Application of quail chicken enterococcus phage in prevention or treatment of colorectal cancer liver metastasis

Abstract

The invention relates to a quail chicken enterococcus bacteriophage PhiEgSY1 and application of the quail chicken enterococcus bacteriophage PhiEgSY1 in prevention or treatment of colorectal cancer liver metastasis. According to the invention, the specific bacteriophage of the quail chicken enterococcus SY2-1 is screened, the form and the splitting effect of the specific bacteriophage are confirmed, and the safety of intervention of the specific bacteriophage in mice is verified through an animal model. According to the invention, a colorectal cancer liver metastasis animal model is also constructed, the phage PhiEgSY1 is utilized to accurately regulate and control quail chicken enterococcus which has a promoting effect on colorectal cancer liver metastasis in a microbiota, and it is verified that the phage PhiEgSY1 can significantly reduce the abundance of Eg in intestinal tracts and can inhibit liver metastasis or reduce the degree of liver metastasis, so that prevention or auxiliary intervention on liver metastasis is realized.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] The present invention claims priority to Chinese patent application No. CN202411268678.8 filed on September 11, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present invention relates to the technical field of biomedicine, and in particular to the application of Enterococcus gallinarum phage in preventing or treating liver metastasis of colorectal cancer. Background Art

[0004] Colorectal cancer is one of the three most frequent cancers worldwide, and metastasis is the leading cause of death in patients. The 5-year survival rate of patients with colorectal cancer metastasis is less than 20%. Among them, the liver is the main distal organ for colorectal cancer metastasis. According to clinical statistics, about 20% of patients newly diagnosed with CRC have metastasis, another 25% of patients gradually develop CRC metastasis during subsequent disease progression, and 15%-25% of patients develop liver metastasis after radical surgery. Due to factors such as the location and size of the tumor, unresectable disease, the presence of extrahepatic disease or comorbidities of the patient, surgery is only applicable to 10-20% of cases of colorectal liver metastasis, with a 5-year survival rate as low as 30%. In addition, patients who do not meet the conditions for surgery have a worse prognosis. In recent years, as the role of the microbiome in colorectal cancer and its liver metastasis has been discovered and clarified, strategies for intervening in the disease using methods based on regulating the microbiome need to be developed.

[0005] The ways to regulate the microbiome can be summarized into three categories: the first is the replacement of the microbiome, which uses fecal microbiota transplantation to replace the disordered microbiome of a patient with a healthy person's microbiome; the second is the addition of the microbiome, which intervenes in the disease by taking in probiotics, prebiotics that promote the growth of probiotics and other substances; the third is the subtraction of the microbiome, including the use of specific antibiotics or bacteria-specific phages to eliminate related bacteria or reduce their abundance. This type of method is suitable for diseases with clear pathogens.

[0006] The current methods of regulating the microbiome have the following shortcomings: Among the ways of microecological regulation of the microbiome, the one that is most similar to phage intervention is antibiotic treatment; as we all know, the development of new narrow-spectrum antibiotics requires a long research and development cycle, and the long-term use of antibiotics can easily lead to an aggravation of the problem of bacterial resistance, especially for enterococci, which are known for their resistance problems. Antibiotics are not a recommended method.

[0007] Regarding Enterococcus gallinarum phage, Junko Nishio et al. (Identification and characterization of a novel Enterococcus bacteriophage with potential to ameliorate murine colitis[J].Scientific reports, (2021) 11: 20231. DOI: 10.1038 / s41598-021-99602-4.) disclosed that a new phage related to Enterococcus gallinarum was isolated from the intestinal contents of mice, named phiEG37k, and its level was increased in C57BL / 6 colitis mice. By inducing colitis in mice colonized with phiEG37k and its host bacteria, the phage was demonstrated to enhance colonic mucus and improve colitis. Specifically, mice colonized with phiEG37k produced more mucin 2 (MUC2) that protects intestinal epithelial cells, indicating that phiEG37k phage has the potential to protect the host by strengthening mucosal integrity, but it does not involve the role of Enterococcus gallinarum phage in intervening in colorectal cancer liver metastasis.

