CN115299438B

CN115299438B - Use of dopamine in anti-phage

Info

Publication number: CN115299438B
Application number: CN202210931078.XA
Authority: CN
Inventors: 张胜婷; 张春婷; 魏云林; 向盈盈
Original assignee: Yunnan Minzu University
Current assignee: Yunnan Minzu University
Priority date: 2022-08-04
Filing date: 2022-08-04
Publication date: 2023-07-21
Anticipated expiration: 2042-08-04
Also published as: CN115299438A

Abstract

The invention discloses application of dopamine in anti-phage, which utilizes three phages of different families as materials to carry out an anti-phage infection experiment, and obtains that the minimum effective concentration of DA on vB-EcoS-IME167, T4 phage and VMY22 is 0.1-1 mg mL ^‑1 Between, i.e., μM levels, and DA is resistant to infection by the long tail phage vB-EcoS-IME167 ₅₀ The value was 0.26mg mL ^‑1 Resistance is improved by 10 ⁵ Doubling; IC for resisting infection of Combretaceae quisqualis thallus T4 phage ₅₀ The value was 0.12mg mL ^‑1 Resistance is improved by 10 ⁴ Doubling; IC resistant to infection by Brevibacterium phage VMY22 ₅₀ The value was 0.73mg mL ^‑1 Resistance is improved by 10 ⁴ Doubling; DA was shown to have a generally efficient ability to resist phage infection.

Description

Use of dopamine in anti-phage

Technical Field

The invention belongs to the field of dopamine application, and particularly relates to application of dopamine in anti-phage.

Background

Phages are a class of bacterial viruses that are diverse on earth, and the process of infecting bacteria with typical phages can be divided into three phases: infection stage, proliferation stage and maturation stage.

Infection stage: the first step in phage infection of host cells is "adsorption", i.e., attachment of the tail of the phage to the cell wall of the bacterium followed by "invasion". The phage firstly opens a gap on the cell wall of the bacterium through the action of lysozyme, the tail sheath contracts like the action of actin to expose the tail shaft and extend into the cell wall, the phage only injects DNA at the head into the cell of the bacterium like the injection action of a syringe, and the protein shell of the phage is left outside the wall and does not participate in the proliferation process.

Proliferation stage: after phage DNA enters bacterial cells, a series of changes are caused: the DNA synthesis of the bacteria ceases, the enzyme synthesis is also repressed, and the phage gradually controls the metabolism of the cell. Phages ingeniously utilize the "machinery" of the host (bacterial) cell to replicate the DNA and proteins of the progeny phage in large quantities and form complete phage particles. Phage formation is manipulated by the nucleic acid material itself by means of the metabolic machinery of the bacterial cell. When phage invade bacterial cells, the cytoplasm of the bacteria fills with DNA filaments soon, starting to develop a complete polygonal head structure in about ten minutes. When the phage is mature, the DNA macromolecules are condensed into polyhedrons, the head proteins surround the polygon DNA condensation bodies through the arrangement and crystallization process, and then the head and the tail are mutually matched to assemble a complete progeny phage.

Maturation stage: after phage maturation, lysozyme which lyses the host cell wall gradually increases at the late stage of incubation, causing the cells to lyse, thereby releasing the progeny phage. The cultured infected cells were observed under an optical microscope, and the cell lysis phenomenon was directly seen. T2 phage can produce 100 to 300 progeny phage at 37℃approximately only in forty minutes. After the progeny phage is released, neighboring bacterial cells are again infected, producing daughter second-generation phage.

And the phage belongs to a class of bacterial viruses, and can crack host bacteria in a short time after infection; they are ubiquitous in all the earth's environment and ecosystem, and can be extracted 10 per gram of soil ⁷ Phage, 10 per ml of sewage ⁸ The phage above. The modern fermentation industry utilizes genetically engineered bacteria products in many fields such as food, chemical industry, energy, enzyme preparation, medicine, materials and the like, but the pollution of phage is an important factor causing economic loss of the fermentation industry every year.

Catecholamines (CA) are a class of biogenic amines containing catechol (catechol) groups and side chain amino groups, mainly Dopamine (DA), norepinephrine (NE) and epinephrine (E). Catecholamines, which are an important neurotransmitter and hormone in animals, are widely available, mainly to regulate neural activity and to enhance the body's ability to adapt under the influence of various stimuli. Catecholamines have been detected in organs and tissues of various plants by researchers in the last century, and have been mainly used to enhance stress resistance and antipathogenic infection activity. In recent years, catecholamine substances are detected in various common microbial communities, and a large number of phage particles are generally also present in environments containing abundant microbial bacteria, so that research on the effect of the catecholamine substances on phage-bacteria systems is particularly important.

Dopamine (DA) is the first catecholamine produced in the catecholamine substance synthesis pathway. There is no report on the effect of dopamine on phage.

Disclosure of Invention

According to the invention, dopamine is used as an experimental material, and whether the DA has an anti-infection effect on 3 kinds of phages is studied from common long-tail phages, myocaudal phages and short-tail phages, so that a specific anti-phage action mechanism is further studied in depth.

The invention aims to study the application of dopamine in anti-phage.

Further, the phage includes a long-tail, a myotail, and a short-tail phage.

Further, the lowest effective concentration of the dopamine to the long tail phage vB-EcoS-IME167, the myocaudal phage TT4phage and the short tail phage VMY22 is 0.1-1 mg mL ^-1 The highest anti-infection concentration is 5-10 mg mL ^-1 。

Further, the dopamine has a semi-inhibitory concentration IC for the long tail phage vB-EcoS-IME167 ₅₀ The value was 0.26mg mL ^-1 。

Further, the dopamine has semi-inhibitory concentration IC to Brevibacterium phage VMY22 ₅₀ The value was 0.73mg mL ^-1 。

Further, the dopamine has semi-inhibitory concentration IC to the myocaudae phage T4phage ₅₀ The value was 0.12mg mL ^-1 。

Further, the application of the dopamine in preventing and treating phage pollution in the fermentation industry.

Furthermore, the dopamine anti-phage mechanism is used for destroying the complete form of phage and then destroying the specific adsorption in the phage process, so as to resist the infection of phage to host bacteria.

Compared with the prior art, the invention has the beneficial effects that:

(1) The general anti-phage infection activity of dopamine is found for the first time; can provide a new research direction for preventing and treating phage pollution in the modern fermentation industry.

(2) The anti-phage infection activity of dopamine can provide a new idea for developing novel antiviral drugs in the field of biological pharmacy.

(3) The research fills the blank of research on the role of catecholamines in microorganisms, and can further research the role played by catecholamines in the defense and anti-defense of the bacteria-phage system.

