Brucella bacteriophage and application thereof

Abstract

The invention discloses a brucella bacteriophage and application thereof, and belongs to the technical field of biology. The Brucella bacteriophage is preserved in the China Center for Type Culture Collection on July 25, 2024, the preservation number is CCTCC NO: M 20241682, the Brucella bacteriophage Y17 is separated from a sheep manure sample collected from Lu Man County in Yunnan province, the Brucella bacteriophage Y17 is separated from a sheep manure sample collected from Lu Man County in Yunnan province, and the Brucella bacteriophage Y17 is separated from a sheep manure sample collected from Lu Man County in Yunnan province. Compared with the prior art, the bovine brucella vaccine strain has the advantages of high specificity, short incubation period, large outbreak amount and certain tolerance to high temperature, acid and alkali, ultraviolet rays and ethanol, shows a very strong splitting effect on the bovine brucella vaccine strain, lays a foundation for research and development of a brucella sterilization biological agent, and provides a new strain and a new idea for prevention and treatment of brucellosis.

Description

Technical Field

[0001] The invention relates to a Brucella phage and application thereof, belonging to the field of biotechnology. Background Art

[0002] Brucella is a highly contagious intracellular parasite that can survive for a long time in the natural environment. The disease it causes is called brucellosis, which is the most important zoonotic infectious disease in the world. The prevalence of brucellosis has caused a large number of deaths in cattle and sheep, miscarriages in female livestock, and decreased production performance, which directly affects the quality of livestock products and causes serious economic losses to farmers. After a person is infected with brucellosis, Brucella can invade various parts of the human body, causing organ lesions, joint swelling, persistent fever, general fatigue, etc., and severe cases can cause lifelong loss of labor. Brucellosis is usually treated with a combination of multiple antibiotics, but it is difficult to cure and can easily become chronic. In addition, with the detection of more and more drug-resistant strains in recent years, brucellosis has become a greater challenge.

[0003] As a virus that can lyse bacteria, bacteriophages have the advantages of host specificity, high safety, and no residue, and have broad prospects in the prevention and treatment of bacterial infections. Bacteriophages can quickly kill specific pathogens without affecting the body's normal flora, and can also play a good disinfecting role when sprayed in the environment. Brucellosis is currently rebounding globally, and my country is a country with a high burden of brucellosis. Phage biological agents have great potential for brucellosis prevention and control. Previously, few Brucella phages have been isolated in the world. Therefore, continuously isolating and purifying new phages targeting Brucella and studying their biological, morphological, and genomic characteristics are of great significance to the development and utilization of phages to prevent Brucella infections. Summary of the invention

[0004] In order to solve the problems existing in the prior art, one of the objects of the present invention is to provide a Brucella phage Y17, which has been deposited in the China Center for Type Culture Collection on July 25, 2024, with the address: Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, with the deposit number: CCTCC NO: M 20241682, and the taxonomic name: Brucella phage.

[0005] Another object of the present invention is to provide an application of Brucella phage Y17, wherein the Brucella phage Y17 can be used to prepare Brucella bactericidal biological preparations.

[0006] Beneficial effects of the present invention:

[0007] (1) The Brucella phage Y17 described in the present invention belongs to the Podoviridae family. The phage has the advantages of strong specificity, a wide lysis spectrum, a short incubation period, a large burst volume, a strong tolerance to high temperature, acid and alkali, ultraviolet rays and organic agents, and good stability. The genome of the phage does not contain virulence factors and antibiotic resistance genes, and has good application potential for the prevention and treatment of brucellosis. The present invention lays a foundation for the research and development of Brucella bactericidal biological agents and provides a new idea for the prevention and treatment of brucellosis.

[0008] (2) The Brucella phage Y17 described in the present invention is a circular double-stranded DNA phage with a full genome size of 38025 bp. It is the Brucella phage with the smallest genome isolated so far and is a completely new phage. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 These are plaques formed by Brucella phage Y17.

[0010] Figure 2 This is a transmission electron micrograph of Brucella phage Y17.

[0011] Figure 3 This is a graph showing the MOI determination of Brucella phage Y17.

[0012] Figure 4 This is a graph showing the results of the one-step growth curve assay of Brucella phage Y17.

