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Novel vibrio alginolyticus phage, its composition and its application

A technology of vibrio alginolyticus and bacteriophage, which is applied in the field of microorganisms, can solve the problems such as the absence of vibrio alginolyticus bacteriophage and the like, and achieve the effects of no toxic side effects, strong acid-base tolerance, and high safety.

Active Publication Date: 2021-07-30
QINGDAO PHAGEPHARM BIO TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, there is currently no effective Vibrio alginolyticus phage, which can be used to prevent and treat various diseases caused by Vibrio alginolyticus infection. Therefore, the existing technology needs to be further improved

Method used

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  • Novel vibrio alginolyticus phage, its composition and its application
  • Novel vibrio alginolyticus phage, its composition and its application
  • Novel vibrio alginolyticus phage, its composition and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: Isolation, purification and amplification of phage

[0039] Sewage samples from the Chengyang Seafood Wholesale Market in Qingdao City, Shandong Province were taken, added to Vibrio alginolyticus logarithmic phase bacterial liquid J20, and an appropriate amount of 2216E medium, and cultured on a shaker at 37°C and 170rpm for 16h. Take out 5ml of the liquid, centrifuge at 11000rpm / min for 10min, and filter the supernatant through a 0.22μm sterile microporous membrane to obtain the phage stock solution, which is stored at 4°C.

[0040] The phage was isolated by the double-layer plate method, and the phage stock solution was diluted 10 times, and 100 μl of the diluted solution was mixed with 200 μl of the corresponding Vibrio alginolyticus bacterial solution, incubated at 37° C. for 5 minutes, and mixed into the upper layer of agar (agar concentration: 0.8%). Spread evenly on the lower layer of 2216E agar (1.5% agar concentration) plates, cool and incubate overn...

Embodiment 2

[0043] Electron microscope observation of embodiment 2 phage

[0044] The phage suspension isolated above was dropped on a copper grid covered with polyvinyl formaldehyde film, stained with 2% phosphotungstic acid for 5-10 minutes, and observed with a Hitachi HT7700 transmission electron microscope after drying.

[0045] Observed under the transmission electron microscope, its shape is as figure 2 As shown, the phage has an icosahedral head with a short but not contractile tail, the head diameter is about 54nm, and the tail length is about 15nm; according to the classification method of the International Committee on Taxonomy of Viruses (ICTV), this application The phage morphology conforms to the characteristics of the Brachyphage family, belongs to the Brachyphage, and is named vB_ValP_PJ32.

Embodiment 3

[0046] Genome Analysis of Example 3 Phage

[0047] Extract the genome of Pseudomonas aeruginosa phage vB_ValP_PJ32, and carry out whole genome sequencing, obtain:

[0048] The full genome length of phage vB_ValP_PJ32 is: 80293bp; among them, the tail protein gene sequence related to phage host recognition is shown in sequence 1 in the sequence table, and the highly conserved terminal enzyme large subunit (terminase large subunit) gene sequence is shown in sequence For sequence 2 in the list, see sequence 3 in the sequence listing for the sequence of DNA polymerase (DNA polymerase) gene. The specific information of the above genes is shown in Table 1 below.

[0049] Using BLAST online tool (http: / / blast.ncbi.nlm.nih.gov / ) to perform sequence similarity comparison analysis on phage genomes. The phage with the highest homology was Vibrio phage Ares1, and the homology between phage vB_ValP_PJ32 and this phage only reached 76.22%. The above results indicated that phage vB_ValP_P...

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Abstract

The invention discloses a new type of Vibrio alginolyticus phage, its composition and its application. The Vibrio alginolyticus phage vB_ValP_PJ32 has been preserved in the General Microbiology Center of China Microbiological Culture Collection Management Committee on November 04, 2019, with the preservation number It is CGMCC No.18860. The above-mentioned novel Vibrio alginolyticus phage not only has a wide lytic spectrum, but also has excellent acid-base tolerance, can adapt to more severe application environments, and is used for the prevention and treatment of Vibrio alginolyticus infection. The above-mentioned phage and the compounded composition using the phage can be used to prepare medicines for preventing and treating diseases caused by Vibrio alginolyticus infection in aquaculture, feed additives for prawns, environmental disinfectants for aquaculture farms, detection kits and for production The biological antibacterial agent for disinfection of fresh food can be used in various links in the process of aquaculture that are prone to losses caused by Vibrio alginolyticus infection, daily disinfection of aquatic products markets and aquaculture environments, etc., which is beneficial to the healthy development of aquaculture industry.

Description

technical field [0001] The invention relates to the technical field of microorganisms, in particular to a novel strain of Vibrio alginolyticus phage, a composition containing the phage and applications thereof. Background technique [0002] Vibrio alginolyticus (Vibrio alginolyticus) belongs to Vibrioceae (Vibrionaceae), Vibrio (Vibrio), Gram-negative short bacilli, no capsule, spores, halophilic mesophilic, facultative anaerobic marine Vibrio , exist alone or end connected to form a "C" or "S" shape. Vibrio alginolyticus is widely found in seawater, estuaries and marine animals all over the world, and its number ranks first among Vibrio in the ocean. [0003] Vibrio alginolyticus is an opportunistic pathogen that can infect a variety of mariculture fish, shrimp and shellfish through the damaged skin and mouth of fish, causing fish canker, ascites, shrimp red body disease or hemorrhage The occurrence and prevalence of gastrointestinal diseases such as gastrointestinal dise...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/00A01N63/40A01P1/00G01N33/569A61K35/76A61P31/04A61P1/00A61P1/04A61P7/04A61P7/10A23K10/18A23K50/80A23L3/3571A23L5/20
CPCA23L3/3571A61K35/76A23K10/18A23K50/80A23L5/28A61P1/00A61P1/04A61P7/04A61P7/10A61P31/04A01N63/40C12N7/00C12N2795/10221G01N33/56983
Inventor 潘强任慧英孙虎芝闫艳新徐帆
Owner QINGDAO PHAGEPHARM BIO TECH CO LTD
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