Drawings

FIG. 1 is a graph showing the effect of treatment of vB-EcoS-IME167 with LB medium and MM medium (mean+ -SD, n=3, a: p >0.05, b: p < 0.05);

FIG. 2 is a bar graph showing the ability of DA to resist vB-EcoS-IME167 infection by LB, MM and LB medium dilution, MM medium dilution in free combination;

wherein: left LB bacteria+lb, MM phage group, right LB, MM bacteria+mm phage group (mean±sd, n=3, a: p >0.05, b: p < 0.05);

FIG. 3 is a bar graph of LB medium composition defect medium verifying the ability to combat vB-EcoS-IME167 infection of host bacteria (mean+ -SD, n=3, a: p >0.05, b: p < 0.05);

fig. 4 is a graph of DA anti-vB-EcoS-IME 167 infectivity versus bar graph in MM medium and saline (mean±sd, n=3, a: p >0.05, b: p <0.05, p <0.01, p <0.001, p < 0.0001);

FIG. 5 results of experiments with different concentrations of DA to treat vB-EcoS-IME 167;

FIG. 6 shows the effect of DA on vB-EcoS-IME167 phage at different incubation times;

FIG. 7 is a bar graph showing the effects of DA on host bacteria and phage, respectively;

FIG. 8 shows phage in the ultracentrifugation tube after ultracentrifugation purification of vB-EcoS-IME 167;

FIG. 9 is a transmission electron micrograph of a vB-EcoS-IME167 phage;

wherein: (a) a normal aqueous solution state, (b) a low concentration DA solution state, and (c) a high concentration DA solution state;

FIG. 10 standard curve of recombinant plasmid standard of vB-EcoS-IME167 phage;

FIG. 11 shows changes in the expression level of vB-EcoS-IME167 phage after treatment in the control group (group A) and the experimental group (group B);

FIG. 12 is a graph showing the effect of treatment of T4 phase with LB medium and MM medium (mean+ -SD, n=3, a: p >0.05, b: p < 0.05);

FIG. 13 is a bar graph showing the ability of DA to resist T4 phase infection by LB, MM and LB medium dilution, MM medium dilution in free combination;

Wherein: left LB bacteria and LB, MM medium combinations, right LB, MM bacteria and MM medium combinations (mean±sd, n=3, a: p >0.05, b: p < 0.05);

FIG. 14 shows the effect of DA treatment on T4 phase in MM medium and saline (mean+ -SD, n=3, a: p >0.05, b: p < 0.05);

fig. 15 shows experimental results of DA treatment T4 phase at different concentrations (DA concentrations: 0, 0.1, 1, 2.5, 5, 10mg mL-1, mean±sd, n=3, < p <0.05, < p <0.01, < p <0.001, < p < 0.0001);

FIG. 16 shows experimental results of DA anti-T4 phage phages at various incubation times;

FIG. 17 shows the anti-infective effect of DA treatment on host bacteria or phages (mean+ -SD, n=3, a: p >0.05, b: p < 0.05);

FIG. 18 phage in the ultracentrifuge tube after ultracentrifugation purification of T4 phase;

FIG. 19 is a transmission electron micrograph of T4 phage;

FIG. 20 is a standard curve of recombinant plasmid standard of T4 phage;

FIG. 21 shows the change in the expression level of T4 phage after treatment in the control group (group A) and the experimental group (group B);

FIG. 22 is the effect of treating VMY22 in LB and MM medium environments (mean+ -SD, n=3, a: p >0.05, b: p < 0.05);

FIG. 23 is a bar graph showing the ability of DA to resist VMY22 infection by LB, MM and LB medium dilution, MM medium dilution in free combination;

FIG. 24 shows the experimental results of DA treatment of VMY22 in MM medium and saline (mean+ -SD, n=3, a: p >0.05, b: p < 0.05);

fig. 25 is an experiment of different concentrations of DA treated VMY22 (DA concentrations: 0, 0.1, 1, 2.5, 5, 10mg mL-1, mean±sd, n=3, < p <0.05, < p <0.01, < p <0.001, < p < 0.0001);

FIG. 26 shows experimental results of DA anti-VMY 22 phage at different incubation times;

FIG. 27 anti-infective effect of DA-treated host bacteria or phages (mean+ -SD, n=3, a: p >0.05, b: p < 0.05);

FIG. 28 shows phage in the ultracentrifuge tube after ultracentrifugation purification of VMY 22;

FIG. 29 is a transmission electron microscope image of VMY22 phage;

FIG. 30 is a standard curve of recombinant plasmid standard of VMY22 phage;

FIG. 31 shows changes in the expression level of VMY22 phage after treatment in control (group A) and experimental (group B).

Detailed Description

The objects, technical solutions and advantages of the present invention will become more apparent by the following detailed description of the present invention when taken in conjunction with the accompanying drawings. It should be understood that the description is only illustrative and is not intended to limit the scope of the invention. In addition, in the following invention, descriptions of well-known structures and techniques are omitted so as not to unnecessarily obscure the concepts of the present invention.

Example 1: anti-phage assay preparation

1. Experimental materials

The experimental strains included:

coli Escherichia coli BW25113 (e.coli BW 25113) from the Lu Tao teacher of the institute of microbiology at the university of yunnan;

coli Escherichia coli (ATCC 11303) (e.coli ATCC 11303) was purchased from American Type Culture Collection (ATCC);

bacillus cereus Bacillus cereus MYB-22 (B.cereus) was isolated from the laboratory in Ming Yongzhuang glacier sample cultures.

The experimental phages included:

the long-tail phage vB-EcoS-IME167, short-tail phage VMY22, and the myotail phage T4 phage purchased from the Cantonese institute of microbiology, inc., isolated from this laboratory.

Plasmid system:

E.coli DH 5. Alpha. Required for recombinant plasmid experiments were purchased from the Optimus Praeparata (Kunming, china);

the pMD-19T vector required for ligation of the desired fragment was purchased from Takara (Beijing, china);

primer fragments were synthesized by the division of Kunming, of the family of the organisms, inc. (Kunming, china);

ex Taq enzyme for experiment was purchased from Takara reagent company (Beijing, china);

viral genome extraction kit e.z.n.a. ^TM The Viral DNA Kit was purchased from Yunnan Chien biotechnology Co., ltd (Kunming, china);

the high purity Plasmid extraction Kit Plasmid Mini Kit was purchased from Yunnan Shuoyang technologies Inc. (Kunming, china).

