Engineering probiotic EcN-Vh as well as construction method and application thereof

Abstract

The invention provides engineering probiotics EcN-Vh as well as a construction method and application thereof, and belongs to the technical field of biological medicines. According to the present invention, the engineering probiotic capable of expressing the BTApep-TAT is constructed through the bioengineering technology, the BTApep-TAT is expressed through the plasmid integration mode, the release of the peptide is achieved by using the arabinose-induced cracking system, and the engineering probiotic is used for the in-vivo sustainable production of the BTApep-TAT. The engineering probiotic EcN-Vh disclosed by the invention can continuously generate an inhibitory peptide BTApep-TAT of a targeted BORIS (Bovine Biosensor), and is used for treating the colorectal cancer. According to the probiotics, by inhibiting ADP-ribosylation and Wnt / beta-catenin signal channels of BORIS, the occurrence rate and progress of tumors are remarkably reduced, and a novel, safe and efficient probiotic delivery system is provided for treatment of colorectal cancer.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an engineered probiotic EcN-Vh, its construction method, and its application. Background Technology

[0002] Colorectal cancer (CRC) is the third most common malignant tumor worldwide, with a complex pathogenesis involving both genetic and epigenetic alterations. BORIS is a transcriptional regulator aberrantly expressed in various cancers and is frequently associated with malignancy and / or drug resistance. Almost all types of cancer express BORIS, including colorectal cancer, lung cancer, breast cancer, prostate cancer, and leukemia. BORIS participates in tumor development by inhibiting apoptosis and promoting cancer cell proliferation.

[0003] Phage display libraries were used to screen for BORIS inhibitory peptides, and BTApep-TAT was identified as a BORIS-targeting inhibitory peptide. BTApep-TAT induces DNA damage in cancer cells and inhibits the progression of NSCLC (Non-Small Cell Lung Cancer) xenograft tumors. Studies on the mechanism of action of BTApep-TAT revealed that BORIS undergoes ADP ribosylation after double-strand or single-strand DNA damage. Replacing five conserved glutamate (E) residues with alanine residues (A) between amino acids (AA) 198 and 228 in BORIS reduces its ADP ribosylation. Inhibition of BORIS ADP ribosylation (ADPr-BORIS) through site-specific mutations or BTApep-TAT treatment blocked its interaction with Ku70 and impaired BORIS's function in DNA damage repair. However, the clinical application of BTApep-TAT is limited by the poor stability and high production cost of peptide drugs. Summary of the Invention

[0004] This invention provides an engineered probiotic EcN-Vh, its construction method, and its application. The engineered probiotic EcN-Vh can continuously produce the inhibitory peptide BTApep-TAT that targets BORIS, for the treatment of colorectal cancer.

[0005] The present invention provides a method for constructing engineered probiotic EcN-Vh, comprising the following steps: (1) transforming a recombinant plasmid expressing BTApep-TAT peptide into Escherichia coli Nissle 1917 to obtain transgenic Escherichia coli; (2) The plasmid controlling EcN lysis is transferred into the transgenic Escherichia coli described in step (1) to obtain the engineered probiotic EcN-Vh.

[0006] In a preferred embodiment of the present invention, the recombinant plasmid in step (1) further includes a GFP tag.

[0007] In a preferred embodiment of the present invention, the backbone vector of the recombinant plasmid expressing the BTApep-TAT peptide includes a pGLO vector, and the gene encoding the BTApep-TAT peptide is inserted into positions 1421-2299 of the pGLO vector by seamless cloning.

[0008] In a preferred embodiment of the present invention, the source of the plasmid controlling EcN lysis in step (2) includes Escherichia coli WM3064.

[0009] The present invention also provides an engineered probiotic EcN-Vh constructed using the above-described construction method.

[0010] This invention also provides the application of the above-mentioned engineered probiotic EcN-Vh in the preparation of drugs that target and inhibit BORIS.

