ACT structural domain protein derived from coprobacterium rodent and application of ACT structural domain protein in preparation of anti-tumor and anti-radiation drugs

Abstract

The invention relates to the technical field of biological medicines, in particular to an ACT structural domain protein derived from coprobacterium rodent and application of the ACT structural domain protein in preparation of anti-tumor and anti-radiation medicines. The invention provides ACT structural domain protein. The amino acid sequence of the ACT structural domain protein is shown as SEQ ID NO: 2. As a functional protein derived from intestinal probiotics, the ACT structural domain protein provided by the invention provides a new molecular probe and a research object for revealing an action mechanism of intestinal flora in tumor generation and development. The protein not only provides an entry point for research on interaction of intestinal microecology and tumor immunity, but also lays a foundation for development of novel biomarkers and drug targets based on microbiome, and has important scientific value in the research field of tumor biology and microorganism-host interaction.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to an ACT domain protein derived from *Bacillus rodentii* and its application in the preparation of antitumor and antiradiation drugs. Background Technology

[0002] The clinical treatment of tumors, especially solid tumors such as colorectal cancer, is facing the dual challenges of limited response rates to existing therapies and high drug resistance. Taking colorectal cancer as an example, its incidence and mortality rates rank among the highest of malignant tumors, and the patient population is showing a trend towards younger ages. Although surgery, radiotherapy, chemotherapy, targeted therapy, and immunotherapy constitute the current main treatment system, most advanced-stage patients eventually develop resistance or intolerance to these therapies, leading to disease progression. This highlights the urgent need for novel anti-tumor strategies with entirely new mechanisms of action that can overcome the limitations of existing therapies.

[0003] The limitations of existing therapies have spurred the exploration of new targets and molecules for combating tumors. In recent years, the interaction between the gut microbiota and tumors has become a research hotspot, with probiotics being found to potentially enhance the effects of chemotherapy or immunotherapy by modulating the tumor immune microenvironment. However, the direct application of live bacterial preparations to tumor treatment faces significant obstacles: the colonization efficiency of strains in the complex human gut is unstable, posing potential translational safety risks (such as infections caused by bacterial translocation), and the significant differences in baseline gut microbiota among individuals make it difficult to predict and standardize efficacy. These fundamental shortcomings severely hinder the translation of probiotic therapy from basic research to stable and controllable clinical applications.

[0004] Therefore, research focus is shifting from intact live bacteria to the specific effector molecules that enable their functions. A key strategy is to bypass the uncertainties of live bacteria applications and directly identify and utilize the functional molecules (such as secreted proteins and metabolites) secreted or released by probiotics that have direct biological activity. These molecules, as the "executors" of probiotic functions, may have advantages such as well-defined components, clear mechanisms of action, and more controllable pharmacokinetics, providing a more ideal starting point for the development of novel drugs.

[0005] In summary, systematically screening and identifying key active molecules from probiotics that can directly inhibit tumor growth or regulate anti-tumor immunity, and elucidating their precise molecular targets and mechanisms of action, is not only crucial for a deeper understanding of the scientific principles of microbe-host interactions in tumor treatment, but also an essential path to transforming gut microbiota research findings into next-generation, highly effective, low-toxicity, and precisely targeted innovative biological agents or treatment strategies. Summary of the Invention

[0006] To address the problems of existing technologies, this invention provides the application of ACT domain proteins derived from *Corynebacterium rodentum* in the preparation of anticancer agents, agents that target and inhibit mitochondrial glutamate transporter SLC25A22, enhance host antitumor immune responses, and agents that prevent ionizing radiation damage; and the application of ACT domain proteins in the preparation of drugs for the research and / or treatment of tumors such as CRC, radiation-induced diseases, and organ transplantation immunity.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides an ACT domain protein, the amino acid sequence of which is shown in SEQ ID NO:2.

[0008] The present invention provides a nucleic acid molecule encoding the ACT domain protein, the nucleotide sequence of which is shown in SEQ ID NO:1.

[0009] This invention provides the application of the ACT domain protein in the preparation of drugs for treating cancer.

[0010] Preferably, the ACT domain protein functions by targeting and inhibiting the mitochondrial glutamate transporter SLC25A22.

[0011] Preferably, the cancer is rectal cancer.

