Enterococcus faecalis and use thereof

Abstract

The present application relates to a kind of enterococcus faecalis and its application, the strain is preserved in China typical culture preservation center (CCTCC) on December 21, 2020, and the preservation number is CCTCC NO:M2020951, address is China, Wuhan, Wuhan University, and is recorded as enterococcus faecalis GZ (Enterococcus faecalis GZ). The strain is high in safety, has no adverse effect on animal health, and the enterococcus faecalis GZ of single use can obtain very significant antitumor effect, and when combined with other antitumor drugs, better antitumor effect can be further obtained.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a type of Enterococcus faecalis and its applications. Background Technology

[0002] Live bacterial drugs are products that use live bacteria as medications to treat diseases; they are also known as live biotherapeutic preparations (LBP). Furthermore, in 2016, the US clinical guidelines explicitly defined them as drugs containing live microorganisms (such as bacteria) that can be used to prevent, treat, or cure human diseases and for their indications, primarily administered orally or by gavage.

[0003] As early as the 1850s, Pasteur discovered lactic acid bacteria, and subsequently researchers isolated lactic acid bacteria from milk. Bifidobacterium and Lactobacillus acidophilus were also subsequently reported. In the early 20th century, it was discovered that these bacteria were actually beneficial to human health, and were subsequently defined as probiotics by the FAO and WHO, with appropriate consumption being beneficial to human health. Early foreign researchers reported that metabolites of Lactobacillus acidophilus and Lactobacillus casei subsp. casei could effectively inhibit the growth of colorectal cancer tumor cells in vitro. Domestically, Chinese scholars intervened in 86 patients with gastrointestinal malignancies using probiotic preparations of Bifidobacterium triple live bacteria capsules and a placebo, observing that probiotic treatment could improve the clinical symptoms of cancer patients by inhibiting the overgrowth of small intestinal bacteria. As a drug, probiotics are mainly used as microbial regulators and adjuvants, and are currently widely used in treating infant jaundice, metabolic diseases, and alleviating side effects such as diarrhea, vomiting, and colitis caused by chemotherapy drugs for cancer. The number of live bacterial drugs developed internationally is only about 20, mainly in the research of diseases of the digestive tract, urinary tract infections and related diseases of the skin system. For example, Mutaflor in Germany is a live biological drug that has been approved. Many other live biological drugs are still in the preclinical and clinical stages, most of which are in Phase 3 and Phase 2, while some live biological drugs that have entered clinical trials are in Phase 1.

[0004] Currently, live bacterial drugs are still in their infancy in China. There are only two products on the domestic market: vaginal lactobacillus (for treating bacterial vaginosis) and the SK08 project from Guangdong Zhiyi Biotechnology, which is in the clinical trial stage. SK08 live bacterial powder received approval from the National Medical Products Administration (NMPA) on November 20, 2019, to enter the clinical trial stage, registered as a Class 1 therapeutic biological product, with the applied indications being irritable bowel syndrome (IBS) and ulcerative colitis (UC). SK08 live bacterial powder is the first live bacterial drug developed internationally using the novel bacterial species *Bacteroides fragilis*. Preclinical studies have shown significant efficacy and excellent safety, providing a new clinical option for IBS / UC patients. This represents a significant breakthrough in the development of live bacterial drugs in China. Therefore, research on live bacterial drugs has great potential and value.

[0005] However, there are still relatively few in vivo biological drugs involved in the field of anti-tumor therapy. Summary of the Invention

[0006] Therefore, it is necessary to provide a type of Enterococcus faecalis with good anti-tumor effects.

[0007] A type of Enterococcus faecalis, with accession number CCTCC NO: M2020951.

[0008] In one embodiment, the 16S ribosomal RNA gene sequence of the Enterococcus faecalis is shown in SEQ ID No: 1.

[0009] The present invention also provides a bacterial fermentation broth, wherein the bacterial fermentation broth is the fermentation broth of Enterococcus faecalis as described above.