[0011] This invention also provides the application of the above-mentioned engineered probiotic EcN-Vh in the preparation of drugs for treating tumor diseases.

[0012] In a preferred embodiment of the present invention, the tumor disease includes colorectal cancer.

[0013] The present invention also provides a pharmaceutical composition comprising the above-described engineered probiotic EcN-Vh and pharmaceutically acceptable excipients.

[0014] The present invention also provides a drug for targeted treatment of colorectal cancer, the active ingredient of which includes the above-mentioned engineered probiotic EcN-Vh.

[0015] Beneficial Effects: This invention provides an engineered probiotic strain, EcN-Vh, which is a probiotic constructed using bioengineering technology capable of expressing BTApep-TAT. The peptide is released using an arabinose-induced cleavage system for the sustainable in vivo production of BTApep-TAT. The engineered probiotic EcN-Vh of this invention can continuously produce the BORIS-targeting inhibitory peptide BTApep-TAT for the treatment of colorectal cancer. This probiotic inhibits ADP-ribosylation and the Wnt / β-catenin signaling pathway in BORIS, suppressing the expression of downstream target genes Ccnd1, Axin2, Mmp7, Myc, and Brca1, significantly reducing tumor incidence and progression. This provides a novel, safe, and efficient probiotic delivery system for the treatment of colorectal cancer, overcoming the shortcomings of poor stability and high production cost of the BTApep-TAT peptide. Attached Figure Description

[0016] Figure 1The diagram shows the construction, colonization ability, and inhibitory effect on CT26 cell proliferation of EcN-Vh. (A) Construction model of EcN-Vh and EcN-Scramble; (B) EcN-Vh has a certain colonization ability; (C) Schematic diagram of the release of BTApep-TAT by the engineered probiotic EcN-Vh; (D) Inhibitory effect of EcN-Vh on CT26 cell proliferation. Figure 2 The therapeutic effect of EcN-Vh in AOM / DSS-induced mouse colorectal cancer model is shown in the following sections: (A) Construction process of AOM-DSS-induced mouse colorectal cancer model; (B) Representative images of colorectal tissue and longitudinal sections of each group, with red arrows indicating tumor locations; (C and D) Comparison of the number of colorectal tumors in each group; (E) Comparison of colorectal length in each group. Figure 3 The effects of EcN-Vh on body weight, survival rate and pathological score of AOM / DSS mice were investigated. (A) Comparison of HE staining pathological tissues of colorectal tissues of mice in each group, EcN-Vh reduced intestinal morphological disorder; (B) Changes in body weight of mice in each group; (C) Changes in survival rate of mice in each group, EcN-Vh reduced mortality of AOM / DSS mice. Figure 4 EcN-Vh inhibits the expression of downstream target genes Ccnd1, Axin2, Mmp7, Myc, and Brca1 of the Wnt / β-catenin signaling pathway in the colorectal tissue of AOM / DSS mice. When the Wnt pathway is activated, the expression of these target genes increases, and EcN-Vh inhibits the increase of these target genes. Figure 5 To investigate the inhibitory effect of BTApep-TAT on BORIS ribosylation and the expression of downstream target genes in the Wnt / β-catenin signaling pathway in CT26 cells, the following findings were made: (AF) Changes in the expression of Ccnd1, Axin2, Mmp7, Myc, and Brca1 in CT26 cells after treatment with BTApep-TAT and His-TAT, with BTApep-TAT significantly inhibiting the expression of the above target genes; (G) After transfection with empty vector plasmid F17 and overexpression plasmid R12, BTApep-TAT and His-TAT were administered, and BTApep-TAT inhibited BORIS ribosylation and Ccnd1 expression. Detailed Implementation

[0017] The present invention provides a method for constructing engineered probiotic EcN-Vh, comprising the following steps: (1) transforming a recombinant plasmid expressing BTApep-TAT peptide into Escherichia coli Nissle 1917 to obtain transgenic Escherichia coli; (2) The plasmid controlling EcN lysis is transferred into the transgenic Escherichia coli described in step (1) to obtain the engineered probiotic EcN-Vh.

