Clostridium butyricum genetically engineered bacterium BHC008-1 for secreting and expressing human vascular endothelial cell growth inhibition factor and application of clostridium butyricum genetically engineered bacterium BHC008-1

Abstract

The invention belongs to the field of biological medicines, and relates to a clostridium butyricum genetically engineered bacterium BHC008-1 for secreting and expressing a human vascular endothelial cell growth inhibition factor and application of the clostridium butyricum genetically engineered bacterium BHC008-1. The invention firstly provides a clostridium butyricum BHD05 capable of preventing and treating colorectal cancer, and the preservation number of the clostridium butyricum BHD05 is CGMCC No. 32029; the invention further provides a clostridium butyricum genetically engineered bacterium BHC008-1, the preservation number of the clostridium butyricum genetically engineered bacterium BHC008-1 is CGMCC No.31490, the engineered bacterium can realize lactose-induced secretory expression of the human vascular endothelial growth inhibition factor VEGI192 under the anaerobic condition at the temperature of 36 DEG C in the colorectal intestinal tract of an organism, and the effects of resisting colorectal cancer and regulating colorectal micro-ecology of the clostridium butyricum are exerted at the same time. The clostridium butyricum engineering bacterium BHC008-1 is applied to colorectal cancer Balb / c mice, treatment is obvious, and a foundation is laid for application of the clostridium butyricum engineering bacterium BHC008-1 to colorectal cancer treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to the genetically engineered Clostridium butyricum strain BHC008-1 that secretes and expresses human vascular endothelial cell growth inhibitory factor and its applications. Background Technology

[0002] Colorectal cancer (CRC) is a common gastrointestinal malignancy with high incidence and mortality rates worldwide. As a crucial component of the gut microbiota, gut microbiota plays a vital role in protecting the intestinal mucosa, maintaining intestinal homeostasis, and even regulating normal bodily functions. Clostridium butyricum is a probiotic that can regulate intestinal dysfunction; studies have also found that it has some therapeutic effects on related inflammation and cancer.

[0003] Clostridium butyricum (CB), also known as butyric acid bacteria, belongs to the genus Clostridium of the family Bacillusceae. It is a strictly anaerobic, Gram-positive spore-forming bacillus. Clostridium butyricum has the following effects: (1) enhancing intestinal barrier function; (2) regulating intestinal flora balance; (3) regulating immunity; (4) assisting host digestion and absorption; and (5) anti-cancer / tumor effects. Currently, probiotics for the prevention and treatment of tumors have become a novel treatment method.

[0004] Studies on tumor development have revealed that angiogenesis is a crucial step in tumor invasion and metastasis; therefore, inhibiting tumor angiogenesis is of great significance in preventing tumor growth and metastasis. Vascular endothelial growth inhibitory factor (VEGI) is an autocrine factor produced by endothelial cells and a member of the tumor necrosis factor (TNF) superfamily. Related studies have shown that VEGI has an inhibitory effect on endothelial cell growth and angiogenesis. Summary of the Invention

[0005] This invention first provides a Clostridium butyricum strain BHD05 for the prevention and treatment of colorectal cancer. Then, based on this strain, an engineered Clostridium butyricum strain capable of temperature-controlled induction of VEGI192 expression was constructed, exploring a new approach to improve the anti-cancer medicinal value of Clostridium butyricum.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a strain of Clostridium butyricum BHD05 for the prevention and treatment of colorectal cancer, the preservation number of which is CGMCC No.32029.

[0008] This invention identified the screened strain BHD05 using Gram staining. The results showed that BHD05 cells appeared blue-purple, indicating it is a Gram-positive bacterium. Malachite green staining showed that BHD05 spores were green and cells were red. 16S rDNA sequencing revealed that the 16S rDNA sequence of BHD05 is shown in SEQ ID NO:1. Based on the morphological characteristics, Gram staining, spore staining, and molecular identification results of strain BHD05, it was identified as *Clostridium butyricum*, named *C. butyricum* BHD05, and deposited with accession number CGMCC No. 32029.

[0009] Secondly, this invention provides a genetically engineered *Clostridium butyricum* strain, BHC008-1, which is an engineered strain formed by transforming *Clostridium butyricum* BHD05 with an expression plasmid containing TlpA36, LacI, *Clostridium butyricum* amylase signal peptide SP, and VEGI192 (TlpA36-LacI-SP-VEGI192) linked sequentially. The nucleotide sequence of the VEGI192 gene is shown in SEQ ID NO:2, the nucleotide sequence of TlpA36 is shown in SEQ ID NO:3, the nucleotide sequence of LacI is shown in SEQ ID NO:4, and the nucleotide sequence of the *Clostridium butyricum* amylase signal peptide SP gene is shown in SEQ ID NO:5. The accession number of this engineered strain, BHC008-1, is CGMCC No. 31490.

[0010] In the colon, this engineered bacterium expressed the exogenous gene VEGI192 through temperature-controlled lactose induction. Specifically, under environmental temperatures above 36°C, the repression effect of the temperature-controlled promoter pTlpA was released, allowing pLacI, downstream of pTlpA, to normally exert its expression regulatory role. Furthermore, under the induction of the lactose structural analogue isopropyl mercaptogalactoside (IPTG), pLacI regulated the smooth expression of the downstream structural gene VEGI192. In summary, the successful expression of the exogenous gene VEGI192 by the engineered Clostridium butyricum bacterium BHC008-1 requires the simultaneous fulfillment of both environmental conditions above 36°C and the presence of lactose or its structural analogue. The dual regulatory effect of environmental conditions above 36°C and lactose or IPTG ensures that the engineered bacterium blocks the expression of the exogenous gene in vitro, improving the stability of the engineered bacterium, and ensuring the induction and secretion of the exogenous gene VEGI192 in vivo, thereby exerting its anti-tumor angiogenesis effect.

[0011] The preparation method of the above-mentioned Clostridium butyricum engineered strain BHC008-1 is as follows: the coding genes of TlpA36, LacI, Clostridium butyricum amylase signal peptide SP, and VEGI192 (TlpA36-LacI-SP-VEGI192) linked sequentially are inserted into the multiple cloning site downstream of the LacZ promoter of the Escherichia coli-Clostaphylococcus shuttle expression vector pMTL82151 to obtain the temperature-controlled induced secretion expression recombinant vector pMTA36-VEGI192. Then, the recombinant vector pMTA36-VEGI192 is transformed into Clostridium butyricum BHD05 to obtain the temperature-controlled induced secretion expression of VEGI192 Clostridium butyricum engineered strain BHC008-1.

[0012] Furthermore, downstream of the VEGI 192 gene is a T7 terminator, the nucleotide sequence of which is shown in SEQ ID NO:6.

[0013] Thirdly, the present invention also provides a microbial inoculant, wherein the microbial inoculant contains the following components:

[0014] (1) Clostridium butyricum BHD05, and / or,

[0015] (2) The genetically engineered butyric acid clostridium BHC008-1 described above.

[0016] Furthermore, the microbial agent contains the following components:

[0017] (1) the fermentation broth, bacterial suspension, or dried bacterial powder of Clostridium butyricum BHD05, and / or,

[0018] (2) The fermentation broth, bacterial suspension or dried bacterial powder of the genetically engineered butyric acid clostridium BHC008-1.

[0019] The preparation method of Clostridium butyricum fermentation broth is as follows:

[0020] Select one slant of Clostridium butyricum and inoculate it into a pre-sterilized stoppered container containing LB medium. Shake well, stopper the container, and place it in an anaerobic incubator at 37–39°C for 36 hours.

[0021] The method for preparing Clostridium butyricum suspension is as follows:

[0022] Take a freshly streaked slant tube of Clostridium butyricum, aseptically pick a single colony from the slant, transfer it to fresh BHI medium, and anaerobic culture at 37℃ for 24 h to obtain a suspension of Clostridium butyricum in the logarithmic phase.

[0023] Furthermore, the dried butyric acid bacteria powder is a lyophilized butyric acid bacteria powder, and the preparation method of the lyophilized butyric acid bacteria powder is as follows:

[0024] Pour the butyric acid bacteria fermentation broth into a clean, sterile, uncovered container and place it in a freeze dryer. Set the temperature of the drying chamber and condensing chamber to -40°C, turn on the vacuum pump to evacuate the pressure to 0.1 mmHg, and the product will be ready after 5–8 hours.