Preparing a culture medium:

LB solid medium: 10g of tryptone powder, 5g of yeast powder, 10g of NaCl, 15g of agar and 1000mL of ultrapure water;

LB semisolid culture medium: 10g of tryptone powder, 5g of yeast powder, 10g of NaCl, 5g of agar and 1000mL of ultrapure water;

MM medium (1L): 5 xMq solution 200mL, 20% glucose 10mL, 1M L ^-1 MgSO ₄ 1mL, ultra pure water 800mL

(Note: 5 times concentrated Mq solution: na) ₂ HPO ₄ ·7H ₂ O64 g、KH ₂ PO ₄ 15 g、NaCl2.5 g、NH ₄ Cl5g, 1000mL of ultrapure water.

Both the liquid and solid media were autoclaved at 121℃for 20 min. Note that: the MM medium is prepared for standby in an ultra clean bench after the components are sterilized separately. )

2. Activation of host bacteria

Taking out the preservation strains of host bacteria corresponding to three phages from an ultralow temperature refrigerator at the temperature of minus 80 ℃, placing E.coli BW25113, E.coli ATCC 11303 and B.cereus MBY41-22 of the three phages on crushed ice for natural melting, dipping a small amount of bacterial preservation solution of E.coli BW25113 on a culture dish containing LB solid medium by using a sterile inoculating loop in a sterile ultra-clean bench after ultraviolet irradiation for 15min, and writing marks, strains, culture medium, date and name for later use; finally, the cells were placed in an incubator (37 ℃ C.) with optimal temperature for E.coli and cultured upside down.

The strain activation procedure for E.coli ATCC 11303 was the same as described above; the strain activation of Bacillus cereus MBY41-22 was slightly different from the above, the optimum growth temperature of Bacillus cereus was 28℃and the strain was finally placed in an incubator at 28℃for cultivation.

3. Phage enrichment

1. Enrichment of Long-tail phage vB-EcoS-IME167

(1) Taking out the activated LB solid medium plate of E.coli BW25113, selecting a single colony in a test tube containing 5mL of sterilized LB liquid medium by using a sterile inoculating loop cooled after burning by an alcohol lamp in a sterile super clean bench, slowly and slightly oscillating, then covering a test tube cover, and writing a mark.

(2) Then placed at 37 ℃ for 160r min ^-1 Culturing in incubator until the bacterial liquid OD ₆₀₀ About 0.8, the culture medium was transferred from the test tube to a sterilized LB liquid culture containing 500mL at a bacterial load of 3%Performing amplification culture in medium until OD ₆₀₀ And after reaching 0.8, the product is ready for use.

(3) Phage of the pre-laboratory stored vB-EcoS-IME167 were removed and transferred to OD at 10% inoculum size ₆₀₀ Operating in a sterile super clean bench in a bacterial liquid conical flask reaching 0.8, and then continuously placing in a 37 ℃ incubator for 160r min ^-1 Culturing.

(4) The culture was continued for about 6-8 hours until the solution in the flask appeared clear and transparent. The solution was removed and centrifuged at 8000g for 15min in a centrifuge, leaving the supernatant, and the pellet was discarded.

(5) The collected supernatant was passed through a sterile 0.22 μm aqueous filter to give phage stock, and stored in a 4℃refrigerator using a sterile 5mL plastic EP tube aliquot, and labeled with phage name, date, name, and so forth, ready for use.

2. Enrichment of myocaudal phage T4 phase

Similar to the above procedure, E.coli BW25113 was replaced with E.coli ATCC 11303 and vB-EcoS-IME167 was replaced with T4 phase.

3. Enrichment of Brevibacterium phage VMY22

Similar to the above procedure, E.coli BW25113 was changed to B.cereus MYB41-22, vB-EcoS-IME167 was changed to VMY22, and the incubator at 37℃was changed to 28 ℃.

Example 2: DA experiment on Long-tail phage vB-EcoS-IME167

1. Incubation environmental impact experiment

The results of experiments performed in LB medium and MM medium environments, respectively, using sterile water as a control group and DA treatment as an experimental group, namely, diluting vB-EcoS-IME167 phage with MM medium and LB medium, respectively, when the double-layer plate method was used, are shown in FIG. 1. In the figure, group a is a group in which phages are diluted by using LB culture medium, and a sterile water control group and a DA treatment group have no obvious difference; group b is a group in which phages were diluted with MM medium, wherein there was a significant difference in the results between the sterile water control group and the DA-treated group, which had a plaque count of about 10 lower than that of the sterile water control group ³ Multiple times. It can be shown that DA was present on vB-EcoS-IME167 phage in the MM medium environmentAt 10 ³ Double resistance to infection (logarithmic difference between PFU/mL in water control and DA treated groups in MM medium group of FIG. 1)>3, it is considered that DA exists 10 in the MM medium environment ³ Multiple anti-infection capability. ) The method comprises the steps of carrying out a first treatment on the surface of the In the LB medium environment, DA was not present against vB-EcoS-IME167 infection.

In order to further confirm the influence of the environment of the host bacteria and phage LB medium in the host bacteria-phage system, experiments were designed on LB culture bacteria, MM culture bacteria and free combination experiments of LB medium dilution and MM medium dilution, and the following results were obtained: FIG. 2 shows the result of experiments of the same LB medium-based host bacteria, vB-EcoS-IME167 phage treated with LB medium and MM medium, and DA shows 10 ³ The capability of resisting vB-EcoS-IME167 infection of host bacteria by more than times; in contrast, the LB host bacteria+LB phage group showed no significant data differences, and DA did not exhibit the ability to combat vB-EcoS-IME167 infection of the host bacteria. The results show that the environment of the LB medium of the host bacteria does not influence the phage infection resistance effect of DA. The right side of FIG. 2 shows that the LB and MM medium culture hosts are mixed with phage in the same MM medium environment, and the result shows that the difference between DA and sterile water is 10 ³ More than two times. The results show that the culture medium environment of DA treatment vB-EcoS-IME167 is an important factor influencing the infection resistance, and the DA can show strong effect of blocking vB-EcoS-IME167 from infecting host bacteria in the MM culture medium environment, and the highest infection resistance can reach 10 ⁴ Multiple times. The experiment shows that the same host bacteria environment, the phage MM medium environment DA has anti-infection activity, and LB does not exist; the same MM culture medium environment and different host bacteria environments DA have anti-infection activity. DA is shown to be only affected by the phage medium environment in phage-host bacteria systems and to exhibit anti-infective activity only in MM medium environment.

