Clostridium ventriculi and use thereof

Abstract

The present invention belongs to the technical field of microorganisms, and relates to Clostridium ventriculi and the use thereof. The Clostridium ventriculi is Clostridium ventriculi SYSU-12 which is deposited in the China General Microbiological Culture Collection Center with a deposit number of CGMCC No. 27761 and a deposit date of 30 June 2023. Dominant strain SYSU-12 in feces is obtained by means of collecting a feces sample from a gastric cancer patient responding to PD-1 antibody treatment, and performing separation by means of anaerobic culture using the serial dilution method and then performing purification, and same is identified as Clostridium ventriculi by means of gram staining, microscopic examination, single colony morphology observation and molecular biology experiments. The Clostridium ventriculi and the component thereof provided can be used in the preparation of an immune activator, a tumor inhibitor and a corresponding drug or health food.

Description

A gastric clostridium and its application Technical Field

[0001] The present invention relates to the field of microbial technology, in particular to a gastric clostridium and an application thereof. Background Art

[0002] In recent years, immunotherapy with PD-1 / PD-L1 blockers has been applied to the treatment of gastric cancer, breaking through the one-year survival bottleneck, but the benefits for most patients remain limited. Gastric cancer has poor antigenicity and minimal immune infiltration, making it a traditionally "cold tumor." However, immune-inflammatory "hot tumors" are more susceptible to treatment with immune checkpoint inhibitors (ICIs). How to transform cold tumors into hot tumors and improve their response rate and therapeutic efficacy to tumor immunotherapy is an urgent problem that needs to be solved.

[0003] In recent years, numerous studies have found that the efficacy of tumor immunotherapy is correlated with the patient's gut microbiome, with a significant association between gut microbial composition and clinical response to immunotherapy. In various animal models, including lung and colon cancer models, bacteria such as Bifidobacterium, Akkermansia, and Faecalibacterium have demonstrated potent ICI therapy. However, these popular bacteria struggle to survive in the stomach. Finding effective single or multiple bacterial species adapted to the gastric ecosystem is a promising approach to enhancing gastric cancer immunotherapy. Summary of the Invention

[0004] The purpose of the present invention is to overcome the shortcomings of the existing technology and provide a gastric clostridium and its application that have a pro-inflammatory effect on immune cells, promote the secretion of chemokines to activate T cells to exert anti-tumor effects, and transform "cold" tumors into "hot" tumors.

[0005] To achieve the above object, the technical solution adopted by the present invention is:

[0006] In a first aspect, the present invention provides a gastric Clostridium, which is Clostridium ventriculi SYSU-12, deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms, with a deposit number of CGMCC No. 27761 and a deposit date of June 30, 2023.

[0007] The present invention collects fecal samples from gastric cancer patients who respond to PD-1 antibody treatment, and isolates and purifies the dominant strain SYSU-12 in the feces through anaerobic culture using the serial dilution method. The dominant strain SYSU-12 is identified as Clostridium ventriculi through Gram staining, microscopy, single colony morphology observation, and molecular biology experiments.

[0008] Clostridium ventriculi, formerly known as Sarcina ventriculi, is a Gram-positive anaerobic bacterium that forms spores and can survive and grow in acidic environments. It has good stress tolerance and is commonly found in the upper gastrointestinal tract. It can colonize and proliferate in the human stomach. Clostridium ventriculi belongs to the Clostridiaceae family and typically exists as tetrads or octads with a diameter of 1.8-3.0 μm.

[0009] As a preferred embodiment of the gastric Clostridium of the present invention, the 16S rRNA sequence of the gastric Clostridium ventriculi SYSU-12 is shown in SEQ ID NO. 1. The present invention identifies SYSU-12 as gastric Sarcina (gastric Clostridium) by PCR amplification, sequencing, alignment, and tree construction of the 16S rRNA of SYSU-12.

[0010] As a preferred embodiment of the gastric Clostridium of the present invention, the gastric Clostridium ventriculi SYSU-12 is isolated from feces of gastric cancer patients who respond to PD-1 antibody treatment.

[0011] In a second aspect, the present application provides a gastric Clostridium culture prepared from the above-mentioned gastric Clostridium ventriculi SYSU-12.

[0012] As a preferred embodiment of the Clostridium gastricis culture of the present invention, the Clostridium gastricis culture contains at least one of live Clostridium gastricis bacteria, Clostridium gastricis bacterial components and derivatives thereof, and Clostridium gastricis metabolites. The Clostridium gastricis culture of the present invention comprises live Clostridium gastricis bacteria, inactivated Clostridium gastricis bacterial bodies, biological materials containing Clostridium gastricis, and metabolites secreted during the growth of Clostridium gastricis.