[0012] This invention provides the application of the ACT domain protein in the preparation of drugs for enhancing the host's anti-tumor immune response.

[0013] This invention provides the application of the ACT domain protein in the preparation of drugs for preventing and treating ionizing radiation damage.

[0014] This invention provides the application of the ACT domain protein in the preparation of drugs for treating immune-related diseases in organ transplantation.

[0015] The present invention provides a pharmaceutical composition comprising the ACT domain protein and a pharmaceutically acceptable carrier.

[0016] The beneficial effects of this invention are as follows: 1. Providing New Research Tools and Functional Molecules: The ACT domain protein of this invention, a functional protein derived from gut probiotics (Bacillus rodentata), provides a new molecular probe and research object for elucidating the role of gut microbiota in tumor development and progression. This protein not only provides an entry point for studying the interaction between gut microbiota and tumor immunity, but also lays the foundation for developing novel microbiome-based biomarkers and drug targets, possessing significant scientific value in the fields of tumor biology and microbiome-host interaction research.

[0017] 2. Targeted inhibition of the mitochondrial glutamate metabolism pathway: The ACT domain protein of this invention can specifically bind to and inhibit the mitochondrial glutamate transporter SLC25A22, thereby interfering with the generation of α-ketoglutarate (α-KG), an intermediate product of glutamate metabolism, in tumor cells. This mechanism of action directly affects the energy metabolism and biosynthetic capacity of tumor cells, providing a new drug candidate molecule for developing targeted therapeutic strategies against tumor metabolic reprogramming, with a clear molecular mechanism and high targeting specificity.

[0018] 3. Enhances the body's anti-tumor immune response: Experiments have shown that ACT domain proteins can significantly promote the infiltration of immune cells into the tumor microenvironment, including increasing CD45. + Total number of immune cells, dendritic cells, tumor-associated macrophages, and CD4 + and CD8 + The number of T cells was also observed. This indicates that the protein not only directly inhibits tumor growth but also enhances the host's own anti-tumor immunity by regulating the tumor immune microenvironment, providing a new synergistic pathway for combined immunotherapy.

[0019] 4. Expanding its application potential in radiation protection and transplant immunology: In addition to its anti-tumor effects, the ACT domain protein also shows significant effects in preventing and treating ionizing radiation damage, improving the survival rate of mice after irradiation. Furthermore, this protein also has potential applications in organ transplant immunomodulation. Therefore, this invention is not limited to tumor treatment but can be extended to multiple clinical fields such as radiation disease protection and transplant immune tolerance induction, demonstrating its multifunctional and multi-application development potential.

[0020] In summary, this invention not only provides a novel protein drug candidate molecule with a clear tumor-suppressing mechanism, but also shows broad application prospects in the fields of immune regulation, radioprotection, and transplantation immunology, and has important scientific research value and clinical translation potential. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0022] Figure 1 The image shows the solid tumors (A) inhibited by *F. rodentium* and *F. rodentium* culture supernatant in Example 1, along with the tumor volume (B) and weight (C) statistics. Figure 2This refers to the identification of ACT domain proteins in the supernatant of *F. coli* culture medium in Example 2; Figure 3 The results show the anti-tumor effect of the recombinant ACT domain protein (rACT protein) in the subcutaneous xenograft mouse model in Example 2, with the tumor solid image (A), tumor volume (B), and weight (C) statistics respectively. Figure 4 It is the receptor identified in Example 3 that binds to the ACT domain protein; Figure 5 This refers to the binding of the ACT domain protein to the mitochondrial glutamate transporter SLC25A22 in Example 3 (A); and the interaction of the rACT protein with SLC25A22-mediated glutamate metabolic intermediates. -ketoglutaric acid ( The effect of -KG) level (C); Figure 6 This refers to the effect of rACT protein on the number of immune cells infiltrating the CRC tumor microenvironment in Example 4; Figure 7 In Example 5, CD117 was reinfused via the tail vein. + Cells, or CD117 cells carrying the ACT protein + Survival rate of wild-type mice after cell irradiation. Detailed Implementation

[0023] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0024] Example 1

[0025] The rodent bacteria used in the embodiments of the present invention are rodent bacteria. F.rodentium The mice were purchased from Beina Biotechnology Co., Ltd.; wild-type mice were purchased from Changzhou Cavens Laboratory Animal Co., Ltd. The modified PYG medium was purchased from Shandong Top Biotechnology Co., Ltd. (reference patent: 2023115007144). Its components are: 5.0 g tryptone, 5.0 g peptone, 10.0 g yeast extract, 5.0 g beef extract, 5.0 g glucose, 2.0 g potassium hydrogen phosphate, 1.0 mL Tween 80, 0.01 g CaCl2-2H2O, 0.02 g MgSO4-7H2O, 0.04 g potassium hydrogen phosphate, 0.04 g potassium dihydrogen phosphate, 0.4 g sodium bicarbonate, 0.08 g sodium chloride, and 1.0 mg resazurin.