[0025] Fourthly, the present invention provides the use of one or more of the following components in the preparation of medicaments for the prevention and / or treatment of colorectal cancer:

[0026] (1) Clostridium butyricum BHD05;

[0027] (2) The fermentation broth, bacterial suspension or dried bacterial powder of Clostridium butyricum BHD05;

[0028] (3) The engineered strain of Clostridium butyricum BHC008-1 described above;

[0029] (4) The fermentation broth, bacterial suspension or dried bacterial powder of the genetically engineered butyric bacterium BHC008-1 described above.

[0030] (5) The microbial agent mentioned above.

[0031] The applications of *Clostridium butyricum* BHD05, or the fermentation broth, suspension, or dried powder of *Clostridium butyricum* BHD05, are as follows:

[0032] Step 1: Construct a Balb / c mouse model of colorectal cancer;

[0033] Step 2: After the model was constructed, the mice were administered the drugs by gavage. The groups were as follows: (1) Blank control group: healthy mice were treated with PBS solution by gavage; (2) Negative control group: model mice were treated with PBS solution by gavage; (3) Clostridium butyricum BHD02, BHD04, BHD05 and BHH07 treatment groups: bacteria were resuspended in PBS by gavage. During the treatment, the mortality, weight change and health index of the experimental mice were recorded.

[0034] Step 3: After treatment, the mice were anesthetized and euthanized. The colorectal region was harvested and the length of the colorectal region was measured. Tumor tissue was harvested and the volume and weight of the tumor were measured to determine the therapeutic effect of BHD05 on the mouse model of colorectal cancer.

[0035] The results showed that the tumor inhibition rate of mice treated with Clostridium butyricum BHD05 reached 90%, demonstrating a good inhibitory effect on the formation of colorectal cancer. Based on all indicators, we believe that BHD05 is a Clostridium butyricum strain with therapeutic effects against colorectal cancer in mice. Therefore, Clostridium butyricum BHD05 can be used to prepare drugs for the prevention and / or treatment of colorectal cancer.

[0036] The applications of the aforementioned *Clostridium butyricum* engineered strain BHC008-1, or the fermentation broth, bacterial suspension, or dried bacterial powder of the aforementioned *Clostridium butyricum* genetically engineered strain BHC008-1, are as follows:

[0037] Step 1: Construct a Balb / c mouse model of colorectal cancer.

[0038] Step 2: After the model was constructed, the mice were administered the drug via gavage. The groups were as follows: (1) Negative control group: treated with PBS solution by gavage; (2) Blank control: treated with PBS solution containing the blank control (BHD05(pMTA36)) bacteria without the target gene by gavage; (3) Clostridium butyricum engineered strain BHC008-1 treatment group: treated with PBS solution containing Clostridium butyricum engineered strain BHC008-1 bacteria by gavage; (4) Positive control: administered capecitabine by body surface area. During the treatment, the mortality, weight changes, and health index of the experimental mice were recorded.

[0039] Step 3: After treatment, blood was collected to measure serum biochemical indicators and serum inflammatory factor IL-6 levels. Mice were anesthetized and euthanized, and the colorectal region was harvested to measure colorectal length; tumor tissue was harvested to measure tumor volume and weight; spleen and thymus were harvested to measure immune organ indices; tumor and colon tissue sections were prepared, H&E stained, and histopathological changes in tissues and tumor tissues were observed; Ki-67 and CD31 immunohistochemical detection of tumor tissue was performed to determine the therapeutic effect of Clostridium butyricum engineered strain BHC008-1 on the mouse model of colorectal cancer.

[0040] The results showed that the supernatant of the fermentation broth of the engineered Clostridium butyricum strain BHC008-1 constructed in this invention could inhibit the proliferation of human umbilical vein endothelial cells, and this inhibition was time- and dose-dependent within a certain range. Therefore, the engineered Clostridium butyricum strain BHC008-1 can be used to prepare drugs for the prevention and / or treatment of colorectal cancer.

[0041] In a fifth aspect, the present invention provides a medicament for the prevention and / or treatment of colorectal cancer, said medicament comprising one or more of the following active ingredients:

[0042] (1) Clostridium butyricum BHD05;

[0043] (2) The fermentation broth, bacterial suspension or dried bacterial powder of Clostridium butyricum BHD05;

[0044] (3) The engineered strain of Clostridium butyricum BHC008-1 described above;

[0045] (4) The fermentation broth, bacterial suspension or dried bacterial powder of the genetically engineered butyric bacterium BHC008-1 described above.

[0046] (5) The microbial agent mentioned above.

[0047] The beneficial effects of this invention are as follows:

[0048] (1) The Clostridium butyricum BHD05 screened in this invention is a Clostridium butyricum strain that has a therapeutic effect on colorectal cancer mice. As a probiotic, it can be used to prepare for the prevention and treatment of colorectal cancer.

[0049] (2) Based on the anti-angiogenic strategy for tumor treatment and the immunomodulatory characteristics of the intestinal probiotic Clostridium butyricum, this invention designs and constructs a recombinant engineered strain expressing the vascular endothelial cell growth inhibitor VEGI 192 using Clostridium butyricum as the host cell, and studies its effects on endothelial cells and its therapeutic efficacy on in situ tumor models. Preliminary experiments demonstrate that using the engineered Clostridium butyricum as an anti-tumor drug has a synergistic therapeutic effect on intestinal tumor models. Furthermore, as an anti-colorectal cancer drug, the engineered Clostridium butyricum can not only treat cancer but also prevent it, with almost no toxic side effects like those of radiotherapy and chemotherapy. We anticipate that this drug, when used in clinical cancer treatment, will have high patient compliance, good prognosis, and significantly reduced treatment costs, undoubtedly making it popular among the general public. Attached Figure Description

[0050] Figure 1 This is the Gram staining microscopic examination result of Clostridium butyricum BHD05 in Example 1 of this invention;

[0051] Figure 2 This is the result of microscopic examination of Clostridium butyricum BHD05 spores in Example 1 of this invention;

[0052] Figure 3 This is a phylogenetic tree of the 16S rDNA sequence of Clostridium butyricum BHD05 in Example 1 of the present invention;

[0053] Figure 4 This refers to the weight and health status of a mouse model of colorectal tumor in Example 3 of this invention after treatment with Clostridium butyricum BHD05. A: Weight change; B: Health index;

[0054] Figure 5 Photographs of in situ tumors in mouse models of colorectal tumors after treatment with Clostridium butyricum BHD05.

[0055] Figure 6 This refers to the length of the colon in the mouse model after dissection following treatment with Clostridium butyricum BHD05 in Example 3 of this invention. *** indicates p<0.001, ** indicates p<0.01, and * indicates p<0.05;

[0056] Figure 7 These are the in situ tumor volumes and weights of mouse colorectal cancer models treated with Clostridium butyricum BHD05 in Example 3 of this invention. A: Average in situ tumor volume in the model mice; B: Average in situ tumor weight; C: Optical photograph of the in situ tumor tissue;

[0057] Figure 8 This is a flowchart illustrating the construction process of the recombinant plasmid pUC19-TlpA36-SP in Example 4 of this invention.

[0058] Figure 9 This is a flowchart of the construction process of the recombinant plasmid pUC19-TlpA36-SP-VEGI-T7 terminator in Example 4 of the present invention;

[0059] Figure 10 This is a flowchart illustrating the construction process of the recombinant plasmid pMTA36-VEGI192 in Example 4 of this invention;

[0060] Figure 11 This is an example of SDS-PAGE analysis of VEGI192 secretion by Clostridium butyricum (pMTA36-VEGI192) in Example 5 of the present invention. M: Pre-stained Protein Marker; Lane 1: Blank plasmid control group; Lane 2: 2mM IPTG, induced for 48h; Lane 3: 1mM IPTG, induced for 48h; Lane 4: 0mM IPTG, induced for 48h; Lane 5: 2mM IPTG, induced for 24h; Lane 6: 1mM IPTG, induced for 24h; Lane 7: 0mM IPTG, induced for 24h.

[0061] Figure 12 This refers to the immunoblotting identification of recombinant VEGI192 in Example 5 of the present invention;

[0062] Figure 13 This is the CCK-8 experimental design diagram in Example 6 of the present invention. 20%, 50%, and 70% represent the three concentrations in each group.