[0013] Figure 5 The temperature sensitivity of Brucella phage Y17.

[0014] Figure 6 UV sensitivity of Brucella phage Y17.

[0015] Figure 7 pH sensitivity of Brucella phage Y17.

[0016] Figure 8 Ethanol sensitivity of Brucella phage Y17.

[0017] Fig. 9 Functional annotation of Brucella phage Y17 genes.

[0018] Fig.10 Phylogenetic tree of Brucella phage Y17. DETAILED DESCRIPTION

[0019] The present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments, but the protection scope of the present invention is not limited to the described contents.

[0020] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those generally understood by those of ordinary skill in the art to which the present invention belongs. Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0021] Example 1

[0022] Isolation, Purification and Observation of Brucella Phage Y17

[0023] 1. Enrichment and Isolation of Brucella Phages

[0024] (1) Sheep feces samples were collected from Ludian County, Yunnan Province as samples for isolating bacteriophages and were soaked in SM buffer at room temperature for 24 h.

[0025] (2) Brucella bovis vaccine strain A19 (approval number: veterinary drug 050157010, batch number: 22940901, Jinyu Baoling Biological Pharmaceutical Co., Ltd.) was inoculated into 5 mL of sterile Brucella broth and cultured overnight at 37°C and 220 r / min with shaking.

[0026] (3) Take 5 ml of the supernatant of the sample soaked in SM buffer in step (1), take 500 ul of Brucella bovis A19 bacterial solution in step (2) and 15 ml of Brucella broth medium to obtain a mixed solution, and culture it at 37°C and 220 r / min for 48 h to enrich Brucella phages.

[0027] (4) After the enrichment, the mixture was centrifuged at 8000 r / min for 5 min, and the supernatant was filtered using a 0.22 um filter membrane to obtain the enrichment filtrate. 400 μL of the enrichment filtrate was mixed with 200 ul of Brucella bovis vaccine strain A19 bacterial solution, and the mixture was placed in a 37°C constant temperature incubator for 24-48 h for plaque screening and purification using the double-layer agar plate method.

[0028] 2. Screening and purification of Brucella phage

[0029] Use a pipette tip to pick up a single plaque and put it into 10 ml Brucella broth, add 200 ul Brucella bovis A19 bacterial solution, and culture overnight at 37°C and 220 r / min. Then use the double-layer plate method to obtain a single plaque. Repeat this step until the plaques are uniform in size and morphology, thus obtaining purified phages.

[0030] like Figure 1 As shown, a Brucella phage was isolated and named Y17. The double-layer agar plate made by mixing the phage suspension with Brucella bovis A19 bacterial liquid showed transparent plaques with a diameter of 4-6 mm.

[0031] 3. Morphological observation of Brucella phage Y17

[0032] 20ul of purified phage was added to the copper grid and allowed to settle for 15 minutes. Then, 1 drop of 2% phosphotungstic acid was added to the copper grid for 10 minutes. Then, the morphology of the phage was observed under a Hitachi HT7700 transmission electron microscope, as shown in Figure 2. Figure 2 As shown, transmission electron microscopy showed that Brucella phage Y17 has a regular polyhedral head with a diameter of about 48.1±2nm and a retractable tail with a length of about 10.8±1nm, belonging to the Podoviridae family.

[0033] Example 2

[0034] Biological characteristics of Brucella phage Y17

[0035] 1. Optimal MOI

[0036] Take 1ml of phage with different dilution concentrations and mix with 1ml of host bacterial solution according to different infection multiplicity (MOI=number of phages / number of bacteria, MOI is: 10, 1, 0.1, 0.01, 0.001 and 0.0001 respectively), and culture at 37℃, 220r / min shaking table for 24h, and detect the number of plaques by double-layer plate method, repeat 3 times, and calculate the phage titer under different infection multiplicity conditions. The ratio of bacteria and phage tested by MOI is shown in Table 1 below.