Table 1 DA groups and results of experiments on the incubation environments of Long tail phage vB-EcoS-IME167

Since the LB medium consisted mainly of yeast powder, peptone and sodium chloride, and the ionic state of sodium chloride in aqueous solution was negligible, it was further intended to simply confirm whether yeast powder or peptone affected DA-treated phages, and a component defect medium validation experiment was prepared, and the results are shown in FIG. 3. The experimental results show that in the environment of the LB defect culture medium of simple yeast-lack powder or peptone, the data difference between DA and sterile water is not obvious. This suggests that DA does not exhibit resistance to infection of host bacteria by vB-EcoS-IME167 in LB medium is not a simple yeast powder or peptone effect, and that it is probably the result of the common effect of complex components in LB medium, and that it is worth later intensive study of differences in medium components and the cause of the effect; it is also demonstrated that the ability of DA to combat vB-EcoS-IME167 infection by host bacteria occurs only in a relatively simple environment.

2. Concentration experiment:

in order to detect whether the quantitative relationship exists between the infection capacity and concentration of DA anti-vB-EcoS-IME 167 phage, experiments were designed to be 0, 0.1, 1, 2.5, 5 and 10mg mL ^-1 Experiments with DA solutions of different concentration gradients for their ability to resist vB-EcoS-IME167 infection. As shown in the experimental results in FIG. 5, the activity of DA solution against vB-EcoS-IME167 infection increases with the increase of concentration in the low concentration range, and the activity of DA solution against vB-EcoS-IME167 infection tends to be stable in the high concentration range; the effective concentration of DA anti-vB-EcoS-IME 167 phage infection activity is 0.1mg mL ^-1 Thereafter, as shown in FIG. 5 (right), the half-inhibitory concentration value IC of DA for vB-EcoS-IME167 was calculated using the concentration and the inhibition ratio ₅₀ The value was 0.26mg mL ^-1 At a concentration of 5mg mL ^-1 After this time, the anti-infective force reached a maximum and remained stable, about 10 compared to the control ⁵ Multiple anti-infection capability.

3. Incubation time experiment

For the long tail phage vB-EcoS-IME167, DA treatment in the MM medium environment for different times may result in different degrees of anti-phage activity. Experiments designed different incubation times of 10, 30, 60, 90, 120min respectivelyResistance experiments, the results of which are shown in FIG. 6 below. As can be seen from the figure, about 10-fold inhibition of the ability of vB-EcoS-IME167 to infect host bacteria was seen in DA in the 10min group compared to the sterile water control group; after a subsequent increase over time, DA showed an increased resistance to vB-EcoS-IME167 infestation of about 10 after 60min ⁴ The doubled anti-vB-EcoS-IME 167 infection effect, after which the resistance tended to stabilize without enhancement. From the overall trend, it was found that DA rapidly developed anti-infective activity to vB-EcoS-IME167 phage in a short period of time, and the anti-infective activity increased over time in a relatively short period of time, eventually reaching about 10 ⁴ Double resistance to vB-EcoS-IME167 infection.

To confirm whether DA acts on the host bacteria or phage, experimental studies were performed in the MM medium environment with either the host bacteria E.coli (BW 25113) or phage vB-EcoS-IME167 as experimental variables. As a result, as shown in FIG. 7, it was found that the difference in the number of group plaques of DA and the sterile water-treated host bacterium E.coli (BW 25113) was not significant, and that the difference in the number of group plaques of DA and the sterile water-treated phage vB-EcoS-IME167 was significant, indicating that DA acted only on phage vB-EcoS-IME167 and did not act on host bacterium E.coli (BW 25113).

Example 3: experimental result of DA on myocaudal phage T4 phase

1. Incubation environmental impact experiment

And (3) selecting the myocaudal phage T4 phase as an experimental material, and carrying out a related experiment of the DA on the research on the infection resistance of the myocaudal phage. First, a related experiment of the infection resistance of T4 hage was performed in an LM medium environment and an MM medium environment using sterile water as a control group and DA as an experimental group, respectively, to obtain the following experimental result diagram of fig. 12. As can be seen from FIG. 12, in the LB medium environment, the sterile water control and the T4 phage of the DA experimental group were pfu.mL ^-1 The difference of the logarithmic values is not obvious, in the large environment of the MM culture medium, the obvious difference of the sterile water control and DA experiment group appears, and PFU mL of the T4phage of the control group ^-1 About 10 ⁸ The DA group was approximately 10 ⁵ Two, between the two groups, are different by 10 ³ Multiple times. This indicates that the medium environment is also a factor limiting the ability of DA to resist T4 phase infection, and that, likewise, DA does not exhibit resistance to T4 phase infection in LB medium environment and exhibits about 10 in MM medium environment ³ More than double the ability to resist T4 phase infestation.

Similarly, in order to confirm the influence of environmental factors of the host bacteria E.coli (ATCC 11303) and phage T4 phase medium, respectively, experiments were designed on LB culture bacteria, MM culture bacteria, and dilution of LB medium, and free combination experiments in which MM medium was used to dilute T4 phase, and the results of the experiments shown in FIG. 13 were obtained. From the left side of FIG. 13, it can be found that the difference between the LB bacteria and the LB phage is not obvious, and the difference between the LB bacteria and the MM phage is obvious, and the difference reaches 10 ³ Multiple times. The right part of FIG. 13 shows that LB bacteria+MM phage and MM bacteria+MM phage data show significant differences in the resulting bacteria, and that experimental groups all showed more than 10 ⁴ The ability to resist T4 phase infestation by a fold. This shows that for the myourological phage T4 phase, the host bacteria medium environment is an independent variable factor of the experiment, the phage medium environment factor is an important factor, and the anti-infectivity ability of DA is only exhibited in the MM medium environment. This experiment was repeated 3 times and the trend of the experimental results was consistent.

2. Concentration experiments

DA has higher effect of inhibiting the infection of the T4phage to host bacteria in the MM culture medium environment, and experiments further develop the method in mu.mol ^-1 －n·mol ^-1 The study of the dose-response relationship in the low concentration range of (1) simply selects 0, 0.1, 1, 2.5, 5 and 10mg mL ^-1 DA solutions of different concentrations were treated with T4phage in a large MM medium environment and the plaque assay was performed on double plates to obtain the following experimental results in FIG. 15. FIG. 15 (right) shows that when the DA concentration is 0.1mg mL ^-1 At the time (i.e., μM level), DA was not active against T4 hage infection when the concentration reached 1mg mL ^-1 At (i.e., mM level), DA has demonstrated significant resistance to T4-phase infection when concentrations reach 10mg mL ^-1 Differential p between DA and sterile Water group<0.0001 to a maximum of about 10 ⁴ Resistance more than double. This indicates that DAThere is a dose-to-effect relationship between concentration and resistance to T4phage infection, and the resistance to infection increases with increasing concentration. FIG. 15 (right) shows that the half inhibitory concentration of DA on the myocaudal phage T4phage, IC, was obtained by further treatment of the concentration of DA and the inhibitory rate on T4phage ₅₀ The value was 0.12mg mL ^-1 。

3. Incubation time experiment

For the myocaudal phage T4phage, a group of experimental researches for different incubation times under the condition of uniform other experimental conditions are simply designed in order to further determine the influence of the incubation time on the capability of DA for resisting the T4phage infection. Time gradient experiments were set for 10, 30, 60, 90, 120min, and the experimental results are shown in fig. 16. The experimental result shows that the anti-infection activity of DA on T4phage is not obvious in the group of 10min, the control group and the experimental group. After 10min, the anti-T4-phase infection capability of DA increases rapidly with time, and after 90min, the anti-infection capability reaches 10 ⁵ Multiple times. This suggests that incubation time is an important factor affecting the ability of DA to resist T4 hage infection and that over a range of time the ability of DA to resist T4 hage infection increases with time.