[0013] As a preferred embodiment of the Clostridium gastricis culture of the present invention, the Clostridium gastricis culture is mainly prepared by at least one of the following methods:

[0014] Method 1: scrape the activated gastric Clostridium SYSU-12 bacteria into a sterile solvent to prepare a gastric Clostridium SYSU-12 bacterial suspension, wherein the gastric Clostridium SYSU-12 bacterial suspension is a gastric Clostridium culture;

[0015] Method 2: scrape and activate a single colony of Clostridium gastricis SYSU-12, inoculate it into the culture medium, and then centrifuge it. The supernatant obtained is the Clostridium gastricis culture;

[0016] Method 3: scrape and activate a single colony of Clostridium gastricis SYSU-12, inoculate it into a culture medium, and then transfer it into a bioreactor for batch fermentation. The resulting fermentation liquid is the Clostridium gastricis culture;

[0017] Method 4: Scrape and activate a single colony of Clostridium gastricis SYSU-12, inoculate it into a culture medium, and then centrifuge. The resulting precipitate is resuspended in a solvent and sterilized at 115-130°C for 15-30 minutes. The resulting inactivated bacterial suspension is the Clostridium gastricis culture. The method for preparing the Clostridium gastricis culture of the present invention is not limited to a single method; any product obtained by culturing Clostridium gastricis is considered a Clostridium gastricis culture.

[0018] As a preferred embodiment of the gastric Clostridium culture of the present invention, in the first method of the preparation method, the solvent includes but is not limited to at least one of phosphate buffer, dimethyl sulfoxide, physiological saline, and water.

[0019] As a preferred embodiment of the gastric Clostridium culture of the present invention, the gastric Clostridium SYSU-12 bacterial cell content in the gastric Clostridium culture is not less than 1×10 6 CFU.

[0020] In a third aspect, the present invention provides the use of the aforementioned Clostridium gastricis or its culture in the preparation of a product for preventing and / or treating tumors. Experimental findings show that inactivated Clostridium gastricis still has certain anti-tumor activity, indicating that Clostridium gastricis and its culture have good anti-tumor activity.

[0021] In a fourth aspect, the present invention provides the use of the aforementioned Clostridium gastricis or a culture thereof in the preparation of a product for the prevention and / or treatment of tumors. Animal experiments have shown that Clostridium gastricis has a significant inhibitory effect on the growth and metastasis of cold tumors, such as microsatellite-stable (MSS) colorectal cancer, melanoma, and gastric cancer. It can effectively prevent the growth and metastasis of tumors, including colorectal cancer, melanoma, and gastric cancer.

[0022] As a preferred embodiment of the application of the present invention, the product includes at least one of medicine, food and health care product.

[0023] As a preferred embodiment of the application of the present invention, the tumor includes at least one of a malignant tumor, a metastatic tumor or a non-metastatic tumor.

[0024] As a preferred embodiment of the application of the present invention, the types of tumors include but are not limited to: adrenocortical carcinoma, bladder urothelial carcinoma, breast cancer, pancreatic cancer, cervical cancer, bile duct cancer, colon cancer, colorectal cancer, diffuse large B-cell lymphoma, multiforme glioma, glioma, head and neck cancer, renal chromophobe cell carcinoma, mixed renal cancer, renal cancer, leukemia, lymphoma, brain cancer, liver cancer, lung adenocarcinoma, lung squamous cell carcinoma, mesothelioma, ovarian cancer, pancreatic cancer, pheochromocytoma, paraganglioma, prostate cancer, rectal adenocarcinoma, sarcoma, skin melanoma, gastric cancer, gastrointestinal stromal tumor, esophageal cancer, testicular cancer, thyroid cancer, thymic cancer, endometrial cancer, uterine sarcoma, uveal melanoma and at least one of soft tissue sarcoma.

[0025] In a fifth aspect, the present invention provides the use of the above-mentioned Clostridium gastricis or its culture in combination with an immune checkpoint inhibitor in the preparation of a drug for treating tumors.

[0026] As a preferred embodiment of the application of the present invention, the immune checkpoint inhibitor is at least one of an inhibitor acting on T cell negative co-stimulatory molecules and / or their ligands, and an inhibitor acting on T cell negative co-inhibitory molecules and / or their ligands.

[0027] As a preferred embodiment of the application of the present invention, the T cell negative co-stimulatory molecule ligand or T cell negative co-inhibitory molecule ligand is selected from at least one of CTLA-4, PD-1, PD-L1, PD-L2, B7-1, B7-2, B7-H3, B7-H4, B7-H6, A2AR, IDO, TIM-3, BTLA, VISTA, TIGIT, LAG-3, CD40, KIR, CEACAM1, GARP, PS, CSF1R, CD94 / NKG2A, TDO, TNFR and DcR3.

[0028] In a sixth aspect, the present invention provides the use of the above-mentioned Clostridium gastricis SYSU-12 or a culture thereof in the preparation of an immunostimulant. The present invention has experimentally found that Clostridium gastricis can stimulate immune cells to secrete chemokines to activate T cell effects, thereby enhancing the infiltration of CD8+ T cells into tumor tissues.

[0029] In a seventh aspect, the present invention provides a Clostridium gastricis preparation comprising the above-mentioned Clostridium gastricis or Clostridium gastricis culture.

[0030] As a preferred embodiment of the preparation of the present invention, the administration of the Clostridium gastricis preparation includes at least one of oral administration, intravenous injection, intratumoral injection and para-cancer injection.