[0010] The present invention also provides a method for preparing bacterial fermentation broth, which includes the following steps: culturing Enterococcus faecalis as described above under suitable conditions, collecting the culture broth and / or the lysate of the Enterococcus faecalis, and then centrifuging to collect the supernatant to obtain the bacterial fermentation broth.

[0011] The present invention also provides the use of Enterococcus faecalis as described above or bacterial fermentation broth as described above in the preparation of drugs for antitumor purposes.

[0012] In one embodiment, the tumor is colon cancer, lung cancer, stomach cancer, liver cancer, melanoma, or cervical cancer.

[0013] The present invention also provides an antitumor drug comprising Enterococcus faecalis as described above and / or bacterial fermentation broth as described above.

[0014] In one embodiment, the Enterococcus faecalis in the antitumor drug is a live bacterium.

[0015] In one embodiment, the antitumor drug also includes a pharmaceutically acceptable carrier.

[0016] The present invention also provides an antitumor pharmaceutical composition comprising the antitumor drugs described above.

[0017] In one embodiment, the antitumor drug composition further includes a combination drug selected from one or more of chemotherapeutic drugs, small molecule targeted drugs, photosensitizers, photothermal agents, antibodies against inhibitory second signaling molecules, and inhibitors against inhibitory second signaling molecules.

[0018] In one embodiment, the inhibitory second signaling molecule is selected from at least one of PD-1, PD-L1, and CTLA-4.

[0019] This invention provides a novel Enterococcus faecalis strain with significant antitumor effects. This strain exhibits high safety and no adverse effects on animal health. Administration of this strain alone (GZ) yields a significant antitumor effect, and its antitumor efficacy can be further enhanced when combined with other antitumor drugs. The Enterococcus faecalis provided by this invention can be used to prepare tumor inhibitors and corresponding drugs, thereby achieving tumor treatment. Attached Figure Description

[0020] Figure 1 This is a Gram-stained microscopic image of Enterococcus faecalis GZ in one embodiment of the present invention;

[0021] Figure 2 This is a phylogenetic tree of Enterococcus faecalis GZ in one embodiment of the present invention;

[0022] Figure 3 This is a growth curve of Enterococcus faecalis GZ in one embodiment of the present invention;

[0023] Figure 4 This is a curve showing the weight change of mice in each group during an experiment using the MC38 colon cancer model in one embodiment of the present invention.

[0024] Figure 5 This is a curve showing the tumor volume change of mice in each group during an MC38 colon cancer model experiment in one embodiment of the present invention;

[0025] Figure 6 This is a curve showing the change in body weight of mice in each group during an H22 lung cancer model experiment in one embodiment of the present invention;

[0026] Figure 7 This is a curve showing the tumor volume change of mice in each group during an H22 lung cancer model experiment in one embodiment of the present invention.

[0027] The Enterococcus faecalis GZ provided by this invention was deposited at the China Center for Type Culture Collection (CCTCC) on December 21, 2020, with accession number CCTCC NO: M2020951, located at Wuhan University, Wuhan, China; and was tested on January 5, 2021, with the result showing it to be viable. Detailed Implementation

[0028] To facilitate understanding of the present invention, a more comprehensive description is provided below, along with preferred embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0030] An embodiment of the *Enterococcus faecalis* strain of this invention was deposited on December 21, 2020, at the China Center for Type Culture Collection (CCTCC), with accession number CCTCC NO: M2020951, located at Wuhan University, Wuhan, China, and designated as *Enterococcus faecalis* GZ. Sequencing results showed that the 16S ribosomal RNA gene sequence of this *Enterococcus faecalis* is shown in SEQ ID No: 1.

[0031] This strain has high safety and no adverse effects on animal health. It can achieve significant anti-tumor effects when used alone as a single-drug treatment of Enterococcus faecalis GZ. When combined with other anti-tumor drugs such as anti-PD-1 antibodies, it can achieve even better anti-tumor effects.

[0032] The bacterial fermentation broth of one embodiment of the present invention is the fermentation broth of the above-mentioned Enterococcus faecalis.