[0008] The inventor's team has previously demonstrated that Enterococcus gallinarum is a bacterium that can promote liver metastasis of colorectal cancer. It can significantly promote liver metastasis of colorectal cancer, while having no significant effect on in situ tumors. Therefore, using bacteriophages to eliminate it may become an auxiliary means of intervention or prevention of liver metastasis of colorectal cancer. However, how to screen and obtain effective bacteriophages is an urgent problem that needs to be solved, and the application of corresponding bacteriophages needs to be verified for functionality and safety. Summary of the invention

[0009] In order to overcome at least one problem existing in the prior art, the present invention screens Gallinarum-specific phages, confirms their morphology and lysis effects, and verifies the safety of their intervention in mice through animal models; the present invention also constructs an animal model of colorectal cancer liver metastasis, intervenes with Gallinarum-specific phages, and verifies that it can inhibit liver metastasis or reduce the degree of liver metastasis.

[0010] To achieve the above object, the present invention adopts the following technical solution:

[0011] The first aspect of the present invention is to provide an Enterococcus gallinarum phage (Enterococcus gallinarumphage), whose host is Enterococcus gallinarum SY2-1, the preservation number of Enterococcus gallinarum SY2-1 is CCTCC NO: M20241929, the preservation date is September 6, 2024, the preservation unit is China Center for Type Culture Collection (abbreviated as CCTCC), and the preservation unit address is Wuhan University, Wuhan, China.

[0012] Furthermore, the Enterococcus gallinarum phage is phage Phi_Eg_SY1, its preservation number is CCTCC NO: M20241928, the preservation date is September 6, 2024, the preservation unit is China Center for Type Culture Collection (CCTCC for short), and the preservation unit address is Wuhan University, Wuhan, China.

[0013] Furthermore, the screening method for Enterococcus gallinarum phage comprises the steps of:

[0014] Step S1, taking hospital wastewater filtrate, Enterococcus gallinarum SY2-1 cultured to the logarithmic phase, and brain heart infusion broth, mixing them in a certain ratio (preferably 19.5 mL: 500 μL: 10 mL), and culturing them in an incubator at 37° C. overnight;

[0015] Step S2, taking the enriched solution (preferably 10 mL), adding 2% volume chloroform, mixing and centrifuging (preferably 3000×g, 10 min, 4° C.), and filtering the supernatant (preferably using a 0.22 μm filter);

[0016] Step S3, taking the filtered enrichment solution and the Enterococcus gallinarum SY2-1 bacterial solution cultured to the logarithmic growth phase in a certain ratio (preferably 200 μL: 200 μL), adding brain heart infusion medium containing agar (preferably brain heart infusion medium containing 0.5% agar, with a total volume of 4 mL), mixing and evenly pouring onto the brain heart infusion plate medium, drying and inverting in a 37° C. constant temperature incubator for overnight culture;

[0017] Step S4: After the plaque is formed, pick up the upper agar in the plaque area with a sterile needle, add physiological saline (preferably 1 mL) and vortex to fully release the phage in the agar;

[0018] Step S5, take the eluate (preferably 100 μL), dilute and plate, dry and then invert and culture in a 37°C constant temperature incubator overnight; after plaques are formed, pick up the upper agar in the plaque area with a sterile needle again to physiological saline (preferably 1 mL); repeat several times to purify and separate the phage, and the obtained Enterococcus gallinarum phage is named Phi_Eg_SY1.

[0019] Furthermore, the bacteriophage Phi_Eg_SY1 has a head with a diameter of about 50nm and a tail with a length of about 220nm, which morphologically conforms to the characteristics of a long-tailed phage; the activity of the bacteriophage Phi_Eg_SY1 is relatively stable in the range of 4-50°C, and is relatively high in the range of pH 5-12; within the co-incubation time of 5 minutes, more than 90% of the phages complete the adsorption of the host bacteria; the phage can produce an effective lysis effect on the host bacteria at multiple infection multiplicities (MOI=0.01, 0.1, 1); the bacteriophage Phi_Eg_SY1 contains a 42,319bp double-stranded circular genome (GenBank: OQ623132.1), encoding structure-related proteins, proteins that play a lysis role, etc.

[0020] The second aspect of the present invention is to provide a preparation containing any one of the Enterococcus gallinarum phages described in the first aspect.

