Application of ganoderma lucidum immunomodulatory protein in preparation of products for targeting TK1

Abstract

The invention discloses application of ganoderma lucidum immunomodulatory protein in preparation of a product for targeting TK1, and belongs to the technical field of biological medicine. The protein can be directly combined with a tumor proliferation marker TK1, and the expression level of TK1 is reduced, so that tumor cell proliferation is inhibited. The invention discloses a molecular mechanism that ganoderma lucidum immunomodulatory protein plays an anti-tumor role through targeting TK1 for the first time, and specifically discloses an amino acid site of the ganoderma lucidum immunomodulatory protein interacting with TK1. Based on the discovery, the invention also provides food, health-care food or medicine which contains the ganoderma lucidum immunomodulatory protein and is used for inhibiting TK1 or tumors, and the food, health-care food or medicine is particularly suitable for preventing and treating breast cancer. The invention provides a new direction for clinical application transformation of the ganoderma lucidum immunomodulatory protein, and has important development value and application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of a Ganoderma lucidum immunomodulatory protein in the preparation of products targeting TK1. Background Technology

[0002] Thymidine kinase 1 (TK1) is a key enzyme in DNA synthesis and damage repair, and its activity is closely related to cell proliferation. In normal cells, TK1 activity is cell cycle-dependent; however, in various malignant tumor cells, TK1 is often abnormally overexpressed, making it an important tumor proliferation marker widely used in the auxiliary diagnosis and prognostic assessment of cancers such as lung cancer, colorectal cancer, breast cancer, and prostate cancer. Studies have shown that TK1 not only provides substrates for DNA replication but also plays an important role in the repair of DNA damage caused by chemotherapy or radiotherapy. Therefore, targeting TK1 to inhibit its function or expression has become a promising strategy in the field of cancer treatment.

[0003] Breast cancer is one of the most common malignant tumors among women worldwide, and its treatment varies depending on the molecular subtype. Although significant progress has been made in existing treatments (such as surgery, radiotherapy, chemotherapy, endocrine therapy, and targeted therapy), limited efficacy, drug resistance, and toxic side effects remain for some subtypes (such as Luminal A) or recurrent / metastatic patients. Therefore, the continuous development of novel anti-tumor drugs or functional products with novel mechanisms of action, high efficacy, and low toxicity is a crucial clinical need that urgently needs to be addressed.

[0004] Reishi mushroom, a traditional medicinal fungus, has had its extracts and active ingredients extensively studied. Among them, the reishi immunomodulatory protein (FIP-glu) has been shown to possess various biological activities, including immunomodulation and anti-tumor activity. However, the specific molecular targets and signaling pathways of FIP-glu's anti-tumor effects are not yet fully elucidated, particularly lacking evidence on its direct action on TK1, a key tumor marker. This gap in understanding severely limits the further development and clinical application of FIP-glu as a precision targeted therapy.

[0005] Therefore, elucidating the deep molecular mechanism of FIP-glu's anti-tumor effect, especially its anti-breast cancer effect, and identifying its direct target, is of great significance for developing novel targeted therapies, health foods, or functional foods based on this protein. Summary of the Invention

[0006] This invention provides the application of Ganoderma lucidum immunomodulatory protein in the preparation of products targeting TK1. Experiments have for the first time demonstrated that FIP-glu can directly bind to TK1 and downregulate its protein expression, thereby inhibiting tumor cell proliferation. Based on this novel mechanism, this invention provides a food, health food, or drug containing FIP-glu for inhibiting TK1 or tumors (such as breast cancer), offering a new strategy for developing novel anti-tumor products.

[0007] On one hand, this invention provides the application of Ganoderma lucidum immunomodulatory protein in the preparation of products targeting thymidine kinase 1 (TK1), using the following technical solution: Application of a Ganoderma lucidum immunomodulatory protein in the preparation of products targeting thymidine kinase 1 (TK1).

[0008] Preferably, Ganoderma lucidum immunomodulatory protein binds to thymidine kinase 1 (TK1).

[0009] Preferably, the amino acid sequence of the Ganoderma lucidum immunomodulatory protein is shown in SEQ ID NO: 1.

[0010] Preferably, amino acid residues L43, T44, S69 and Q73 of the A chain of Ganoderma lucidum immunomodulatory protein and amino acid residues N24, R27, D88, Q109 and N111 of the B chain interact with amino acid residues Q20, R38, R42, Y48, R130, E144, R158 and R186 of TK1, respectively.

[0011] Preferably, Ganoderma lucidum immunomodulatory protein can be used to reduce the expression level of TK1.

[0012] The present invention also provides a composition for inhibiting thymidine kinase 1 (TK1) comprising the above-mentioned Ganoderma lucidum immunomodulatory protein.

[0013] The present invention also provides a composition for inhibiting tumors, comprising the above-mentioned Ganoderma lucidum immunomodulatory protein.

[0014] Preferably, the composition is a food, health food, or drug.

[0015] Preferably, the tumor is breast cancer.

[0016] In summary, the beneficial effects of the present invention are as follows: This invention reveals for the first time the molecular mechanism by which Ganoderma lucidum immunomodulatory protein (FIP-glu) directly targets and binds to thymidine kinase 1 (TK1) and downregulates its expression. This discovery not only provides a new and specific modern scientific explanation for the traditional anti-tumor effects of Ganoderma lucidum, but also provides key theoretical and experimental basis for its transformation from a functional component to a precision targeted therapy drug.