[0018] The BTApep-TAT peptide described in this invention has been previously disclosed in an article (BTApep-TAT peptide inhibits ADP-ribosylation of BORIS to induce DNA damage in cancer), and will not be repeated here. This invention first constructs an expression plasmid by inserting the coding gene of the BTApep-TAT peptide into a vector. In one embodiment, the coding gene is shown in SEQ ID No. 1: atgGTGCATTGGGATTTTCGGCAGTGGTGGCAGCCTTCTGGTGGTCGTAAAAAACGTCGTCAGCGTCGTCGTGGTtaa. In one embodiment, the pGLO vector is selected as the backbone vector, and the coding gene and... gfp The gene (encoding the GFP tag) was inserted into the 1421-2299 bp site of the backbone vector via seamless cloning.

[0019] After constructing the expression plasmid, this invention further uses a one-step cloning method to insert the GFP tag into the synthesized plasmid at an insertion site of 1421-2299 bp. After obtaining the complete plasmid, this invention transforms the complete plasmid into Dh5α competent cells and sends it to a company for sequencing confirmation of successful construction. Then, plasmid mini-prep is performed; for example, in one embodiment, plasmid mini-prep is performed according to the instructions of the Tiangen plasmid mini-prep kit to obtain the recombinant plasmid pGLO-BTApep-TAT- expressing the BTApep-TAT peptide. gfp In one embodiment of the invention, BTApep-TAT- is inserted into the expression plasmid. gfp The complete gene sequence is shown in SEQ ID No. 2: TTGACGGCTAGCTCAGTCCTAGGTACAGTGCTAGCactagaaagaggagaaatactagatgGTGCATTGGGATTTTCGGCAGTGGTGGCAGCCTTCTGGTGGTCGTAAAAAACGTCGTCAGCGTCGTCGTGGTtaaactagaaagaggagaaatactagatgGCTAGCAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCTACATACGGAAAGCTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTCTCTTATGGTGTTCAATGCTTTTCCCGTTATCCGGATCATATGAAACGGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAACGCACTATATCTTTCAAAGATGACGGGAACTACAAGACGCGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATCGTATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTCGGACACAAACTCGAGTACAACTATAACTCACACAATGTATACATCACGGCAGACAAACAAAAGAATGGAATCAAAGCTAACTTCAAAATTCGCCACAACATTGAAGATGGATCCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCGACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGCGTGACCACATGGTCCTTCTTGAGTTTGTAACTGCTGCTGGGATTACACATGGCATGGATGAGCTCTACAAAtaa。

[0020] The recombinant plasmid pGLO-BTApep-TAT expressing the BTApep-TAT peptide in the present invention - gfpTransfected into E. coli Nissle 1917 (EcN), yielding EcN-pGLO-BTApep-TAT- gfp In one embodiment of the present invention, a control strain was also constructed, namely, the recombinant plasmid pGLO-Scramble-TAT- expressing the control peptide Scramble. gfp Transferring to EcN, we obtain EcN-pGLO-Scramble-TAT- gfp The present invention does not specifically limit the method of conversion; in the embodiments, an electroconversion method is used.

[0021] This invention utilizes a bacterial conjugation method to control the lysis of EcN using pHG101-P. BAD - lysin The plasmids were transferred from the conjugation donor Escherichia coli WM3064 into the process described in step (1) to obtain engineered probiotic EcN-Vh and engineered probiotic EcN-Scramble as a control.

[0022] In this invention, due to the control of EcN cleavage by pHG101-P BAD - lysin Plasmids are difficult to transfer into E. coli (EcN), therefore bacterial conjugation was chosen, and pHG101-P was transferred into the plasmid. BAD - lysin The plasmid was transferred into EcN. In one embodiment of the present invention, *Escherichia coli* WM3064 was selected as the intermediate strain, and pHG101-P was first transferred into EcN. BAD - lysin The conversion to WM3064 yields WM3064-pHG101-P BAD - lysin strain, and then with the WM3064-pHG101-P strain. BAD - lysin Using the strain as a donor, pHG101-P BAD - lysin Transfer it into EcN.

