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Vibrio bacteriophage PC-Liy1 with cross-species lysis capability, preparation method and application

A technology of phage and Vibrio, applied in the field of biological antibacterial agents, can solve the problem that there is no efficient cracking of pathogenic Vibrio phages, and achieve the effect of efficient cracking ability

Pending Publication Date: 2022-05-31
青岛百奥安泰生物科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because of the high specificity of phages, most of them can only infect one kind of host bacteria, and there are many types of Vibrio in the actual breeding environment. At present, phages with high specificity cannot meet the needs of diversity and complexity of the breeding environment. There is also no report of efficient cracking of various pathogenic Vibrio phages

Method used

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  • Vibrio bacteriophage PC-Liy1 with cross-species lysis capability, preparation method and application
  • Vibrio bacteriophage PC-Liy1 with cross-species lysis capability, preparation method and application
  • Vibrio bacteriophage PC-Liy1 with cross-species lysis capability, preparation method and application

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Experimental program
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Effect test

Embodiment 1

[0038] Embodiment 1: Separation and purification of Vibrio phage PC-Liy1

[0039] The samples were collected from seafood water samples from various aquatic farmers' markets in Nanning City, Guangxi Province.

[0040] Take 100mL of water sample, add 100mL of 2×LB liquid medium and 1.5mL of Vibrio cholerae VC1 in logarithmic growth phase (preserved by our research group), and culture at 180r / min at 37°C for 12h to enrich phages. The enriched solution was sterilized by centrifugation at 10,000 r / min for 15 minutes, and the supernatant was passed through a 0.22 μm sterile filter membrane. Then it was diluted with PBS buffer and mixed with Vibrio cholerae VC1 to prepare a double-layer agar plate. Put it in a 37°C incubator and incubate for 6 hours to observe whether there are phage plaques. A single plaque was picked for multiple double-layer plate method to isolate and purify the phage. Until the phage forms plaques of the same shape and size on the plate, such as figure 1 sh...

Embodiment 2

[0042]Example 2: Genome-wide characterization of Vibrio phage PC-Liy1

[0043] The DNA of Vibrio phage PC1-Liy1 was extracted and used as a template to sequence the entire phage genome. Vibrio phage PC1-Liy1 is a linear double-stranded DNA with a genome size of 246,348bp and a GC% content of 42.6%. According to the whole genome and proteomic analysis, the sequence has no virulence factors and antibiotic resistance genes. The results of genome similarity comparison by NCBI BLASTn are shown in Table 1. The maximum similarity between the phage and the broad-spectrum phage KVP40 was 98%, and the coverage rate was 97%.

[0044] Table 1 Genome homology comparison of Vibrio phage PC1-Liy1

[0045] strain type coverage Similarity serial number Spectrum phage KVP40 97％ 98％ AY283928 Vibrio phage V09 96％ 98％ MT135026 Vibrio phage V07 96％ 98％ MT135025 Vibrio phage VH1_2019 95％ 98％ MN794232

[0046] The Vibrio phage PC-Liy1 was pre...

Embodiment 3

[0047] Embodiment 3: Biological characteristic experiment of Vibrio phage PC-Liy1

[0048] (1) Host range detection

[0049] The lysability of phage to vibrio, yeast, spore, lactobacillus and enterobacter was determined by spot method. The bacteria to be tested that were cultured to the logarithmic growth phase were made into a double-layer plate, and 5 μL of phage liquid (10 9 PFU / mL) was dropped on the surface of the solidified double-layer plate, and cultured in a 37°C incubator for 8 hours to observe whether there were plaques.

[0050] The drop method was used to determine the effect of phage on Vibrio isolated from diseased shrimp samples and water samples in Shandong Qingdao, Shandong Rizhao, Shandong Yantai, Guangxi Nanning, Jiangsu Nantong, Jiangsu Rudong, Zhejiang Ningbo and other places, as well as Escherichia coli and E. Lyticity of various Gram-positive bacteria.

[0051] The host range results of bacteriophage PC-Liy1 are shown in Table 2-Table 4. In addition ...

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Abstract

The invention discloses a vibrio bacteriophage PC-Liy1 with cross-species lysis capability as well as a preparation method and application of the vibrio bacteriophage PC-Liy1. The bacteriophage belongs to the myophage family (Myoviridae). The vibrio phage disclosed by the invention has a wide host range and a relatively high splitting rate, and can be used for splitting vibrio alginolyticus, vibrio parahaemolyticus, vibrio cholerae, vibrio mimicus, vibrio neokarst and vibrio shewanii. The bacteriophage is high in environmental adaptability and has stable and good biological characteristics, and a biological bacteriostatic agent prepared from the bacteriophage can effectively control growth of vibrio in water and other environments.

Description

technical field [0001] The invention belongs to the field of biological technology, and in particular relates to a vibrio phage PC-Liy1, a biological antibacterial agent prepared by the vibrio phage PC-Liy1 and an application thereof. Background technique [0002] Vibrio is an aerobic facultative anaerobic Gram-negative bacteria, one of the most common bacterial groups in the marine environment, widely present in seawater and marine organisms in coastal waters. In recent years, with the continuous increase of human demand and consumption of seafood, my country's aquaculture industry has flourished, and the degree of intensive aquaculture has become higher and higher. The expansion of breeding density has increased the incidence of farmed animals. The common and most harmful bacterial disease of mariculture animals is mainly vibriosis caused by Vibrio. Vibriosis can cause mass mortality in shrimp, fish, shellfish and crustaceans. Vibrio cholerae, Vibrio alginolyticus, Vibri...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00A01N63/40A01P1/00C12R1/92
CPCC12N7/00A01N63/40C12N2795/10121C12N2795/10131Y02A50/30
Inventor 何增国李映汤伟吴春光付静芸张军孙晓雯唐涛
Owner 青岛百奥安泰生物科技有限公司
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