[0017] Based on the aforementioned core findings, this invention expands the application prospects of FIP-glu, developing it into a food, health food, or drug capable of targeting TK1. This provides a novel candidate product with a clear mechanism and natural origin for the prevention and treatment of tumors (especially breast cancer), possessing good development potential and application value. Attached Figure Description

[0018] Figure 1 Co-IP electrophoresis image using FIP-glu as decoy; Figure 2 Images showing co-localization of FIP-glu with TK1 cells; Figure 3 The Co-IP results for FIP-glu and TK1 are shown in the figure; Figure 4 The pull-down results for FIP-glu and TK1 are shown in the image. Figure 5 The simulation diagram shows the interaction between FIP-glu and TK1; Figure 6 Figure showing the TK1 protein expression level; Figure 7 In Figure A, the subcutaneous tumor tissue growth curve was obtained from the experiment on the inhibition of breast cancer cell growth in mice by FIP-glu. Figure 7 Image B shows a solid subcutaneous tumor. Figure 7 C represents the subcutaneous tumor mass; Figure 7 D represents the volume of the subcutaneous tumor; Figure 7 E represents the weight change curve of the mice during the treatment process. Detailed Implementation

[0019] The present invention will be further described in detail below with reference to the embodiments.

[0020] Example Example 1 The specific steps for preparing the Ganoderma lucidum immunomodulatory protein FIP-glu are as follows: S1, Carrier Construction Gene design and synthesis: based on Ganoderma lucidum ( Ganoderma lucidum The amino acid sequence of Ganoderma lucidum immunomodulatory protein (SEQ ID NO: 1) from the source was determined according to the Pichia pastoris (…). Pichia pastoris The preferred codons were optimized, and the DNA sequence encoding the protein (SEQ ID NO: 2) was chemically synthesized and cloned into the pUC-57 cloning vector.

[0021] Plasmid extraction and restriction enzyme digestion: The constructed pUC-57-FIP-glu plasmid was transformed into E. coli DH5α, amplified, and then the plasmid was extracted. Simultaneously, restriction endonucleases were used. Eco RI and Apa I performed double enzyme digestion on the expression vector pPIC9K and the extracted FIP-glu gene fragment.

[0022] Ligation and transformation: The linearized pPIC9K vector and FIP-glu insert fragment, after enzyme digestion, were purified and ligated overnight at 16°C using T4 DNA ligase. The ligation product was transformed into E. coli DH5α competent cells, plated on LB agar plates containing the appropriate antibiotics, and positive clones were screened and sequenced to obtain the recombinant plasmid pPIC9K-FIP-glu.

[0023] S2, yeast conversion Preparation of competent yeast cells: Pichia pastoris GS115 strain, stored at -80°C, was streaked and revived. Single colonies were picked and inoculated into 10 mL of YPD liquid medium and cultured at 30°C with shaking at 200 rpm until late logarithmic growth. An appropriate amount of the bacterial culture was then transferred to fresh YPD medium for further culture. When the OD... 600 Bacterial cells were collected when the saturation point was 0.8 ~ 1.0, washed sequentially with sterile water and 100 mmol / L LiCl solution, and finally resuspended in 400 μL LiCl.

[0024] Chemical transformation: Take 50 μL of the above-mentioned competent yeast cells and add 360 μL of 50% PEG3350, 240 μL of 1 mol / L LiCl, 10 μg of linearized pPIC9K-FIP-glu plasmid, and 5 μL of 10 mg / mL salmon sperm DNA (carrier DNA) sequentially. After gently mixing, incubate at 30°C for 30 minutes, and then heat shock at 42°C for 20-25 minutes. Centrifuge to collect the cells, resuspend them, and spread them on MD deficient medium plates. Incubate at 30°C for 2-3 days until transformants appear.

[0025] Positive clone identification: Select well-grown single colonies and perform colony PCR or plasmid extraction verification to ensure that the FIP-glu gene has been integrated into the yeast genome.

[0026] S3. Recombinant protein expression and purification Methanol-induced expression: Verified positive transformants were inoculated into BMGY medium and cultured at 30°C until OD600. 600 = 2~6. Centrifuge to collect bacterial cells, and resuspend in BMMY induction medium to OD. 600 = 1. Add anhydrous methanol every 24 hours to a final concentration of 1%, and continue to induce expression for 5 days.

[0027] Protein collection and preliminary processing: Collect the fermentation broth and centrifuge at 4°C and 5000×g for 20 minutes to remove the cells. The supernatant was concentrated using an ultrafiltration tube with a molecular weight cutoff of 3.5 kDa and the system was replaced with lysis buffer.

[0028] Affinity purification: The concentrated sample was loaded onto a pre-equilibrated Ni-NTA affinity chromatography column (His60 NiSuperflow Resin), and non-specific contaminating proteins were removed with 10 column volumes of wash buffer (containing 20 mM imidazole). Finally, the target protein was collected in fractions with elution buffer containing 250 mM imidazole.

[0029] Desalting and Preservation: Combine the high-purity elution fractions and place them in a 3.5 kDa dialysis bag. Dialyze overnight at 4°C in PBS buffer (pH 7.4). After dialysis, the samples are freeze-dried into powder or directly aliquoted and stored at -80°C for later use. Protein concentration can be quantified using the BCA method, and purity can be detected by SDS-PAGE.

[0030] Table 1. Ganoderma lucidum immunomodulatory protein FIP-glu and its coding sequence

[0031] Example 2 The specific steps for obtaining the Ganoderma lucidum immunomodulatory protein FIP-glu target are as follows: Prokaryotic expression and purification of S1, FIP-glu-Flag fusion protein Vector construction: The gene encoding FIP-glu (SEQ ID NO: 2) was cloned into the multiple cloning site of the prokaryotic expression vector pQE-60, and a Flag tag sequence was introduced at its C-terminus to construct the recombinant plasmid pQE60-FIP-glu-Flag. After confirmation by sequencing, it was transformed into Escherichia coli M15 expression strain.

[0032] Induction of expression: Positive single clones were selected and inoculated into LB liquid medium containing ampicillin, and cultured at 37°C with shaking at 220 rpm until OD was reached. 600 ≈ 0.6. Isopropyl-β-D-thiogalactoside (IPTG) was added to a final concentration of 1 mmol / L, and expression was induced at 37°C for 4 hours.