[0033] A method for preparing the bacterial fermentation broth according to an embodiment of the present invention includes the following steps: culturing the above-mentioned Enterococcus faecalis under suitable conditions, collecting the culture broth and / or the lysate of Enterococcus faecalis, and then centrifuging to collect the supernatant to obtain the bacterial fermentation broth. Optionally, suitable conditions are: BHI liquid medium, static culture at 37°C.

[0034] This invention provides the use of the aforementioned Enterococcus faecalis or the fermentation broth of the aforementioned bacteria in the preparation of a drug for antitumor purposes. Optionally, the aforementioned tumor is colon cancer, lung cancer, gastric cancer, liver cancer, melanoma, or cervical cancer, but is not limited thereto.

[0035] An antitumor drug according to an embodiment of the present invention includes the above-mentioned Enterococcus faecalis and / or the above-mentioned bacterial fermentation broth.

[0036] Optionally, the Enterococcus faecalis in the antitumor drug is a live bacterium, that is, the antitumor drug is a live bacterium drug.

[0037] In a specific example, antitumor drugs also include pharmaceutically acceptable carriers, such as excipients, diluents, buffers, etc.

[0038] An antitumor drug composition according to an embodiment of the present invention includes the above-mentioned antitumor drug.

[0039] In one specific example, the antitumor drug composition also includes a combination drug selected from one or more of chemotherapeutic drugs, small molecule targeted drugs, photosensitizers, photothermal agents, antibodies against inhibitory second signaling molecules, and inhibitors of inhibitory second signaling molecules. Thus, combining the aforementioned antitumor drug with other drugs can enhance the antitumor effect. It is understood that the aforementioned antitumor drug and combination drug can be mixed together in the antitumor drug composition, or presented as a combination product or kit.

[0040] Optionally, the inhibitory second signaling molecule is selected from at least one of PD-1, PD-L1, and CTLA-4, but is not limited thereto.

[0041] A tumor treatment method according to an embodiment of the present invention includes the following steps: treatment with the above-mentioned anti-tumor drug.

[0042] The present invention will now be described in further detail with reference to specific embodiments and accompanying drawings.

[0043] I. Isolation and Identification of Enterococcus faecalis

[0044] 1. Reagents and Instruments

[0045] Culture media: MRS broth (Qingdao Haibo Biotechnology Co., Ltd., catalog number: HB0384-1); BHI broth (Qingdao Haibo Biotechnology Co., Ltd., catalog number: HB8297-5).

[0046] Main reagents: Agar powder (Beijing Solarbio Science & Technology Co., Ltd., catalog number: A8190-500g); Gram staining solution (Zhuhai Beso Biotechnology Co., Ltd., catalog number: BA-4012); Streptococcus biochemical coding identification tubes (Hangzhou Binhe Microbial Reagent Co., Ltd.); Gram-positive bacteria antimicrobial susceptibility testing discs (Hangzhou Microbial Reagent Co., Ltd.); 2*EasyTaqPCRSuperMix (Beijing TransGen Biotechnology Co., Ltd., catalog number: AS111-11); Agarose (Beijing Solarbio Science & Technology Co., Ltd., catalog number: 91622); Trans2K DNA Marker (Beijing TransGen Biotechnology Co., Ltd., catalog number: BM101-01); Antimicrobial susceptibility testing discs for 30 types (Hangzhou Microbial Reagent Co., Ltd., catalog number: S1100).

[0047] Instruments: Upright microscope (Ningbo Yongxin Optics Co., Ltd., model: N-117M); inverted microscope; PCR instrument (Zhuhai Heima Medical Instrument Co., Ltd., model: Hema9600).

[0048] 2. Separation and Identification

[0049] Under aseptic conditions, the prepared YK donor fecal bacterial suspension was streaked onto MRS medium using an inoculation loop and incubated anaerobically at 37°C for 72 hours. Suspected colonies were picked and purified by repeated streaking in BHI, followed by Gram staining and microscopic examination to observe bacterial morphology. Figure 1 As shown in (prs-217-02). Next, 16S rRNA sequencing was performed using the following primer sequences: universal primer 27F: AGAGTTTGATCCTGGCTCAG; 1429R: GGTTACCTTGTTACGACTT.