[0021] Furthermore, the dosage form of the preparation includes liquid suspension preparation, phage solid preparation and the like.

[0022] Furthermore, the preparation contains 10 8～ 10 10 PFU / mL phage particles; for example, containing 1×10 8 ~5×10 8 PFU / mL phage particles, 5×10 8 ~1×10 9 PFU / mL phage particles, 1×10 9 ~5×10 9 PFU / mL phage particles, 5×10 9 ~1×10 10 PFU / mL phage particles. Preferably, the preparation contains 10 9 PFU / mL phage particles.

[0023] Further, the preparation method of the preparation comprises the steps of: inoculating the bacterial liquid of Enterococcus gallinarum SY2-1 into brain heart infusion broth at a predetermined ratio, culturing on a 37°C shaker to the logarithmic phase, adding purified phage liquid, and placing it on a 37°C shaker again; collecting the mixed culture after a predetermined time, centrifuging, and filtering the supernatant; subjecting the filtrate to ultra-high speed centrifugation, discarding the supernatant after centrifugation, and resuspending the phage particles at the bottom of the tube with physiological saline to adjust its concentration. Specifically, the amplification and culture step of the above-mentioned phage Phi_Eg_SY1 comprises: inoculating the bacterial liquid of Enterococcus gallinarum SY2-1 in the logarithmic growth phase into 200mL brain heart infusion broth at a ratio of 1:100, culturing on a 37°C shaker to the logarithmic phase, adding 100μL purified phage liquid, and placing it on a 37°C shaker again. After 24h, the mixed culture is collected, centrifuged (3000×g, 10min, 4°C), and the supernatant is filtered through 0.22μm. The filtrate was subjected to ultrahigh-speed centrifugation (110,000×g, 2 h, 4°C), the supernatant was discarded after centrifugation, and the phage particles at the bottom of the tube were resuspended with 1 mL of physiological saline to obtain an injection suspension of a predetermined concentration.

[0024] The third aspect of the present invention is to provide an application of any of the Enterococcus gallinarum phages described in the first aspect, or any of the preparations described in the second aspect, which is selected from at least one of the following applications: application in the preparation of a drug for reducing the abundance of Enterococcus gallinarum SY2-1, and application in the preparation of a drug for preventing or treating a disease caused by Enterococcus gallinarum SY2-1.

[0025] Furthermore, the disease caused by Enterococcus gallinarum SY2-1 includes liver metastasis of colorectal cancer. Specifically, the bacteriophage Phi_Eg_SY1 can inhibit liver metastasis or reduce the degree of liver metastasis, thereby achieving prevention or auxiliary intervention of liver metastasis.

[0026] Furthermore, in the above application, the bacteriophage Phi_Eg_SY1 has no obvious toxic side effects after intervention and is highly safe.

[0027] Furthermore, the drug is in the form of an injection suspension, and a single dose of the drug contains 10 7～ 10 9 PFU phage particles, preferably containing 10 8 PFU phage particles.

[0028] Furthermore, the method of administering the drug is: intraperitoneal injection of 100 μL of a concentration of 10 9 PFU / mL Phi_Eg_SY1 phage particles in normal saline (i.e., the injection solution contains 10 8 PFU Phi_Eg_SY1 phage particles), the injection times are 2 to 4 times, preferably 3 times.

[0029] Furthermore, the drug also includes a pharmaceutically acceptable carrier.

[0030] Furthermore, the medicine also includes other medicines for treating Enterococcus gallinarum SY2-1 infection, such as antibiotics, Enterococcus gallinarum antagonists, etc.

[0031] "Pharmaceutically acceptable" means that when Phi_Eg_SY1 phage is appropriately administered to animals or humans, the carrier or excipient will not produce adverse, allergic or other adverse reactions. "Pharmaceutically acceptable carriers or excipients" should be compatible with the active ingredient, that is, they can be mixed with it without significantly reducing the effect of the drug under normal circumstances. The above carriers or excipients are all substances commonly used in the art. Suitable carriers or excipients are selected according to actual needs to help the stability of the formulation or help improve the activity or biological effectiveness or produce an acceptable taste or smell when taken orally.