In order to further confirm whether DA acts on host bacterium E.coli (ATCC 11303) or phage T4phage, a grouping experiment was performed in which DA was used to treat host bacterium E.coli (ATCC 11303) and phage T4phage, respectively, and the experimental results are shown in FIG. 17. As a result, it was found that DA-treated host bacterium E.coli (ATCC 11303) did not exhibit an anti-infective effect, and that the treated phage T4phage exhibited 10 ³ Multiple anti-infective ability. This suggests that the site of action of DA against T4-phase infection is the phage and not the host.

Example 4: experimental results of DA on brachycarpus phage VMY22

1. Incubation environmental impact experiment

After determining that DA has anti-infection capability for both the long-tail phage vB-EcoS-IME167 and the myocaudal phage T4phage, the short-tail phage VMY22 separated in a laboratory is selected as an experimental material to verify whether the anti-phage infection capability of DA has universality. First, like the previous vB-EcoS-IME167 and T4 phaseSterile water is used as a control group, DA is used as an experimental group, and whether DA has different VMY22 infection resistance in different culture medium environments is analyzed. As a result of the experiment, as shown in FIG. 22, it was found that DA showed anti-infective activity in the middle of LB medium environment but showed about 10 in MM medium environment ³ Multiple anti-infection capability. This suggests that the demonstration of the ability of DA to resist VMY22 infection also requires that complex environments would mask the ability of DA to resist VMY22 infection by host B.cereus MBY41-22 in relatively simple environments.

In order to further confirm whether the culture medium environment of the host bacteria B.cereus MBY41-22 affects the ability of DA to resist VMY22 infection, experiments were designed on LB medium, MM medium and free combination experiments of LB medium VMY22, MM medium VMY 22. The experiment is shown in fig. 23, and the left result shows that the difference of LB bacteria and LB phage data is not obvious, and the difference of LB bacteria and MM phage data is obvious; the data difference of LB bacteria, MM phage and MM bacteria, MM phage all show great difference, and the difference is 10 ⁴ Multiple times. This shows that for VMY22, only the phage media processing environment is the decisive factor for the experiment, as long as DA is processing phage in MM media, the experimental results all show 10 ³ More than one-fold resistance to VMY22 infestation.

2. Concentration experiments

To further confirm the effect of concentration on the ability of DA to resist VMY22 infestation, experiments were performed with DA solutions of different concentrations, and the experimental results are shown in fig. 25. For VMY22 phage, the resistant minimum onset concentration of DA is between 0.1-1mg mL ^-1 In between, and as the concentration increases, the ability of DA to resist VMY22 infestation increases. At 10mg mL ^-1 The resistance of DA against VMY22 infection reaches 10 ⁴ More than two times, this suggests that there is a dose-response relationship between DA concentration and resistance to VMY22 infestation. The right half of FIG. 25 shows the analysis of DA concentration and half inhibitory concentration obtained by inhibiting infection rate, and shows that DA has an IC for VMY22 ₅₀ The value was 0.73mg mL ^-1 And the IC for the long tail phage obtained by the above experiment ₅₀ Value 0.26mg mL ^-1 IC for myocaudal phage ₅₀ Value 0.12mg mL ^-1 In contrast, DA versus Brevibacterium IC ₅₀ The value is larger.

3. Incubation time experiment

For the Brevibacterium phage VMY22, previous series of experiments have examined the effect of environmental and concentration factors on the ability of DA to resist VMY22 infection. And when other experimental conditions are uniform and optimal, further designing an influence experiment of a time factor on the VMY22 infection resistance of DA. The experimental results are shown in fig. 26, which shows that the incubation time is an important influence factor for affecting the anti-VMY 22 infectivity of DA, and that DA hardly shows the ability of resisting the VMY22 infection of host bacteria within 30 min; however, by 60min, the DA has a VMY22 infestation resistance of up to 10 ³ Doubling; after 60min, the ability of DA to resist VMY22 infestation did not increase to a large extent with increasing incubation time, reaching the highest 10 at 120min ⁴ Multiple anti-VMY 22 infection capacity.

To further confirm the ability of DA to combat VMY22 infection, either by acting on the host bacterium B.cereus MYB41-22 or on the phage VMY22, experimental studies were continued. As shown in the experimental results 2.28, it was found that the difference between the result of DA-treated host bacterium B.cereus MYB41-22 and the control group was not significant, and 10 was found between the result of DA-treated phage VMY22 and the control group ² Fold difference. This suggests that the DA-expressing site of action against VMY22 infection is consistent with both phage models described above, and acts on phage rather than host bacteria.

Example 5: concentrating and purifying high concentration phage particles

1. Purification and enrichment of long tail phage particles

1) From a culture dish plate containing E.coli BW25113, a single colony of host bacteria is picked up in a sterile super clean bench after 15min of ultraviolet irradiation by using an inoculating loop, and the single colony is placed in a sterile test tube containing 5mL of LB liquid culture medium after sterilization, marked and placed in a shaking table at 37 ℃ for culture. 6 tubes were inoculated simultaneously.

2) After the bacterial liquid in the test tube is cultured to a turbid state, the bacterial liquid is transferred into a 1L conical flask containing 500mL LB liquid culture medium according to an inoculation amount of 3 percent. Placing in a shaking table corresponding to the optimal culture temperature for culturing the host bacteria.

3) Detecting bacterial liquid OD ₆₀₀ Reaching 1.0 (about 8h of incubation), the corresponding pre-simply enriched vB-EcoS-IME167 phage solution (phage: host bacteria = 1:10) was added and the shake culture continued until the solution in the Erlenmeyer flask was clear and transparent (about 4-6 h).

4) The flask was taken out and placed at room temperature, after the solution therein had cooled to room temperature, 50. Mu.L of pancreatic DNase I and 50. Mu.L of RNase enzyme were added for digestion treatment for 30 minutes in order to digest out the nucleic acid substances released after lysis of the host bacteria.

