CN115725446A

CN115725446A - Enterococcus faecium for resisting rectal tumors and application thereof

Info

Publication number: CN115725446A
Application number: CN202210987632.6A
Authority: CN
Inventors: 朱永亮; 穆晓静; 刘丹; 马梦楠; 邱娜
Original assignee: Suzhou Preyson Biotechnology Co ltd
Current assignee: Suzhou Preyson Biotechnology Co ltd
Priority date: 2021-12-22
Filing date: 2022-08-17
Publication date: 2023-03-03
Also published as: CN114107133A; WO2023116283A1

Abstract

The invention provides an anti-colorectal tumor enterococcus faecium and application thereof, wherein the preservation number is CCTCC NO: M20211220, and the enterococcus faecium is preserved in China center for type culture collection. The enterococcus faecium provided by the invention can still ensure the survival rate of 77.60% after being incubated for 3 hours in the environment of artificial gastric juice with the pH value of 4.0. In mouse CT26 and MC38 models, the enterococcus faecium has significant effect in inhibiting tumor volume. After the enterococcus faecium and the PD-1 monoclonal antibody are used together, the growth rate of tumor cells can be better inhibited under the condition of not treating by antibiotic medicines, and the effect of inhibiting the tumor volume is obvious.

Description

Anti-colorectal tumor enterococcus faecium and application thereof

The present application claims priority from the chinese patent application entitled "an anti-rectal neoplasm enterococcus faecium and its uses" filed by the chinese patent office at 22.12 months 22/2021 under application number 202111580351.0, the entire contents of which are incorporated herein by reference.

Technical Field

The invention relates to the technical field of microorganisms, and particularly relates to anti-colorectal-tumor enterococcus faecium and application thereof.

Background

Colorectal cancer is a common malignant tumor of the digestive system, the incidence of the cancer is hidden, and the mortality rate is high. For the treatment of advanced colorectal cancer, chemotherapy with 5-FU or capecitabine plus oxaliplatin or irinotecan regimen is mainly combined with targeted drugs such as bevacizumab, cetuximab, panitumumab and the like. Although chemotherapy combined with targeted therapy improves the overall survival rate of patients with advanced colorectal cancer, few drugs can be used for patients with colorectal cancer after the progress of multi-line chemotherapy, and adverse reactions such as gastrointestinal reactions, bone marrow suppression, neurotoxicity and the like caused by chemotherapy often occur.

The regulation of intestinal homeostasis is involved by microorganisms, some of the commensals and pathogenic organisms of the human intestinal microbiome being essential in the pathogenesis of colorectal cancer. Thus, manipulation of intestinal bacterial composition and local metabolites through the use of probiotics has been demonstrated to be useful for colorectal cancer therapeutic intervention. Probiotics are live microbial feeding additives that beneficially affect the health of the host.

Chinese patent 201910909859.7 discloses a probiotic formula for inhibiting colorectal cancer pathogenic bacteria and a screening method thereof, and the probiotic formula comprises: clostridium butyricum 1.3X 10 ¹⁰ CFU/mL, enterococcus faecalis 1X 10 ⁹ CFU/mL, lactobacillus brevis 1X 10 ⁹ CFU/mL, lactobacillus plantarum 1X 10 ⁹ CFU/mL, lactobacillus rhamnosus 1X 10 ⁹ CFU/mL, lactobacillus sake 1X 10 ⁹ CFU/mL, leuconostoc mesenteroides 1X 10 ⁹ CFU/mL. The probiotic formula of the invention does not generate drug resistance, is safe and effective, has excellent performance, but has more related strains and complex scheme.

Chinese patent 201480080834.6 discloses the use of microorganisms, in particular eubacterium strains, in the treatment and prevention of colorectal cancer related diseases. Compositions comprising a eubacterium strain are described. The results of animal experiments demonstrated the ability of Eubacterium ventriosum (Eubacterium ventriosum) and Eubacterium shigella (Eubacterium elegans) to effectively prevent and treat colitis and colorectal cancer.

The enrichment of microbial germplasm resources for treating colorectal cancer is very beneficial to the treatment and prognosis of colorectal cancer.

In view of this, the invention is particularly proposed.

Disclosure of Invention

In order to solve the problems, the invention separates a enterococcus faecium resisting colorectal tumors from healthy human fecal bacteria liquid, and applies the enterococcus faecium to the treatment of colorectal tumors so as to provide a medicine with more convenient and faster obtaining way and better effect for the treatment of colorectal tumors.

In one aspect, the invention provides a strain of enterococcus faecium.

The enterococcus faecium 16S rRNA sequence has more than 99.93 percent of identity with SEQ ID NO. 1.

The enterococcus faecium 16S rRNA sequence is SEQ ID NO.1, the preservation number is CCTCC NO. M20211220, and the enterococcus faecium is preserved in the China center for type culture collection with the preservation date of 2021, 9 months and 26 days.