[0031] As a preferred embodiment of the preparation of the present invention, the preparation further comprises a pharmaceutically acceptable carrier and excipients.

[0032] As a preferred embodiment of the preparation of the present invention, the dosage form of the preparation includes but is not limited to at least one of lyophilized powder, powder, tablet, capsule, granule and injection.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] 1. The present invention collects fecal samples from gastric cancer patients who respond to PD-1 antibody treatment, and isolates and purifies the dominant strain SYSU-12 in the feces through anaerobic culture by serial dilution method. The dominant strain SYSU-12 is identified as Clostridium ventriculi through Gram staining, microscopy, single colony morphology observation, and molecular biology experiments.

[0035] 2. The gastric Clostridium of the present invention is a spore-forming bacterium with natural stress resistance, heat resistance, gastric acid and bile salt resistance. It can be retained in the stomach and intestines after oral administration and is sensitive to multiple antibiotics.

[0036] 3. This study used Clostridium ventriculi SYSU-12 alone or in combination with immune checkpoint inhibitors to treat tumors in mice implanted with these cells. The results showed that the strain screened for this purpose, Clostridium ventriculi SYSU-12, stimulated an anti-tumor immune response, reshaped the tumor microenvironment, and transformed "cold" tumors into "hot" tumors. Furthermore, Clostridium ventriculi SYSU-12 significantly enhanced the therapeutic efficacy of immune checkpoint inhibitors for gastric cancer, inhibiting both in situ tumor growth and metastasis with a good safety profile. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 shows the basic morphology of the gastric Clostridium isolated and purified in Example 1, wherein A is the Gram staining microscopic examination result (scale bar is 10 μm), B is the electron microscopic result (scale bar is 0.5 μm), and C is the colony morphology on the culture medium;

[0038] FIG2 is a phylogenetic tree of the 16S rRNA of Clostridium gastricis isolated and purified in Example 1;

[0039] FIG3 is the results of the acid resistance test of Clostridium gastricis in Effect Example 1;

[0040] FIG4 is the bile salt tolerance test results of Clostridium gastricis in Effect Example 1;

[0041] Figure 5 is a schematic diagram of the experiment of gastric Clostridium into tumors in Example 2. Figure A shows the results of live bacterial culture of tumor homogenate and microscopic examination of culture smear (scale bar 10 μm);

[0042] FIG6 shows the lung tissue weight of mice in the experimental study on Clostridium gastricis against B16-F10 tumors in Effect Example 3;

[0043] Figure 7 is a schematic diagram (Figure A) and results of the experiment on Clostridium gastricis against B16-F10 tumors in Effect Example 3 (Figure B shows the changes in spleen and lung in different treatment groups, and Figure C shows HE-stained sections of major organs);

[0044] FIG8 is a schematic diagram of the anti-MFC tumor experiment of oral live Clostridium gastricis in Effect Example 4 (Figure A) and a statistical diagram of tumor volume results (Figure B);

[0045] FIG9 is a schematic diagram of the anti-CT26 tumor experiment of oral administration of killed Clostridium gastricis bacteria in Effect Example 5 (Figure A) and a statistical diagram of tumor volume results (Figure B);

[0046] FIG10 is a graph showing the results of CXCL10 production by macrophages stimulated by live and dead Clostridium gastricis in Effect Example 6;

[0047] Figure 11 is a schematic diagram of the Transwell experiment for evaluating CD8+ T cell tumor tropism in Example 6;

[0048] FIG12 is the statistical results of the number of CD8+ T cells in the Transwell chamber with different treatments and different cancer cells in Effect Example 6;

[0049] In the above figure, "*" indicates that there is a significant difference between the two groups (p < 0.05); "**" indicates that there is a significant difference between the two groups (p < 0.01); and "***" indicates that there is a significant difference between the two groups (p < 0.005). DETAILED DESCRIPTION

[0050] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0051] In the following examples, comparative examples and experimental examples, the experimental methods used are conventional methods unless otherwise specified.

[0052] In the following examples, comparative examples and experimental examples, “Clostridium ventriculi”, “Gastrostomyces spp”, “Gastrostomyces spp SYSU-12”, “C. ventriculi” and “Cv” all refer to Gastrostomyces spp.

[0053] Unless otherwise specified, other materials, reagents, etc. used in the Examples, Comparative Examples, and Experimental Examples can be obtained from commercial sources.

[0054] MRS medium: 66.2 g of MRS medium powder was added to 1000 mL of water, fully dissolved, and sterilized by autoclaving at 121°C for 20 min. The powder was cooled and then used. MRS medium powder was purchased from HKM with the catalog number 027312.

[0055] BHI medium: 37 g of BHI medium powder was added to 1000 mL of water, fully dissolved, and then sterilized by autoclaving at 121° C. for 20 min. The powder was cooled and then used. BHI medium powder was purchased from HKM with the product number BR250 g.

[0056] CBA medium: 41.2 g of CBA medium powder was added to 1000 mL of water, fully dissolved, and sterilized by autoclaving at 121°C for 20 min. The mixture was cooled and then used for further use. CBA medium powder was purchased from HKM with the catalog number 024064.