[0023] Specifically, in this invention, the donor and the EcN strain are cultured at 37°C for approximately 4 hours until OD (Organic Demand). 600When the concentration of bacteria is 0.4–0.6, 1.5 mL of donor bacteria and 0.75 mL of recipient bacteria (volume ratio 2:1) are taken, and the bacterial cells are collected by centrifugation. The bacterial precipitate is washed with antibiotic-free LB medium to remove antibiotics. The bacterial cells are then resuspended in light LB medium containing DAP. The donor and recipient bacteria are mixed and concentrated to 100 μL, which is then added dropwise to a light LB medium plate containing DAP. After the bacterial suspension dries, it is incubated at 37 °C for 4 h. The bacterial block is washed with 1 mL of LB medium, the bacterial suspension is collected and serially diluted 10-fold, and plated onto LB plates containing Ampicillin and Kanamycin antibiotics. The plates are incubated at 37 °C until colonies grow. The samples are sent to Zhejiang Youkang Biotechnology Co., Ltd. for single-colony sequencing, and the successfully constructed engineered probiotic EcN-Vh is finally obtained.

[0024] In this invention, the composition of the light LB medium is 10 g tryptone, 5 g yeast extract, 5 g sodium chloride, and deionized water to a final volume of 1 L. A stock solution of 5.7 mg / mL DAP (diaminopimelic acid solution) is diluted 1:100 for use. The WM3064 strain is a DAP auxotroph, and DAP (final concentration 57 μg / mL) needs to be added to the light LB medium during cultivation.

[0025] The present invention also provides an engineered probiotic EcN-Vh constructed using the above-described construction method.

[0026] The engineered probiotic EcN-Vh described in this invention can express and release the inhibitory peptide BTApep-TAT targeting BORIS, which is released through an arabinose-induced cleavage system.

[0027] This invention also provides the application of the above-mentioned engineered probiotic EcN-Vh in the preparation of drugs that target and inhibit BORIS.

[0028] The engineered probiotic EcN-Vh described in this invention can release peptides using an arabinose-induced cleavage system, thus sustainably producing BTApep-TAT in vivo, thereby targeting and inhibiting BORIS. This invention also provides the application of the aforementioned engineered probiotic EcN-Vh in the preparation of drugs for treating tumor diseases.

[0029] In one embodiment, the tumor disease described in this invention is colorectal cancer, and the engineered probiotic EcN-Vh shows significant anti-tumor effects in a colorectal cancer model, specifically: inhibiting the proliferation of mouse colorectal cancer cells; reducing the incidence and severity of AOM / DSS-induced mouse colorectal cancer; and exerting a therapeutic effect by blocking ADP-ribosylation of BORIS and inhibiting the Wnt signaling pathway in mouse colorectal cancer.

[0030] The present invention also provides a pharmaceutical composition comprising the above-described engineered probiotic EcN-Vh and pharmaceutically acceptable excipients.

[0031] 2. In this invention, the engineered probiotic EcN-Vh cells are used as the active ingredient. In one embodiment, when the cells are administered to an animal model via gavage, the cell count is required to be 10. 8 ~10 10 CFU / each.

[0032] The present invention also provides a drug for targeted treatment of colorectal cancer, the active ingredient of which includes the above-mentioned engineered probiotic EcN-Vh.