[0032] Compared with the prior art, the present invention adopts the above technical solution to have the following beneficial effects:

[0033] The present invention screens specific Enterococcus gallinarum phages and verifies their functions in colorectal cancer liver metastasis; using the above-mentioned Enterococcus gallinarum phages to intervene in colorectal cancer liver metastasis is an attempt to use phage therapy in chronic disease intervention, and is also a new attempt at a microecological intervention system for colorectal cancer liver metastasis.

[0034] The present invention utilizes bacteriophages to precisely regulate Enterococcus gallinarum in the microbiota that has a promoting effect on liver metastasis of colorectal cancer. Compared with antibiotics, the host spectrum is narrower and the normal structure of the microbiota will not be destroyed. Bacteriophages are viruses that target host bacteria and are relatively safe in host animals or humans. This indicates that prevention or auxiliary intervention of liver metastasis can be achieved by intervening with Enterococcus gallinarum phage Phi_Eg_SY1 in patients who have already developed intestinal cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are only used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0036] Figure 1 Schematic diagram of the result of gradient dilution counting of Enterococcus gallinarum phage Phi_Eg_SY1 in one embodiment of the present invention; wherein the phage concentration is 2.5×10 9 PFU / mL;

[0037] Figure 2 The stained transmission electron microscopic morphology of the Enterococcus gallinarum phage Phi_Eg_SY1 in one embodiment of the present invention;

[0038] Figure 3 1 is a result schematic diagram of the biological characteristics of the Enterococcus gallinarum phage Phi_Eg_SY1 in one embodiment of the present invention; wherein, part a is the thermal stability of the phage Phi_Eg_SY1, the horizontal axis represents the Celsius temperature, and the vertical axis represents the activity rate of the phage at different temperatures compared with that at 4°C; part b is the pH stability of the phage Phi_Eg_SY1, the horizontal axis represents the pH value, and the vertical axis represents the activity rate of the phage at different pH environments compared with that at pH 7; part c is the adsorption curve of the phage Phi_Eg_SY1 to the host Enterococcus gallinarum SY2-1 strain; part d is the infection curve of the phage Phi_Eg_SY1 to the host Enterococcus gallinarum SY2-1 strain;

[0039] Figure 4 1 is a schematic diagram of the results of the genome characteristics of Enterococcus gallinarum phage Phi_Eg_SY1 in one embodiment of the present invention; wherein different colors represent different functional modules;

[0040] Figure 5 1 is a schematic diagram of the safety evaluation results of the Enterococcus gallinarum phage Phi_Eg_SY1 in one embodiment of the present invention; wherein, part a is the H&E staining of the large intestine, small intestine and spleen of healthy mice after the intervention of phage Phi_Eg_SY1; part b is the weight change of mice after the intervention of phage Phi_Eg_SY1;

[0041] Figure 6 It is a schematic diagram of the results of the relative abundance of Enterococcus gallinarum (Eg) in mouse fecal samples after different times of intervention with Enterococcus gallinarum bacteriophage Phi_Eg_SY1 in one embodiment of the present invention;

[0042] Figure 7 This is a schematic diagram of the results that the Enterococcus phage Phi_Eg_SY1 of Gallinarum can significantly inhibit liver metastasis of colorectal cancer in one embodiment of the present invention; wherein, part a is the in vitro tumor signal imaging of the liver at the end point of the experiment of the colorectal cancer liver metastasis model intervened by spleen injection of Enterococcus phage Phi_Eg_SY1; part b is the statistics of tumor signals in part a; *p<0.05.

[0043] The above-mentioned Enterococcus gallinarum phage (Enterococcus gallinarum phage) Phi_Eg_SY1 has been deposited, and its deposit number is CCTCC NO: M20241928. The deposit date is September 6, 2024. The depository is China Center for Type Culture Collection (CCTCC for short), and the depository address is Wuhan University, Wuhan, China. DETAILED DESCRIPTION

[0044] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work belong to the scope of protection of the present invention. The experimental methods without specific conditions in the following embodiments are usually measured according to national standards. The experimental materials and reagents without the source in the following embodiments are all commercially available raw materials. The equipment used in each step in the following embodiments is conventional equipment. If there is no corresponding national standard, it is carried out according to the general international standards, conventional conditions, or according to the conditions recommended by the manufacturer. Unless otherwise specified, all parts are weight parts and all percentages are mass percentages. Unless otherwise defined or specified, all professional and scientific terms used in the present invention have the same meaning as those familiar to those skilled in the art. In addition, any method and material similar or equal to the recorded content can be applied to the method of the present invention.