[0057] Modified MRS liquid medium: 5 g yeast powder, 5 g anhydrous sodium acetate, 30 g anhydrous glucose, 10 g peptone, 0.1 g magnesium sulfate heptahydrate, 2.6 g dipotassium hydrogen phosphate trihydrate, 0.05 g manganese sulfate monohydrate, 2 g diammonium hydrogen citrate, 1 g Tween-80, and 1 g L-cysteine ​​hydrochloride were added to 1000 mL of water. After fully dissolving, the mixture was sterilized in an autoclave at 121°C for 20 min and cooled for later use. Modified MRS solid medium was prepared by adding 15 g / L agar powder to the liquid medium.

[0058] The freeze-drying protective agent includes 0.5-2.5 wt% skim milk powder, 2-6 wt% trehalose polymer, 0.2-0.4 wt% sodium glutamate and water.

[0059] The composite protective agent comprises 5 wt% of calcium salt, 0.25-0.35 wt% of gum arabic, 5 wt% of maltodextrin and 0.15-0.25 wt% of silicon dioxide.

[0060] The drying protective agent comprises 10 wt % of skimmed milk powder, 10 wt % of inulin, 10 wt % of oligosaccharide and 0.15-0.25 wt % of silicon dioxide.

[0061] The bacterial genomic DNA extraction kit was purchased from Tiangen Biochemical Technology Co., Ltd. with the catalog number DP302-02.

[0062] C57BL / 6 and Balb / c mice were purchased from the Laboratory Animal Center, East Campus, Sun Yat-sen University. 615 mice were purchased from the Hematology Hospital, Chinese Academy of Medical Sciences.

[0063] Melanoma cells B16-F10 were purchased from Fenghbio (catalog number CL0039).

[0064] Rectal cancer cells CT26 were purchased from iCell with the catalog number iCell-m014; colon cancer cells MC38 were purchased from iCell with the catalog number iCell-m032; forestomach cancer cells MFC were purchased from iCell with the catalog number iCell-m035; macrophages RAW264.7 were purchased from iCell with the catalog number iCell-m047; and gastric cancer cells AGS were purchased from iCell with the catalog number iCell-h016.

[0065] CXCL10 RT-qPCR primers were synthesized by Shanghai Bioengineering Co., Ltd., and the sequences are as follows (5'-3'):

[0066] Forward-ATCATCCCTGCGAGCCTATCCT;

[0067] Reverse-GACCTTTTTTGGCTAAACGCTTC.

[0068] Example 1

[0069] This embodiment provides a method for isolating and purifying Clostridium ventriculi SYSU-12, comprising the following steps:

[0070] 1. Fecal Sample Collection and Bacterial Isolation

[0071] 1.1 Place the cotton swab with the stool sample into a cell culture flask containing 20 mL of bacterial freezing solution, seal the flask in an anaerobic bag, and store and transport it in an ice box. The stool should be collected from gastric cancer patients who have responded to PD-1 therapy (anti-PD-1 therapy response, PR) and should be freshly collected within 10 minutes of ex vivo collection.

[0072] 1.2 Shake the cell culture flask containing the stool sample to allow the stool sample to dislodge into the bacterial cryopreservation solution. Serially dilute the bacterial cryopreservation solution containing the stool sample 10 times in a 10-fold gradient. Spread 20 μL of each dilution onto MRS, BHI, or CBA culture media. Incubate at 37°C under anaerobic conditions for 48-72 hours. Select plates with 5-100 single colonies for purification. Use PBS buffer as the dilution solution.

[0073] 2 Purification of fecal bacteria

[0074] 2.1 Randomly pick a single colony and transfer it to 20 μL PBS. Mix by pipetting and spread 15 μL of the bacterial suspension onto the same plate. Incubate under the same conditions for 48-72 hours. The remaining 5 μL of the bacterial suspension is used for Gram staining.

[0075] 2.2 Further purify the plate with single colonies obtained in step 2.1 and transfer the plate continuously 3-5 times until all colonies on the same plate have the same morphology. The same culture medium and culture conditions should be used for each transfer.

[0076] 2.3 Pick the single colony purified in step 2.2 and place it in sterile freezing medium, mix it by pipetting, and store it at -80°C. At the same time, retain an appropriate amount of bacterial liquid for extracting bacterial genomic DNA; the sterile freezing medium is prepared by 30% glycerol + high-temperature sterilized BHI liquid culture medium.

[0077] 3 Identification of Clostridium gastricum

[0078] 3.1 Gram stain the remaining 5 μL of bacterial suspension from step 2.1. Add 5 μL of the bacterial solution prepared when picking a single colony to the center of the slide. Use an alcohol burner to intermittently bake and fix. Add crystal violet stain to cover for 1 minute, rinse with running water, then add Lugol's iodine solution to stain for 1 minute, rinse with running water, and then cover the slide surface with decolorizing alcohol for 20-30 seconds. Gently shake the slide during this time. Rinse with running water to remove the alcohol, then counterstain with safranin stain for 1 minute, rinse with running water, and observe bacterial morphology under an optical microscope after the slide is dry. If the colony is impure and more than one bacterial morphology is present, streak the sample on the plate, incubate, and re-pick a single colony (repeat steps 2.1-2.3).