[0050] The PCR reaction parameters are as follows:

[0051]

[0052] The amplified PCR product was sequenced using 16S rRNA, and the sequence was analyzed by NCBI blast.

[0053] 3. Drug resistance testing (safety evaluation)

[0054] The antimicrobial susceptibility testing was performed using the disk diffusion method. Liquid-cultured Enterococcus faecalis were centrifuged at 4000 rpm for 15 min, the supernatant was discarded, and the cultured bacteria were resuspended in PBS, thoroughly mixed, and the OD value was adjusted to approximately 0.5, resulting in a viable count of approximately 5 × 10⁻⁶. 7CFU / mL was evenly spread on BHI solid culture dishes. After the bacterial solution was completely absorbed, antimicrobial susceptibility test discs were placed on the plates. One plate contained one type of antimicrobial susceptibility test disc (3 discs) and one blank control (1 disc). The plates were inverted and incubated at 37°C. After 24 hours, the diameter of the inhibition zone was measured with a ruler and the data was recorded. The results were interpreted according to the antimicrobial drug test standard of the disc method (WS / T125—1999).

[0055] The results are shown in the table below:

[0056]

[0057] In summary, this strain of Enterococcus faecalis is mainly resistant to aminoglycosides and sulfonamides, with a narrow resistance spectrum. It is sensitive or intermediate to penicillins, chloramphenicol, macrolides, and cephalosporins, sensitive to tetracyclines, and sensitive to vancomycin.

[0058] 4. Whole genome sequencing and construction of phylogenetic trees

[0059] BLAST alignment of the sequencing results was performed to construct a phylogenetic tree, and the results are as follows: Figure 2 As shown.

[0060] 5. Tolerance to artificial gastrointestinal fluids

[0061] The results of the tolerance of this strain of Enterococcus faecalis to artificial gastrointestinal fluid are shown in the table below:

[0062]

[0063] When this bacterium was added to a pH 2.0 artificial gastric fluid with a pepsin concentration of 1 g / L, the viable bacterial count decreased by three orders of magnitude after 1 hour of treatment. After 2 hours of treatment, dilution and plating resulted in almost no growth of viable bacterial colonies, with a survival rate of approximately 0%. After 3 hours of treatment in a pH 3.0 artificial gastric fluid, the survival rate was also approximately 0%. After 3 hours of treatment in a pH 4.0 artificial gastric fluid, the survival rate remained at 95.87%. Artificial intestinal fluid had virtually no effect on the survival rate of this bacterium.

[0064] 6. Growth curve

[0065] The growth curves of this strain of Enterococcus faecalis are shown in the table below. Figure 3 As shown:

[0066]

[0067] II. MC38 colon cancer model experiment

[0068] 1. Laboratory animals

[0069] Female C57BL / 6 mice, 6-7 weeks old (estimated age of mice at the time of tumor cell inoculation), with an estimated weight range of 18-22g at the time of cell inoculation, were purchased from Junke Biotechnology Co., Ltd.

[0070] 2. Environmental conditions of the experimental animal housing

[0071] All experimental animals were housed in individually ventilated enclosures with constant temperature and humidity, and were allowed to acclimatize to the environment for at least 7 days. The enclosure temperature was 20℃~26℃, humidity 40%~70%, with 10~20 air changes per hour. A day-night cycle was implemented, with light from 7:00 AM to 7:00 PM and darkness from 7:00 PM to 7:00 AM the following day. They were continuously provided with cobalt-60 radioactively sterilized complete pelleted rat feed, with unlimited access. Tap water (sterilized by autoclaving) was provided continuously from water bottles. The enclosures were polysulfone cages, autoclaved, and measured 325mm×210mm×180mm. The bedding was autoclaved corn cobs. Each cage contained 5 animals. Cage tags indicated the IACUC approval number, experiment number, experiment start time, project leader, researchers, animal origin, group, and animal number. The animals were marked with ear tags.

[0072] 3. Experimental drugs and reagents

[0073] Enterococcus faecalis (self-made); Anti-mouse PD-1 (B7-H1) (supplier: BioXcell; catalog number: BE146; batch number: 186520N1B). Total drug required (calculated at 25g per mouse, including 50% surplus): 27mg (5mg / kg × 6 doses × 10 mice / group × 0.025kg body weight × 3 treatment groups × 1.2 times (20% surplus) = 27mg).