In another aspect, the invention provides the use of enterococcus faecium in the manufacture of a product or medicament for the diagnosis, prevention and/or treatment of tumors.

Enterococcus faecium is the aforementioned enterococcus faecium.

Tumors include, but are not limited to: tumors of digestive system, respiratory system, reproductive system, motor system, nervous system, endocrine system, circulatory system, urinary system, and reproductive system.

Preferably, the tumor is a tumor of the digestive system, and the tumor of the digestive system is one or more of tumor of esophagus, tumor of stomach, tumor of cardia, tumor of intestine, tumor of liver and tumor of gallbladder.

Further preferably, the tumor is a colorectal tumor, including one or more of a polyp-like type, a stenosis type, and an ulcer type.

In an alternative embodiment, good therapeutic results may be obtained without prior administration at the time of therapeutic application. For example, without treatment with antibiotic drugs and/or microbial agents (enterococcus faecium), the growth rate of tumor cells can be better inhibited, and the tumor volume can be significantly inhibited.

In a further aspect, the present invention provides a formulation for the aforementioned use.

The preparation comprises the enterococcus faecium and/or the preparation thereof.

The preparation is dead bacteria, cell disruption product, fermentation supernatant, fermentation precipitate, modified bacteria, mutant bacteria and/or mutant bacteria of enterococcus faecium.

The preparation method of the fermented supernatant of the enterococcus faecium comprises the following steps:

s1, inoculating enterococcus faecium CCTCC NO of M20211220;

s2, carrying out conditional culture;

and S3, removing the thallus to obtain the microbial inoculum.

The preparation method of the enterococcus faecium cell disruption product comprises the following steps:

s1, inoculating enterococcus faecium CCTCC NO of M20211220;

s2, performing conditional culture;

s3, breaking the somatic cells.

Conditioned cultures include, but are not limited to: anaerobic culture, aerobic culture and facultative anaerobic culture.

Preferably, the preparation is a liquid preparation comprising enterococcus faecium in an amount of 5 × 10 ⁹ CFU/mL。

Preferably, the preparation is a solid or semi-solid preparation, and the enterococcus faecium is included in the preparation in an amount of 5 × 10 ⁹ CFU/g。

Preferably, the formulation is a lyophilized powder.

In yet another aspect, the invention provides a gene-encoded product.

The gene-encoded product is encoded by a gene on the genome of enterococcus faecium as described above, and is capable of diagnosing, preventing and/or treating colorectal tumors.

In another aspect, the present invention provides a method for culturing enterococcus faecium, comprising the following steps:

s1, inoculating the enterococcus faecium;

s2, culturing under conditions.

In still another aspect, the invention provides the use of the aforementioned formulation in the manufacture of a product or medicament for the diagnosis, prevention and/or treatment of colorectal neoplasms.

In a further aspect, the present invention provides a medicament for the aforementioned use.

The medicine comprises the enterococcus faecium or the preparation thereof.

Alternatively, the medicament may comprise a formulation as hereinbefore described.

The above medicines contain enterococcus faecium in an amount of 5 × 10 ⁹ CFU/mL。

The medicine also comprises one or more of 5-FU, capecitabine, oxaliplatin, irinotecan, bevacizumab, cetuximab and panitumumab.

The medicine also comprises other pharmaceutically acceptable carriers or excipients.

Preferably, the medicament is an oral medicament.

The invention also provides a pharmaceutical composition which comprises the enterococcus faecium and/or a preparation thereof; alternatively, the pharmaceutical composition includes the above-described formulation.

In a preferred embodiment of the present invention, the pharmaceutical composition further comprises a combination drug, wherein the combination drug is selected from at least one of the following drugs:

chemotherapeutic drugs, photosensitizers, photothermal agents, antibodies inhibiting the second signaling molecule, inhibitors inhibiting the second signaling molecule, PD-L1 inhibitors, PD-1/PD-L1 monoclonal antibody drugs.

In an alternative embodiment, the chemotherapeutic agent is selected from any one or more of taxanes, vinca alkaloids, anthracyclines, epipodophyllotoxins, tyrosine kinase inhibitors, fraxidin, irinotecan and its metabolite SN-38, topotecan, teniposide, etoposide, imatinib, gefitinib, dannousertib, doruofenacin, daunorubicin, mitoxantrone, methotrexate, camptothecin, and saquinavir.

The photosensitizer is selected from: (1) 5-aminolevulinic acid (ALA) or a derivative thereof; (2) photosensitive compounds containing tetrapyrrole rings; (3) traditional Chinese medicine photosensitizer; or (4) ALA or its derivative and the compound in (2) or (3).

The photo-thermal agent is selected from the group consisting of IR-780, IR-783, IR-805, IR-808, IR-825, IR-1045, IR-1048, IR-1061, and IR-26.