[0079] 3.2 Extract bacterial genomic DNA from the appropriate amount of bacterial solution retained in step 2.3 according to the instructions of the bacterial genomic DNA extraction kit. Use the universal primer 16S rRNA for bacterial molecular identification and the diluted genomic DNA as templates in the presence of PCR mix to perform PCR amplification. The PCR amplification program is shown in Table 1 to obtain PCR products.

[0080] 3.3 Take 5 μL of the obtained PCR product and identify it as specific amplification by agarose gel electrophoresis. Send the remaining PCR product to Shanghai Bioengineering for sequencing to obtain the nucleic acid sequence of the PCR product.

[0081] 3.4 The nucleic acid sequences of the PCR products were assembled using ChromasPro software and aligned against the NCBI database (blast.ncbi.nlm.nih.gov). Based on the alignment results, the bacterium numbered SYSU-12 was identified as Clostridium ventriculi. A phylogenetic tree of SYSU-12 was constructed using MEGA7 software using the Newton-Joint Joint method with 1000 repetitions. The basic morphology is shown in Figure 1, and the phylogenetic tree is shown in Figure 2.

[0082] Table 1 PCR amplification program

[0083] SYSU-12, identified as Clostridium ventriculi, was deposited in the General Microbiology Center of the China Culture Collection Administration. The deposit date was June 30, 2023, the deposit number was CGMCC No. 27761, and the address of the depository was No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0084] Example 2

[0085] This embodiment provides a Clostridium gastricis culture and a preparation method thereof, wherein the preparation method of the Clostridium gastricis culture comprises the following steps:

[0086] (1) 20 μL of the frozen Clostridium gastricis solution obtained in step 2.3 was applied to CBA solid culture medium and cultured at 37°C for 36 h to obtain activated Clostridium gastricis;

[0087] (2) Scrape the activated gastric Clostridium obtained in step (1) into 100 μL of sterile phosphate buffer and resuspend it to prepare a gastric Clostridium suspension. The obtained gastric Clostridium suspension is the gastric Clostridium culture.

[0088] Example 3

[0089] This embodiment provides a Clostridium gastricis culture and preparation and a preparation method thereof, wherein the preparation method comprises the following steps:

[0090] (1) inoculating gastric Clostridium into a modified MRS solid medium, culturing at a constant temperature, inoculating the obtained colonies into a modified MRS liquid medium at an inoculum amount of 2-3 wt% and continuing to culture at a constant temperature of 37° C. for 10-30 h to obtain a seed solution; inoculating the obtained seed solution into a liquid medium at an inoculum amount of 3-4% (v / v), culturing at a constant temperature of 37° C. for 10-20 h to obtain a bacterial solution, centrifuging the bacterial solution, discarding the supernatant, and collecting the bacterial mud, which is the gastric Clostridium culture;

[0091] (2) resuspending the bacterial sludge obtained in step (1) in physiological saline at a mass ratio of bacterial sludge to physiological saline = 1:1-5, adjusting the pH to 6-7 with a 1-3wt% sodium hydroxide aqueous solution, adding a lyophilization protectant at a mass ratio of lyophilization protectant to bacterial sludge = 2-5:1, mixing evenly under sterile conditions, and freeze-drying to obtain a lyophilized powder of Clostridium gastricum. The obtained lyophilized powder of Clostridium gastricum is a Clostridium gastricum preparation.

[0092] Example 4

[0093] This embodiment provides a Clostridium gastricis culture and preparation and a preparation method thereof, wherein the preparation method comprises the following steps:

[0094] (1) After adding the modified MRS liquid culture medium to the fermentation tank, sterilize it at 115-121°C for 15-20 minutes, stir and cool it; pass nitrogen until the dissolved oxygen returns to zero, and then cool it to 35-40°C; inoculate Clostridium gastricis into the above-mentioned modified MRS liquid culture medium, and then pass nitrogen at 1 L / min for 40 minutes; stop ventilation and exhaust, and maintain a positive pressure of 0.015-0.025 MPa, a temperature of 35-37°C, and a rotation speed of 150 rpm to ferment. When anaerobic fermentation reaches the late stage of fermentation and spore production, the tank is released 36 hours later to obtain Clostridium gastricis seed liquid;

[0095] (2) Add the modified MRS liquid culture medium to the fermentation tank and sterilize at 115-121°C for 15-20 minutes, cool to 85-95°C, pass nitrogen until the dissolved oxygen returns to zero, cool to 35-40°C and adjust the pH to 6.8-7.0, transplant the Clostridium gastric seed liquid obtained in step (1) into the culture medium, and then sterilize at a flow rate of 20-50m 3 / h nitrogen is introduced for 20-30min;

[0096] (3) stopping ventilation and exhaust, and maintaining a positive pressure of 0.015-0.025 MPa, fermenting at 35-37°C and 150 rpm until spores are formed, stopping fermentation, centrifuging, and retaining the precipitate. The resulting precipitate is the gastric Clostridium culture; during the above fermentation process, ammonia water is introduced to adjust the pH to 8.0;

[0097] (4) The precipitate obtained in step (3) is mixed with the composite protective agent and spray-dried at an air inlet temperature of 170-180° C. and an air outlet temperature of 70-80° C. to obtain a spray-dried bacterial powder, which is a gastric Clostridium preparation.