[0074] The abbreviations used below are shown in the table below:

[0075]

[0076] 4. Experimental Methods

[0077] This embodiment evaluates the antitumor effects of the test drugs Enterococcus faecalis and anti-PD-1 antibody, administered alone or in combination, in a C57BL / 6 mouse animal model with xenografted colon cancer cell line from MC38 mice.

[0078] (1) Experimental grouping

[0079] The number of animals in each group, along with detailed routes of administration, dosages, and regimens, are shown in the table below:

[0080]

[0081] Note: n: number of animals; administration volume is 10 μL / g.

[0082] (2) Cell culture

[0083] MC38 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum. MC38 cells in the exponential growth phase were collected and resuspended in PBS to a suitable concentration for subcutaneous tumor inoculation in mice.

[0084] (3) Tumor inoculation

[0085] The experimental mice were subcutaneously inoculated with 1×10⁻⁶ spores on their right back 6 One MC38 cell was resuspended in PBS (0.1 mL / cell), and tumor growth was observed regularly until the tumor reached an average volume of 96 mm². 3 Mice were randomly assigned to groups based on tumor size and body weight for drug administration. Detailed administration methods, dosages, and routes are shown in the table above. Tumor inoculation was recorded as day 0.

[0086] (4) Random grouping

[0087] Before administration, all animals were weighed and tumor volume was measured using calipers. Since tumor volume affects treatment effectiveness, a randomized controlled trial was conducted, grouping mice according to their tumor volume to ensure similar tumor volumes across different groups.

[0088] (5) Preparation of drug solution

[0089] The compound solutions are prepared in a biosafety cabinet or clean bench and used immediately after preparation. The preparation method is shown in the table below:

[0090]

[0091] Mix well before use to ensure the formulation is homogeneous.

[0092] (6) Administration

[0093] On day 9 after tumor inoculation, the drug was administered according to the experimental design.

[0094] (7) Experimental observation and data collection

[0095] Following tumor cell inoculation, routine monitoring included monitoring tumor growth and the impact of treatment on normal animal behavior. Specific monitoring included the animal's activity level, food and water intake, weight gain or loss, and any abnormalities observed in the eyes, coat, or other areas. Clinical symptoms observed during the experiment were recorded in the raw data. After drug administration began, mouse weight and tumor size were measured on days 6, 8, 10, 12, and 14. Tumor size was calculated using the formula: Tumor volume (mm²) 3 = 0.5 × (tumor long diameter × tumor short diameter) 2 ).

[0096] 5. Results

[0097] like Figure 4 The figures show the weight change curves of mice in each group. No significant changes in mouse weight were observed, indicating that the above treatment methods have no adverse effects on animal health.

[0098] like Figure 5 The figure shows the tumor volume change curves of mice in each group. It can be seen that compared with the control group, the administration of this strain of Enterococcus faecalis GZ alone can achieve a very significant anti-tumor effect, and when combined with anti-PD-1 antibody, it can achieve an even better anti-tumor effect.

[0099] III. H22 Lung Cancer Model Experiment

[0100] 1. Laboratory animals

[0101] Female BALB / c mice, 6-7 weeks old (estimated age of mice at the time of tumor cell inoculation), with an estimated weight range of 18 kg to 22 g at the time of cell inoculation, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (VRL).

[0102] 2. Environmental conditions of the experimental animal housing

[0103] All experimental animals were housed in individually ventilated enclosures with constant temperature and humidity, and were allowed to acclimatize to the environment for at least 7 days. The enclosure temperature was 20℃~26℃, humidity 40%~70%, with 10~20 air changes per hour. A day-night cycle was implemented, with light from 7:00 AM to 7:00 PM and darkness from 7:00 PM to 7:00 AM the following day. They were continuously provided with cobalt-60 radioactively sterilized complete pelleted rat feed, with unlimited access. Tap water (sterilized by autoclaving) was provided continuously from water bottles. The enclosures were polysulfone cages, autoclaved, and measured 325mm×210mm×180mm. The bedding was autoclaved corn cobs. Each cage contained 5 animals. Cage tags indicated the IACUC approval number, experiment number, experiment start time, project leader, researchers, animal origin, group, and animal number. The animals were marked with ear tags.