[0033] Protein purification: Bacterial cells were collected, resuspended in lysis buffer (50 mmol / L Tris-HCl, 300 mmol / L NaCl, pH 8.0), and sonicated on ice. The lysis buffer was centrifuged at 12,000×g for 20 minutes at 4°C, and the supernatant was collected. The supernatant was incubated with pre-equilibrated Ni-NTA resin at 4°C for 1 hour, followed by washing with wash buffer containing 50 mmol / L imidazole, and finally eluting the target protein with elution buffer containing 250 mmol / L imidazole. The eluent was dialyzed against PBS buffer (pH 7.4) and concentrated using ultrafiltration to obtain high-purity FIP-glu-Flag protein, which was stored at -80°C for later use.

[0034] S2. Cell Culture and Processing Cell culture: Human breast cancer T-47D cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin antibiotics and placed in a cell culture incubator at 37°C and 5% CO2.

[0035] Cell seeding and treatment: When cells reach 80%–90% confluence, digest with 0.25% trypsin, count, and then seed at 1.5 × 10⁻⁶ cells / year. 6 Cells were seeded at a density of 10 cells / well in 6-well plates. After culturing for 24 hours, the old medium was aspirated and replaced with fresh complete medium containing 1 μg / mL FIP-glu-Flag protein (experimental group) or an equal volume of PBS (control group), and the culture was continued for 6 hours.

[0036] S3. Sample preparation for co-immunoprecipitation (Co-IP) Cell lysis: After treatment, wash cells twice with pre-chilled PBS. Add 200 μL of non-denaturing IP lysis buffer containing a protease inhibitor complex to each well and lyse on ice for 30 minutes. Collect the lysis buffer and centrifuge at 14,000 × g for 10 minutes at 4°C, and collect the supernatant.

[0037] Immunoprecipitation: Take a small amount of supernatant (40 μL) and mix it with 5×SDS loading buffer. Heat at 95°C for 5 minutes as the "Input" control group. Divide the remaining supernatant into two equal parts and add 20 μL of Anti-Flag magnetic beads (experimental group) or isotype control IgG magnetic beads (negative control group) equilibrated with lysis buffer to each part. Incubate at 4°C for 2 hours.

[0038] Magnetic bead washing: After incubation, collect the magnetic beads using a magnetic rack and discard the supernatant. Wash the magnetic beads 5 times with pre-cooled TBST buffer (containing 0.05% Tween-20) for 5 minutes each time to completely remove non-specific binding.

[0039] S4. Protein separation and staining identification SDS-PAGE electrophoresis: Add 40 μL of 1×SDS loading buffer to the washed magnetic beads and heat at 95°C for 10 minutes to fully elute the bound proteins. Perform 10% SDS-PAGE electrophoresis on the eluent and "Input" sample (loading volume 10 μL). The initial voltage is 80 V, and after the sample enters the separating gel, adjust it to 120 V until the bromophenol blue reaches the bottom of the gel.

[0040] Silver staining: After electrophoresis, carefully cut off the separating gel. Perform the following steps sequentially: fixation (40% ethanol, 10% glacial acetic acid, 30 min), sensitization (30% ethanol, 0.2% sodium thiosulfate, 10 min), rinsing, silver staining (0.1% silver nitrate, 20 min), rinsing again, and finally developing with a solution containing 2% sodium carbonate and 0.04% formaldehyde until the bands are clear. Immediately terminate the reaction with 5% glacial acetic acid.

[0041] Mass spectrometry identification: Based on the silver staining results, the experimental group and the negative control group were compared, and specific bands were excised from the lanes of the experimental group. The gel strips were sent to a professional biotechnology service company (such as Beijing Biotech) for in-gel enzymatic digestion and liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis. Finally, the protein that specifically binds to FIP-glu-Flag was identified as thymidine kinase 1 (TK1).

[0042] like Figure 1 As shown, the specific protein gel strips in lane 2 were cut and sent to Beijing Biotech Biotechnology Co., Ltd. for protein identification.

[0043] Example 3 The cellular co-localization analysis of Ganoderma lucidum immunomodulatory protein (FIP-glu) and thymidine kinase 1 (TK1) was performed using the following steps: S1. Construction of fluorescently labeled expression vectors Construction of the TK1-mCherry vector: Using genomic DNA from human breast cancer T-47D cells as a template, the full-length coding sequence of the TK1 gene was amplified by PCR using specific primers (TK1-pc-F, sequence SEQ ID NO: 3; TK1-pc-R, sequence SEQ ID NO: 4). The purified PCR product was then mixed with... Nhe I and Hin The pcDNA3.1(+)-mCherry vector, double-digested with d III enzymes, was ligated overnight at 16°C using T4 DNA ligase. The ligation product was transformed into E. coli DH5α, and positive clones were screened and sequenced to obtain the recombinant plasmid pcDNA3.1-TK1-mCherry.

[0044] Construction of the FIP-glu-GFP vector: The synthesized FIP-glu gene was cloned into a vector that had been modified with the appropriate restriction endonuclease (e.g., FIP-glu-GFP). Nde I and Xho I) In the linearized pET-30a(+)-GFP vector, the recombinant plasmid pET-30a-FIP-glu-GFP was constructed and sequenced for verification.

[0045] Prokaryotic expression and purification of S2, FIP-glu-GFP fusion protein Expression and Induction: The validated pET-30a-FIP-glu-GFP plasmid was transformed into *E. coli* BL21(DE3) competent cells. Single colonies were picked and inoculated into LB medium containing kanamycin and cultured at 37°C with shaking until OD200. 600 ≈ 0.6. Add IPTG to a final concentration of 0.5 mmol / L and induce expression at 37°C for 4 hours.

[0046] Protein purification: Bacterial cells were collected, resuspended in binding buffer (20 mmol / L Tris-HCl, 500 mmol / L NaCl, 20 mmol / L imidazole, pH 7.9), and then sonicated. The supernatant was collected after centrifugation and loaded onto a Ni-NTA agarose affinity chromatography column pre-equilibrated with binding buffer. The cells were washed with wash buffer containing 50 mmol / L imidazole, and finally eluted with elution buffer containing 250 mmol / L imidazole. The eluent was dialyzed in PBS buffer (pH 7.4) to desalt, concentrated using ultrafiltration, and the concentration was determined. The eluent was then aliquoted and stored at -80°C.