[0098] Example 5

[0099] This embodiment provides a Clostridium gastricis preparation and a preparation method thereof, wherein the preparation method of the Clostridium gastricis preparation comprises the following steps:

[0100] (1) Prepare bacterial sludge according to step (1) of Example 3;

[0101] (2) resuspending the bacterial sludge obtained in step (1) in physiological saline at a mass ratio of bacterial sludge: physiological saline = 1:1-5, sterilizing at high temperature, adding a drying protective agent at a mass ratio of bacterial sludge: drying protective agent = 1:2-5, mixing evenly under aseptic conditions, and spray drying at an inlet air temperature of 170-180°C and an outlet air temperature of 70-80°C to obtain inactivated gastric Clostridium powder, which is a gastric Clostridium preparation.

[0102] Effect Example 1

[0103] Physiological and biochemical experiments were performed on the gastric Clostridium isolated in Example 1, and the specific steps were as follows:

[0104] 1. Acid resistance test.

[0105] Pick up the activated gastric Clostridium single colony and put it into CBA liquid culture medium, and culture it at 37℃ and 150rpm shaking until the growth phase (the bacterial liquid concentration is about 1×10 7 CFU / mL), pipette 10 μL of bacterial solution into 990 μL of acid solution with pH 0, 1, and 2, respectively. Take another 10 μL of bacterial solution and add it into 990 μL of sterile PBS as a blank control. Mix the bacterial solution with acid solution or PBS and let it stand at 37°C for 1 h.

[0106] After standing, centrifuge at 12000 rpm for 1 min, discard the supernatant, add 990 μL PBS to resuspend, repeat the centrifugation step until no acid solution remains, take 20 μL of bacterial suspension and spread it on CBA medium for plate counting. The statistical results are shown in Figure 3.

[0107] As shown in FIG3 , the gastric Clostridium of the present invention can still grow normally under the condition of pH=1-2 and has good acid resistance.

[0108] 2. Bile salt tolerance test.

[0109] Pick up the activated gastric Clostridium single colony and put it into CBA liquid culture medium, and culture it at 37℃ and 150rpm shaking until the growth phase (the bacterial liquid concentration is about 1×10 7 CFU / mL), 10 μL of bacterial solution was pipetted into 990 μL of 0.5 wt% ox bile salt solution, and another 10 μL of bacterial solution was added into 990 μL of sterile PBS as a blank control. The bacterial solution was mixed with the ox bile salt solution or PBS, and then incubated at 37°C under anaerobic conditions for 0.5, 1, and 2 h, respectively;

[0110] After standing, centrifuge at 12000 rpm for 1 min, discard the supernatant, add 990 μL PBS to resuspend, repeat the centrifugation step until no ox bile salt remains, take 20 μL of bacterial suspension and spread it on CBA medium for plate counting. The statistical results are shown in Figure 4.

[0111] As shown in FIG4 , the gastric Clostridium of the present invention can still grow normally after being placed in a 0.5% ox bile salt solution for 0.5-2 hours, and has good bile salt tolerance.

[0112] The above results show that the gastric Clostridium of the present invention has good acid and bile salt resistance, good stress resistance in the gastrointestinal tract, and has excellent industrial development potential.

[0113] 3. Drug sensitivity test.

[0114] Pick one loop of activated gastric Clostridium colony and add it to 2 mL of sterile PBS. Use a sterile cotton swab to repeatedly dip the bacterial solution and evenly spread it on a CBA plate.

[0115] After the bacterial solution on the plate is absorbed, place the drug-sensitive paper on the plate with tweezers and incubate it upright at 37°C for 36 hours. Measure the size of the inhibition zone near the drug-sensitive paper. The results are shown in Table 2.

[0116] The drug sensitive papers used contained penicillin (PEN), gentamicin (GEN), ceftriaxone (CTR), ciprofloxacin (CIP), chloramphenicol (CLM), erythromycin (E), tetracycline (TET), lincomycin (LIN), and trimethoprim-sulfamethoxazole (T / S).

[0117] Table 2 Statistical results of drug sensitivity test

[0118] As shown in Table 2, the antibiotics PEN, CIP, CLM, E and TET all have strong inhibitory effects on the gastric Clostridium of the present invention, indicating that the gastric Clostridium of the present invention has good sensitivity to common antibiotics and the risk of drug resistance during use is relatively low.