[0037] Table 1

[0038] Brucella A19 viable count Titer of Brucella phage Y17 MOI <![CDATA[1.0×10 8 CFU / ml]]> <![CDATA[1.0×10 9 PFU / ml]]> 10 <![CDATA[1.0×10 8 CFU / ml]]> <![CDATA[1.0×10 8 PFU / ml]]> 1 <![CDATA[10×10 8 CFU / ml]]> <![CDATA[1.0×10 7 PFU / ml]]> 0.1 <![CDATA[1.0×10 8 CFU / ml]]> <![CDATA[1.0×10 6 PFU / ml]]> 0.01 <![CDATA[1.0×10 8 CFU / ml]]> <![CDATA[1.0×10 5 PFU / ml]]> 0.001 <![CDATA[1.0×10 8 CFU / ml]]> <![CDATA[1.0×10 4 PFU / ml]]> 0.0001

[0039] like Figure 3 As shown, the optimal MOI of Brucella phage Y17 is 0.001.

[0040] 2. One-step growth curve

[0041] Take 500ul of Brucella bovis A19 bacterial suspension and 500ul of Brucella phage Y17 at the optimal MOI, adsorb at 37℃ for 10min, centrifuge at 4℃, 5500r / min for 15min, discard the supernatant, resuspend the precipitate with 8mL of Brucella broth medium, place in an air shaker at 37℃, 220r / min for 2h, take samples at 10min intervals to detect the phage titer; draw a one-step growth curve with time as the horizontal axis and the measured titer as the vertical axis. Phage lysis amount = phage titer at the end of the outbreak / host bacteria concentration at the beginning of infection; if Figure 4As shown, the number of Brucella phage Y17 infecting the host bacteria remained unchanged within 40 minutes and was in a latent state; the number of Brucella phage Y17 increased sharply within 40-80 minutes of infection and entered the burst lysis period, with an outbreak time of 40 minutes and an outbreak volume of 187 pfu / cell; after 80 minutes of infection, the number of Brucella phage Y17 tended to stabilize and entered a plateau period.

[0042] 3. Temperature sensitivity of Brucella phage Y17

[0043] Take 1ml titer as 10 10 pfu / cell Brucella phage Y17 suspension was placed in a water bath at 4°C, 28°C, 37°C, 42°C, 50°C, 60°C, 70°C, and 80°C, and the samples were taken out after 1 hour, and the titer was measured by the double-layer agar method; Figure 5 As shown in the figure, the titer of Brucella phage Y17 remained basically stable at temperatures below 50°C, and gradually began to decrease when the temperature was above 50°C until it was completely inactivated at 80°C, indicating that Brucella phage Y17 has a strong tolerance to high temperatures.

[0044] 4. UV sensitivity of Brucella phage Y17

[0045] Take 5ml and the potency is 10 10 pfu / ml Brucella phage Y17 suspension was placed in a 9-cm diameter dish, which was placed 15 cm away from the UV lamp and irradiated continuously for 1 hour. During this period, 300 ul of sample was taken every 5 minutes and its titer was measured using the double-layer agar method; Figure 6 As shown, the titer of the phage decreased after ultraviolet irradiation, but it still had strong activity after 1 hour of irradiation, indicating that Brucella phage Y17 has strong tolerance to ultraviolet rays.

[0046] 5. pH sensitivity of Brucella phage Y17

[0047] Take 10ul and the titer is 10 10 pfu / ml Brucella phage Y17 suspension was added to 990ul of a solution with a pH value of 0-14, mixed and incubated at 37°C for 1h, and then the titer was measured using the double-layer agar method; Figure 7 As shown, the titer of Brucella phage Y17 remained basically unchanged at a high level when the pH value was 3-11. When pH ≤ 2 or pH ≥ 12, the activity of Brucella phage Y17 completely disappeared, indicating that the suitable pH range of Brucella phage Y17 was relatively wide.

[0048] 6. Ethanol sensitivity of Brucella phage Y17

[0049] Take the titer as 10 10pfu / ml Brucella phage Y17 suspension was mixed with anhydrous ethanol to make the final volume concentration of ethanol reach 10%, 25%, 50%, 75%, and 90%, and then incubated at 37°C for 1 hour, and then the titer was measured using the double-layer agar method; Figure 8 As shown, the titer of Brucella phage Y17 decreased under treatment with different concentrations of ethanol, but it was not completely inactivated, so Brucella phage Y17 has a certain tolerance to ethanol.