5) Solid NaCl particles (29.2 g/500 mL) were added to each flask of culture, dissolved with stirring with a glass rod, and the flask was placed in ice-cubes for 60min. The solid NaCl is added to facilitate the separation of phage particles and bacterial cell debris afterwards.

6) At the end of the ice bath, a high capacity cryocentrifuge (4 ℃,11000g,15 min) separates bacterial cell debris from phage supernatant. The total volume of phage supernatant was measured with a measuring cylinder.

7) Phage supernatant was transferred in its entirety into a 2L beaker and 50g PEG per 500mL was added in total volume measured ₆₀₀₀ Particle ratio, adding corresponding amount of PEG ₆₀₀₀ And (3) particles. PEG was dissolved by slow stirring at room temperature using a magnetic stirrer ₆₀₀₀ And (3) particles.

8)PEG ₆₀₀₀ After the particles were completely dissolved, the solution was transferred to a new conical flask and placed in an ice-water bath for overnight sedimentation.

9) After the sedimentation was completed, the solution was centrifuged using a high capacity refrigerated centrifuge (4 ℃,11000g,15 min) and the supernatant was fully discarded, leaving only the vB-EcoS-IME167 phage pellet.

10 Lightly re-suspending the pellet in SM solution with a sterilized 5mL pipette tip; the addition was performed in a proportion of 8mL of SM solution per 500mL according to the volume obtained in step 6. (SM solution preparation method: naCl 5.8g L) ^-1 、MgSO ₄ ·7H ₂ O2 g L ^-1 、1moL L ^-1 Tris-Cl(PH＝7.5)50mL L ^-1 The mixture is sterilized at high temperature and high pressure by an autoclave and then is divided into small parts for use, and can be stored in a refrigerator at 4 ℃; to prevent contamination, only every small portion can makeThe medicine is used once. )

11 After 60min at room temperature, transferring the resuspended liquid into a sterilized 50mL centrifuge tube by using a pipette, adding chloroform solution with the same volume according to the volume of the resuspended liquid, and extracting the residual PEG in the solution ₆₀₀₀ And bacterial cell debris. After mixing well, the mixture was centrifuged at 3000g for 15min at 4℃to separate the organic phase and the hydrophilic phase, leaving the hydrophilic phase with phage particles in the upper part.

12 Phage solution volume was determined by adding the desired cesium chloride in a ratio of 0.75g solid cesium chloride per mL, and shaking gently until cesium chloride was completely dissolved.

13 Transferring the solution into a super-separation tube matched with a super-speed centrifuge, and centrifuging at 4 ℃ for 160000g and 12h.

14 At the end of centrifugation, the super-tube was carefully removed to give a bluish high concentration vB-EcoS-IME167 phage layer as shown in FIG. 8.

The concentrated high concentration vB-EcoS-IME167 phage particles were aspirated by syringe insertion into phage layer, labeled, and stored at 4deg.C for further use.

2. Purification and enrichment of myocaudal phage particles

The purification and enrichment operation of the myocaudal phage is consistent with the purification and enrichment operation of the long-tailed phage, and only the corresponding strain E.coli BW25113 and phage vB-EcoS-IME167 are required to be changed into the corresponding E.coli (ATCC 11303) and phage T4 phage, and the other operations are consistent, so that a bluish layer shown in figure 18 can be obtained, namely the T4 phage particles obtained through concentration, enrichment and purification.

3. Purification and enrichment of Brevibacterium phage particles

The purification and enrichment operation of the short-tail phage is consistent with that of the long-tail phage, only the corresponding strain E.coli BW25113 and phage vB-EcoS-IME167 are needed to be changed into the corresponding bacillus cereus B.cereus MBY41-22 and phage VMY22, the operation temperature is changed to 28 ℃, and the rest operations are consistent, so that the thin and thin layer which presents a slight blue color as shown in figure 28 is VMY22 phage particles.

4. Purification of concentrated phage solution: the phage layer solution extracted from the ultra-high speed centrifuge tube by the syringe is dialyzed by a dialysis bag. The main operation is as follows:

i. selecting a dialysis bag with a pore diameter capable of intercepting the molecular weight of 6000-8000 daltons, and boiling for later use.

Dialysis solvent, 3L sterile water+25.5g NaCl, final concentration 0.85% salt solution.

Transferring phage solution into dialysis bag, placing into dialysis solvent, and dialyzing and purifying on magnetic stirrer.

iv.3h of replacement of the dialysis solvent with saline for 4 total times.

v. after dialysis, sucking out phage from the dialysis bag, and preserving at 4 ℃ for standby.

5. Transmission electron microscope slide:

i. the purified high concentration vB-EcoS-IME167 or T4 phase or VMY22 phage solution is gently sucked into 20 mu L to 100 mu L sterilized plastic EP tube with a pipetting gun, 3 groups are synchronized, 20 mu L of sterile water and 0.01mg mL are added respectively ^-1 DA solution, 10mg mL ^-1 DA solution, incubating for 60min;

sucking 5 mu L of the mixed solution, lightly dripping the mixed solution on a carbon coating surface of a copper mesh, and standing and adsorbing for 10min at room temperature under the action of self gravity.

And III, sucking the residual liquid on the copper mesh by using a dry and clean filter paper, airing for 1min, and then dropwise adding 5 mu L of 1% phosphotungstic acid solution for negative dyeing for 2min.

And iv, sucking the residual liquid on the copper net by using a dry and clean filter paper sheet to finish tabletting.

Transmission electron microscope detection results: from FIG. 9 (a), it can be seen that vB-EcoS-IME167, which was a normal sterile aqueous solution, is an elongate phage with good individual integrity, having a head of about 40 x 40nm and a tail of about 150 nm. From FIG. 9 (b), it was found that the low concentration DA solution-treated vB-EcoS-IME167 phage exhibited a clear head and cross-linking at the tail. Map 9 (c) to high concentration DA solution it was found that vB-EcoS-IME167 phage displayed an aggregation phenomenon with head connected and tail tightly wrapped around head. Overall, the DA treatment resulted in a significant change in the morphological characteristics of the vB-EcoS-IME167 phage, i.e., the DA's anti-invasion mechanism for vB-EcoS-IME167 was responsible for the specific adsorption of vB-EcoS-IME 167.