[0063] Figure 14 The growth inhibition rate of HUVECs cells by the fermentation supernatant of engineered bacteria BHC008-1 in Example 6 of this invention;

[0064] Figure 15 This describes the therapeutic effect of engineered bacteria BHC008-1 powder on tumor model mice in Example 7 of this invention. A: Health index of the model mice; B: Abdominal image of the model mice; C: Photograph of in situ colonic tumor in the model mice;

[0065] Figure 16 These are the volume and weight of the engineered bacteria BHC008-1 used in Example 7 of this invention to treat orthotopic tumors in mice. A: Optical photograph of the tumor; B: Volume of the orthotopic tumor; C: Weight of the orthotopic tumor;

[0066] Figure 17These are the thymus index and spleen index of mice treated with engineered bacteria BHC008-1 in Example 7 of this invention. A: Thymus index of mice in each treatment group; B: Spleen index of mice in each treatment group; *** indicates p<0.001;

[0067] Figure 18 This refers to the serum IL-6 content of mice treated with engineered bacteria BHC008-1 in Example 7 of this invention. **** indicates p<0.0001; *** indicates p<0.001;

[0068] Figure 19 This is a graph showing the levels of three important liver function indicators in mice treated with engineered bacteria BHC008-1 in Example 7 of this invention. A: ALP represents alkaline phosphatase; B: ALT represents alanine aminotransferase; C: AST represents aspartate aminotransferase.

[0069] Figure 20 This is a graph showing the levels of two important renal function indicators in mice treated with engineered bacteria BHC008-1 in Example 4 of this invention. A: BUN represents blood urea nitrogen; B: CREA represents creatinine;

[0070] Figure 21 This is an H&E section image of mouse tumor tissue treated with engineered bacteria BHC008-1 in Example 7 of this invention.

[0071] Figure 22 These are immunohistochemical results of Ki67 and CD31 in mouse tumor tissue treated with engineered bacteria BHC008-1 in Example 7 of this invention. A: Immunohistochemical results of Ki67 in tumor tissue; B: Immunohistochemical results of CD31 in tumor tissue;

[0072] Figure 23 This is an H&E section image of the colon in Example 7 of this invention. The black arrows indicate tumor cells within the intestinal wall; the white arrows indicate thickening of the colonic wall.

[0073] Preservation information:

[0074] Preserved biological material: Clostridium butyricum BHD05

[0075] Accession number: CGMCC No. 32029;

[0076] Classification and nomenclature: Clostridium butyricum;

[0077] Preservation institution: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Culture Collections;

[0078] Address of the depository: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing;

[0079] Date of preservation: September 20, 2024.

[0080] Preserved biological material: Clostridium butyricum genetically engineered strain BHC008-1

[0081] Accession number: CGMCC No. 31490;

[0082] Classification and nomenclature: Clostridium butyricum;

[0083] Preservation institution: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Culture Collections;

[0084] Address of the depository: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing;

[0085] Preservation date: July 29, 2024. Detailed Implementation

[0086] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of the embodiments of this invention will be clearly described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0087] Example 1: Identification of Clostridium butyricum

[0088] BHD05 strain was isolated from healthy human subjects and then optimized and rejuvenated. The following identification procedures were then performed:

[0089] 1. Gram staining microscopy

[0090] The microscopic examination results are shown below. Figure 1 When observed under an oil lens microscope, the BHD05 bacteria appeared blue-purple, indicating that it is a Gram-positive bacterium.

[0091] 2. Spore staining and microscopic examination

[0092] The microscopic examination results are shown below. Figure 2 When observed under an oil immersion microscope, the BHD05 spores appeared green, while the bacterial cells were red.

[0093] 3. Molecular biological identification of strain BHD05

[0094] (1) PCR amplification of 16S rRNA gene

[0095] Total genomic DNA was extracted using the Fast Pure Microbiome DNA Isolation Kit (Nanjing Novozymes Biotechnology Co., Ltd.). 16S rRNA gene-specific primers (upstream primer 27F) were used.

[0096] PCR amplification was performed using primer 1492R (5'-AAGTTTTGATCCTGCTCA-3') and downstream primer 1492R (5'-GGTACCTTGTTACGACTT-3') to identify the species of strain BHD05.

[0097] PCR amplification was performed using the 2×Es Taq Master Mix (Dye) kit (Kangwei Century Biotechnology Co., Ltd., Beijing). A 25 μL PCR amplification system was used, and the system was prepared as follows: 12.5 μL of 2×Es Taq Master Mix (Dye), 1 μL each of forward and reverse primers, 1 μL of Template DNA, and 9.5 μL of ddH2O.

[0098] The PCR reaction conditions were set as follows: First, pre-denaturation was performed at 94℃ for 2 minutes, followed by a cycling phase. Each cycle consisted of 30 seconds of denaturation at 94℃, 30 seconds of annealing at 60℃, and 30 seconds of extension at 72℃, for a total of 35 cycles. Finally, a final extension at 72℃ for 2 minutes was performed to complete the reaction. The amplified products were detected by 1.5% agarose gel electrophoresis and then sent to BGI Genomics Co., Ltd. for sequencing. The 16S rDNA sequence of BHD05 is shown in SEQ ID NO:1.

[0099] (2) Construction of phylogenetic tree

[0100] The 16S rRNA sequence of strain BHD05 was compared with known sequences in the NCBI database using nucleotide BLAST. Specifically, the rRNA / ITS database BLAST was used, and similar sequences were downloaded for phylogenetic analysis in MEGA11. The phylogenetic tree was inferred using the Neighbor-Joining method, and the optimal tree was displayed. The percentage of duplicate trees where related taxa clustered together in the bootstraptest is shown below the branches. The evolutionary distance was calculated using the maximum composite likelihood method.

[0101] Phylogenetic tree based on 16S rRNA sequence analysis Figure 3 It can be seen that strains BHD05 and C...

[0102] Butyricum ATCC19398 belongs to the same branch, with a confidence level of 98%, indicating the highest homology. Based on the morphological characteristics, Gram staining and spore staining results, and molecular identification results of strain BHD05, strain BHD05 was identified as Clostridium butyricum and named C. butyricum BHD05.

[0103] Example 2: Cultivation of Clostridium butyricum

[0104] Preparation of Clostridium butyricum suspension:

[0105] Take a freshly streaked slant tube of Clostridium butyricum, wipe its outer surface with a 75% alcohol swab, and place it in a sterile operating table. Near an alcohol lamp flame, use a sterile inoculation loop to pick up a single colony from the slant, transfer it to fresh BHI medium, and anaerobic incubate at 37°C for 24 hours to obtain a suspension of Clostridium butyricum in the logarithmic growth phase.

[0106] Preparation of butyric acid lyophilized powder:

[0107] (1) Preparation of Clostridium butyricum fermentation broth: Select one slant culture of Clostridium butyricum, inoculate it with Platinum Ear fungus into a pre-sterilized 1000ml Erlenmeyer flask containing LB medium, shake well, stopper and place in an anaerobic incubator, incubate at 37-39℃ for 36h, and the broth is obtained. (2) Preparation of Clostridium butyricum lyophilized powder: Pour 500ml of butyric acid bacteria fermentation broth into a clean and sterile enamel dish and place it in a small freeze dryer. Lower the temperature of the drying chamber and condensing chamber to -40℃, turn on the vacuum pump to evacuate the pressure to 0.1mmHg, and the broth is obtained after 5-8 hours.

[0108] Example 3: Anti-colorectal cancer activity of Clostridium butyricum

[0109] The test sample was a suspension of Clostridium butyricum.

[0110] Animals: SPF-grade Balb / c mice were purchased from the Experimental Animal Center of Zhengzhou University and were routinely housed in the animal laboratory. Mice were given feed and drinking water and allowed to acclimatize to the environment for one week prior to the experiment.

[0111] 1. Culture of CT26-LUC tumor cells

[0112] Tumor cells CT26-LUC (purchased from ATCC) were cultured in DMEM containing 1% penicillin and 10% premium fetal bovine serum in a cell culture incubator until they reached confluent levels. The culture conditions were set at 37°C and 5% CO2.

[0113] 2. Establishment of a subcutaneous CT26-LUC tumor model in Balb / c mice

[0114] Step 1: Prepare tumor cells. Collect CT26-LUC cells in the logarithmic growth phase (generally passage 3-5) using 0.25% trypsin to obtain a single-cell suspension. Centrifuge at 1000 rpm for 3 min, discard the supernatant, and carefully resuspend the cells in 1 mL of DMEM complete medium (containing 1% penicillin and streptomycin and 10% premium fetal bovine serum) while avoiding air bubbles. Take 2 μL of the cell suspension, dilute to 1 mL with PBS, mix thoroughly by pipetting, and then take 20 μL of the cell suspension for hemocytometer counting. Based on the counting results, dilute the tumor cell concentration to 3-5 × 10⁻⁵ cells by adding a certain volume of PBS. 7 The concentration should be around 10 cells / ml. After dilution, place the solution on ice immediately to maintain cell viability.