[0045] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other. The present invention is further described below in conjunction with the drawings and specific embodiments, but is not intended to be a limitation of the present invention.

[0046] In the following embodiments, the preparation method of the Enterococcus gallinarum phage Phi_Eg_SY1, its genome feature analysis, and partial function verification have been disclosed in the inventor's previous research. For details, please refer to the document: Isolation and characterization of a novel Enterococcus phage Phi_Eg_SY1. Virus Res. 2023 Jul 15; 332: 199132.

[0047] In the following embodiments, the Enterococcus gallinarum used is Enterococcus gallinarum SY2-1, whose deposit number is CCTCC NO: M20241929, the deposit date is September 6, 2024, the depository is China Center for Type Culture Collection (CCTCC for short), and the depository address is Wuhan University, Wuhan, China.

[0048] In the following embodiments, the mouse intestinal cancer liver metastasis cell line used is the mouse colon cancer cell clone CT26-TL-CL5, whose preservation number is CCTCC NO: C2024307, the preservation date is September 5, 2024, the preservation unit is the China Center for Type Culture Collection (abbreviated as CCTCC), and the preservation unit address is Wuhan University, Wuhan, China.

[0049] The following embodiments are only used to illustrate the technical solutions of the present invention.

[0050] Example 1 - Screening, enrichment, separation, purification and amplification of Enterococcus gallinarum phage

[0051] This embodiment provides a method for screening, enriching, isolating, purifying and amplifying Enterococcus gallinarum phage, which specifically includes the following steps:

[0052] (1) Take 50 mL of hospital wastewater (for phage enrichment), centrifuge (4000 rpm, 15 min), and filter the supernatant through a 0.22 μm filter. Take 19.5 mL of the filtrate, 500 μL of Enterococcus gallinarum SY2-1 cultured to the logarithmic phase, and 10 mL of 3× brain heart infusion broth (OXOID, CM1135B), mix them, and incubate them in a 37°C incubator overnight for phage enrichment. The recovery process of Enterococcus gallinarum SY2-1 is as follows: take out the cryopreserved tube from the -80°C refrigerator, place it on ice, transfer it to a biosafety cabinet, open it, scrape an appropriate amount of frozen bacterial culture with a sterile inoculation loop, inoculate it onto a Columbia blood plate (CHROMagar) by the three-zone streak method, and incubate it in a 37°C incubator overnight; the next day, pick a single clone and inoculate it into 5 mL of brain heart infusion broth for culture and subsequent use.

[0053] (2) Take 10 mL of the enriched solution after the above mixed culture, add 2% volume chloroform, mix well and centrifuge (3000×g, 10 min, 4°C), and filter the supernatant with a 0.22 μm filter.

[0054] (3) Take 200 μL of the filtered enrichment solution and 200 μL of Enterococcus gallinarum SY2-1 cultured to the logarithmic growth phase into a 5 mL EP tube, add 4 mL of brain heart infusion medium containing 0.5% agar, mix well and evenly pour onto the brain heart infusion plate medium, dry and invert in a 37°C constant temperature incubator for overnight culture. Observe the plate for plaque formation the next day.

[0055] (4) After plaques are formed, use a sterile needle to pick up the upper agar in the plaque area, add 1 mL of saline and vortex to fully release the phages in the agar.

[0056] (5) 100 μL of eluate was diluted and plated, and then inverted and incubated at 37°C for overnight incubation after drying. After the plaques were formed, the upper agar in the plaque area was picked up again with a sterile needle to 1 mL of saline. Repeat 3 times to purify and isolate the phage (for specific methods, see the literature: Isolation and characterization of a novel Enterococcus phage Phi_Eg_SY1. Virus Res. 2023 Jul 15; 332: 199132.), and the phage isolated above was named Phi_Eg_SY1.