[0047] S3, Cell Transfection and Fluorescent Protein Treatment Cell preparation: Human breast cancer T-47D cells and MCF-7 cells were cultured at 1.5 × 10⁶ cells per well. 5 Cells were seeded at a density of approximately 70% in laser confocal microscopy dishes (such as 35 mm glass dishes) using complete culture medium containing 10% FBS and cultured at 37°C and 5% CO2 for 24 hours to achieve a cell confluence of approximately 70%.

[0048] TK1-mCherry plasmid transfection: Before transfection, replace the medium with Opti-MEM medium containing 2% FBS and free of antibiotics. Mix the plasmid pcDNA3.1-TK1-mCherry with the transfection reagent Lipofectamine 2000 in serum-free medium according to the recommended ratio in the liposome transfection reagent instructions. After incubating at room temperature for 15 minutes, add the mixture dropwise to the cell culture dish. Gently mix and return to the incubator for 6 hours. Then replace with complete medium and continue culturing for 24 hours to fully express the TK1-mCherry fusion protein.

[0049] S4. Fluorescence treatment and sample preparation FIP-glu-GFP treatment: 24 hours after transfection, discard the old culture medium and gently wash the cells once with preheated PBS. Add serum-free culture medium containing 30 μg / mL of purified FIP-glu-GFP fusion protein to each dish and treat the cells at 37°C for 4 hours.

[0050] Cell fixation and nuclear staining: After treatment, aspirate the culture medium and wash the cells three times with PBS. Add 1 mL of 4% paraformaldehyde (dissolved in PBS) to each dish and fix for 15 minutes at room temperature. Discard the fixative and wash three times with PBS. Add PBS containing 0.1% Triton X-100 and permeate for 5 minutes at room temperature. After washing with PBS, add 1 μg / mL DAPI staining solution (dissolved in PBS) and stain for 10 minutes at room temperature in the dark. Finally, wash thoroughly three times with PBS.

[0051] S5. Confocal fluorescence microscopy observation Image acquisition: Add an appropriate amount of anti-fluorescence quenching mounting medium to a glass dish and immediately place it under a laser scanning confocal microscope for observation. Use the following channel settings for image acquisition: DAPI (excitation wavelength 405 nm, emission band 420 ~ 480 nm, blue) to label cell nuclei; GFP (excitation wavelength 488 nm, emission band 500 ~ 550 nm, green) to label FIP-glu; mCherry (excitation wavelength 561 nm, emission band 570 ~ 620 nm, red) to label TK1.

[0052] Colocalization analysis: Single-channel images were acquired separately and then overlaid using microscopy system software or image processing software such as ImageJ. The distribution of green fluorescence (FIP-glu-GFP) and red fluorescence (TK1-mCherry) within the cells was observed. The overlapping area of ​​the two will be displayed in yellow, thus visually demonstrating the spatial colocalization of FIP-glu and TK1 within the cells.

[0053] The results are as follows Figure 2 As shown, the green FIP-glu interacts with the red TK1, causing color superposition and resulting in yellow.

[0054] Example 4 The interaction between FIP-glu and TK1 in cells was verified by co-immunoprecipitation (Co-IP), and the specific steps are as follows: S1. Construction of recombinant expression vector Construction of the TK1-Flag vector: Using T-47D cell genomic DNA as a template, the complete coding sequence of the TK1 gene was amplified by high-fidelity PCR using primers (TK1-FC-F, sequence SEQ ID NO: 5; TK1-FC-R, sequence SEQ ID NO: 6). The purified PCR product was then mixed with... Sac I and Hin The 3×Flag-CMV-14 vector, after being double-digested with d III enzymes, was ligated at 16°C for 16 hours using T4 DNA ligase. The ligation product was transformed into DH5α competent cells, and positive clones were selected for double digestion and sequencing verification to obtain the correct recombinant plasmid pCMV-TK1-Flag.

[0055] S2, Cell Transfection and Protein Processing Cell preparation: Human breast cancer T-47D cells and MCF-7 cells were cultured at 1.5 × 10⁶ cells per well. 6 Cells were seeded at a density of 10% in 6-well cell culture plates and cultured in a complete medium containing 10% fetal bovine serum for 24 hours at 37°C in a 5% CO2 incubator until the cell confluence reached 70% to 80%.

[0056] TK1-Flag plasmid transfection: Before transfection, replace the culture medium with antibiotic-free Opti-MEM medium containing 2% FBS. Dilute and mix the plasmid pCMV-TK1-Flag and the transfection reagent Lipofectamine 2000 separately in serum-free medium according to the ratio recommended in the liposome transfection reagent instructions. After incubating at room temperature for 15 minutes, add the mixture dropwise to the cell culture dish. Gently shake well and return to the incubator for 6 hours. Then replace with complete culture medium and continue culturing for 48 hours to ensure sufficient expression of TK1-Flag protein.

[0057] FIP-glu treatment: 24 hours after transfection, the old culture medium was discarded. The experimental group was replaced with complete culture medium containing 1 μg / mL of purified recombinant FIP-glu protein (prepared in Example 1), and the control group was replaced with an equal volume of complete culture medium treated with PBS. The treatment continued for 6 hours.

[0058] S3, cell lysis and immunoprecipitation Cell collection and lysis: After treatment, wash cells three times with pre-chilled PBS. Add 300 μL of pre-chilled Western lysis buffer (Beyotime, P0013) containing 1× protease inhibitor complex and phosphatase inhibitor to each well and lyse on ice for 30 minutes, gently shaking the culture plate every 10 minutes during lysis.