[0115] Step 2: Subcutaneous Injection. Determine the injection site in advance. Disinfect the skin at the injection site on the mouse's back with an alcohol swab. Use a 1 mL sterile insulin syringe to draw 100 μL of single-cell suspension and inject subcutaneously. To ensure a sufficient number of subcutaneous tumors, inoculate two subcutaneous tumor sites on each mouse for later use.

[0116] 3. Constructing a mouse model of orthotopic colon tumor

[0117] Step 1: Prepare the tumor mass. When the tumor volume for subcutaneous transplantation with CT26-LUC is approximately 100mm... 3 Mice with subcutaneous tumors were euthanized by cervical dislocation. The skin at the tumor site was disinfected with alcohol, and the mice were fixed on a sterile board in a laminar flow hood. The subcutaneous tumor was carefully removed using sterile ophthalmic surgical scissors, fresh tumor tissue was separated, and cut into 1mm pieces. 3 Place the tumor, approximately the size of a lumps, in a disposable sterile dish containing 0.9% NaCl and keep it on ice. Use within 3 hours.

[0118] Step 2: In situ tumor implantation. Before constructing the in situ model, mice were grouped as follows: Mice were weighed and those within the same weight range were placed in one cage. Then, mice within each weight range were randomly assigned to different experimental groups. After grouping, the right abdomen of the mice was immediately shaved. The mice were fasted for 12 hours and then anesthetized briefly by intraperitoneal injection. After successful anesthesia, the mice were fixed supine on a sterile operating table. After routine skin disinfection, a 1cm incision was made in the lower right abdomen. The skin was gently separated from the peritoneum. The peritoneum was lifted with ophthalmic forceps and cut open with ophthalmic scissors. The colon was pulled out of the body, and a 1mm tumor seed was implanted using the tip of a 1mL sterile syringe. 3The tumor tissue from both sides was inserted into the mesentery adjacent to the colon wall. The injection site was gently scraped with a syringe needle to ensure the tumor mass was firmly fixed. The mouse's colon was carefully returned to the abdominal cavity, and the peritoneum and skin were sutured layer by layer with absorbable surgical sutures. The surgical site was then disinfected with povidone-iodine. After surgery, the model mouse was placed on gauze and observed until it was fully awake. It was then fed and watered normally.

[0119] 4. Treatment regimen for mouse orthotopic tumor models

[0120] BHD02, BHD04, and BHD07 were isolated from healthy human bodies, kimchi, and mammalian feces, respectively, and identified as Clostridium butyricum.

[0121] On the third day after the model was established, the mice were administered medication by gavage. The specific dosages were as follows: ① Blank control group (healthy mice) and negative control group (subcutaneous CT26-LUC tumor model mice): Each mouse was administered 200 μL of PBS solution by gavage once daily; ② Clostridium butyricum BHD02, BHD04, BHD05, and BHH07 treatment groups: After counting the cultured bacteria, an appropriate amount was centrifuged at 8000 rpm, the bacterial pellet was resuspended in PBS, centrifuged again, and washed twice. Finally, the bacterial pellet was resuspended in PBS. Each mouse was administered Clostridium butyricum suspension by gavage once daily, with a volume of 200 μL. The total bacterial count of Clostridium butyricum was 2 × 10⁻⁶. 7 CFU. The animal experiment concluded after a total of 14 days of treatment.

[0122] 5. Monitoring of mouse weight and health status, and tumor tissue condition after treatment.

[0123] During treatment, the survival, weight, and activity level of each mouse were observed and recorded daily. Mice were scored based on their health status, with scores ranging from 0 to 5, representing different health conditions, with higher scores indicating better condition. Specific criteria were as follows: 5 points indicated the mouse was basically healthy; 4 points indicated disheveled fur and decreased activity; 3 points indicated increased respiratory rate; 2 points indicated the mouse was depressed and inactive; 1 point indicated the mouse was near death and had essentially lost its ability to move; 0 points indicated death.

[0124] The results showed that during the 14-day treatment period, the mice in the blank control group remained healthy (health index = 5), had normal food intake, and experienced a slight increase in body weight. In contrast, the mice in the negative control group showed a continuous decline in health and body weight due to the influence of intestinal tumors. Compared to the negative control group, the mice in the Clostridium butyricum treatment group showed improvements in both health and body weight. (See attached results). Figure 4 .

[0125] During treatment, a hard mass and significant swelling were palpable in the abdomen of mice in the negative control group, indicating successful establishment of the in situ tumor model. After treatment, the survival rate of mice in all groups was 100% (n=8). Mice were euthanized by cervical dislocation, and their abdominal cavities were dissected. Images of the dissected intestines showed that the tumors in the model mice were mainly concentrated in the colon. Figure 5 No metastatic tumors were found in other parts of the intestine. The tumor inhibition rate among the treatment groups was calculated based on the number of orthotopic tumors in each group (as shown in Table 1). The results show that in the Clostridium butyricum treatment group, the tumor inhibition rate of the Clostridium butyricum BHD05 treatment group reached 90%, demonstrating a good inhibitory effect on the formation of colorectal cancer.

[0126] Table 1. Tumor inhibition rate of different Clostridium butyricum strains

[0127] BHD05 Treatment Group BHD04 Treatment Group BHD02 Treatment Group BHH07 Treatment Group negative control group Tumor inhibition rate (%) 90 37.5 50 12.5 0

[0128] After treatment, mice were euthanized by cervical dislocation, and a portion of the intestine from the cecum to the anus was removed and photographed to document the in situ tumor model. The in situ tumor tissue was then completely excised using ophthalmic surgical scissors, and the volume and weight of the tumor were photographed and measured. Tumor volume was calculated using V / mm². 3 = [A / mm × (B / mm)] 2 [2], where A is the long diameter of the tumor and B is the short diameter. The length of the intestine from the colon to the anus was measured and recorded as an indicator of the degree of inflammation in mice.

[0129] The results showed that, compared with the blank control group (9.20±0.16 cm), orthotopic inoculation of colorectal tumor tissue significantly shortened the colon in the model mice (colon length in the negative control group: 6.15±0.65 cm). Colon length was often negatively correlated with the degree of intestinal inflammation in mice, such as... Figure 6 As shown, the colon length of mice treated with Clostridium butyricum recovered, indicating that it can significantly alleviate intestinal inflammation. Among different treatment groups, Clostridium butyricum BHD05 and BHD07 showed the most significant improvement in colon length in mice, while there was no significant difference in the average colon length among the groups.

[0130] Orthotopic tumors were harvested from the colon of mice, and their volume and weight were measured and calculated. Quantitative analysis was performed on the tumor volume and weight of five groups of mice with orthotopic tumor models. Figure 7 From A and 7B, we can conclude that the average tumor volume in the negative control group model mice was 249.62 mm. 3 (±109.05), with an average tumor weight of 370.82 mg (±171.8). The tumor volume and weight in the Clostridium butyricum gavage group were lower than those in the control group, with the BHD05 treatment group showing the smallest in situ tumor volume and weight. Figure 7As can be clearly seen from C, Clostridium butyricum treatment not only inhibits the formation of in situ tumors, but also reduces the volume and weight of in situ tumors in the colon of mice, thereby significantly inhibiting the progression of colorectal cancer.

[0131] Based on a comprehensive assessment of all indicators, we believe that BHD05 is a Clostridium butyricum strain with therapeutic effects on colorectal cancer in mice.

[0132] Example 4: Construction of recombinant plasmid pMTA36-VEGI192

[0133] The recombinant plasmid pMTA36-VEGI192 was constructed using enzyme digestion and ligation methods. First, the desired target gene fragment was ligated using the multiple cloning site on the *E. coli* cloning vector pUC19. Finally, the ligated complete target fragment was ligated into the *Clostridium* expression vector pMTL82151. Details are as follows:

[0134] 1. Obtaining the target gene sequence

[0135] Based on the sequence information of VEGI 192, TlpA36, LacI, Clostridium butyricum amylase signal peptide SP gene, and T7 terminator gene from NCBI, the recombinant plasmid pMTA36-VEGI192 was synthesized by Beijing BGI Genomics Co., Ltd. to construct the target gene. The target gene was cloned into the pUC57 plasmid, namely pUC57-VEGI 192 and pUC57-TlpA36(pUC57...