[0057] (6) The purified phage Phi_Eg_SY1 was amplified and cultured. First, the bacterial liquid of Enterococcus gallinarum SY2-1 in the logarithmic growth phase was subcultured into 200 mL of brain heart infusion broth at a ratio of 1:100, and cultured in a 37°C shaker until the logarithmic phase. 100 μL of purified phage liquid was added and placed in a 37°C shaker again. After 24 hours, the mixed culture was collected and centrifuged (3000×g, 10 min, 4°C), and the supernatant was filtered through 0.22 μm. The filtrate was subjected to ultrahigh-speed centrifugation (110000×g, 2h, 4°C), the supernatant was discarded after centrifugation, and the phage particles at the bottom of the tube were resuspended with 1 mL of physiological saline.

[0058] (7) Perform 10-fold gradient dilutions on the amplified and concentrated phages and plate them separately. Count the number of plaques on the plates after overnight culture. Quantify the phage concentration accordingly (e.g. Figure 1 shown).

[0059] (8) The phage Phi_Eg_SY1 was photographed using a transmission electron microscope to observe its morphological characteristics. The results are as follows: Figure 2 As shown, the phage has a head with a diameter of about 50nm and a tail with a length of about 220nm, and its morphology conforms to the characteristics of a long-tailed phage.

[0060] (9) Analysis of the biological characteristics and genomic characteristics of the phage Phi_Eg_SY1: The thermal stability, acid-base stability, adsorption curve and infection curve of the phage were measured according to the previous literature (Isolation and characterization of a novel Enterococcus phage Phi_Eg_SY1. Virus Res. 2023 Jul 15; 332: 199132.). The results showed that the activity of the phage was relatively stable in the range of 4-50°C ( Figure 3 The activity is relatively high in the pH range of 5-12 ( Figure 3Part b); within 5 minutes of co-incubation, more than 90% of the phages completed adsorption to the host bacteria SY2-1 ( Figure 3 The phage was able to effectively lyse the host bacteria SY2-1 at multiple infection multiplicities (MOI = 0.01, 0.1, 1) set in the experiment ( Figure 3 d part); phage Phi_Eg_SY1 contains a 42,319 bp double-stranded circular genome (GenBank: OQ623132.1), encoding structural related proteins, proteins that play a lytic role, etc. ( Figure 4 ).

[0061] The above-isolated and identified Enterococcus gallinarum phage Phi_Eg_SY1 has been deposited, and its deposit number is CCTCC NO: M20241928. Its host is Enterococcus gallinarum SY2-1, and the culture conditions are brain heart infusion broth, aerobic culture at 37°C.

[0062] Example 2-Safety Assessment of Enterococcus gallinarum Phage Phi_Eg_SY1

[0063] This example uses healthy mice to verify the safety of the Enterococcus gallinarum phage Phi_Eg_SY1. Six-week-old BALB / c female mice (from Shanghai Xipul-Bikai Experimental Animal Co., Ltd.) are used for the safety verification of the phage, which specifically includes the following steps:

[0064] (1) The bacteriophage Phi_Eg_SY1 obtained in Example 1 was diluted to 10 9 PFU / mL, intraperitoneal injection of 100 μL containing 10 8 PFU Phi_Eg_SY1 phage particles in normal saline, and the control group was injected with an equal amount of normal saline.

[0065] (2) After 9 consecutive days of injection once a day, the mice were euthanized. The large intestine, small intestine and spleen were removed, placed in 4% paraformaldehyde, and sent to a related company for histochemical testing. The mice were also weighed.

[0066] The above experimental results are as follows Figure 5 As shown, the results proved that there was no obvious pathological reaction in the large intestine, small intestine and spleen of mice after phage treatment, and there was no significant difference in body weight compared with the control group, which indicates that phage intervention has no obvious toxic side effects on mice and is highly safe.

[0067] Example 3- Functional verification of Enterococcus gallinarum phage Phi_Eg_SY1

[0068] This example uses a mouse model of colorectal cancer liver metastasis to verify the function of Enterococcus gallinarum phage Phii_Eg_SY1, which specifically includes the following steps:

[0069] (1) Five 6-week-old BALB / c female mice (from Shanghai Xipu-Bikai Laboratory Animal Co., Ltd.) were used and injected intraperitoneally with 100 μL of 10 8 The control group was injected with an equal amount of saline containing PFU Phi_Eg_SY1 phage particles. After 1-4 injections, qRT-PCR was used to detect the relative abundance of Enterococcus gallinarum (Eg) in the intestinal contents to confirm the number of phage interventions. The experimental method is briefly described as follows:

[0070] 1) After phage intervention, the feces of mice were collected and DNA was extracted using a kit (Qiagen, 51804). The DNA concentration was detected using NanoDrop2000 (Thermo Scientific). The DNA was diluted to 50 ng / μL using ddH2O.