The inhibitory second signaling molecule may be PD-1; CTLA-4; PD-1 and CTLA-4.

In a preferred embodiment of the invention, the PD-1/PD-L1 monoclonal antibody drug is at least one selected from the following group: nivolumab (Nivolumab), pembrolizumab (Pembrolizumab), pidilizumab (Pidilizumab), lambertilizumab (lambilizumab), lambertilizumab (Lambrolizumab), BMS-936559, atelizumab (Atezolizumab), AMP-224, AMP224, AUNP12, BGB108, MCLA134, MEDI0680, PDROOl, REGN2810, SHR1210, STIAl ox, STIAl llO, TSR042, BMS-936558, BGB-a317, BCD-100, and JS001.

The inventor finds that compared with single treatment of PRS-15 monoclonal antibody or PD-1 monoclonal antibody of enterococcus faecium, PRS-15 monoclonal antibody and PD-1 monoclonal antibody can better inhibit the growth rate of tumor cells without antibiotic drug treatment after being used together, and has obvious effect on inhibiting the tumor volume. The PRS-15 and PD-1 monoclonal antibodies of the enterococcus faecium have good synergistic effect.

The inventors carried out tumor inoculation 7 days before administration, and found that the injection had a significant effect of inhibiting tumor volume well from 4 days after administration. Compared with the method of administering the medicament before tumor inoculation, the method provided by the invention can more accurately simulate the tumor inhibition effect of the patient after the disease.

In a preferred embodiment of the invention, the PD-L1 inhibitor is selected from the group consisting of DOVAMAb, ATTRAUzumab or Abamectin.

In a preferred embodiment of the present invention, the above mentioned drugs further comprise one or more of 5-FU, capecitabine, oxaliplatin, irinotecan, bevacizumab, cetuximab, and panitumumab.

The invention has the following beneficial effects:

the enterococcus faecium CCTCC NO: M20211220 provided by the invention can still ensure the survival rate of 77.60% after being incubated for 3 hours in an artificial gastric juice environment with the pH value of 4.0. In a mouse MC38 model, the enterococcus faecium has a remarkable effect in inhibiting tumor volume.

After the enterococcus faecium and the PD-1 monoclonal antibody are combined, the growth rate of tumor cells can be better inhibited under the condition of not treating antibiotic medicines, and the effect of inhibiting the tumor volume is obvious.

Deposit description

And (4) storage address: university of Wuhan, china

The preservation date is as follows: 26/09/2021

The strain name is as follows: enterococcus faecium PRS-15

Latin name: enterococcus faecalium PRS-15

The preservation organization: china center for type culture Collection

The preservation organization is abbreviated as: CCTCC (China center for cell communication)

The registration number of the collection center: CCTCC NO: M20211220

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 is a plot of the change in tumor size of MC38 mouse model after dosing.

FIG. 2 is a graph of the change in body weight of the MC38 mouse model after administration;

FIG. 3 is a graph showing the statistical results of the combination of PRS-217-15 and anti-PD-1 in example 5 on the tumor volume of mouse MC 38;

FIG. 4 is a graph showing the statistical results of the combination of PRS-217-15 and anti-PD-1 in example 5 on the body weight of mice;

FIG. 5 is a graph of statistics on CT26 tumor volume in mice after PRS-217-15 treatment in example 6;

FIG. 6 is a graph showing the statistical results of the PRS-217-15 treatment in example 6 on the body weight of mice;

FIG. 7 is a graph of statistical results for CT26 tumor volume in mice treated with PRS-217-15 and PRS-217-05 prior to tumor inoculation in example 7;

FIG. 8 is a graph of the statistics of CT26 tumor volume in mice after PRS-217-15 and PRS-217-05 treatments after tumor inoculation in example 8;

FIG. 9 is a graph of statistics on mouse MC38 tumor volume after PRS-217-15 and PRS-217-05 treatments prior to tumor inoculation in example 9;

FIG. 10 is a graph of statistics on mouse MC38 tumor volume after PRS-217-15 and PRS-217-05 treatments after tumor inoculation in example 10;

FIG. 11 is a graph of statistics on H22 tumor volume in mice after PRS-217-15 treatment after tumor inoculation in example 11;

FIG. 12 is a graph of statistics for H22 body weight in mice treated with PRS-217-15 after tumor inoculation as in example 11.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The features and properties of the present invention are described in further detail below with reference to examples.

Example 1

Separation of enterococcus faecium for resisting colorectal tumor.

Under the aseptic condition, 10 times of gradient dilution is carried out on the prepared healthy human fecal bacteria liquid, three concentration gradients are selected, streaking is carried out on NA and MRS culture media, three repeated plates are placed in a 37 ℃ constant-temperature incubator for anaerobic culture for 72h, colonies in different forms are selected for gram staining microscopy, streaking purification is carried out for 2 times in the MRS culture media, staining microscopy is carried out again, the bacterial form and purification are observed, 16srRNA sequencing is carried out, and the used primer sequence is as follows:

27F：SEQ ID NO.2；

1429R：SEQ ID NO.3。

the PCR reaction system is as follows:

the PCR reaction conditions were as follows:

the PCR products obtained by amplification were subjected to 16srRNA sequencing and then aligned at NCBI.