[0136] The gene fragments contained in TlpA36 are TlpA36-LacI, pUC57-SP, and pUC57-T7 terminator. The nucleotide sequence of the VEGI192 gene is shown in SEQ ID NO:2, the nucleotide sequence of TlpA36 is shown in SEQ ID NO:3, the nucleotide sequence of LacI is shown in SEQ ID NO:4, the nucleotide sequence of the Clostridium butyricum amylase signal peptide SP gene is shown in SEQ ID NO:5, and the nucleotide sequence of the T7 terminator is shown in SEQ ID NO:6.

[0137] 2. Primer design and synthesis

[0138] The upstream and downstream primers required for gene cloning during the construction of recombinant plasmids were designed by SnapGene (6.0.2) and synthesized by Beijing BGI Genomics Co., Ltd. The primer sequences are shown in Table 2 below.

[0139] Table 2. Primers used in the experiment

[0140]

[0141]

[0142] 3. Construct the recombinant plasmid pUC19-TlpA36-SP

[0143] The construction flowchart of recombinant plasmid pUC19-TlpA36-SP is shown below. Figure 8 The specific experimental procedures are as follows:

[0144] Step 1: Extraction of pUC19 plasmid. The plasmid was extracted from E. coli DH5α (pUC19) strain, following the instructions of the plasmid extraction kit. The extracted plasmid was stored at -20°C.

[0145] Step 2: TlpA36-LacI and SP gene amplification. Using pUC57-TlpA36 and pUC57-SP provided by BGI Genomics Co., Ltd. as templates, the TlpA36-LacI and SP gene fragments were amplified, respectively. The products were purified using a gel extraction kit and stored at -20°C for later use.

[0146] The PCR reaction system for TlpA36-LacI consisted of: 25 μL of 2×PhantaMax Buffer, 4 μL of pUC57-TlpA36 template, 1 μL of dNTP Mix, 2 μL of 10 μM TlpA36-F, 2 μL of 10 μM TlpA36-R, and 1 μL of PhantaMax Super-Fidelity DNA Polymerase, with the volume brought to 50 μL by ddH2O. The PCR program for TlpA36-LacI was as follows: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 15 s, 60℃ annealing for 15 s, 72℃ extension for 90 s, 30 cycles, and a final extension at 72℃ for 5 min. The PCR reaction system for the SP gene was as follows: 25 μL of 2×PhantaMax Buffer, 3 μL of pUC57-SP template, 1 μL of dNTP Mix, 2 μL of 10 μM SP-F, 2 μL of 10 μM SP-R, and 1 μL of PhantaMax Super-Fidelity DNA Polymerase, with the volume brought to 50 μL by ddH2O. The PCR reaction program for TlpA36-LacI was as follows: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 15 s, 54℃ annealing for 15 s, 72℃ extension for 10 s, 30 cycles, and a final extension at 72℃ for 5 min.

[0147] Step 3: Enzyme ligation and transformation. The plasmid pUC19 and TlpA36-LacI fragments were simultaneously digested with restriction endonucleases EcoRI and BamHI. The digestion products were purified using a gel extraction kit, ligated overnight with T4 DNA Ligase at 16°C, and then transformed into competent E. coli cells and cultured overnight on LB agar plates containing ampicillin.

[0148] Step 4: Colony PCR Validation. Add 20 μL of sterile ddH2O to a centrifuge tube, transfer the picked single colonies to a PCR tube in a laminar flow hood, and label them.

[0149] Colony PCR reaction system: 20 μL bacterial culture, 2 μL 10 μM upstream primer TlpA36-F, 2 μL 10 μM downstream primer TlpA36-R, 25 μL 2×Es Taq MasterMix (Dye), and ddH2O to make up to 50 μL.

[0150] Mix the reaction solution by pipetting and aspirating. Centrifuge briefly at low speed to allow the reaction solution to pool at the bottom of the centrifuge tube. Set the reaction program as follows: 94℃ pre-denaturation for 7 min, 94℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 50 s, 30 cycles, and a final extension at 72℃ for 2 min. Perform colony PCR.

[0151] Step 5: Plasmid restriction enzyme digestion verification. The PCR products were verified by agarose gel electrophoresis. Successfully verified positive clones were inoculated into 10 mL of LB broth containing Amp resistance, and the culture was incubated overnight at 37°C with the shaker set to 220 rpm. Plasmids were extracted from the cultured bacterial solution and further verified by double digestion with EcoRI and BamHI. Successfully verified positive clones were sent to BGI Genomics for sequencing verification.

[0152] Step 6: Construct plasmid pUC19-TlpA36-SP. The successfully validated recombinant plasmid pUC19-TlpA36 and SP were simultaneously double-digested with restriction endonucleases BamHI and HindIII. The digestion products were recovered using a gel extraction kit, and an enzyme ligation system was established. Positive clones were screened after transformation into *E. coli*, and colony PCR and plasmid digestion verification were performed. Successfully validated recombinant plasmids were sent to BGI Genomics for sequencing verification.

[0153] 4. Construct the recombinant plasmid pUC19-TlpA36-SP-VEGI-T7 terminator

[0154] The construction process of the pUC19-TlpA36-SP-VEGI-T7 terminator plasmid is as follows: Figure 9 The specific steps are as follows:

[0155] Step 1: Cloning of VEGI 192 and T7 terminator genes. Using pUC57-VEGI 192 and pUC57-T7 terminators as templates, the VEGI 192 and T7 terminator gene fragments were amplified, respectively.

[0156] The PCR reaction system for VEGI 192 consisted of: 25 μL of 2×Phanta Max Buffer, 4 μL of pUC57-VEGI 192 template, 1 μL of dNTP Mix, 2 μL of 10 μM VEGI-F, 2 μL of 10 μM VEGI-R, and 1 μL of Phanta Max Super-Fidelity DNA Polymerase, with the volume brought to 50 μL by ddH2O. The PCR program for VEGI 192 was as follows: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 15 s, 61℃ annealing for 15 s, 72℃ extension for 90 s, 30 cycles, and a final extension at 72℃ for 5 min. The PCR reaction mixture for the T7 terminator consisted of: 25 μL of 2×Phanta Max Buffer, 4 μL of pUC57-T7 Terminator template, 1 μL of dNTP Mix, 2 μL of 10 μM T7 Terminator-F, 2 μL of 10 μM T7 Terminator-R, and 1 μL of Phanta Max Super-Fidelity DNA Polymerase, brought to a final volume of 50 μL with ddH2O. The PCR reaction procedure for the T7 Terminator was the same as that for VEGI 192.

[0157] The target fragment amplified by PCR was detected by 1.5% agarose gel electrophoresis, and the PCR product was then recovered using a kit. For specific steps, please refer to the instruction manual.

[0158] Step 2: SOE PCR amplification of the VEGI-T7 terminator. PCR amplification was performed using VEGI 192 and the T7 terminator as templates for each other. The SOE PCR reaction mixture consisted of: 25 μL of 2×Phanta Max Buffer, 1 μL of dNTP Mix, 1 μL of VEGI 192, 1 μL of the T7 terminator, and 1 μL of Phanta Max Super-Fidelity DNA Polymerase, with the volume adjusted to 50 μL by ddH2O. The SOE PCR reaction program was: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 15 s, 67℃ annealing for 15 s, 72℃ extension for 90 s, 8 cycles, and a final extension at 72℃ for 5 min.

[0159] After the reaction, 2 μL of VEGI-F and T7 terminator-R were added directly for PCR amplification (reaction program: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 15 s, 58℃ annealing for 15 s, 72℃ extension for 2 min, 30 cycles, 72℃ final extension for 5 min). A 1.5% agarose gel was prepared, and electrophoresis was used to check whether the two fragments were correctly ligated.

