiment 3

[0051] Example 3 Determination of Vibrio alginolyticus bacteriophage VAP21 to the optimal multiplicity of infection (MOI) of Vibrio alginolyticus

[0052] A single colony of Vibrio alginolyticus was picked, inoculated into a test tube filled with 3 ml of TSB (2% NaCl) culture solution, and cultured with shaking at 150 rpm in a shaker at 30° C. for 12 hours to obtain a host bacterial suspension. The bacterial suspension was transferred to 10ml TSB (2% NaCl) culture solution at a ratio of 1:100, and cultured at 30°C with shaking at 150rpm to the pre-logarithmic phase. Add the pure culture solution of phage VAP21 (prepared by Example 1) and host bacteria (MOI=number of phages / number of bacteria) according to the multiplicity of infection ratio, and add TSB liquid medium to make the total volume of each tube the same. Shake overnight at 150 rpm in a shaker at 30°C. After the culture was completed, centrifuge at 5000 g for 10 min and collect the supernatant to measure the phage ti...

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Abstract

The invention belongs to the technical field of research and development of vibrio alginolyticus phage, and in particular, relates to an alkali-resistant vibrio alginolyticus phage and a composition thereof, and a kit. The vibrio alginolyticus phage is vibrio alginolyticus phage VAP21, and has the preservation number of CCTCC NO:M 2018768. The invention also discloses an application of the vibrioalginolyticus phage, or a vibrio alginolyticus phage composition, or a reagent or kit containing the vibrio alginolyticus phage or the vibrio alginolyticus phage composition in killing and / or preventing vibrio microorganisms. The vibrio alginolyticus phage has wide host range, still has high toxicity to host bacteria at low concentration, good proliferation on non-pathogenic bacteria host and large-scale industrial production, and can provide excellent strain resources for an application of phagotherapy.

Description

technical field [0001] The invention belongs to the technical field of research and development of vibrio alginolyticus phage, and in particular relates to an alkali-resistant vibrio alginolyticus phage and its composition, kit and application. Background technique [0002] Vibrio alginolyticus is a halophilic Gram-negative short bacillus without spores and capsules, belonging to the family Vibrio and the genus Vibrio. the first. It can cause wound infection, food poisoning, otitis media and other diseases to humans. At the same time, it is also an opportunistic pathogen for fish, shrimp, shellfish and other marine aquaculture animals. When the environment deteriorates, the body's immune function decreases, and alginolytic vibriosis is prone to outbreaks. [0003] Bacteriophage is a type of virus that specifically lyses bacteria. Its main chemical components are composed of protein and nucleic acid. It is widely found in soil, air, water and organisms, and has strong speci...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00A61K35/76A61P31/04
CPCA61K35/76A61P31/04C12N7/00C12N2795/00021
Inventor 乔欢陈海徐旭凌黄杰费文斌胡怿林何四龙丛郁
Owner PHAGELUX (NANJING) BIO-TECH CO LTD
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