The sequencing result is shown in SEQ ID NO. 1.

Example 2 drug resistance detection

The method is carried out by adopting a drug sensitive paper diffusion method, centrifuging a bacterial solution cultured by an MRS liquid culture medium (purchased from Qingdao Haibo biotechnology limited, the product number is HB 0384-1) for 15min at 4000rpm, removing supernatant, suspending by PBS, blowing uniformly, adjusting OD value to about 0.6, uniformly coating the bacterial solution in a culture dish containing an MRS solid culture medium, placing drug sensitive paper (containing antibacterial drugs or antibiotics) after the bacterial solution is completely absorbed, placing a drug sensitive paper (3 sheets) and a blank control (1 sheet) on a flat plate, inversely culturing in an incubator at 37 ℃, measuring the diameter of a bacteriostatic ring by a ruler for 24h, recording data, and judging the result according to a paper method antibacterial drug test standard (WS/T125-1999). The results were as follows:

remarking: s = sensitivity, I = mediation, R = drug resistance,/is no zone of inhibition, — is the zone of inhibition of elimination.

The strain is mainly resistant to quinolones, nitrofurans antibacterial drugs, polymyxin B, lincomycins, compound sulfamethoxazole and gentamicin; exhibit sensitivity to penicillin, chloramphenicol, macrolide, tetracycline, cephalosporin, and glycopeptide antibiotics.

Example 3 detection of resistance to Artificial gastrointestinal fluids

(1) Preparing an MTT solution: weighing 0.5g MTT (purchased from Beijing Solibao, cat # M8180-250), adding 100mL PBS to dissolve (the final concentration is 5 mg/mL), filtering and sterilizing, and subpackaging in 15mL centrifuge tubes, freezing at-20 ℃ for standby, wherein the validity period is half a year.

(2) Simulated Gastric Fluid (SGF) experiment

1. 2.0g NaCl and 3.2g pepsin (Solebao pepsin, 1.

2. Adjusting pH to 1.2 (simulating human fasting intestinal juice pH), 2.0, 3.0 (simulating human postprandial intestinal juice pH and mouse fasting intestinal juice pH) and 4.0 respectively, filtering and sterilizing;

3. bacteria collection: statically culturing the strain at 37 ℃ for 8h to reach logarithmic phase, sub-packaging the strain liquid in a 50mL sterile EP tube, centrifuging at 4000rpm for 15min at room temperature, discarding the supernatant, re-suspending the strain with PBS, and taking OD of the strain liquid ₆₀₀ The number of bacteria obtained was about 2X 10 ⁹ CFU/mL, then taking 17 sterile EP tubes with the volume of 15mL, respectively adding 6mL of bacterial liquid with the adjusted OD value, centrifuging (conditions are the same as above), removing PBS (PBS) by 16 centrifuge tubes (experimental group), collecting bacterial precipitates for later use, and blowing and resuspending 1 (control group);

4. artificial gastric juice culture: adding the artificial gastric juice with different pH values into 16 centrifugal tubes with 15mL of bacterial precipitates collected after washing, respectively, uniformly pumping, and respectively incubating and culturing for 1h, 2h and 3h;

5. MTT detection: firstly, respectively taking 3mL of a control group bacterial liquid and a bacterial liquid with different incubation pH values and different times into a new centrifugal tube, respectively adding 1mL of MTT reagent, incubating overnight (the step can be that the bacterial liquid is centrifuged at 3500rpm for 8min, removing supernatant artificial gastric juice, adding the MTT reagent, reducing the MTT reagent to 500ul, and shortening the incubation time), after incubation, then carrying out centrifugal treatment (3500rpm for 8 min), removing supernatant, adding 600 mu L of DMSO (national drug analysis purity 98.5%), blowing, dissolving, respectively adding into a deep-hole plate, then transferring into a 96-hole sterile culture plate by using a discharging gun, adding 150 mu L of DMSO into each hole (marking the order and name of each hole for adding samples clearly), then detecting by using an enzyme labeling instrument, selecting a wavelength of 570nm or 630nm to obtain an absorbance value, and calculating the proportion of live bacteria;

6. and (4) performing data processing, and calculating the corresponding survival rate, wherein the bacterial survival rate = OD value of the experimental group/OD value of the control group multiplied by 100%, so as to obtain the tolerance degree of the strain after simulating the action of the artificial gastric juice for different time.