[0059] Pretreatment of lysates: Collect the lysates with a cell scraper and transfer them to pre-chilled 1.5 mL centrifuge tubes. Centrifuge at 4°C, 14,000×g for 15 minutes, and carefully aspirate the supernatant into a new tube, avoiding aspiration of precipitate.

[0060] Input sample preparation: Take 40 μL of supernatant, add 10 μL of 5×SDS-PAGE protein loading buffer, vortex thoroughly to mix, heat in a 95°C metal bath for 10 minutes as the "Input" control, and store at -20°C.

[0061] Immunoprecipitation reaction: Divide the remaining lysis supernatant into two equal portions and transfer them to new centrifuge tubes containing 25 μL of Protein A / G magnetic beads (pre-equilibrated by washing three times with lysis buffer). Add 2 μg of anti-Flag monoclonal antibody (experimental group) to one tube and an equal volume of isotype control IgG antibody (negative control group) to the other tube. Place the mixture on a rotary mixer and incubate gently at room temperature for 2 hours.

[0062] Magnetic bead capture and washing: After incubation, place the centrifuge tube on a magnetic rack and let it stand for 1 minute until the magnetic beads are completely adsorbed. Then carefully discard the supernatant. Wash the magnetic beads five times with pre-cooled TBST buffer (20 mmol / L Tris-HCl, 150 mmol / L NaCl, 0.1% Tween-20, pH 7.5), adding 500 μL of washing buffer each time. After swirling and washing for 5 minutes, place the tube on a magnetic rack to separate and discard the supernatant.

[0063] S4. Western Blot Detection Sample elution and denaturation: After the final wash, aspirate as much residual liquid as possible. Add 40 μL of 1×SDS-PAGE protein loading buffer to the magnetic beads, vortex thoroughly to mix, and heat at 95°C for 10 minutes to elute and denature the bound proteins from the magnetic beads. After brief centrifugation, place the centrifuge tube on a magnetic rack, aspirate the supernatant as the IP sample, and load it immediately or store it at -20°C.

[0064] SDS-PAGE electrophoresis: Load the "Input" and IP samples (20 μL each) along with the pre-stained protein marker onto a 15% sodium dodecyl sulfate-polyacrylamide gel. Electrophore at a constant voltage of 80V in 1×Tris-Glycine electrophoresis buffer until the sample enters the separating gel, then adjust the voltage to 120V and continue electrophoresis until the bromophenol blue front reaches the bottom of the gel.

[0065] Transfer: Proteins were transferred to a 0.22 μm PVDF membrane using wet transfer. Transfer conditions were as follows: transfer was performed at a constant current of 300 mA for 90 minutes in pre-cooled 1×Tris-Glycine transfer buffer (containing 20% ​​methanol).

[0066] Blocking and antibody incubation: After transfer, the PVDF membrane was quickly rinsed once with TBST, then immersed in 2% BSA (dissolved in TBST) and blocked by slow shaking on a shaker at room temperature for 2 hours. After blocking, the membrane was washed three times with TBST for 5 minutes each time.

[0067] Primary antibody incubation: Incubate the membrane with the corresponding primary antibody dilution buffer overnight at 4°C. Specific primary antibody and dilution conditions are as follows: Anti-His polyclonal antibody (for detecting FIP-glu-His): 1:5000 diluted in 2% BSA / TBST.

[0068] Anti-Flag monoclonal antibody: 1:3000 diluted in 2% BSA / TBST.

[0069] Secondary antibody incubation: The next day, wash the membrane three times with TBST, 10 minutes each time. Incubate the membrane with horseradish peroxidase-labeled goat anti-rabbit secondary antibody at room temperature for 2 hours.

[0070] Chemiluminescence development: After incubation with secondary antibody, wash the membrane three times with TBST, 10 minutes each time. Mix equal volumes of ECL chemiluminescence substrate solution A and solution B, and add the mixture evenly to the membrane. Incubate at room temperature for 1-2 minutes. Expose and acquire images using a chemiluminescence imaging system.

[0071] By comparing the Western blot results of the experimental group (Anti-Flag IP) and the negative control group (IgG IP), if both the TK1-Flag band and the FIP-glu-His band were detected only in the IP product of the experimental group, while neither band was detected in the negative control group, it indicates that FIP-glu can undergo specific immunoprecipitation with TK1 in cell lysate, thus confirming the direct interaction between the two in the intracellular environment.

[0072] The results are as follows Figure 3 As shown, in T-47D cells and MCF-7 cells, after treatment with FIP-glu, FIP-glu binds to TK1, and the target location shows a corresponding band.

[0073] Example 5 Pull-down experiments were conducted to verify the direct in vitro interaction between FIP-glu and TK1. The specific steps are as follows: Construction of expression vectors for S1, GST and GST-TK1 fusion proteins Plasmid construction: Using T-47D cell genomic DNA as a template, the full-length coding sequence of the TK1 gene (excluding the stop codon) was amplified by PCR using specific primers (TK1-pG-F, SEQ ID NO: 7; TK1-pG-R, SEQ ID NO: 8). The purified PCR product was then compared with... Eco RI and Not The pGEX-4T-1 prokaryotic expression vector, digested with enzyme I, was ligated to construct the recombinant plasmid pGEX-4T-1-TK1, which encodes the GST-TK1 fusion protein. Simultaneously, the empty pGEX-4T-1 plasmid served as a control, encoding the GST tag protein. Both plasmids were transformed into *E. coli* DH5α for amplification, and the results were verified by sequencing.

[0074] S2, Prokaryotic expression and purification of recombinant proteins (GST, GST-TK1, FIP-glu-His) Protein expression: The validated pGEX-4T-1 (control) or pGEX-4T-1-TK1 plasmid was transformed into Escherichia coli BL21(DE3) expression strain. Single colonies were picked and inoculated into LB medium containing ampicillin and cultured at 37°C with shaking until OD. 600 ≈ 0.6. Add IPTG to a final concentration of 1 mmol / L, and induce expression at 37°C for 4 hours to obtain GST or GST-TK1 protein in soluble form.