The morphology features of phages of the control group and the experimental group were observed by a transmission electron microscope using the purified concentrated enriched T4 phase described above. FIG. 19 (a) is a morphology of T4 phase in sterile aqueous solution, showing that the phage is in tail sheath contracted state at the time of negative staining, but from the figure we can still see that the phage has the complete components of myocaudal phage, tail sheath, tail tube, substrate, tail nail and tail fiber of head, neck and tail; FIG. 19 (b) is a T4 phase electron micrograph of a low concentration DA solution, showing that the phage head begins to deform, tail fiber has been lost, and T4 phase has lost its original intact morphology. Further increasing the concentration of the DA solution, and obtaining a completely deformed T4 phase electron microscope image; FIG. 19 (c) shows that the head of T4 phase has been completely deformed and even partially broken to release the contents, the head and tail begin to separate, and the tail protein is mostly deformed and shed, and the integrity has been completely lost. In order to put it together, the morphological characteristics of T4 phase were significantly changed like vB-EcoS-IME167 described above, and the anti-invasion mechanism of DA for T4 phase was also achieved by affecting specific adsorption.

For the Brevibacterium phage VMY22, the phage particles obtained by the above experiment were simply subjected to sterile water and DA treatment, and morphological features were observed by transmission electron microscopy. Experimental results as shown in fig. 29, it can be seen that fig. 29 (a) is a morphological view of the VMY22 of the sterile water control group, in which the hexagonal prism-like head, neck, and short straight tail of the VMY22 can be clearly distinguished. About 60 nm in head size, about 10nm in neck collar protein and about 40nm in tail length fig. 29 (b) is an electron microscopy image of low concentration DA treated VMY22 phage, short straight tail loss of most VMY22 phage can be observed, head and neck proteins are left, and morphology begins to be incomplete; FIG. 29 (c) is an electron micrograph of the VMY22 after high concentration DA treatment, where the VMY22 phage is observed to have only deformed head shells, and the deformed head shells aggregate to shrink and crush, and the original morphological features of the VMY22 phage have not been seen at all. In combination with the electron microscopy of vB-EcoS-IME167 and T4 phase, we reason to believe that the mechanism of action of DA on VMY22 is to destroy the specific adsorption during its phagocytosis by affecting the intact morphology of VMY22, resulting in an efficient resistance against VMY22 infection by host bacteria.

In summary, the action mechanism of DA on phage is to destroy the specific adsorption in the phage process by affecting the whole form of phage, thus resulting in efficient capability of preventing phage from infecting host bacteria.

Example 6 phage adsorption Rate experiment

1) Phage genome extraction, phage DNA was extracted using the visual DNA Kit of OMEGA;

2) Preparation of recombinant plasmids

1. Primer design and Synthesis

i. Selecting a target gene: the NCBI website database was searched for downloaded phage whole genome sequences according to GeneBank accession number of each phage genome. Appropriate gene sequence fragments of interest were selected for the following procedure.

Conventional PCR specific primer design: after inputting the target gene sequence by using Primer 5 software, clicking search to automatically search for a proper Primer group. And sequentially checking whether the primers meet the requirements from high score to low score, selecting the primer which meets the requirements best, and preferably selecting two primers for standby.

Primer design for real-time fluorescent quantitative PCR (RT-PCR): the method comprises the steps of selecting a target gene sequence by utilizing a full genome sequence downloaded by an NCBI website, designing primers by utilizing Primer 5 software, setting the length of a PCR product to be 70-200 bp, automatically generating specific primers after clicking search, sequentially checking whether the primers meet the requirements from high score to low score, and selecting the primers which meet the conditions best, and preferably selecting two primers for standby.

2. PCR amplification system and program

The PCR amplification system was as follows (20. Mu.L):

3. PCR product gel recovery

The product was recovered using agarose gel DNA recovery kit (tin-free, china) from Jiangsu tin-free Baitaike Biotechnology Co., ltd, and stored in a-20deg.C refrigerator.

4. The target fragment was ligated to a vector (15 ℃ C. 12 h)

The connection system is as follows:

5. ligation product conversion

i. 5. Mu.L of the ligated mixture was transferred to a sterile 1.5mL microcentrifuge tube, and then 50. Mu.L of E.coli DH 5. Alpha. Competent cells were added for mixing.

ii, placing on crushed ice for 30min.

And iii, immediately transferring the mixture into a water bath kettle at 42 ℃ to heat for 80s.

iv, placing on ice for 1min.

v. adding sterilized 900. Mu.L LB liquid medium into a centrifuge tube, mixing, and standing at 37deg.C for 120r min ^-1 Culturing for 60min.

And vi, taking out the centrifuge tube after finishing the culture, and centrifuging for 4min at a state of 2000 g.

Only 100. Mu.L of supernatant is left, the rest is discarded, and the suspension is carefully blown with a pipette.

Transfer 100. Mu.L of the suspension to ampicillin-resistant LB selective medium in a sterile super clean bench, spread with a glass spreading bar, and incubate at 37℃for 12h with inversion.

6. Positive clone validation

10 single colonies growing on LB solid plates selectively cultured by ampicillin were randomly selected, and inoculated into 10 test tubes containing 5mL of sterilized LB medium, respectively, and cultured for 6-8 hours. 20 mu L of the sample are taken out of each test tube and thermally cracked in a PCR instrument at 98 ℃ for 10min, 10000g of the sample is centrifuged for 10min, and the genome in the supernatant after centrifugation is taken for PCR verification. (the PCR system is identical to the above-described operation)

7. Recombinant plasmid extraction (recombinant plasmid DNA was collected using StarPrep rapid plasmid extraction kit. The collected recombinant plasmid DNA was stored at-20℃under low temperature).

8. Construction of recombinant plasmid Standard

i. The concentration and purity of the recombinant plasmid were determined by a spectrophotometer, and the copy number of the recombinant plasmid was calculated.

Use of sterile ddH for recombinant plasmid ₂ O dilution 10 ^-1 、10 ^-2 、10 ^-3 、10 ^-4 、10 ^-5 、10 ^-6 、10 ^-7 、10 ^-8 、10 ^-9 ngμL ^-1 Making into standard template diluted with standard substance, and storing at-20deg.C.

9. Establishing a standard curve

The diluted standard template is subjected to RT-PCR amplification, and an amplification system (12 mu L) is adopted:

(Note: 3 times per concentration duplicate detection.)

Step temperature: step constant temperature time at 1 ℃): 20s

The standard for each concentration was tested for 3 sets of parallel data and a standard curve was drawn for phage plasmid standards based on Ct values for recombinant plasmid standards. And (3) drawing a standard curve of a recombinant plasmid standard of the vB-EcoS-IME167 phage shown in figure 10, a standard curve standard of a recombinant plasmid standard of the T4 phage shown in figure 20 and a standard curve of a recombinant plasmid standard of the VMY22 phage shown in figure 30 respectively by taking Ct values as an abscissa and Lg values of copy numbers of standard templates as an ordinate.