(3) Simulated Intestinal Fluid (SIF) -Simulated Intestinalfluid (SIF) experiment

1. Taking 6.8g of monopotassium phosphate, adding 500mL of water for dissolving, and adjusting the pH value to 6.8 by using 0.1mol/L sodium hydroxide solution; taking 1g of pancreatin (Solebao trypsin, 1.

2. The bacteria collection method is similar to the Simulated Gastric Fluid (SGF) experiment;

3. artificial intestinal juice culture: adding the artificial intestinal juice with the pH value of 6.8 into 8mL of washed bacterial precipitates in 4 15mL centrifuge tubes respectively, blowing and stirring uniformly, and incubating and culturing for 0h, 1h, 2h and 3h;

4. MTT detection: the gastric juice (SGF) experiment was simulated as above.

5. And (4) performing data processing, and calculating the corresponding survival rate, wherein the bacterial survival rate = OD value of the experimental group/OD value of the control group multiplied by 100%, so as to obtain the tolerance degree of the strain after simulating the artificial intestinal juice for acting for different time.

(4) Experiment for simulating human body internal bile salt environment

1. Preparing bile: adding the ox gall powder into a culture medium (MRS or BHI) solution of target bacteria, setting three final concentrations of 10g/L (1% of ox gall powder), 20g/L (2% of ox gall powder) and 40g/L (4% of ox gall powder), sterilizing under high pressure for later use, and simultaneously taking a culture medium solution without the added gall powder for 0h as a reference. In this example, MRS medium was used.

2. Strains were collected as above.

3. And (3) culturing the ox gall powder: MRS or BHI culture media containing 0%, 1%, 2% and 4% of cow bile powder (purchased from Hefei Ba-Si-Fu-Biotech Co., ltd., product number: N0101-100 g) are respectively added into 10mL of centrifuge tubes of 20 15mL of bacteria precipitates collected after washing for incubation culture for 0h, 1h, 2h, 3h and 4h, 0h of 0% cow bile powder culture medium solution is used as a control group, and the MRS culture medium is selected in the embodiment.

4. Pouring culture: the gastric juice (SGF) experiment was simulated as above.

5. Calculating gastric acid tolerance of bacteria: counting plates, recording the colony number of each plate, and processing data to obtain the tolerance degree of the strain to the bovine bile salt.

The results were as follows:

the result shows that the enterococcus faecium CCTCC NO is M20211220: the survival rate can be kept better in the gastric juice environment with lower pH value; the survival rate of 77.60 percent can still be ensured after 3 hours of incubation in the environment of artificial gastric juice with the pH value of 4.0; can normally grow in 1% -4% of the cow bile powder culture medium, and the number of the living bacteria is increased along with the increase of the incubation culture time, which shows that the strain has a certain tolerance degree to cow bile salt and can normally grow and reproduce.

Example 4 mouse model experiment

The mice selected in this example were female C57BL/6 or mice, with a week age of 6-7 weeks (the expected week age of the mice when tumor cells were inoculated), and the weight range of the mice when cells were inoculated was 18-22g, purchased from sovereikon biotechnology limited.

The mouse MC38 model is used for drug effect verification, and the model construction method comprises the following steps:

MC38 cells (Shanghai Fuji Biotech Co., ltd., product No. FH-0125) were cultured in RPMI1640 medium containing 10% fetal bovine serum. The MC38 cells in exponential growth phase were collected and resuspended in PBS to 1X 10 ⁷ one/mL was used for subcutaneous tumor inoculation in mice. 0.1mL MC38 cells are inoculated to the subcutaneous part of the right back of an experimental mouse, the growth condition of the tumor is observed periodically, and the tumor grows to the average volume of 96mm ³ At this time, the tumor size and mouse body weight were randomly divided into groups and tumor inoculation was recorded as day 0.

Before the start of dosing, the animals were weighed and tumor volumes were measured.

The experimental groups were as follows:

the preparation method of the enterococcus faecium living bacteria comprises the following steps: PBS +0.25% glycerol suspended enterococcus faecium.

The administration is started on the 9 th day of tumor inoculation, and is carried out at the 6 th day after the administration,8. Mice were measured for 10, 12, 14, 16, 19, 21, 24, 27 days for body weight (g) and tumor size (mm) ³ ). Tumor size calculation formula: tumor volume (mm) ³ ) =0.5 × (tumor major axis × tumor minor axis) ² )。

The results are shown in fig. 1-2, and show that: in a mouse MC38 model, the enterococcus faecium has a remarkable effect in inhibiting tumor volume.

