CN114774370A

CN114774370A - Catfish Edwardsiella phage EIP-1 and application thereof

Info

Publication number: CN114774370A
Application number: CN202210348497.0A
Authority: CN
Inventors: 刘如铟; 寸淑娟; 韩岗华; 刘新春; 余志晟
Original assignee: University of Chinese Academy of Sciences
Current assignee: University of Chinese Academy of Sciences
Priority date: 2022-04-01
Filing date: 2022-04-01
Publication date: 2022-07-22

Abstract

The invention discloses a catfish Edwardsiella phage EIP-1 and application thereof, belonging to the technical field of biology. The catfish Edwardsiella phage EIP-1 has the preservation number of CGMCCNo.45077; the separation and identification not only enriches the phage germplasm resource library, but also provides a selectable potential scheme for solving the problem of fish death caused by the infection of the Edwardsiella catfish in aquaculture; through sequencing the whole genome of the phage EIP-1, functional genes and functional proteins of the phage EIP-1 can be further deeply excavated, one or more enzymes with a broad-spectrum cracking effect are provided for phage treatment and biological control of catfish Edwardsiella infection in aquaculture, and the universality and effectiveness of phage application are improved; the bacteriophage EIP-1 can effectively crack the Edwardsiella catfish, and is used for preventing fish infection death caused by the Edwardsiella catfish infection in aquaculture.

Description

Catfish Edwardsiella phage EIP-1 and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to an Edwardsiella catfish phage EIP-1 and application thereof.

Background

The catfish Edwardsiella is a common fish pathogenic bacterium and can infect various aquaculture fishes such as turbot, eel, catfish, zebra fish, yellow catfish, channel catfish, rainbow trout, tilapia and the like. Edwardsiella catfish mainly infects the digestive tract and nervous system of fish. The specific form of the infection of the digestive tract is as follows: the Edwardsiella catfish enters the blood of the fish through the alimentary canal and then infects various tissues by utilizing a blood circulation system, so that various organs have symptoms of inflammation, congestion, hemorrhage, fester and the like; the specific form of nervous system infection is: the catfish Edwardsiella enters the olfactory bulb through the nasal cavity of the fish, then enters the brain through the olfactory bulb, and finally infects meninges, cranium and head skin of the fish, so that the cranium and head skin of the fish are subjected to necrosis, ulceration and other symptoms. The catfish Edwardsiella infection mainly erupts in 6-10 months every year, the suitable water temperature for the disease attack is 22-28 ℃, the disease incidence is more than 50 percent, and the mortality rate after the disease attack is up to 70-100 percent. Aquatic diseases caused by the catfish Edwardsiella are devastated in countries such as America, China, Thailand, Vietnam, Australia and the like, and in China, the aquatic diseases caused by the catfish Edwardsiella occur in regions such as Sichuan, Guangdong, Hunan, Hubei, Jiangxi and the like, which causes great economic loss to the aquaculture industry and seriously affects the healthy development of the aquaculture industry at home and abroad. At present, the catfish Edwardsiella infection is mainly prevented and treated by chemical sterilization medicines such as disinfectants and antibiotics, but the residue of antibiotic components and the spread of Antibiotic Resistance Genes (ARGs) generally exist in aquaculture environments, which undoubtedly aggravates the difficulty of preventing and controlling aquatic diseases and also poses great threats to ecological environment and human health. Therefore, there is a need for new antibacterial agents that combine therapeutic effects and reduce environmental risks.

Bacteriophage, as a potential, novel bio-antibacterial agent, has the following advantages over antibiotics: the specificity is strong, the damage to normal flora is small, and the bacteriophage only acts on a single species or bacterial strain; coexisting with the host and positively correlating with the number of hosts; co-evolution exists between the phage and the host, and the phage can adapt to and infect bacterial resistance mutants appearing during the treatment process; a large number of phages exist only at the infection site, and the phages generally can only replicate by using the pathogenic host in the focus and have a slow migration rate to other organs. The phage is proved to have good biological safety and tolerance, and has almost no toxic and side effects on animal cells. However, the few kinds of phage that have been isolated are an important factor limiting the further development of phage therapy.

Therefore, the problem to be solved by the technical personnel in the field is to provide the catfish Edwardsiella phage EIP-1 and the application thereof.

Disclosure of Invention

In view of the above, the invention provides an Edwardsiella catfish phage EIP-1 and application thereof.