[0033] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, provides an engineered probiotic EcN-Vh, its construction method, and its applications. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0034] Example 1 (1) Construction of engineered probiotics Using SnapGene software, suitable primers were designed for BTApep-TAT. The linearized pGLO vector and the synthesized BTApep-TAT expression fragment were amplified separately by polymerase chain reaction (PCR), as were the linearized pHG101 vector and the lysed gene. lysin The fragment was purified and recovered using the sodium acetate-ethanol method and DpnI restriction enzyme digestion. The purified and recovered linearized pGLO vector and the fragment expressing BTApep-TAT were configured in a specific ratio to form a one-step cloning system (ClonExpress® II One Step Cloning Kit C112), and in vitro recombination was performed to obtain the target recombinant plasmid. The complete plasmid was transformed into Dh5α competent cells and sent to the company for sequencing to confirm successful construction, followed by plasmid mini-prep. The primers used are shown in Table 1 below (bold text indicates homologous recombination regions with the vector). The one-step cloning system is shown in Table 2.

[0035] Table 1 Primers used for the construction of engineered probiotics

[0036] pHG101-P BAD - lysin The plasmid was transformed into WM3064 to obtain WM3064-pHG101-P BAD - lysin (Donor bacteria). pHG101-P BAD - lysin Plasmid transformation was transferred to the recipient bacterium EcN-pGLO-BTApep-TAT- via conjugation. gfpand EcN-pGLO-Scramble-TAT- gfp The conjugates, namely the engineered probiotics EcN-Vh and EcN-Scramble, were obtained. Single colonies were picked and cultured overnight in LB medium containing dual resistance to Kanamycin and Ampicillin. Glycerol was added to a final concentration of 15% and stored at -80°C.

[0037] Table 2 One-step cloning system

[0038] (2) The effect of engineered bacteria on inhibiting colorectal cancer in vitro Mouse CRC cell line CT26, grown to the logarithmic growth phase, was seeded at 1000 cells per well in 1640 medium in 96-well clear plates. Two revived engineered bacteria were inoculated at a 1:50 ratio into sterile shake flasks (containing Ampicillin and Kanamycin in LB liquid) and shaken until OD was reached. 600 Approximately 1 hour later, the bacterial culture was aliquoted into 50 mL centrifuge tubes and centrifuged at 5000 rpm for 5 min. The supernatant was discarded, and the culture was resuspended in 1640 medium for further incubation. The cells were then divided into experimental and control groups. Arabinose was added to the experimental group to induce bacterial lysis, while the control group received no treatment. The culture was prepared overnight. The next day, the overnight culture from both the experimental and control groups was centrifuged at 12000 rpm for 1 min three times. After the last centrifugation, the supernatant was filtered using a 0.22 μm syringe to avoid cell contamination. The prepared culture was then added to cell wells, and the cell plates were placed in an incubator. At 24 h, 48 h, 72 h, and 96 h, 10 μL of CCK-8 reagent was added directly to the cell wells, and the cells were incubated at 37℃ for 0.4–2 h. The OD was then measured using a microplate reader. 450 Value, result as Figure 1 As shown in Figure E, EcN-Vh inhibits the proliferation of colorectal cancer cells in CT26 mice.

[0039] (3) Determination of the colonization ability of engineered bacteria in mouse intestine, construction of AOM / DSS colorectal cancer model and verification of the anti-cancer ability of engineered bacteria. Mice from the same batch with similar growth and development were randomly divided into two groups for parallel comparison of the colonization effects of EcNs containing pMUT1 and pMUT2 recombinant plasmids, respectively. Ampicillin (1:1000) was added to the mice's drinking water before gavage to inhibit the native gut microbiota. After pretreatment, both groups of mice were given the corresponding bacterial suspensions daily, and feces were collected daily for colony counting. After stopping the administration of bacterial suspensions, feces were still collected and colony counts were performed. Results are as follows: Figure 1 As shown in Figure C, EcN-Vh has a certain intestinal colonization ability.

[0040] AOM / DSS colorectal cancer model: Azomethine (AOM) (10 mg / kg) was injected intraperitoneally. Two days later, the mice were fed 2% DSS (diethylstilbestrol) instead of drinking water for 8 days, followed by 2 weeks of regular drinking water. This cycle was repeated three times. During this period, the bacterial strains were resuscitated weekly, shaken overnight, centrifuged at 12,000 rpm for 5 min, resuspended in sterile PBS, and then mixed with an appropriate amount of 2M arabinose before gavage. This was performed twice weekly. After each DSS treatment, antibiotics containing the corresponding bacterial strain were added to the drinking water to inhibit the mice's gut microbiota.