[0160] Step 3: Construct the pUC19-TlpA36-SP-VEGI-T7 terminator recombinant plasmid. Using pUC19-TlpA36-SP as a template, the TlpA36-LacI-SP gene fragment was amplified by PCR using primers TlpA36-F and TlpA36-SP-R. A SmaⅠ restriction site was introduced at its 3' end, and the fragment was digested and ligated with restriction endonucleases EcoRI and SmaⅠ to construct a new pUC19 plasmid, resulting in the new pUC19-TlpA36-SP. The recombinant plasmid pUC19-TlpA36-SP and the VEGI-T7 terminator were newly constructed using stepwise double digestion with BglII and SmaI. Specifically, BglII was used to digest the new pUC19-TlpA36-SP and VEGI-T7 terminator for 3 hours at 37°C. A small amount of digestion product was collected for detection. After successful detection, the digestion product was recovered and digested with SmaI at 30°C for 3 hours. After gel electrophoresis detection, the digestion product was recovered, ligated overnight, and transformed into E. coli. Positive clones were screened, and plasmid digestion and sequencing were performed for verification. The successfully verified plasmid was stored at -20°C for later use. The pUC19-TlpA36-SP-VEGI-T7 terminator was obtained.

[0161] 5. Construct the recombinant plasmid pMTA36-VEGI192

[0162] The pMTA36-VEGI 192 plasmid construction procedure can be found in [link to documentation]. Figure 10 The specific steps are as follows:

[0163] Plasmids pMTL82151 and pUC19-TlpA36-SP-VEGI-T7 were rapidly digested with EcoRI and SmaI restriction endonucleases, reacted at 37℃ for 30 min, and detected by agarose gel electrophoresis. The target fragment was purified according to the DNA gel extraction kit instructions. Ligation and screening were then performed to obtain the recombinant plasmid pMTA36-VEGI192.

[0164] Example 5: Obtaining the engineered Clostridium butyricum strain BHC008-1 and detecting VEGI192 expression.

[0165] 1. Recombinant plasmid pMTA36-VEGI192 was transformed into Clostridium butyricum BHD05.

[0166] Step 1: The expression plasmid was transformed into E. coli conjugation transfer strain CA434 (purchased from Hangzhou Baosai Biotechnology Co., Ltd.) using the heat shock method. Successfully identified positive clones, E. coli CA434 (pMTA36-VEGI 192), were selected and cultured overnight in LB medium supplemented with chloramphenicol and kanamycin. Simultaneously, C. butyricum BHD05 was cultured overnight in TGY medium.

[0167] Step 2: Take 3 mL of overnight cultured engineered E. coli CA434 in a laminar flow hood.

[0168] (pMTA36-VEGI 192), centrifuge at 6000 rpm and discard the supernatant. Resuspend the bacterial pellet in 3 mL of fresh LB medium, centrifuge again and discard the supernatant. Repeat the washing process three times to completely remove the antibiotic. Add 400 μL of overnight culture medium (C4).

[0169] The butyricum BHD05 bacterial culture was thoroughly mixed by aspiration. The cell mixture was then spread onto antibiotic-free TGY agar plates and incubated in an anaerobic environment at 37°C for conjugation.

[0170] Step 3: After culturing for 24 hours, add 1 mL of fresh TGY medium to each plate to wash and collect the cells. Transfer the cell solution to a TGY plate containing 30 μg / mL thiamphenicol and 250 μg / mL D-cyclic serine (to eliminate residual E. coli CA434 donor cells). Incubate in an anaerobic chamber for 48-96 hours until transformant colonies can be observed.

[0171] Step 4: Take a PCR tube, add 20 μL of sterile water, and select the positive clone C. butyricum BHD05 (pMTA36-VEGI 192) for colony PCR verification (primers used: upstream primer TlpA36-F; downstream primer T7 terminator-R). Successfully verified transformants are then sequenced. The successfully verified C. butyricum BHD05...

[0172] The (pMTA36-VEGI 192) positive clone was named Clostridium butyricum engineered strain BHC008-1.

[0173] 2. SDS-PAGE detection of VEGI 192 secretory expression and screening of expression conditions

[0174] Step 1: Sample Preparation. The engineered *Clostridium butyricum* strain BHC008-1 was inoculated into RCM liquid medium (containing 30 μg / mL thiamphenicol) and anaerobically cultured overnight at 37°C. The overnight culture was then inoculated at a 10% inoculation rate into fresh RCM liquid medium (containing 30 μg / mL thiamphenicol) and cultured anaerobically until OD (Organic Discharge). 600IPTG inducer was added at final concentrations of 0, 1 mM, and 2 mM, respectively, and the mixture was anaerobically cultured at 36°C with shaking for 24 h and 48 h, respectively. The fermentation supernatant was collected, filtered through a 0.22 μm filter, and then PMSF was added to a final concentration of 1% and stored at -20°C. 1 mL of the fermentation supernatant was taken, and the protein was concentrated using trichloroacetic acid precipitation. The concentrated protein sample was mixed thoroughly with a certain volume of 5× loading buffer, boiled in a water bath for 10 min, cooled, and centrifuged at 3000 rpm for 30 s. The supernatant was then used for gel electrophoresis.

[0175] Step 2: SDS-PAGE detection

[0176] See results Figure 11 As shown in the figure. This result indicates that VEGI 192 expression level was highest after induction with 1 mM IPTG for 48 h at 36 °C.

[0177] 3. Western Blot detection of proteins

[0178] The results are as follows Figure 12 As shown, the results indicate that VEGI 192 expression levels were highest after 48 hours of induction with 1 mM IPTG at 36 °C.

[0179] Example 6: Cellular-level antitumor activity

[0180] The effect of the fermentation supernatant of *Clostridium butyricum* engineered strain BHC008-1 on the proliferation of human umbilical vein endothelial cells (HUVECs) was detected using the Cell Counting Kit-8 (CCK-8). The specific procedures are as follows:

[0181] Step 1: Preparation of fermentation broth supernatant. Anaerobic culture *Clostridium butyricum* BHD05 and engineered *Clostridium butyricum* strain BHC008-1 using RCM liquid medium containing a final concentration of 30 μg / mL thiamphenicol. After overnight culture, take 3 mL of the bacterial suspension and centrifuge at 7000 rpm for 10 min. Discard the supernatant, add an equal volume of PBS (pH 7.0) to resuspend the bacterial pellet, and centrifuge at 7000 rpm for 10 min. Repeat the above steps to wash the bacterial cells three times. Discard the supernatant, add 3 mL of fresh RCM liquid medium, resuspend the bacterial cells by pipetting, and then add fresh RCM liquid medium to a final volume of 30 mL. Incubate anaerobically until OD (dose-free ratio) reaches 100%. 600 The culture medium was approximately 0.6, and IPTG solution was added to a final concentration of 1 mM. Anaerobic fermentation was carried out at 36°C for 72 h. The control group consisted of fresh RCM medium with a final IPTG concentration of 1 mM added, fermented under the same conditions. After fermentation, the bacterial culture was centrifuged at 7000 rpm for 10 min, and the supernatant was filtered through a 0.22 μL filter membrane and reserved for later use.

[0182] Step 2: Culture of human umbilical vein endothelial cells (HUVECs). Select well-grown HUVECs, add trypsin-EDTA digestion solution, and digest at 37°C for approximately 5 minutes. Immediately discard the digestion solution and add fresh cell culture medium (DMEM complete medium containing 10% fetal bovine serum) to terminate the digestion. Repeatedly pipette to completely detach the cells from the dish, collect the cell slurry, and centrifuge at 1000 rpm for 3 minutes. Discard the supernatant, resuspend the cells in fresh complete culture medium (DMEM complete medium containing 10% fetal bovine serum), and count the cells.

[0183] Step 3: Experimental Design. Divide the 96-well plate into 3 sections: an RCM liquid culture medium control group (Ctrl group), a BHD05 fermentation broth group, and an engineered bacteria BHC008-1 fermentation broth group. For example... Figure 13 As shown, each group was prepared with three different concentrations (20%, 50%, and 70% by volume) of PBS (100 μL per well). The control group received 20 μL, 50 μL, and 70 μL of RCM liquid medium per well, followed by 80 μL, 50 μL, and 30 μL of fresh PBS. The BHD05 fermentation broth group and the engineered BHC008-1 fermentation broth group received 20 μL, 50 μL, and 70 μL of PBS containing 5 × 10⁻⁶ ppm, respectively. 4 BHD05 or BHC008-1 fermentation broth for 1 cell were added, followed by 80 μL, 50 μL, and 30 μL of fresh PBS. Fresh PBS was added to one well around the edge of the plate as a control. The cells were incubated together at 37°C and 5% CO2 for 12 h and 24 h.