Example 5

This example demonstrates the anti-tumor effect of enterococcus faecium in combination with PD-1 monoclonal antibody drugs. The method comprises the following specific steps:

the mice selected in this example were female C57BL/6 mice, 5-6 weeks old (the expected week old of the mice when tumor cells were inoculated), 16-20g of the body weight of the mice when cells were inoculated, and purchased from jiangsu jiejiao kang bio-technology corporation. anti-PD-1 is: BE0146-100MG, inVivoMab anti-mouse PD-1 (CD 279) antibody, cat #: BE0146-100M; the supplier: shanghai Youning vitamin science & technology GmbH

MC38 cells (Nanjing Ke Bai Biotech Co., ltd., product number CBP 60825) were cultured in RPMI1640 medium containing 10% fetal bovine serum. MC38 cells in exponential growth phase were collected and PBS resuspended to 1X 10 ⁷ one/mL was used for subcutaneous tumor inoculation in mice. 0.1mL MC38 cells are inoculated in the subcutaneous part of the right back of an experimental mouse, and the growth of the tumor is observed periodically until the tumor grows to the average volume of 113mm ³ At this time, the tumor size and mouse body weight were randomly divided into groups and tumor inoculation was recorded as day 0.

The dosing groups were as follows: control group: 7 mice were given 0.2mL of physiological saline in p.o; dosing time QD × 3 weeks; experimental group-1: 7 mice were given 0.2mL of PRS-217-15, viable bacteria 5X 10 ⁹ CFU/mL, the administration mode is p.o; dosing time QD × 3 weeks; experimental group-2: 7 mice were given 0.2ml 5mpk anti-PD-1, i.p.; the administration time was QW × 3 weeks; experimental group-3: 7 mice were given 0.2ml of 5mpk anti-PD-1, i.p.; the administration time is QW × 3 weeksWhile 0.2mL of PRS-217-15 and 5X 10 viable bacteria were administered ⁹ CFU/mL, administration was p.o; the dosing time was QD × 3 weeks.

The experimental groups were as follows:

the preparation method of the enterococcus faecium living bacteria comprises the following steps: PBS +15% glycerol suspended enterococcus faecium.

Administration was started on day 7 of tumor inoculation, and the body weight (g) and tumor size (mm) of the mice were measured on

days

3, 7, 10, 14, 17, and 20 after administration ³ ). Tumor size calculation formula: tumor volume (mm) ³ ) =0.5 × (tumor major diameter × tumor minor diameter) ² )。

The results are shown in fig. 3 and 4, and show that: in the mouse MC38 model, the enterococcus faecium has a remarkable effect on inhibiting the tumor volume. And the combination of the anti-PD-1 and the anti-PD-1 has obvious synergistic effect.

Example 6

This example is a mouse model experiment for another colon cancer cell (CT 26),

the mice selected in the example are female BALB/c mice, the week age is 7-8 weeks (the expected week age of the mice when tumor cells are inoculated), the weight range of the mice when the cells are inoculated is 18-22g, and the mice are purchased from Jiangsu Jiejiao Kangshengki Co., ltd.

The mouse CT26 model is used for drug effect verification, and the model construction method comprises the following steps:

CT26 cells (Nanjing Ke Bai Biotech Co., ltd., product number CBP 61189) were cultured in RPMI1640 medium containing 10% fetal bovine serum. CT26 cells were collected in exponential growth phase and resuspended in PBS to 5X 10 ⁶ one/mL was used for subcutaneous tumor inoculation in mice. 0.1mL of CT26 cells are inoculated to the subcutaneous part of the right back of an experimental mouse, and the growth of the tumor is observed periodically until the tumor grows to the average volume of 133mm ³ At the time, the administration was randomly divided into groups according to the tumor size and the mouse body weightTumor inoculation was scored as day 0.

Before the start of dosing, animals were weighed and tumor volumes were measured.

The experimental groups were as follows:

days

4, 7, 11, 14, and 18 after administration ³ ). Tumor size calculation formula: tumor volume (mm) ³ ) =0.5 × (tumor major axis × tumor minor axis) ² )。

The results of the experiment are shown in fig. 5 and 6, and the results show that: in a mouse CT26 model, the enterococcus faecium PRS-217-15 provided by the invention has a remarkable effect on the aspect of inhibiting the tumor volume.

Example 7

This example was conducted with a tumor volume suppression experiment on PRS-217-05, a viable enterococcus faecium strain, screened from healthy persons, and with a pre-tumor inoculation treatment with CT26 cells.

CT26 cells (Nanjing Ke Bai Biotech Co., ltd., product number CBP 61189) were cultured in RPMI1640 medium containing 10% fetal bovine serum. CT26 cells were collected in exponential growth phase and resuspended in PBS to 5X 10 ⁶ one/mL was used for subcutaneous tumor inoculation of mice.

0.1mL of CT26 cells are inoculated to the subcutaneous part of the right back of an experimental mouse, and the growth of the tumor is observed periodically until the tumor grows to the average volume of 133mm ³ At this time, the tumor size and mouse body weight were randomly divided into groups and tumor inoculation was recorded as day 7.