[0075] GST fusion protein purification: Collect the induced bacterial cells, resuspend them in pre-cooled PBS, and sonicate. Centrifuge the lysis buffer at 12,000×g for 30 minutes at 4°C and collect the supernatant. Incubate the supernatant with Glutathione Sepharose 4B resin equilibrated with PBS at 4°C for 2 hours.

[0076] Resin washing and elution: The resin was packed into the chromatography column and washed thoroughly with 10 column volumes of PBS to remove non-specific conjugates. Finally, the target protein GST or GST-TK1 was eluted with elution buffer containing 10 mmol / L reduced glutathione (dissolved in 50 mmol / L Tris-HCl, pH 8.0). The eluent was dialyzed in PBS to remove glutathione, concentrated using ultrafiltration tubes, and the concentration was determined. After aliquoting, the eluent was stored at -80°C.

[0077] Preparation of FIP-glu-His protein: Recombinant Ganoderma lucidum immunomodulatory protein (FIP-glu-His) with the His tag was prepared and purified according to the method in Example 1, and quantified for later use.

[0078] S3, GST Pull-down Experiment Binding reaction: Take 1 μg of purified FIP-glu-His protein and mix it with an equal amount of GST protein (negative control group) or GST-TK1 fusion protein (experimental group) in 400 μL binding buffer (PBS, containing 0.1% Triton X-100, 1 mmol / L DTT, 1× protease inhibitor).

[0079] Affinity capture: Add 5 μL of pre-equilibrated GST magnetic beads with binding buffer to each binding reaction system. Continue incubation at 4°C for 2 hours to allow GST or GST-TK1 protein to bind to the magnetic beads via their GST tags, while simultaneously capturing any interacting FIP-glu-His protein.

[0080] Washing: After incubation, use a magnetic rack to pick up the magnetic beads and carefully discard the supernatant. Wash the resin 5 times with pre-cooled TBST buffer, adding 500 μL of buffer each time, gently inverting to mix, then use a magnetic rack to pick up the magnetic beads and discard the supernatant to completely remove non-specifically adsorbed proteins.

[0081] S4. Sample Preparation and Western Blot Detection Sample elution and denaturation: After the final wash, aspirate as much residual liquid as possible. Add 40 μL of 1×SDS-PAGE protein loading buffer to each resin tube, vortex thoroughly to mix, and heat at 95°C for 10 minutes to elute and denature all proteins bound to the resin.

[0082] SDS-PAGE and Transfer: The elution supernatant (Pull-down product) and the pre-reserved "Input" sample (a portion of the pre-reaction mixture or purified protein, denatured with loading buffer) were subjected to 12% SDS-PAGE electrophoresis. Subsequently, the protein was transferred to a PVDF membrane using wet transfer.

[0083] Immunoblotting: After transfer, the membrane was blocked with 2% BSA / TBST at room temperature for 1 hour. Incubation with specific primary antibodies was then performed to detect different proteins. The FIP-glu-His protein was detected using an anti-His tag antibody (1:5000 dilution) to verify whether it was pulled down by GST-TK1.

[0084] GST or GST-TK1 protein was detected using an anti-GST tag antibody (1:4000 dilution) as a control for pull-down efficiency and loading volume.

[0085] After incubation with the corresponding HRP-labeled secondary antibody (1:10000 dilution), the cells were developed using an ECL chemiluminescent substrate.

[0086] like Figure 4 As shown, the FIP-glu-His protein was successfully pulled down when the GST-TK1 fusion protein was used, but not in the negative control group using only the GST-tagged protein. This result directly confirms in vitro the existence of a specific protein-protein interaction between FIP-glu and TK1.

[0087] Table 2 Primers required for the three vectors

[0088] Example 6 Molecular docking simulations were used to predict the binding mode of Ganoderma lucidum immunomodulatory protein (FIP-glu) to thymidine kinase 1 (TK1). The specific steps are as follows: S1. Acquisition and preprocessing of target protein three-dimensional structure Structural data download: Download the crystal structure files of Ganoderma lucidum immunomodulatory protein FIP-glu (PDB ID: 3F3H) and human thymidine kinase 1 (TK1) (PDB ID: 1XBT) from the RCSB protein database (https: / / www.rcsb.org).

[0089] Initial structure processing (using Schrödinger Maestro software): Protein preparation: Load the two PDB files separately and use the “Protein Preparation Wizard” module for systematic preprocessing.

[0090] Structural optimization process: Preprocess: Checks and adds missing side chain atoms, optimizes protonation state, and removes all water molecules and irrelevant ligand ions.

[0091] Refine: Under the OPLS4 force field, the protein structure is energy minimized with the root mean square deviation of convergence (RMSD) set to 0.30 Å to eliminate spatial conflicts between atoms and obtain a stable conformation.

[0092] Output: Save the processed and structure-optimized FIP-glu and TK1 proteins as “prepared_FIP-glu.pdb” and “prepared_TK1.pdb” files respectively for docking.

[0093] S2, Protein-protein molecular docking simulation Platform setup: In the Schrödinger Maestro software, use the “Protein-Protein Docking” module (such as algorithms based on shape complementarity and electrostatic interaction).

[0094] Receptor and ligand designation: The pretreated TK1 protein is designated as the "receptor" and the FIP-glu protein is designated as the "ligand".

[0095] Connection parameter configuration: Docking sampling: Set “Number of ligand rotations to probe” to 70,000 to ensure a full search of the ligand rotation conformation space.

[0096] Output: Set "Maximum poses to return" to 30 to collect the 30 candidate binding conformations with the highest scores.

[0097] Mesh generation: The system will automatically generate a sufficiently large docking mesh on the surface of the receptor protein to cover possible binding sites.

[0098] Run the docking: Submit the computation task. The software will simulate the translation, rotation, and conformational changes of the ligand protein on the surface of the receptor protein, and evaluate and rank all predicted binding modes based on scoring functions such as complementarity, desolvation energy, and electrostatic interactions.