In order to achieve the purpose, the invention adopts the following technical scheme:

the catfish Edwardsiella phage EIP-1 has the preservation number of CGMCC No.45077, is preserved in China general microbiological culture Collection center of China Committee for culture Collection of microorganisms (CGMCC for short), is deposited at the institute of microbiology of China academy of sciences No. 3, Xilu No. 1, North Chen province, Kyoho, the Beijing, and has the preservation date of 2022 years, 01 months and 10 days, and is named as the catfish Edwardsiella phage in a classified manner.

Further, the application of the catfish Edwardsiella phage EIP-1 in the lysis of catfish Edwardsiella.

Further, the application of the catfish Edwardsiella phage EIP-1 in preparing a medicament for preventing and treating aquatic diseases caused by catfish Edwardsiella in aquaculture.

According to the technical scheme, compared with the prior art, the catfish Edwardsiella phage EIP-1 and the application thereof are disclosed and provided, the separation and identification of the catfish Edwardsiella phage EIP-1 not only enrich the phage germplasm resource library, but also provide a selectable potential scheme for solving the problem of fish death caused by infection of the catfish Edwardsiella in aquaculture; through sequencing the whole genome of the phage EIP-1, functional genes and functional proteins of the phage EIP-1 can be further deeply excavated, one or more enzymes with a broad-spectrum cracking effect are provided for phage treatment and biological control of frequent catfish Edwardsiella infection in aquaculture, so that the application range of the phage is enlarged, and the treatment effect is improved; the bacteriophage EIP-1 can effectively crack the catfish Edwardsiella sp, is used for preventing and treating fish infection death caused by catfish Edwardsiella sp infection in aquaculture, and is especially used in the background of poor antibiotic treatment effect and antibiotic resistance gene flooding.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments or the prior art descriptions will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.

FIG. 1 is a drawing showing the plaque morphology of the bacteriophage EIP-1 of Edwardsiella catfish of the present invention on a double-layer plate;

FIG. 2 is a transmission electron microscope of Edwardsiella catfish phage EIP-1; the scale bar is 200 nm;

FIG. 3 is the drawing of the catfish Edwardsiella phage EIP-1 gene map of the invention;

FIG. 4 is a drawing of a phylogenetic tree of bacteriophage EIP-1 from Edwardsiella catfish according to the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.

The phage host bacteria Edwardsiella catfish MCCC 1K00244 is purchased from China marine microorganism culture collection center and preserved by the laboratory.

The brain-heart leachate broth culture medium and the brain-heart leachate agar culture medium are purchased from Qingdao Hi-Tech industrial Garden Haibo biotechnology Limited; the 0.22 μm microporous filter membrane is purchased from Jinteng laboratory equipments Ltd of Tianjin; chloroform was purchased from biotechnology limited of beijing dingguo.

EXAMPLE 1 isolation and purification of phage

Taking 38.5g of brain-heart leachate broth culture medium, and placing in 1L of ultrapure water to obtain brain-heart leachate broth culture medium.

Taking 38.5g of brain-heart leachate broth culture medium, putting into 1L of ultrapure water, and adding 7.5g of agar powder to obtain the brain-heart leachate semisolid culture medium.

Taking 52g of brain-heart leachate agar culture medium, and placing in 1L of ultrapure water to obtain a brain-heart leachate solid culture medium.

The above culture medium is mixed and autoclaved at 121 deg.C for 15 min.

The water sample for the test is collected in a fishing ground in Beijing City in 9 months in 2020, and the water sample is used as a matrix for separating the phage.

10mL of an environmental water sample is filtered and sterilized by a 0.22-micron microporous filter membrane, and the catfish Edwardsiella MCCC 1K00244 is cultured to the logarithmic growth phase overnight. 5mL of the filtered water sample is added with 1mL of host bacterium liquid in logarithmic phase and 25mL of brain-heart leaching liquid culture medium, and shake culture is carried out overnight at 30 ℃ and 120 rpm. The next day, centrifuging at 4 deg.C and 6000g for 10min, collecting supernatant, and filtering to obtain filtrate which is the stock solution enriched with bacteriophage. The filtrate was examined for the presence of plaques by double-plate method. If clear and bright plaques appear on the plate, the existence of the phage in the stock solution is indicated, otherwise, the phage is not separated, and the sampling and separation are required again.