[0071] 2) Using 16S rRNA as an internal reference, qRT-PCR (the reagent is TB Green Premix DimerEraserTM, TAKARA, RR091Q) was used to detect the relative abundance of Enterococcus gallinarum in the sample. For detailed PCR system and reaction process, please refer to the reagent manual. Add 20μL of reaction system to a 96-well PCR reaction plate (Monad, MQ50301S): 10μL of TB Green Premix DimerEraser (2X), 0.6μL of each of the upstream and downstream primers (10μM) of PCR, 2μL of diluted DNA, and 6.8μL of ddH2O. In LightCycler96 (Roche LifeScience). Set the program in the LightCycler (Roche LifeScience) qRT-PCR instrument: pre-denaturation, 95℃ (30s); PCR reaction, 95℃ (5s), 55℃ (30s), 72℃ (30s), set 40 cycles.

[0072] Primers used:

[0073] ① Detection of Enterococcus gallinarum (reference: Translocation of a gut pathobiont drives autoimmunity in mice and humans. Science. 2018 Mar 9; 359 (6380): 1156-1161.):

[0074] EG-fwd 5'-TTACTTGCTGATTTTGATTCG-3'

[0075] EG-rev 5'-TGAATTCTTCTTTGAAATCAG-3'

[0076] ②16S rRNA (Reference: Tumor-resident intracellular microbiota promotes metastatic colonization in breast cancer. Cell. 2022Apr 14; 185(8):1356-1372.e26.):

[0077] Forward 5'-CGGTGAATACGTTCYCGG-3'

[0078] Reverse 5'-GGWTACCTTGTTACGACTT-3'

[0079] The above experimental results are as follows Figure 6 As shown in the figure, after 3 intraperitoneal injections (5 days), the abundance of Enterococcus gallinarum (Eg) decreased significantly, and 3 / 5 samples detected Eg with a Ct value > 35. After increasing the number of interventions to 4 times, there was no significant difference in the relative abundance of Eg. The above results indicate that 3 treatments with bacteriophage Phi_Eg_SY1 can significantly reduce the abundance of Eg in the intestine, and even reduce its abundance to below the minimum detection limit of qRT-PCR.

[0080] (2) After three injections of phage, the spleens of mice in the experimental and control groups were injected with 10 5 Mouse colorectal cancer liver metastasis cell line CT26-TL-CL5 (accession number: CCTCC NO: C2024307) was used to construct a mouse model of colorectal cancer liver metastasis. The model construction process is briefly described as follows:

[0081] 1) Digest CT26-TL-CL5 cells and filter through a 70 μm filter. The obtained single cell suspension was washed twice with PBS. After the last centrifugation (1000 rpm, 5 min), the cells were resuspended in an appropriate volume of PBS to adjust the cell concentration to 10 6 / mL.

[0082] 2) Induce and maintain anesthesia in the pre-depilated mice with 1-3% isoflurane, and cut the epidermis and peritoneum about 0.5 cm at the lower edge of the left rib cage of the mouse with a scalpel. Gently pick out the spleen of the mouse with a sterile cotton swab and place it on a sterile gauze moistened with saline to prepare for cell injection. Mix the cell suspension thoroughly and draw 100 μL of the cell suspension with a 29G insulin syringe. Inject the needle into the spleen about 1 cm at a straight angle and slowly inject the cell suspension.

[0083] 3) Gently press with a cotton swab, and remove the insulin needle after the blister produced by the injection disappears. Continue to press the needle hole for 2-3 minutes. Ligate the blood vessels at both ends of the spleen with No. 4 sutures and remove the spleen. Suture the wound of the mouse, and after suturing, place the mouse on a 37℃ heating pad to keep warm and wait for it to recover. Put the recovered mouse into a sterile cage, mark the date of surgery, and return it to the ventilated cage for breeding.