[0104] 3. Experimental drugs and reagents

[0105] Enterococcus faecalis (self-made); Anti-mouse PD-1 (B7-H1) (supplier: BioXcell; catalog number: BE146; batch number: 186520N1B). Total drug required (calculated at 25g per mouse, including 50% surplus): 27mg (5mg / kg × 6 doses × 10 mice / group × 0.025kg body weight × 3 treatment groups × 1.2 times (20% surplus) = 27mg).

[0106] The abbreviations used below are shown in the table below:

[0107]

[0108] 4. Experimental Methods

[0109] This embodiment evaluates the antitumor effects of the test drugs Enterococcus faecalis and anti-PD-1 antibody, administered alone or in combination, in a BALB / c mouse animal model with xenografted H22 mouse lung cancer cell line.

[0110] (1) Experimental grouping

[0111] The number of animals in each group, along with detailed routes of administration, dosages, and regimens, are shown in the table below:

[0112]

[0113] Note: n: number of animals; administration volume is 10 μL / g.

[0114] (2) Cell culture

[0115] H22 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum. H22 cells in the exponential growth phase were collected and resuspended in PBS to a suitable concentration for subcutaneous tumor inoculation in nude mice.

[0116] (3) Tumor inoculation

[0117] The experimental mice were subcutaneously inoculated with 1×10⁻⁶ spores on their right back 6 One H22 cell was resuspended in PBS (0.1 mL / cell), and tumor growth was observed regularly until the tumor reached an average volume of 96 mm². 3 Mice were randomly assigned to groups based on tumor size and body weight for drug administration. Detailed administration methods, dosages, and routes are shown in the table above. Tumor inoculation was recorded as day 0.

[0118] (4) Random grouping

[0119] Before administration, all animals were weighed and tumor volume was measured using calipers. Since tumor volume affects treatment effectiveness, a randomized controlled trial was conducted, grouping mice according to their tumor volume to ensure similar tumor volumes across different groups.

[0120] (5) Preparation of drug solution

[0121] The compound solutions are prepared in a biosafety cabinet or clean bench and used immediately after preparation. The preparation method is shown in the table below:

[0122]

[0123] Mix well before use to ensure the formulation is homogeneous.

[0124] (6) Administration

[0125] On day 9 after tumor inoculation, the drug was administered according to the experimental design.

[0126] (7) Experimental observation and data collection

[0127] Following tumor cell inoculation, routine monitoring included monitoring tumor growth and the impact of treatment on normal animal behavior. Specific monitoring included the animal's activity level, food and water intake, weight gain or loss, and any abnormalities observed in the eyes, coat, or other areas. Clinical symptoms observed during the experiment were recorded in the raw data. After drug administration began, mouse weight and tumor size were measured on days 6, 8, 10, 12, and 14. Tumor size was calculated using the formula: Tumor volume (mm²) 3 = 0.5 × (tumor long diameter × tumor short diameter) 2 ).

[0128] 5. Results

[0129] like Figure 6 The figures show the weight change curves of mice in each group. No significant changes in mouse weight were observed, indicating that the above treatment methods have no adverse effects on animal health.

[0130] like Figure 7 The figure shows the tumor volume change curves of mice in each group. It can be seen that compared with the control group, the administration of this strain of Enterococcus faecalis GZ alone can achieve a very significant anti-tumor effect, and when combined with anti-PD-1 antibody, it can achieve an even better anti-tumor effect.