[0119] Effect Example 2

[0120] In order to verify the tropism of Clostridium gastricis to anaerobic tumor sites, an animal experiment was conducted to allow Clostridium gastricis to enter the tumor. The specific protocol is as follows:

[0121] Male C57BL / 6 mice aged 4-6 weeks were selected as experimental subjects and divided into two groups, with 8 mice in each group. The specific procedures are shown in Figure 5A and Table 3. 96 hours after treatment with C. gastricis, the mice were killed by cervical dislocation. The tumors were homogenized in a clean bench to prepare a tumor suspension, which was then spread onto CBA medium for live bacterial culture and microscopic examination. The results are shown in Figure 5B.

[0122] Table 3 Treatment of mice in each group

[0123] As shown in Figure 5B, gastric Clostridium SYSU-12 (as indicated by the arrow) can be observed in the tumor smear by both oral administration and tail vein injection. The number of bacteria enriched in the tumor by tail vein injection is much higher than that in the gastric gavage group, indicating that the gastric Clostridium of the present invention can enter the blood circulation system from the gastrointestinal tract ectopically to reach the tumor site through the in vivo pathway, and has a certain tropism for the tumor site.

[0124] Effect Example 3

[0125] In order to verify the efficacy and safety of Clostridium gastricum in treating tumors, animal experiments were conducted using melanoma as a representative tumor, as follows:

[0126] 1. Experimental design.

[0127] Male C57BL / 6 mice aged 4-6 weeks were used as experimental subjects and were divided into two groups, with 5 mice in each group. The specific procedures are shown in Figure 7A and Table 4. The body weight of the mice was observed regularly after tumor implantation. The mice were euthanized 30 days after tumor implantation, and tumor tissues and major organ tissues were removed. The lung and spleen tissues were weighed, and major organs such as the heart, liver, spleen, and kidney were sectioned and stained with HE. The results are shown in Figure 6, Figure 7B, and Figure 7C.

[0128] Table 4 Treatment of mice in each group

[0129] 2. Experimental results.

[0130] As shown in Figures 6 and 7B, lung metastasis was significantly reduced in mice treated with C. gastricis compared to the control group. While the control mice developed melanomas in their lungs, the treated mice had fewer melanomas and significantly lower lung weights than the control group, demonstrating that C. gastricis of the present invention is effective in preventing tumor metastasis. As shown in Figure 7C, HE sections of major organs showed no organic changes, demonstrating the good safety of C. gastricis alone.

[0131] Effect Example 4

[0132] To further verify the efficacy of oral live Clostridium gastricis combined with immune checkpoint inhibitors in treating tumors, an anti-tumor experiment was conducted on gastric cancer cell MFC mice using PD-1 inhibitors as a representative of immune checkpoint inhibitors combined with Clostridium gastricis. The specific protocol is as follows:

[0133] 1. Male 615 mice aged 4-6 weeks were used as experimental subjects and divided into four groups, with 5 mice in each group. The specific operation and process are shown in Figure 8A and Table 5.

[0134] Table 5 Treatment of mice in each group

[0135] The tumor volumes were recorded on days 11, 13, 15, 17, 12, and 19 after tumor implantation. The results are shown in FIG8B .

[0136] As shown in Figure 8B, compared with the PD-1 inhibitor and oral administration of Clostridium sysutum alone, the tumor volume of the combined Clostridium sysutum and PD-1 inhibitor group grew more slowly, indicating that under oral administration, Clostridium sysutum SYSU-12 has a certain enhancing effect on the anti-tumor effect of PD-1 inhibitors.

[0137] Effect Example 5

[0138] To verify whether oral administration of inactivated Clostridium gastricis has the effect of treating tumor metastasis, animal experiments were conducted using CT26 colorectal cancer cells as a representative tumor. The specific protocol is as follows:

[0139] 1. Male Balb / c mice aged 3-5 weeks were used as experimental subjects and divided into two groups, with 5 mice in each group. The specific operation and process are shown in Figure 9A and Table 6.

[0140] Table 6 Treatment of mice in each group

[0141] The tumor volumes were recorded on days 6, 9, 12, 15, 18, 21, and 24 after tumor implantation. The results are shown in FIG9B .

[0142] As shown in Figure 9B, the tumor volume of the oral inactivated Clostridium gastricis group grew more slowly than that of the control group, indicating that the inactivated Clostridium gastricis SYSU-12 also has certain anti-tumor activity under oral administration.

[0143] Effect Example 6

[0144] To prove that Clostridium gastricis SYSU-12 has the effect of activating immunity, SYSU-12 was co-cultured with mouse macrophages (RAW264.7) and the supernatant was taken to measure the content of CXCL10.

[0145] The specific operations are as follows:

[0146] (1) Macrophages were co-cultured with live or inactivated Clostridium gastricis at an MOI (multiplicity of infection) of 1:100 for 24 h, and the cells and supernatant were collected for subsequent operations;

[0147] (2) After extracting cell RNA and reverse transcription, RT-qPCR was performed to detect the expression level of CXCL10 or ELISA content in the supernatant was measured using a CXCL10 ELISA kit.

[0148] (3) CXCL10 expression level RT-qPCR results are shown in Figure 10A, and ELISA content detection results are shown in Figure 10B.