[0184] Step 4: CCK-8 assay. At the corresponding reaction time point, slowly aspirate the liquid from the well with the pipette tip close to the edge of the well wall, and wash three times with 100 μL of fresh PBS solution. Discard the liquid from the well, add 10 μL of CCK-8 reaction solution and 90 μL of complete culture medium to each well, wrap with aluminum foil, and place in a 37°C cell culture incubator in the dark for 2 hours (the reaction time is fixed for each time point). After the reaction, immediately measure the absorbance at 450 nm using a microplate reader. Results are as follows: Figure 14 As shown, different concentrations of BHC008-1 fermentation supernatant can inhibit the proliferation of endothelial cells, and the inhibitory effect increases with the increase of fermentation broth content, which is dose-dependent; moreover, the inhibitory effect after 24 h of treatment is stronger than that after 12 h, indicating that it is time-dependent.

[0185] Example 7: Anti-colorectal cancer activity of Clostridium butyricum engineered strain BHC008-1 at the animal level.

[0186] The test sample was lyophilized Clostridium butyricum engineered strain BHC008-1 (prepared using the method in Example 2). Animals: 6-8 week old SPF-grade male Balb / c mice (weighing 17-22g) were purchased from the Experimental Animal Center of Zhengzhou University and were routinely housed in the animal laboratory. The mice were given feed and drinking water and allowed to acclimatize to the environment for one week before the experiment.

[0187] 1. Culture of CT26-LUC tumor cells

[0188] Same as Example 3.

[0189] 2. Establishment of a subcutaneous CT26-LUC tumor model in Balb / c mice

[0190] Same as Example 3.

[0191] 3. Constructing a mouse colonic orthotopic tumor model

[0192] Same as Example 3.

[0193] 4. Treatment regimen for mouse orthotopic tumor models

[0194] Three days after the establishment of the in situ tumor model, administration of the bacterial preparation began. The experimental design was as follows: ① Negative control group: Each mouse was administered 200 μL of PBS solution by gavage once daily; ② Blank control group (BHD05(pMTA36)) without the target gene: 200 μL of lyophilized control Clostridium butyricum containing the BHD05(pMTA36) plasmid was dissolved in PBS solution and administered by gavage once daily to each mouse, with a bacterial count of 2 × 10⁻⁶. 7 CFU; BHD05(pMTA36) was prepared by transforming the plasmid pMTA36, which does not contain the exogenous gene, into Clostridium butyricum BHD05. ③ Clostridium butyricum engineered strain BHC008-1 treatment group: The lyophilized powder of engineered strain BHC008-1 was dissolved in PBS solution and administered to each mouse once a day by gavage at a volume of 200 μL, with a total bacterial count of 2 × 10⁻⁶. 7 CFU; ④ Capecitabine group: Administration was based on body surface area. The dosage was converted from the adult clinical capecitabine dosage to the mouse dosage using the equivalent dose factor conversion method. The specific conversion method is as follows: Body surface area is calculated according to Xu Wensheng's formula: Body surface area (m²) 2 The formula is: 0.0061 × height (cm) + 0.0128 × weight (kg) - 0.1529. Using an average adult weight of 61.75 kg and an average height of 161.45 cm, the average adult body surface area is calculated to be 1.62 m². 2 According to the capecitabine instructions, the body surface area is 1.62 m². 2At that time, the clinical dosage of capecitabine was 2000 mg, which is equivalent to an average adult clinical dose of 32.39 mg / kg, administered once daily for 2 weeks before discontinuation. The average dose for mice is calculated as 32.39 mg / kg × 70 kg × 0.0026 / 20 g = 294.75 mg / kg. For example, if the average weight of mice at the start of administration was 17 g, the single dose of capecitabine would be 5 mg. Mice were weighed daily to calculate the gavage dose. Treatment lasted for a total of 14 days. Due to the small diameter of the mouse gastric tube, capecitabine tablets were ground into powder and prepared as a suspension with PBS for gavage. Therefore, in subsequent experiments, capecitabine may irritate the digestive tract of mice, producing side effects.

[0195] 5. Monitoring of mouse weight and health status, and tumor tissue condition after treatment.

[0196] During treatment, the survival, weight, and activity level of each mouse were observed and recorded daily. Mice were scored based on their health status, with scores ranging from 0 to 5, representing different health conditions, with higher scores indicating better condition. Specific criteria were as follows: 5 points indicated the mouse was basically healthy; 4 points indicated disheveled fur and decreased activity; 3 points indicated increased respiratory rate; 2 points indicated the mouse was depressed and inactive; 1 point indicated the mouse was near death and had essentially lost its ability to move; 0 points indicated death.

[0197] After the experiment, mice were euthanized by cervical dislocation, dissected, and a portion of the intestine from the cecum to the anus was removed. The in situ tumor model was then photographed and recorded. The in situ tumor tissue was then completely removed using ophthalmic surgical scissors, and the volume and weight of the in situ tumor in the mice were photographed and measured. Tumor volume was calculated using V / mm². 3 = [A / mm × (B / mm)] 2 [2], where A is the long diameter of the tumor and B is the short diameter. The length of the intestine from the colon to the anus was measured and recorded as an indicator of the degree of inflammation in mice.

[0198] The results are as follows Figure 15 and Figure 16 As shown in the figure, after treatment with the engineered *Clostridium butyricum* strain BHC008-1, the health status of the mice was significantly better than that of the negative control group. Dissection of the abdominal cavity after treatment revealed tumor masses in the colon of all mice in the negative control group, and the tumor tissue in the treatment group was smaller than that in the negative control group. The results indicate that the engineered *Clostridium butyricum* strain BHC008-1 has a therapeutic effect on colorectal cancer, with efficacy comparable to that of capecitabine.

[0199] Post-treatment tumor condition Figure 16As shown in B and 16C, the average volume and weight of tumor tissue in the negative control group mice treated with PBS by gavage were larger, while the average volume and weight of tumor tissue in the treatment groups of engineered Clostridium butyricum BHC008-1 and capecitabine were significantly reduced. The blank control group of Clostridium butyricum without plasmids had no significant inhibitory effect on tumors.

[0200] 6. Immune organ index

[0201] The spleen and thymus are two important immune organs, and immune organ indices are often used as indicators of the strength of the body's immune response. After dissecting a mouse, the spleen and thymus are removed with forceps, excess water is absorbed, and the organs are weighed and recorded. The ratio of organ weight (mg) to mouse body weight (g) is the corresponding immune organ index. Figure 17 It can be concluded that the thymus index and spleen index of the negative control group mice were at a very high level, which is presumably because the growth of the in situ tumor stimulated the mice's autoimmunity to produce a large number of immune cells; while after treatment with the engineered butyric acid bacterium BHC008-1, both immune indices of the mice decreased, which may be due to the reduction of inflammation in the mice after the treatment ended, which no longer stimulated the proliferation of thymic immune cells.

[0202] 7. Serum inflammatory factor IL-6 level

[0203] IL-6 (Interleukin-6) is a typical pro-inflammatory cytokine, and its level can reflect the body's inflammation level. It shows significantly high expression levels in colorectal cancer and colitis. The secretion of cytokines in mouse serum was detected using ELISA, and a gradient concentration standard solution was used for enzyme-linked immunosorbent assay (ELISA). The results are as follows: Figure 18 As shown, the level of the pro-inflammatory cytokine IL-6 was the highest in the negative control group, significantly higher than that in the treatment group with the engineered Clostridium butyricum strain BHC008-1 (p<0.0001). These results indicate that the engineered Clostridium butyricum strain BHC008-1 can reduce the secretion of the pro-inflammatory cytokine IL-6 in mice and alleviate the inflammatory response caused by colorectal cancer.

[0204] 8. Serum biochemical indicators

[0205] Blood biochemical indicators are not only important indicators for diagnosing the normal functioning of the body in human clinical medicine, but also important references in physiological, pathological, and toxicological studies. For example, alkaline phosphatase (ALP), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) are related to the normal functioning of liver metabolism; blood urea nitrogen (BUN) and creatinine (CRE) are used in medicine to reflect kidney function. After treatment in the experimental mouse model, mice were captured and blood was collected from their eyeballs. Whole blood was collected and allowed to stand at room temperature for 30 minutes. Then, it was centrifuged at 3000 rpm and 4°C for 15 minutes, and the supernatant was retained. Three liver indicators—alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP)—and two kidney indicators—blood urea nitrogen (BUN) and creatinine (CRE)—were measured using a fully automated biochemical analyzer. The results are as follows: Figure 19 and Figure 20 As shown, compared with the negative control group, there were no significant differences in the five serum biochemical indicators of mice in the Clostridium butyricum engineered strain BHC008-1 treatment group, which means that Clostridium butyricum engineered strain BHC008-1 has no toxic effect on mouse organs.