The experimental groups were as follows:

Administration was started 7 days before tumor inoculation, and the body weight (g) of mice and the size of tumor (mm) were measured on

days

0, 4, 7, 11, 14, and 18 after administration ³ ). Tumor size calculation formula: tumor volume (mm) ³ ) =0.5 × (tumor major axis × tumor minor axis) ² )。

The results are shown in FIG. 7 and show that: in a mouse model administered before inoculation of CT26 cells, PRS-217-15 and PRS-217-05 had a significant effect in suppressing tumor volume, and PRS-217-15 had a superior inhibitory effect.

Example 8

The mice selected in the example are female BALB/c mice, the week age is 7-8 weeks (the expected week age of the mice when tumor cells are inoculated), the weight range of the mice when the cells are inoculated is 18-22g, and Jiangsu Jiejiao Kangshengji Biotech GmbH is purchased. In this example, the administration treatment after the inoculation of CT26 cell tumor was carried out.

CT26 cells (Nanjing Ke Bai Biotech Co., ltd., product number CBP 61189) were cultured in RPMI1640 medium containing 10% fetal bovine serum. CT26 cells were collected in exponential growth phase and resuspended in PBS to 5X 10 ⁶ one/mL was used for subcutaneous tumor inoculation in mice. 0.1mL of CT26 cells were inoculated subcutaneously into the right dorsal part of experimental mice, and the growth of tumors was observed periodicallyWhen the tumor grows to an average volume of 133mm ³ At this time, the tumor size and mouse body weight were randomly divided into groups and tumor inoculation was recorded as day 0.

The experimental groups were as follows:

Administration was started on day 7 after tumor inoculation, and the body weight (g) and tumor size (mm) of the mice were measured on

days

4, 7, 11, 14, and 18 after administration ³ ). Tumor size calculation formula: tumor volume (mm) ³ ) =0.5 × (tumor major diameter × tumor minor diameter) ² )。

The results are shown in FIG. 8 and show that: in a mouse model administered after inoculation of CT26 cells, PRS-217-15 and PRS-217-05 had a significant effect in suppressing tumor volume, and PRS-217-15 had a superior suppression effect.

Example 9

The mice selected in the example are female C57BL/6 mice, the week age is 5-6 weeks (the expected week age of the mice when tumor cells are inoculated), the weight range of the mice when the cells are inoculated is 16-20g, and the mice are purchased from Jiangsu Jiejiao Kangkang biotech GmbH. This example was performed with the administration treatment prior to the inoculation of the MC38 cell tumor.

MC38 cells (Nanjing Ke Bai Biotech Co., ltd., product number CBP 60825) were cultured in RPMI1640 medium containing 10% fetal bovine serum. MC38 cells in exponential growth phase were collected and PBS resuspended to 1X 10 ⁷ one/mL was used for subcutaneous tumor inoculation in mice. 0.1mL MC38 cells are inoculated in the subcutaneous part of the right back of an experimental mouse, and the growth of the tumor is observed periodically until the tumor grows to the average volume of 113mm ³ At the same time, the medicine is randomly divided into groups according to the size of the tumor and the weight of the mouseTumor inoculation was scored as day 7.

The experimental groups were as follows:

Administration was started 7 days before tumor inoculation, and the body weight (g) and tumor size (mm) of the mice were measured on

days

0, 3, 7, 10, 14, 17, and 20 after administration ³ ). Tumor size calculation formula: tumor volume (mm) ³ ) =0.5 × (tumor major axis × tumor minor axis) ² )。

The results are shown in fig. 9 and indicate that: in a mouse model administered before inoculation of MC38 cells, PRS-217-15 and PRS-217-05 had a significant effect in suppressing tumor volume, and PRS-217-15 had a superior inhibitory effect.

Example 10

The mice selected in the example are female C57BL/6 mice, the week age is 5-6 weeks (the expected week age of the mice when tumor cells are inoculated), the weight range of the mice when the cells are inoculated is 16-20g, and the mice are purchased from Jiangsu Jiejiao Kangkang biotech GmbH. This example was carried out by administration after inoculation of MC38 cell tumors.

MC38 cells (Nanjing Kebai Biotechnology Co., ltd., product No. CBP 60825) were cultured in RPMI1640 medium containing 10% fetal bovine serum. The MC38 cells in exponential growth phase were collected and resuspended in PBS to 1X 10 ⁷ one/mL was used for subcutaneous tumor inoculation in mice. 0.1mL MC38 cells are inoculated in the subcutaneous part of the right back of an experimental mouse, and the growth of the tumor is observed periodically until the tumor grows to the average volume of 113mm ³ At this time, the tumor size and mouse body weight were randomly divided into groups and tumor inoculation was recorded as day 0.