[0099] S3. Analysis and Visualization of Docking Results Conformation evaluation: After docking, analyze the 30 docking conformations and their corresponding docking scores. Select 1-2 optimal conformations with the highest scores (usually indicating the most stable docking) and reasonable spatial positions for in-depth analysis.

[0100] Combining interface and interaction analysis: In the optimal conformation, the key interactions formed between the FIP-glu and TK1 molecules are precisely identified using the software's "Contact Analysis" tool or manual analysis. This includes: Hydrogen bond: Measure the distance (typically <3.5 Å) and angle between donor and acceptor atoms.

[0101] Salt bridges: recognize residue pairs with opposite charges (such as arginine and aspartic acid / glutamic acid).

[0102] Hydrophobic interactions: Observe the aggregation regions of nonpolar residues.

[0103] Key residue identification: Based on the above interaction analysis, the key amino acid residues involved in the binding were identified. For example, in this embodiment, residues L43, T44, S69, and Q73 of the FIP-glu A chain and residues N24, R27, D88, Q109, and N111 of the B chain were identified, forming a complex interaction network with residues Q20, R38, R42, Y48, R130, E144, R158, and R186 of TK1.

[0104] Results visualization (using PyMOL software): The optimal docking complex structure was loaded into PyMOL, and the FIP-glu and TK1 proteins were displayed in a "cartoon" mode, distinguished by different colors. The key interacting residues identified above were highlighted in a "stick" mode. The distances between key atom pairs were measured and labeled, and hydrogen bonds or ionic interactions were indicated using dashed lines to generate a final high-resolution schematic diagram that visually demonstrates the predicted binding mode of the two proteins.

[0105] The results are as follows Figure 5 As shown, amino acid residues L43, T44, S69, and Q73 of the FIP-glu A chain and amino acid residues N24, R27, D88, Q109, and N111 of the B chain interact with amino acid residues Q20, R42, R38, Y48, R130, E144, R158, and R186 of TK1, respectively.

[0106] Example 7 The effect of FIP-glu on the expression level of thymidine kinase 1 (TK1) protein was detected by Western blot. The specific steps are as follows: S1. Cell Culture and Drug Treatment Cell preparation: Human breast cancer cell lines T-47D and MCF-7 were cultured at 1.5 × 10⁶ cells per well. 6 Cells were seeded at a density of 100 cells per well in 6-well cell culture plates and cultured in RPMI-1640 complete medium containing 10% fetal bovine serum and 1% penicillin-streptomycin antibiotics for 24 hours at 37°C in a 5% CO2 incubator until the cells reached approximately 70% to 80% confluence.

[0107] FIP-glu treatment: Discard the old culture medium and gently wash the cells once with preheated PBS. The experimental group was replaced with fresh complete culture medium containing 1 μg / mL of purified recombinant Ganoderma lucidum immunomodulatory protein (FIP-glu, prepared in Example 1); the control group was replaced with an equal volume of complete culture medium containing PBS. The cells were returned to the incubator and treated for 0 hours (i.e., untreated control), 1 hour, 2 hours, and 4 hours, with three replicates at each time point.

[0108] S2, Total Cell Protein Extraction Cell collection and lysis: Upon reaching each predetermined time point, immediately discard the culture medium and wash the cells twice with pre-chilled PBS. Add 150 μL of pre-chilled RIPA whole-cell lysis buffer (containing 1× protease inhibitor complex and 1× phosphatase inhibitor) to each well and lyse on ice for 30 minutes. During this time, scrape off cells with a cell scraper and transfer the lysis buffer to pre-chilled 1.5 mL centrifuge tubes.

[0109] Protein sample preparation: Centrifuge the lysis buffer at 14,000×g for 15 minutes at 4°C. Carefully aspirate the supernatant into a new tube, avoiding aspiration of precipitate. Determine the protein concentration using the BCA method with a small amount of supernatant. Based on the measured concentration, adjust all samples to a uniform concentration using the lysis buffer. Take a solution containing 20 μg of total protein and mix it with 5×SDS-PAGE protein loading buffer at a volume ratio of 4:1. Vortex thoroughly and heat in a 95°C metal bath for denaturation for 10 minutes. After brief centrifugation, place on ice for later use or store at -20°C.

[0110] S3, SDS-PAGE electrophoresis Prepare a 10% separating gel and a 5% stacking gel. Load the denatured protein samples and pre-stained protein markers into the wells. In 1×Tris-Glycine-SDS electrophoresis buffer, initially perform electrophoresis at a constant voltage of 80 V. After the bromophenol blue indicator enters the separating gel, increase the voltage to 120 V and continue electrophoresis until the bromophenol blue front reaches the bottom of the gel.

[0111] S4, Wet Transfer Coating Activate the PVDF membrane: Immerse the PVDF membrane in methanol for 30 seconds to activate it, and then equilibrate it together with the filter paper and sponge pad in pre-cooled 1× transfer buffer (25 mmol / L Tris, 192 mmol / L Glycine, 20% methanol) for 15 minutes.

[0112] Transfer assembly: Assemble the "sandwich" structure in the transfer clamp in the order of "anode plate → sponge pad → 3 layers of filter paper → gel → PVDF membrane → 3 layers of filter paper → sponge pad → cathode plate" to ensure that there are no air bubbles between the layers.

[0113] Transfer: Place the transfer clamp into the transfer tank and add pre-cooled transfer buffer. Transfer the membrane at a constant current of 300 mA for 90 minutes under ice bath conditions.

[0114] S5. Western Blot Detection Blocking: After the transfer is complete, remove the PVDF membrane and rinse it once quickly with TBST buffer. Immerse the membrane in 2% BSA (dissolved in TBST) and block it by gently shaking on a shaker at room temperature for 2 hours.

[0115] Primary antibody incubation: After blocking, wash the membrane three times with TBST for 5 minutes each time. Incubate the membrane overnight at 4°C with rabbit anti-TK1 polyclonal antibody and GAPDH monoclonal antibody diluted with 2% BSA / TBST.