[0131] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0132] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims. sequence list <110> Suzhou Purison Gene Technology Co., Ltd. <120> Enterococcus faecalis and its applications <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1469 <212> DNA <213> Artificial Sequence <400> 1 nnnnnnnnnn gggcgngcta tacatgcagt cgaacgcttc tttcctcccg agtgcttgca 60 ctcatttgga aagaggagtg gcggacgggt gagtaacacg tgggtaacct acccatcaga 120 gggggataac acttggaaac aggtgctaat accgcataac agtttatgcc gcatggcata 180 agagtgaaag gcgctttcgg gtgtcactga tggatggacc cgcggtgcat tagctagttg 240 gtgaggtaac ggctcaccaa ggccacgatg catagccgac ctgagagggt gatcggccac 300 actgggactg aagacacggc ccagactcct acgggaggca gcagtaggga atcttcggca 360 atggacgaaa gtctgaccga gcaacgccgc gtgagtgaag aaggttttcg gatcgtaaaa 420 ctctgttgtt agagaagaac aaggacgtta gtaactgaac gtcccctgac ggtatctaac 480 cagaaagcca cggctaacta cgtgccagca gccgcggtaa tacgtaggtg gcaagcgttg 540 tccggattta ttgggcgtaa agcgagcgca ggcggtttct taagtctgat gtgaaagccc 600 ccggctcaac cggggagggt cattggaaac tgggagactt gagtgcagaa gaggagagtg 660 gaattccatg tgtagcggtg aaatgcgtag atatatggag gaacaccagt ggcgaaggcg 720 gctctctggt ctgtaactga cgctgaggct cgaaagcgtg gggagcaaac aggattagat 780 accctggtag tccacgccgt aaacgatgag tgctaagtgt tggagggttt ccgcccttca 840 gtgctgcagc aaacgcatta agcactccgc ctggggagta cgaccgcaag gttgaaactc 900 aaaggaattg acgggggccc gcacaagcgg tggagcatgt ggtttaattc gaagcaacgc 960 gaagaacctt accaggtctt gacatccttt gaccactcta gagatagagc tttcccttcg 1020 gggacaaagt gacaggtggt gcatggttgt cgtcagctcg tgtcgtgaga tgttgggtta 1080 agtcccgcaa cgagcgcaac ccttattgtt agttgccatc atttagttgg gcactctagc 1140 gagactgccg gtgacaaacc ggaggaaggt ggggatgacg tcaaatcatc atgcccctta 1200 tgacctgggc tacacacgtg ctacaatggg aagtacaacg agtcgctaga ccgcgaggtc 1260 atgcaaatct cttaaagctt ctctcagttc ggattgcagg ctgcaactcg cctgcatgaa 1320 gccggaatcg ctagtaatcg cggatcagca cgccgcggtg aatacgttcc cgggccttgt 1380 acacaccgcc cgtcacacca cgagagtttg taacacccga agtcggtgag gtaacctttt 1440 ggagccagcc gcctaaggng anncnnnnt 1469

Claims

1. The use of *Enterococcus faecalis* in the preparation of drugs for treating colon cancer and / or lung cancer, characterized in that... The preservation number of the *Enterobacter faecalis* is CCTCC NO: M2020951; The 16S ribosomal RNA gene sequence of Enterococcus faecalis is shown in SEQ ID No:

1. The anti-colon cancer and / or lung cancer drug composition further includes a combination drug, wherein the combination drug is a PD-1 antibody.

2. A bacterial fermentation broth, characterized in that, The bacterial fermentation broth is the fermentation broth of Enterococcus faecalis as described in claim 1.

3. A method for preparing bacterial fermentation broth, characterized in that, Includes the following steps: The Enterococcus faecalis of claim 1 is cultured under suitable conditions, and the culture medium is collected to obtain the bacterial fermentation broth.

4. The use of the bacterial fermentation broth according to claim 2 in the preparation of drugs for treating colon cancer and / or lung cancer.

5. A drug for treating colon cancer and / or lung cancer, characterized in that, The drug includes the Enterococcus faecalis of claim 1 or the bacterial fermentation broth of claim 2, and the anti-colon cancer and / or lung cancer drug further includes a combination drug, wherein the combination drug is a PD-1 antibody.

6. The anti-colon cancer and / or lung cancer drug according to claim 5, characterized in that, The anti-colon cancer and / or lung cancer drugs also include pharmaceutically acceptable carriers.

Images