[0041] Experimental results are as follows Figure 2 and Figure 3 As shown, EcN-Vh reduces the incidence of AOM / DSS-induced colorectal cancer in mice ( Figure 2 (BD), alleviated the shortening of the colon and rectum caused by colorectal cancer ( Figure 2 E), alleviated the decrease in body weight in mice ( Figure 3 (B), reduced the mortality rate of mice ( Figure 3 (C) reduces the severity of colorectal cancer. Figure 3 (A)

[0042] (4) Cell passage, plating and transfection Cell passage and plating: When the cell density in the T25 cell culture flask reaches 80-90%, passage is performed. For CT26 adherent cells, the culture medium is first aspirated, and the cells are gently washed with PBS buffer. After aspirating the PBS, 1 mL of trypsin digestion solution is added. After digestion, the cells are returned to the clean bench and 3 mL of complete culture medium is added to stop the digestion. At the same time, the cells are gently resuspended at the bottom of the culture flask in complete culture medium by pipetting. The cells are then transferred to new T25 culture flasks pre-filled with complete culture medium at a ratio of 1:2 or 1:3. For plating, when the cells are resuspended in complete culture medium, a certain amount of cell suspension is taken and counted using a cell counter. Then, according to the experimental requirements, the cells are added to the wells of a plate, and the cells are evenly distributed to the bottom of the wells using the figure-eight method. The plate is then placed in an incubator for culture.

[0043] Cell transfection: After cell confluence is adequate after plating and adhesion, transfect the cells with overexpression plasmids. F17 is an empty vector plasmid, and R12 is a BORIS overexpression plasmid. In a six-well plate, discard the original culture medium and add 2 mL of complete culture medium. Prepare a transfection suspension by adding 125 mL of incomplete culture medium, 2.5 μg of plasmid, and 4 μL of Lip8000 transfection reagent to each well. Gently pipette to mix well and let stand for 5 minutes to allow the liposomes and plasmids to fully bind. Finally, add 125 mL of the mixture dropwise to each well and mix thoroughly. Other procedures can be performed 24 hours after transfection.

[0044] (5) Extraction of total RNA and reverse transcription PCR Cellular RNA extraction: Remove cells from the cell culture incubator, discard the culture medium, wash the cell surface with 1×PBS, add 1 mL Trizol to each well of a 6-well plate and 700 μL Trizol to each well of a 12-well plate, lyse on ice for 3 minutes, thoroughly pipetting and mixing in each well until the liquid is no longer viscous, transfer to labeled 1.5 mL EP tubes, extract RNA according to the RNA extraction kit (#AG21024) instructions, and finally centrifuge at room temperature for 2 minutes to elute the RNA on the membrane. The eluted RNA can be directly measured for concentration and used for reverse transcription or frozen at -80℃ for 1-2 years.

[0045] RNA extraction from animal tissue samples: Fresh mouse colorectal tissue, or tissue flash-frozen in liquid nitrogen at -80°C, was placed in a 1.5 mL EP tube. One clean, pre-chilled steel ball and 1 mL of Trizol were added. The EP tubes were symmetrically arranged in a high-throughput tissue homogenizer and homogenized at 60 Hz for 120 seconds. The homogenized EP tubes were then centrifuged at 12,000 pm for 5 minutes in a pre-chilled centrifuge at 4°C. The supernatant was transferred to a new EP tube for the next step. Subsequent RNA extraction procedures were the same as those for cell sample extraction.