[0206] 9. H&E sections of tumor and colon tissue

[0207] Hematoxylin and eosin (H&E) staining is a commonly used staining method in paraffin sectioning. This method primarily utilizes two solutions: alkaline hematoxylin, which stains intracellular nucleic acids a purplish-blue, and acidic eosin, which stains other components red. By observing the proportions of the staining colors, the proliferation status of cells can be determined. A higher proportion of blue-purple stains indicates denser tissue cells, suggesting that tumor cells are in a proliferative state. Figure 21 As shown, the negative control group mice had a large nucleus-to-cytoplasm ratio in their tumors, indicating that the tumor cells were still proliferating. The tumor tissue staining results of the engineered bacteria treatment group showed mostly pinkish-purple cytoplasm and other components, meaning that the engineered bacteria could inhibit tumor cell proliferation and thus achieve a tumor-suppressing effect. The capecitabine treatment group mice had sparsely arranged tumor cells, reduced cell proliferation levels, and damaged tumor tissue.

[0208] 10. Immunohistochemical detection of Ki-67 and CD31 in tumor tissue.

[0209] Ki67 is a nuclear protein involved in the cell cycle. It is expressed during cell division but not in G0 phase resting cells. Ki67 immunohistochemical staining can be used to determine cell state; strong Ki67 expression indicates that tumor cells are in the division phase. Figure 22 As can be seen from A, the tumor cells in the negative control group and the blank control group were mostly in the division phase, while the tumor tissue in the capecitabine group and the engineered bacteria BHC008-1 treatment group consisted of resting cells, which were in a non-dividing state.

[0210] CD31, also known as the platelet-endothelial cell adhesion molecule, is typically located in vascular endothelial cells. Tumor growth and metastasis require energy supply, necessitating the formation of numerous capillaries by endothelial cells. Therefore, in immunohistochemistry, CD31 is primarily used to demonstrate the presence of endothelial cell tissue, assess tumor angiogenesis, and thus determine the extent of tumor growth. Strong CD31 immunohistochemical staining indicates active angiogenesis within the mouse tumor, suggesting further tumor growth. Figure 22 As can be seen from B, compared with the negative control group, the expression level of CD31 in the tumor tissue of the engineered bacteria BHC008-1 and capecitabine treatment groups decreased, which means that the amount of angiogenesis in the tumor tissue of these two groups was reduced and the tumor growth was inhibited.

[0211] 11. Analysis of colonic intestinal tissue structure in mouse models

[0212] Intestinal morphology is an important indicator for evaluating intestinal health. Villus height and crypt depth reflect the efficiency of nutrient digestion and absorption, and are important indicators of intestinal function. Colonic sections often reveal significant structural damage and changes in colonic cells, such as crypt height and goblet cell count. Therefore, we prepared colonic sections from orthotopic tumor inoculation sites to determine the occurrence and progression of lesions in a colorectal cancer model. Figure 23 H&E-stained transverse sections of the colon were observed under an optical microscope. In the capecitabine-treated group, tumor cells were located entirely within the muscular and serosa layers of the colonic wall, with two sites showing infiltration of tumor cells into the intestinal wall. In the Clostridium butyricum engineered strain BHC008-1-treated group, the colonic structure was intact, crypts were clearly visible, goblet cells were neatly arranged, and the serosa layer at the interface between the tumor tissue and the colon was significantly thickened. In the negative control group, a large area of ​​goblet cells was reduced and crypts were shortened, indicating damage to the intestinal mucosa in this group; tumor cells were mostly located in the muscular and serosa layers of the colonic wall, and tumor cells showed signs of penetrating the intestinal wall. In conclusion, the Clostridium butyricum engineered strain BHC008-1-treated group of mice maintained a relatively normal colonic structure and function and could prevent tumor cell infiltration by increasing intestinal wall thickness.

[0213] In summary, the supernatant of the fermentation broth of the engineered Clostridium butyricum strain BHC008-1 constructed in this invention can inhibit the proliferation of human umbilical vein endothelial cells, and this inhibition is time- and dose-dependent within a certain range. A 70% concentration of fermentation supernatant, after 24 hours of incubation, showed an 85% inhibition rate against endothelial cells. In vivo animal experiments investigated the therapeutic effect of engineered strain BHC008-1 on a mouse model of tumors. Compared with untreated mice, mice treated with BHC008-1 by gavage showed higher health indices and lower levels of the pro-inflammatory factor IL-6; in situ tumors were inhibited, and the colonic wall at the tumor site thickened, without tumor cell infiltration. Serum biochemical results indicated that BHC008-1 treatment had no toxic effects on mouse organs.

[0214] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A strain of Clostridium butyricum BHD05 for the prevention and treatment of colorectal cancer, characterized in that, The preservation number of this strain is CGMCC No.32029.

2. A genetically engineered strain of Clostridium butyricum, BHC008-1, characterized in that, This engineered bacterium was created by transforming Clostridium butyricum BHD05 with an expression plasmid containing TlpA36, LacI, Clostridium butyricum amylase signal peptide SP, and VEGI192 linked together. The nucleotide sequence of the VEGI192 gene is shown in SEQ ID NO:2, the nucleotide sequence of TlpA36 is shown in SEQ ID NO:3, the nucleotide sequence of LacI is shown in SEQ ID NO:4, and the nucleotide sequence of the Clostridium butyricum amylase signal peptide SP gene is shown in SEQ ID NO:

5. The preservation number of Clostridium butyricum BHD05 is CGMCC No. 32029.

3. The *Clostridium butyricum* genetically engineered strain BHC008-1 according to claim 2, characterized in that, The preservation number of this engineered bacteria is CGMCC No. 31490.

4. The method for preparing the genetically engineered Clostridium butyricum strain BHC008-1 according to claim 2 or 3, characterized in that, include: The coding genes for TlpA36, LacI, Clostridium butyricum amylase signal peptide SP, and VEGI192, which are sequentially linked, are inserted into the multiple cloning site downstream of the LacZ promoter of the Escherichia coli-Clostaphylococcus shuttle expression vector pMTL82151 to obtain the recombinant vector pMTA36-VEGI192; then, the recombinant vector pMTA36-VEGI192 is transformed into Clostridium butyricum BHD05 to obtain the engineered Clostridium butyricum strain BHC008-1.

5. The preparation method according to claim 4, characterized in that, Downstream of the VEGI 192 gene is the T7 terminator.

6. The preparation method according to claim 4, characterized in that, The nucleotide sequence of the T7 terminator is shown in SEQ ID NO:

6.

7. A microbial inoculant, characterized in that, The microbial inoculant contains the following components: (1) Clostridium butyricum BHD05 as described in claim 1, and / or, (2) The butyric acid Clostridium butyricum genetically engineered strain BHC008-1 as described in claim 2 or 3.

8. The microbial agent according to claim 7, characterized in that, The microbial inoculant contains the following components: (1) The fermentation broth, suspension, or dried powder of Clostridium butyricum BHD05 as described in claim 1, and / or, (2) The fermentation broth, bacterial suspension or dried bacterial powder of Clostridium butyricum genetically engineered strain BHC008-1 as described in claim 2 or 3.

9. The use of one or more of the following components in the preparation of drugs for the prevention and / or treatment of colorectal cancer: (1) Clostridium butyricum BHD05 as described in claim 1; (2) The fermentation broth, bacterial suspension or dried bacterial powder of Clostridium butyricum BHD05 as described in claim 1; (3) The engineered Clostridium butyricum strain BHC008-1 as described in claim 2 or 3; (4) The fermentation broth, bacterial suspension or dried bacterial powder of Clostridium butyricum genetically engineered strain BHC008-1 as described in claim 2 or 3; (5) The microbial agent according to claim 4 or 5.

10. A drug for the prevention and / or treatment of colorectal cancer, characterized in that, The drug contains one or more of the following ingredients: (1) Clostridium butyricum BHD05 as described in claim 1; (2) The fermentation broth, bacterial suspension or dried bacterial powder of Clostridium butyricum BHD05 as described in claim 1; (3) The engineered Clostridium butyricum strain BHC008-1 as described in claim 2 or 3; (4) The fermentation broth, bacterial suspension or dried bacterial powder of Clostridium butyricum genetically engineered strain BHC008-1 as described in claim 2 or 3; (5) The microbial agent according to claim 4 or 5.

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