The experimental groups were as follows:

The administration was started on day 7 after tumor inoculation, and the body weight (g) and the tumor size (mm) of the mice were measured on

days

3, 7, 10, 14, 17, and 20 after the administration ³ ). Tumor size calculation formula: tumor volume (mm) ³ ) =0.5 × (tumor major axis × tumor minor axis) ² )。

The results are shown in FIG. 10 and show that: the results show that: in a mouse model administered after inoculation of MC38 cells, PRS-217-15 and PRS-217-05 had significant effects in inhibiting tumor volume.

Example 11

This example demonstrates the significant effect of PRS-217-15 on inhibiting the size of liver cancer.

The mice selected in the example are female Balb/c mice, the week age is 6-7 weeks (the expected week age of the mice when the tumor cells are inoculated), the weight range of the mice when the cells are inoculated is 18-22g, and the mice are purchased from Jiangsu Jiejiao Kangsheng Biotech Co., ltd.

The mouse H22 model is used for drug effect verification, and the model construction method comprises the following steps:

h22 cells (Nanjing Ke Bai Biotech Co., ltd., product No. CBP 60230) were cultured in RPMI1640 medium containing 10% fetal bovine serum. The exponential growth phase of H22 cells were collected and PBS resuspended to 1X 10 ⁷ one/mL was used for subcutaneous tumor inoculation in mice. 0.1mL of H22 cells were inoculated subcutaneously into the right inguinal region of the experimental mice, and the growth of the tumor was periodically observed until the tumor grew to an average volume of 113mm ³ In the method, the medicine is randomly divided into groups according to the size of the tumor and the weight of the mouse,tumor inoculation was scored as day 0.

Animal body weights were weighed before dosing began.

The experimental groups were as follows:

the preparation method of the enterococcus lactis living bacteria comprises the following steps: PBS + 8X 1011CFU/g fungal powder (0.011 g fungal powder/mL).

Dosing was started 7 days before tumor inoculation, and mouse body weight (g) and tumor size (mm) were measured on

days

0, 3, 6, 9, 12, and 16 after tumor inoculation ³ ). Tumor size calculation formula: tumor volume (mm) ³ ) =0.5 × (tumor major axis × tumor minor axis) ² )。

The results are shown in FIGS. 11-12, and show that: in the mouse H22 model, the enterococcus lactis has a remarkable effect in inhibiting tumor volume.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. An enterococcus faecium characterized in that its 16S rRNA sequence has an identity of 99.93% or more as compared with the sequence shown in SEQ ID NO. 1.

2. The enterococcus faecium according to claim 1, wherein the 16S rRNA sequence is SEQ ID NO.1, the preservation number is CCTCC NO. M20211220, and the preservation date is 2021, 9 and 26 days.

3. Use of enterococcus faecium according to claim 1 or 2 in the manufacture of a product for the diagnosis, prevention and/or treatment of tumors;

preferably, the product is a medicament.

4. The use of claim 3, wherein the tumor is one or more of a tumor of the digestive system, a tumor of the respiratory system, a tumor of the reproductive system, a tumor of the motor system, a tumor of the nervous system, a tumor of the endocrine system, a tumor of the circulatory system, a tumor of the urinary system, a tumor of the reproductive system;

preferably, the tumor is a tumor of the digestive system;

more preferably, the tumor of the digestive system is one or more of tumor of esophagus, tumor of stomach, tumor of cardia, tumor of intestine, tumor of liver and tumor of gallbladder;

more preferably, the tumor is a colorectal tumor comprising one or more of a polyp-like type, a stenosis type, and an ulcer type;

preferably, the administration is not preceded by a dose.

5. A preparation comprising enterococcus faecium and/or a preparation thereof according to claim 1 or 2; the preparation is selected from at least one of dead bacteria, cell disruption products, fermentation supernatant, fermentation precipitates, modified bacteria, mutant bacteria and mutagenic bacteria of enterococcus faecium.

6. A pharmaceutical composition comprising enterococcus faecium and/or a preparation thereof according to claim 1 or 2; alternatively, the pharmaceutical composition comprises the formulation of claim 5.

7. The pharmaceutical composition of claim 6, further comprising a combination selected from at least one of the following:

8. The pharmaceutical composition of claim 7, wherein the PD-1/PD-L1 mab drug is selected from at least one of the following groups: nivolumab (Nivolumab), pembrolizumab (Pembrolizumab), pidilizumab (Pidilizumab), lambertizumab (lambilizumab), BMS-936559, atelizumab (Atezolizumab), AMP-224, AMP224, AUNP12, BGB108, MCLA134, MEDI0680, PDROOl, REGN2810, SHR1210, stialox, STIAl lO, TSR042, BMS-936558, BGB-317 a, BCD-100, and JS001.

9. The pharmaceutical composition of claim 7, wherein the PD-L1 inhibitor is selected from the group consisting of DOVACUMAB, ATTACUMAB, and Avermectin.

10. The medicament of claim 9, further comprising one or more of 5-FU, capecitabine, oxaliplatin, irinotecan, bevacizumab, cetuximab, and panitumumab.