The separated phage is not necessarily single phage, and the obtained phage is purified by a double-layer plate method, which comprises the following steps: the plaques on the plate were picked and immersed in a sterile tube containing 1mL of SM and left at room temperature for 1h to dissolve out the phage. Diluting the dissolved phage by 10 times, mixing 500 mu L of the diluted phage with 20 mu L of host bacteria suspension, adding the mixture into a 5mL tube sterile centrifuge tube, then adding 3.8mL brain-heart leachate semisolid culture medium, then quickly pouring the culture medium into the brain-heart leachate solid culture medium, coating the culture medium on a whole double-layer plate, carrying out inverted culture at 30 ℃ overnight, and observing the plaque form the next day. Single plaques were picked and purified 5-6 times until the plaques were substantially identical in size and morphology in the bilayer plate, see FIG. 1.

Example 2 morphological Observation of phages

And (3) dripping 20 mu L of phage suspension on a copper net, naturally precipitating for 10min, sucking dry the phage suspension from the side by using dry filter paper, airing for about 1min, adding 1 drop of 1% uranyl acetate on the copper net, dyeing for 2min, carefully sucking dry the redundant dye from the side by using the dry filter paper, naturally airing in a dark place for 30min, and observing by using a transmission electron microscope (JEM-1400).

As a result of transmission electron microscopy, as shown in FIG. 2, phage EIP-1 had an icosahedral head and a contractible tail, the head diameter was about 45.74nm, the tail length was about 96.61nm, and the tail diameter was about 24.49 nm. According to the eighth report of the International Classification of viruses (ICTV), the strain of bacteriophage EIP-1 can be preliminarily classified as Caudovirales (Caudovirales), Myoviridae (Myoviridae).

Example 3 phage genome analysis identification

Extracting bacteriophage DNA by a phenol-chloroform method, dissolving DNA precipitate by TE buffer solution, and performing whole genome sequencing, wherein the sequencing platform is IlluminaNovaSeq. The DNA of a sample is randomly broken into small fragments of about 350bp before the sequencing on a computer, then a NEB standard library building kit is used for selecting a specific joint, the sample is subjected to library preparation, the size of the library fragment is detected by using aglent2100, then the molar concentration of the library is detected by using qPCR, and PE 2 x 150 sequencing is carried out by using Illumina NovaSeq after the library is qualified. Splicing the optimized sequence by using spades v.3.11.1 splicing software to obtain an optimal assembly result; predicting whether the bacteriophage EPP-1 genome contains Antibiotic resistance genes and virulence Factors on line by using CARD (the Comprehensive antibacterial Research database) and VFDB (viral Factors database) databases; performing predictive analysis on the open reading frame of the genome by using biological software PHASTER; and using NCBI Blastp to complete the preliminary annotation of the functional gene; the CGView Server software (http:// CGView. ca /) is used for completing the drawing of the whole genome circus (see figure 3), and the MEGA X software is used for constructing a phylogenetic tree (see figure 4) based on the whole genome data.

The phage EIP-1 is a circular phage with a genome size of 43485bp and a GC content of 52.3%. According to CARD and VFDB prediction, the bacteriophage EIP-1 genome does not contain antibiotic resistance genes and virulence factors. The PHASTER prediction showed 67 CDS in phage EIP-1, and no tRNA structure was found. From the matching results, the genome has a typical phage modular structure. Comprises a DNA replication module, a structural protein gene, a lysis module and a DNA packaging gene. Through experimental determination of a host spectrum (see table 1), the phage EIP-1 can crack catfish Edwardsiella and most of Edwardsiella piscicola, and the host spectrum is relatively wide.

TABLE 1 host Spectroscopy assay of bacteriophage P69

Note: "+" indicates plaque presence, "-" indicates no plaque; host indicates the host from which the phage was isolated.

CGMCC＝ChinaGeneral Microbiological Culture CollectionCenter。

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. The catfish Edwardsiella phage EIP-1 is characterized in that the preservation number is CGMCC No. 45077.

2. The use of the Edwardsiella catfish phage EIP-1 of claim 1 for lysing Edwardsiella catfish.

3. The use of the Edwardsiella catfish phage EIP-1 of claim 1 in the preparation of a medicament for controlling aquatic diseases caused by Edwardsiella catfish in aquaculture.