[0084] (3) Three weeks after the establishment of the colorectal cancer liver metastasis mouse model, the mice were injected with D-luciferin sodium salt, and the livers were removed after euthanasia to image the tumor bioluminescence signal in vitro ( Figure 7 Part a of the study), and quantitative and comparative analysis of the bioluminescent signal intensity of liver tumors ( Figure 7 Part b) was used to evaluate the effect of phage Phi_Eg_SY1 intervention on liver metastasis of colorectal cancer in mice. The above experimental results prove that phage Phi_Eg_SY1 significantly inhibits liver metastasis of colorectal cancer.

[0085] The specific embodiments of the present invention are described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions made to the present invention are also within the scope of the present invention. Therefore, the equalization changes and modifications made without departing from the spirit and scope of the present invention should be included in the scope of the present invention.

Claims

1. An Enterococcus gallinarum phage, characterized in that: The host of the Enterococcus gallinarum bacteriophage is Enterococcus gallinarum SY2-1, and the preservation number of the Enterococcus gallinarum SY2-1 is CCTCC NO: M20241929.

2. The Enterococcus gallinarum phage according to claim 1, characterized in that The Enterococcus gallinarum phage is phage Phi_Eg_SY1, which is preserved in China Center for Type Culture Collection with a preservation number of CCTCC NO: M20241928.

3. The Enterococcus gallinarum phage according to claim 1, characterized in that The screening method of the Enterococcus gallinarum phage comprises the following steps: Step S1, taking hospital wastewater filtrate, Enterococcus gallinarum SY2-1 bacterial solution cultured to the logarithmic phase, and brain heart infusion broth, mixing them in a certain proportion, and culturing them in an incubator at 37° C. overnight; Step S2, adding 2% volume chloroform to the enriched solution, mixing and centrifuging, and filtering the supernatant; Step S3, taking the filtered enrichment solution and the bacterial solution of Enterococcus gallinarum SY2-1 cultured to the logarithmic growth phase in a certain proportion, adding the brain heart infusion medium containing agar, mixing and evenly pouring the mixture onto the brain heart infusion plate medium, drying and inverting the mixture in a 37° C. constant temperature incubator for overnight culture; Step S4, after the plaque is formed, pick up the upper agar in the plaque area with a sterile needle, add physiological saline and vortex to fully release the phage in the agar; Step S5, take the eluate, dilute and plate, dry it, and then invert it in a 37°C constant temperature incubator for overnight culture; after the plaque is formed, pick up the upper agar in the plaque area with a sterile needle and transfer it to physiological saline; repeat several times to purify and separate the phage, and the obtained Enterococcus gallinarum phage is named Phi_Eg_SY1.

4. A preparation containing the Enterococcus gallinarum phage according to any one of claims 1 to 3.

5. The preparation according to claim 4, characterized in that The preparation contains 10 8～ 10 10 PFU / mL phage particles.

6. The preparation according to claim 5, characterized in that The preparation method of the preparation comprises the following steps: inoculating the bacterial liquid of Enterococcus gallinarum SY2-1 into brain heart infusion broth at a predetermined ratio, culturing on a shaking platform at 37°C to a logarithmic phase, adding purified phage liquid, and placing on a shaking platform at 37°C again; collecting the mixed culture after a predetermined time, centrifuging, and filtering the supernatant; performing ultra-high-speed centrifugation on the filtrate, discarding the supernatant after centrifugation, and resuspending the phage particles at the bottom of the tube with physiological saline to adjust the concentration thereof.

7. A use of the Enterococcus gallinarum phage according to any one of claims 1 to 2, or the preparation according to any one of claims 3 to 6, characterized in that: The application is selected from at least one of the following applications: application in the preparation of a drug for reducing the abundance of Enterococcus gallinarum SY2-1, and application in the preparation of a drug for preventing or treating a disease caused by Enterococcus gallinarum SY2-1.

8. The use according to claim 7, characterized in that: The diseases caused by Enterococcus gallinarum SY2-1 include colorectal cancer liver metastasis.

9. The use according to claim 7, characterized in that: A single dose of the drug contains 10 7～ 10 9 PFU phage particles.

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