3) Real-time quantitative PCR adsorption rate test

1. Two groups of experiments were set up AB (a as control group and B as experimental group).

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Experimental treatment of groups a and B

i. 5 sterilized 2mL centrifuge tubes are taken from each group, and labels are made.

Adding 900 mu L of phage solution with different dilutions according to experimental design, adding 100 mu L of sterile water or DA according to requirements, and incubating for 30min.

After incubation, 1000. Mu.L of the mixture was transferred to a sterilized 5mL centrifuge tube, 1000. Mu.L was added and incubated to OD ₆₀₀ The culture was incubated for 5min at 0.8 strain.

After incubation, centrifugation is performed for 8000g,5min, and the supernatant is retained.

v. supernatant was filtered with 0.22 μl sterile filter membrane in a fresh sterilized 2mL centrifuge tube, and the filtrate was kept labeled.

3. And extracting phage genome from the treated filtrate of the group A and the group B.

4. An RT-PCR amplification system (12. Mu.L) was used (consistent with the q-PCR amplification system used in the construction of the recombinant plasmid standard curve described above).

5. Real-time RT-PCR program settings (consistent with the q-PCR program settings in the recombinant plasmid standard curve construction described above).

6. And according to the real-time quantitative PCR result, combining a standard curve, and comparing the change of the adsorption rate.

4) Adsorption Rate results of RT-PCR

Phage genetic material was extracted with the Viral DNA Kit from OMEGA after phage treatment according to the experimental design of control (group A) and experimental (group B) in the experimental plan described above. The Ct value of each dilution in the A group and the B group is obtained through RT-PCR technology, the Ct value is converted into gene copy number through a standard curve equation, and the difference of the adsorption rates of the A group and the B group is determined through comparing the gene copy numbers of the A group and the B group.

FIG. 11 is the copy number of vB-EcoS-IME167 after treatment of groups A and B, and it can be found that the initial template amount of group A is higher than that of group B at each dilution, which indicates that there is indeed a huge significant difference in adsorption rate results between group A and group B, and it can be primarily confirmed that the anti-invasion mechanism of DA on vB-EcoS-IME167 is caused by affecting the specific adsorption during the phagocytosis process. The experiment was repeated 3 times and the trend of the results obtained from the experiment was consistent.

FIG. 21 shows the results of the experiments after treatment of groups A and B, and shows that the initial template amount of group A is significantly higher than that of group B at each dilution, which indicates that the difference of adsorption between groups A and B is significant, and that the anti-invasion mechanism of DA on the T4 phage of the sarcodaceae phage is caused by influencing the specific adsorption of host bacteria in the process of phage like the above-mentioned long tail phage vB-EcoS-IME 167. The experiment was repeated at least 3 times and the trend of the experimental results was consistent.

FIG. 31 is a comparison of the adsorption rates of VMY22 after treatment of groups A and B, and the data of groups A and B can be found to be very different at each dilution, which indicates that there is a significant difference in adsorption rates of groups A and B, and it can be confirmed that the infection resistance of DA to VMY22 is obtained by affecting the specific adsorption during the phagocytosis process. And the experiment was repeated at least 3 times, the trend of the experimental results was consistent.

In summary, the anti-infection of phage by DA is caused by affecting specific adsorption of the host bacteria and during the phage process.

Example 7: resistance of DA to phages in a physiological saline Water Environment

1. As shown in FIG. 4, DA still shows high-strength capability of inhibiting bacteria infected by vB-EcoS-IME167 phage in a physiological saline environment, and the inhibition capability reaches 10 ² The above-mentioned factors, which are comparable to the degree of anti-phage of DA in MM medium environment, indicate that DA has the basic requirement of studying the effect of anti-phage vB-EcoS-IME167 in living animals.

2. The experiment uses MM medium environment as control group, and the experimental result is shown in FIG. 14, in physiological saline water ringIn-the-environment DA still exists at about 10 ³ The ability to resist T4 phage infection by times is comparable with the environmental results of MM medium, which shows that DA has the basic requirement of researching the action of resisting phage T4 phage in living animals.

3. The result shows that DA still shows the capability of effectively blocking the VMY22 from infecting host bacteria in a physiological saline water environment; comparing the ability of DA to resist VMY22 infection in a physiological saline environment and in an MM medium environment, it can be found that DA resistance to VMY22 in both environments is up to 10 ² More than two times. This suggests that DA has the fundamental requirement to study the resistance of the Brevibacterium in the body.

From the above data, the DA has the basic requirement of experiment in the organism for the anti-infection capability of the long-tail phage vB-EcoS-IME167, the myocaudal phage T4 phase and the short-tail phage VMY22, and can provide a new idea for developing novel antiviral drugs in the biopharmaceutical field.

It is to be understood that the above-described embodiments of the present invention are merely illustrative of the invention or serve to explain the principles of the invention and are not to be construed as limiting the invention. Accordingly, any modification, equivalent replacement, improvement, etc. made without departing from the spirit and scope of the present invention should be included in the scope of the present invention. Furthermore, the appended claims are intended to cover all such changes and modifications that fall within the scope and boundary of the appended claims, or equivalents of such scope and boundary.

Claims

1. The use of dopamine for the preparation of an anti-phage product, characterized in that said phage is a phage of the family long, the family myococcyx or the family short; the phage is cultured in an MM culture medium environment, and the ratio of 1L of MM culture medium is as follows: 5 xMq solution 200mL, 20% glucose 10mL, 1M L ^-1 MgSO ₄ 1mL, 800mL of ultrapure water.

2. The use according to claim 1, wherein said dopamine has its lowest onset on the long-tail phage vB-EcoS-IME167, the myotail phage TT4 phase and the short-tail phage VMY22The effective concentration is 0.1-1 mg mL ^-1 The highest anti-infection concentration is 5-10 mg mL ^-1 。

3. The use according to claim 2, wherein said dopamine has a semi-inhibitory concentration IC for the long tail bacteriophage vB-EcoS-IME167 ₅₀ The value was 0.26 mg mL ^-1 。

4. The use according to claim 2, wherein said dopamine has a semi-inhibitory concentration IC for the brachycarpus phage VMY22 ₅₀ The value was 0.73 mg mL ^-1 。

5. The use according to claim 2, characterized in that said dopamine has a half inhibitory concentration IC on the sarcodaceae phage T4 phage ₅₀ The value was 0.12 mg mL ^-1 。

6. The use according to claim 1, characterized in that the use of the anti-phage product for controlling phage contamination in the fermentation industry.

7. The use according to claim 1, wherein said dopamine anti-phage mechanism is a mechanism that disrupts the whole form of the phage and thereby disrupts the specific adsorption during its phage process, and is resistant to phage infection by the host bacteria.