[0046] Reverse transcription PCR: The RNA samples from the previous step were reverse transcribed using the EvoM-MLV Reverse Transcription Kit (#AG11734). First, take out the gDNA, and calculate the total RNA concentration for each sample. Take 14g of total RNA and add it to an RNase-free eight-tube container. Add 2 μL of 5×gDNA Clean Reaction Mix, and finally add RNase-free Water to bring the reaction volume to 16μL. Centrifuge to mix the liquid in the tube at 42℃ for 2 minutes. After the reaction is complete, centrifuge again, add 4μL of 5×EvoM-MLV RT Reaction Mix to each tube, centrifuge to mix the liquid, and centrifuge at 85℃ for 5 seconds and then at 37℃ for 15 minutes. The cDNA after reverse transcription needs to be diluted 2-5 times before quantitative real-time PCR detection and stored at -20℃.

[0047] Real-time quantitative PCR: Semi-quantitative gene expression was detected using the SYBR Green Pro Taq HS premixed qPCR kit. The reaction mixture consisted of 5 μL of 2×SYBR Green Mix, 0.4 L of primers (F+R, 10 μM), 2 μL of template cDNA, and 2.6 μL of RNase-free water, for a total volume of 10 L. The reaction program was followed according to the manufacturer's instructions. After the reaction, the relative mRNA expression level was calculated based on the cycle value (Cq value) provided by the instrument. The results are shown below. Figure 4 and Figure 5As shown, EcN-Vh inhibited the mRNA expression levels of downstream target genes Ccnd1, Axin2, Mmp7, Myc, and Brca1 in the Wnt / β-catenin signaling pathway in colorectal cancer tissues of mice with colorectal cancer. Figure 4 BTApep-TAT inhibited the expression of the aforementioned target genes in CT26 mouse colorectal cancer cells. Figure 5 (AE). The primers used for each gene in this paper are shown in Table 13 below.

[0048] Table 3 Primers used for qPCR

[0049] (6) Extraction, preparation and Western Blot of protein samples Cell protein extraction: RIPA lysis buffer preparation: 1×RIPA Buffer, 50×cocktail protease inhibitor, 100mM PMSF. The final concentrations of the latter two are 1× and 1mM, respectively. Remove the cell plate from the incubator, wash with pre-chilled 1×PBS on ice, then discard the PBS. Add an appropriate amount of cell lysis buffer to each well. Incubate on ice for 2-3 minutes. Use a clean cell scraper to scrape the cells from the wells and transfer them to EP tubes. Place the tubes on ice for 20 minutes to allow complete cell lysis. Then centrifuge at 12000 pm for 20 minutes at 4°C. After centrifugation, aspirate the supernatant and transfer it to a new EP tube. Add 10 μL to a 96-well plate, add 300 mL of G250 staining solution, incubate at room temperature for 10 minutes, and measure the absorbance of each well at 595 nm using a microplate reader. Calculate the concentration based on the standard curve. Add one-quarter of the remaining protein sample volume after final concentration testing to 5× protein loading buffer to dilute the buffer to 1×. Boil in a metal bath at 100°C for 10 minutes to ensure complete protein denaturation. Centrifuge briefly; the sample can be directly loaded for Western blotting or stored at -80°C.

[0050] Western blotting: Preparing the adhesive: Place the cleaned and dried long glass plate and short glass plate together in the adhesive preparation clamp, ensuring the bottom ends are aligned, and fix them in place. Mix pure water, 30% acrylamide, 1.5 M Tris-HCl (pH=8.8), 10% ammonium persulfate, and TEMED in a specific ratio and vortex. Use a pipette to inject the lower layer of adhesive into the gap between the long and short glass plates to the appropriate position. Then add isopropanol to the top and press the adhesive in place. After the lower layer of adhesive has solidified, absorb the isopropanol with absorbent paper. Mix pure water, 30% acrylamide, 1 M Tris-HCl (pH=6.8), 10% ammonium persulfate, and TEMED in a specific ratio and vortex. Use a pipette to inject the upper layer of adhesive. Slide a cleaned 10 / 15-well comb diagonally into the gap and wait for the upper layer of adhesive to solidify.