[0050] 7. Determination of host spectrum of Brucella phage Y17

[0051] The lysis spectrum of phage was determined by spot assay.

[0052] (1) 30 Brucella melitensis clinical isolates (from Kunming, Qujing, and Yuxi, Yunnan Province), Brucella melitensis M5 vaccine strain, Brucella bovis 104M vaccine strain, and Brucella bovis A19 vaccine strain. After activation, single colonies of all the strains were picked and inoculated into Brucella broth, and cultured at 37°C and 220 r / min in a shaker for 48 h to obtain bacterial suspension.

[0053] (2) Take 200ul of bacterial liquid to make a double-layer plate. After it is completely solidified, take 20ul of Brucella phage Y17 droplet and place it at the corresponding position of the plate. Incubate at 37°C for 48h to observe whether there is plaque formation. Compare it with the existing Brucella phage Tb. The results are shown in Table 2. The bactericidal effects of Brucella phage Y17 and Brucella phage Tb are different. Brucella phage Y17 mainly shows a strong lysis effect on bovine Brucella vaccine strains and has a certain lysis ability on most melitensis Brucella. However, the plaques are generally blurred and only have a strong lysis effect on individual melitensis Brucella. Therefore, Brucella phage Y17 can be used to prepare Brucella bactericidal biological agents.

[0054] Table 2

[0055]

[0056] Note: + clear plaque; ± fuzzy plaque; - no plaque. Numbers 1-30 are Brucella strains isolated from clinical samples, numbers 31-33 are Brucella vaccine strains, and biotype N is not detected.

[0057] Example 3

[0058] Whole genome sequencing analysis of Brucella phage Y17

[0059] Brucella phage Y17 was propagated to a high titer (>10 10pfu / ml), the DNA of Brucella phage Y17 was extracted using the steps of the lambda phage DNA extraction kit (purchased from Beijing Aibogen Biotechnology Co., Ltd.), and Guangdong MegaGene Technology Co., Ltd. was commissioned to sequence the whole genome of Brucella phage Y17. BWA (v0.7.17) software was used to align and clean reads related to the host genome, and the host genome sequence was removed. Megahit software was used for de novo assembly (v1.1.2SCIME). Open reading frames (ORFs) were identified by RAST, and then compared with all other lytic Brucella phage sequences using NCBI BLAST+ software (version 2.16.0) to further predict the function of each protein, and then the whole genome map was drawn by SnapGene (v6.0.2). PhageLeads checked the lysogenicity, antibiotic resistance genes, and virulence genes in the genome, and Orthofinder was used to construct a phylogenetic tree of the whole genome amino acid sequence based on the maximum likelihood method.

[0060] The results showed that Brucella phage Y17 is a circular double-stranded DNA phage with a full genome size of 38025bp, which is the smallest Brucella phage isolated so far, with a GC content of 48.2%. RAST predicted 56 ORFs in the complete genome of Brucella phage Y17. Using blastp to predict the functions of the 56 ORFs of Brucella phage Y17, 26 functional proteins were predicted, and one gene product of unknown function prediction had no homology with the sequence published in the database. The 26 functional proteins of Brucella phage Y17 were divided into 5 modules: metabolism and management, DNA packaging, structure, host lysis, DNA replication and modification. No proteins related to the lysogeny module were found. The gene function annotations are shown in Fig. 9 PhageLeads was used to examine the genome of Brucella phage Y17, and it was predicted that there were no genes encoding lysogeny, antibiotic resistance, and bacterial virulence-related genes. The results showed that Brucella phage Y17 is a lytic Brucella phage and is also safe. Fig.10 As shown in the figure, the phylogenetic tree of Brucella phage Y17 and 21 strains of Brucella phage constructed based on the whole genome is divided into two branches. Brucella phage Y17 and Iz are closely related. At the same time, it can be seen that Brucella phage Y17 has a close relationship with Bk2, Pr, R / C, Bk, Wb, and S708 phages.

Claims

1. Brucella phage Y17, characterized in that: The deposit number of the Brucella phage Y17 is CCTCC NO:M 20241682.

2. Use of the Brucella phage Y17 according to claim 1 in the preparation of Brucella bactericidal biological preparations.

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