[0116] Secondary antibody incubation: The next day, the primary antibody was recovered, and the membrane was washed three times with TBST for 10 minutes each time. The membrane was then incubated with the corresponding horseradish peroxidase-labeled secondary antibodies: HRP-labeled goat anti-rabbit IgG (for TK1) and HRP-labeled goat anti-mouse IgG (for GAPDH), both diluted 1:10000 in 2% BSA / TBST, at room temperature for 2 hours.

[0117] S6. Chemiluminescence Development and Result Analysis Development: After incubation with secondary antibody, wash the membrane three times with TBST, 10 minutes each time. Mix equal volumes of ECL chemiluminescent substrate solutions A and B, and evenly coat the protein surface of the membrane. Incubate at room temperature for 1 minute. Use a chemiluminescence imaging system, set an appropriate exposure time, and acquire images.

[0118] Gray-scale analysis: Image analysis software such as ImageJ was used to measure the gray-scale values ​​of each band (TK1 and β-actin). The gray-scale value of the TK1 band was standardized using the gray-scale value of GAPDH as an internal reference (TK1 gray-scale value / corresponding GAPDH gray-scale value).

[0119] The results are as follows Figure 6 As shown, compared with the untreated group (0 hours), after treatment with 1 μg / mL FIP-glu for 1 hour, 2 hours, and 4 hours, the intensity of the TK1 protein band in T-47D and MCF-7 cells gradually decreased over time. Quantitative analysis of grayscale values ​​(corrected using β-actin as an internal reference) confirmed that the TK1 protein expression level was significantly downregulated after 4 hours of treatment. This result directly demonstrates that FIP-glu can effectively reduce the TK1 protein expression level in breast cancer cells in a time-dependent manner.

[0120] Example 8 After acclimatizing 6-8 week old Balb / c-nu nude mice for one week, T-47D cell line was subcutaneously injected into the right side of each mouse at a dose of 2 × 10⁻⁶ cells. 6 Individual cells. When the tumor diameter is 3-5 mm (volume approximately 80-100 mm²), 3At that time, mice were randomly divided into four groups of six each, based on tumor volume and animal weight. The control group was intraperitoneally injected with PBS; the low-dose group was intraperitoneally injected with 40 mg / kg FIP-glu; the high-dose group was intraperitoneally injected with 80 mg / kg FIP-glu; and the positive control group was intraperitoneally injected with 10 mg / kg paclitaxel. The administration period was 14 days, with administration every two days. Tumor size and mouse weight were measured on days 0, 2, 4, 6, 8, 10, 12, and 14. The tumor volume was calculated as follows: Tumor volume = (L × W) / (L × W) 2 (L) / 2, where L is the length of the tumor and W is the width of the tumor. On day 15, the tumor-bearing mice were sacrificed, and the tumor samples were taken, weighed, and photographed.

[0121] like Figure 7 As shown in Figure A, the tumor growth curves indicate that, compared to the PBS control group, the tumor growth rate in the FIP-glu treatment group (especially the high-dose group of 80 mg / kg) was significantly slowed, and this slowed growth in a dose-dependent manner. Figure 7 As shown in Figure B, the solid tumor image clearly demonstrates that the tumor mass significantly shrank after FIP-glu treatment; as... Figure 7 As shown in Figure C, tumor quality statistics confirm that high-dose FIP-glu can significantly reduce tumor weight; Figure 7 As shown in Figure D, the quantitative data on tumor volume further support the above conclusions, showing that FIP-glu can effectively inhibit tumor volume growth; as Figure 7 As shown in Figure E, the mouse body weight did not change significantly compared to the control throughout the entire administration process, indicating that FIP-glu has no toxicity to mice. Overall, the results demonstrate that FIP-glu effectively inhibits breast cancer growth in vivo, with high-dose efficacy approaching that of the positive control drug paclitaxel; and it also exhibits safety.

[0122] The above are all preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Therefore, all equivalent changes made in accordance with the structure, shape and principle of the present invention should be covered within the scope of protection of the present invention.

Claims

1. The application of a Ganoderma lucidum immunomodulatory protein in the preparation of a product targeting thymidine kinase 1.

2. The application of the Ganoderma lucidum immunomodulatory protein according to claim 1 in the preparation of a product targeting thymidine kinase 1, characterized in that, The Ganoderma lucidum immunomodulatory protein binds to thymidine kinase 1.

3. The application of the Ganoderma lucidum immunomodulatory protein according to claim 1 or 2 in the preparation of a product targeting thymidine kinase 1, characterized in that, The amino acid sequence of the Ganoderma lucidum immunomodulatory protein is shown in SEQ ID NO:

1.

4. The application of the Ganoderma lucidum immunomodulatory protein according to claim 3 in the preparation of a product targeting thymidine kinase 1, characterized in that, The amino acid residues L43, T44, S69, and Q73 of the A chain of the Ganoderma lucidum immunomodulatory protein and the amino acid residues N24, R27, D88, Q109, and N111 of the B chain interact with the amino acid residues Q20, R38, R42, Y48, R130, E144, R158, and R186 of TK1, respectively.

5. The use of a Ganoderma lucidum immunomodulatory protein according to any one of claims 1-4 in the preparation of a product for targeting thymidine kinase 1, characterized in that, The Ganoderma lucidum immunomodulatory protein can be used to reduce the expression level of TK1.

6. A composition for inhibiting thymidine kinase 1, characterized in that, The composition comprises the Ganoderma lucidum immunomodulatory protein according to any one of claims 1-5.

7. A composition for inhibiting tumors, characterized in that, The composition comprises the Ganoderma lucidum immunomodulatory protein according to any one of claims 1-5.

8. The composition according to claim 7, characterized in that, The composition is a food, health food, or drug.

9. The composition according to claim 7 or 8, characterized in that, The tumor is breast cancer.

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