[0051] Sample loading for electrophoresis: After placing the gel plate on the electrophoresis clamp and ensuring no leakage of electrophoresis solution, remove the comb and load the samples slowly to avoid sample diffusion. The loading volume for each sample is 20-30 μg. After all samples have been loaded, fill the electrophoresis clamp with electrophoresis solution and add electrophoresis solution to the appropriate level in the electrophoresis tank. Run the stacking gel at 80V for 20 minutes, then run the separating gel at 150V for 60 minutes until the protein markers are completely separated.

[0052] Transfer: Gently pry open the long and short glass plates with a glue cutter, cut off the blank parts around the edges, and place them in pure water for later use. Activate the PVDF membrane with methanol, then rinse it in the transfer equilibration solution specifically for the rapid transfer apparatus. The order of placement on the positive electrode of the transfer clamp, from bottom to top, is: sponge pad, PVDF membrane, PAGE glue, sponge pad again. After confirming the order is correct and there are no air bubbles between the glue and the membrane, close the transfer clamp tightly and insert it into the rapid transfer apparatus tank. Select the appropriate program to begin the transfer.

[0053] Blocking: After the membrane transfer is complete, pour 10 mL of rapid blocking solution into the blocking box, immerse the membrane in it, and place it on a horizontal shaker to slowly block for 10-20 minutes.

[0054] Incubation with primary and secondary antibodies and membrane washing: Cut the blocked membrane to the appropriate size, incubate with the corresponding primary antibody, and shake overnight on a 4-degree side-shake incubator. After incubation, wash the membrane three times with 1×TBST, 10 minutes each time. Then incubate with the corresponding species' secondary antibody at room temperature on a side-shake incubator for 1 hour. Wash the membrane again with 1×TBST three times, 10 minutes each time.

[0055] Development: Prepare the developer solution according to the ratio of A:B = 1:1, and perform the development operation on the developing instrument; analyze the grayscale value of the developed band using ImageJ software. Results are as follows: Figure 5 As shown, BTApep-TAT inhibited ADP ribosylation of BORIS in HCT116 cells overexpressing BORIS. Figure 5 (F), inhibited the expression of Ccnd1 protein in CT26 cells ( Figure 5(G).

[0056] 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 method for constructing engineered probiotic EcN-Vh, characterized in that, Includes the following steps: (1) Transgenic Escherichia coli was obtained by transforming the recombinant plasmid expressing BTApep-TAT peptide into Escherichia coli Nissle 1917; (2) The plasmid controlling EcN lysis is transferred into the transgenic Escherichia coli described in step (1) to obtain the engineered probiotic EcN-Vh.

2. The construction method according to claim 1, characterized in that, The recombinant plasmid described in step (1) also includes a GFP tag.

3. The construction method according to claim 2, characterized in that, The backbone vector of the recombinant plasmid expressing the BTApep-TAT peptide includes a pGLO vector, and the gene encoding the BTApep-TAT peptide is inserted into positions 1421-2299 of the pGLO vector.

4. The construction method according to claim 1, characterized in that, The source of the plasmid controlling EcN lysis in step (2) includes Escherichia coli WM3064.

5. The engineered probiotic EcN-Vh constructed using the construction method described in any one of claims 1 to 4.

6. The use of the engineered probiotic EcN-Vh according to claim 5 in the preparation of a drug that targets and inhibits BORIS.

7. The use of the engineered probiotic EcN-Vh according to claim 5 in the preparation of a medicament for treating tumor diseases.

8. The application according to claim 7, characterized in that, The tumor diseases mentioned include colorectal cancer.

9. A pharmaceutical composition, characterized in that, It includes the engineered probiotic EcN-Vh as described in claim 5 and pharmaceutically acceptable excipients.

10. A drug for targeted treatment of colorectal cancer, characterized in that, The active ingredient includes the engineered probiotic EcN-Vh as described in claim 5.

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