[0149] As shown in Figure 10, after a period of culture, both live and dead Clostridium gastricis SYSU-12 bacteria significantly increased macrophage CXCL10 expression. This suggests that Clostridium gastricis can activate key cytokines that promote the conversion of "cold" tumors to hot tumors. Since both live and dead Clostridium gastricis bacteria have this effect, it suggests that the active ingredient is a component of the Clostridium gastricis bacteria.

[0150] Effect Example 7

[0151] To prove that Clostridium gastricis SYSU-12 has the effect of increasing immune infiltration of tumor tissue and converting "cold" tumors into hot tumors, taking colorectal cancer cells (MC38) and gastric cancer cells (AGS) as examples, the cancer cells were co-cultured with Clostridium gastricis and the supernatant was taken to act on primary CD8+T cells extracted from human peripheral blood. The Transwell experiment was used to observe whether the tumor tropism of CD8+T cells was increased.

[0152] The experimental principle diagram is shown in Figure 11. The specific operations are as follows:

[0153] (1) Cancer cells and bacteria were co-cultured at an MOI (multiplicity of infection) of 1:100 for 24 h, and the supernatant was collected for subsequent operations;

[0154] (2) CD8+ T cells were isolated from human peripheral blood by magnetic beads;

[0155] (3) Transwell experiment: The cell density was adjusted to 3×10 5 500 μL of control medium and bacterial co-culture medium were added to the lower wells, and 1640 medium containing 0.3% v / v FBS and 6×10 7 cell / mL single cell suspension and culture for 6-8 hours.

[0156] (4) Cell counting: Take photos of the cells in the lower chamber under a fluorescence microscope. Take 9 fields of view in each well and count them. The statistical results are shown in Figure 12.

[0157] As shown in Figure 12, after a period of culture, some cells migrated through the Transwell chamber into the lower chamber, and these cells were observed under a microscope. In both experiments, the number of cells in the lower chamber of the co-culture group with Clostridium gastricis was higher than that of the control group, indicating that Clostridium gastricis can enhance the infiltration of CD8+ T cells into tumor tissues. This indicates that Clostridium gastricis SYSU-12 has the ability to increase immune infiltration of tumor tissues and convert "cold" tumors into hot tumors.

[0158] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A Clostridium ventriculi, characterized in that, The Clostridium ventriculi is Clostridium ventriculi SYSU-12, which is deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, with the deposit number of CGMCC No. 27761 and the deposit date of June 30, 2023.

2. The Clostridium ventriculi according to claim 1, wherein, The 16S rRNA sequence of the Clostridium ventriculi SYSU-12 is as shown in SEQ ID NO.

1.

3. The Clostridium ventriculi according to claim 1, characterized in that, The Clostridium ventriculi SYSU-12 is isolated from the feces of a gastric cancer patient responsive to PD-1 antibody treatment.

4. A Clostridium pylori culture, characterized in that, It is prepared from the Clostridium ventriculi SYSU-12 according to any one of claims 1-3.

5. The Clostridium pylori culture according to claim 4, characterized in that, The Clostridium ventriculi culture contains at least one of viable Clostridium ventriculi bacteria, bacterial components and their derivatives, and Clostridium ventriculi metabolites.

6. The Clostridium pylori culture according to any one of claims 4-5, characterized in that, The Clostridium ventriculi culture is mainly prepared by at least one of the following methods: Method 1: Scraping the activated Clostridium ventriculi SYSU-12 cells into a sterile solvent to prepare a Clostridium ventriculi SYSU-12 cell suspension, and the Clostridium ventriculi SYSU-12 cell suspension is the Clostridium ventriculi culture; Method 2: Scraping the activated single colony of Clostridium ventriculi SYSU-12 and inoculating it into a culture medium, culturing, centrifuging, and the obtained supernatant is the Clostridium ventriculi culture; Method 3: Scraping the activated single colony of Clostridium ventriculi SYSU-12 and inoculating it into a culture medium, culturing, then transferring it to a bioreactor for batch fermentation, and the obtained fermentation broth is the Clostridium ventriculi culture; Method 4: Scraping the activated single colony of Clostridium ventriculi SYSU-12 and inoculating it into a culture medium, culturing, centrifuging, resuspending the obtained precipitate in a solvent, and sterilizing at 115-130 °C for 15-30 min, and the obtained inactivated cell suspension is the Clostridium ventriculi culture.

7. Use of the Clostridium ventriculi according to any one of claims 1-3 or the Clostridium ventriculi culture according to claim 4 in the preparation of a tumor prevention and / or treatment product.

8. Use of the Clostridium ventriculi according to any one of claims 1-3 or the Clostridium ventriculi culture according to claim 4 in combination with an immune checkpoint inhibitor in the preparation of an anti-tumor drug.

9. Use of the Clostridium ventriculi according to any one of claims 1-3 or the Clostridium ventriculi culture according to claim 4 in the preparation of an immune stimulant.

10. A Clostridium ventriculi preparation, characterized in that, The Clostridium ventriculi preparation comprises the Clostridium ventriculi according to any one of claims 1-3 or the Clostridium ventriculi culture according to claim 4.

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