[0026] Example 1: *Corynebacterium valerate* F.rodentium and its culture medium supernatant intervention in subcutaneous xenograft tumor models

[0027] 1. Establishment of MC-38 xenograft mouse model: 6-8 week old C57BL / 6 female mice (Changzhou Cavens Laboratory Animal Co., Ltd.) were shaved on their backs, and the shaved areas were disinfected with alcohol. MC-38 cells (Shanghai Fuheng) were digested with 1 mL of 0.25% trypsin for 1 min, and the cells were collected. After centrifugation at 500 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in 1 mL of phosphate-buffered saline (0.01 M, pH 7.2-7.4, Nanjing Shenghang Biotechnology Co., Ltd.), repeated twice. The cells were then resuspended in RPMI complete medium. 1×10⁶ cells were subcutaneously inoculated on the ipsilateral side of the back of each mouse. 6 Each cell.

[0028] 2. *Factobacillus rodentii* F.rodentium Culture and model intervention: Bacillus faecalis F.rodentium Culture (refer to patent: 2023115007144); until the tumor volume in step 1 reaches 50 mm. 3 At the same time, tumor-bearing mice were given 100 mg of medication every other day. L control medium (Ctrl group), *Corynebacterium tumefaciens* (100...) L contains 1.5 × 10 7 CFU / each, Fr group), 100 L-type *Factobacillus cuspidatum* culture supernatant (FCM group) or 100 L is the high-pressure sterilized rodent bacteria solution (containing rodent bacteria and rodent bacteria culture supernatant, HFr group), and the long diameter, short diameter, and height of the tumor are measured with vernier calipers and recorded as a, b, and c respectively. The tumor volume is calculated according to the formula: V=a×b×c.

[0029] Depend on Figure 1 It can be known that: F.rodentium live bacteria group and F.rodentium Treatment with the culture supernatant significantly inhibited the growth of subcutaneous tumors in tumor-bearing mice. Figure 1 A is a photograph of the tumor; B is the tumor volume; C is the tumor weight.

[0030] Example 2: *Bacillus faecalis* F.rodentium Antitumor activity of ACT domain proteins derived from this source

[0031] 1. SDS-PAGE electrophoresis: SDS-PAGE electrophoresis was performed on the control medium and the supernatant of *F. dentata*. The procedure is briefly described below: Protein lysis buffer containing 1% PMSF, 1% PI, and 1% SDS was added to modified PYG medium and the supernatant of *F. dentata* collected by centrifugation (4℃, 13200 rpm, 10 min), and mixed thoroughly. 12 μL of protein lysis buffer was taken from each group, and 3 μL of 5× loading buffer (Yisheng Biotechnology (Shanghai) Co., Ltd.) was added. After vortexing and brief centrifugation, the mixture was heated at 95℃ for 3 min and centrifuged at 4℃, 13200 rpm for 15 s to ensure no residual protein lysis buffer remained on the tube wall. The precast gel (GenScript Biotechnology Co., Ltd.) was fixed in the electrophoresis tank (Bio-Live Biomedical Products Co., Ltd.), and sufficient MOPS electrophoresis buffer (GenScript Biotechnology Co., Ltd.) was poured in. The gel comb was gently removed. 15 μL of protein marker (Novozymes) or protein sample was added to the wells, and electrophoresis was performed at 80 V.

[0032] 2. Gel fixation: After electrophoresis, place the gel in 200 mL of fixative (40% methanol / 10% acetic acid by volume) and gently shake at room temperature for 1 hour.

[0033] 3. Staining: Discard the fixative, add 200 mL of 0.1% Coomassie Brilliant Blue R-250 staining solution (R-250 dissolved in an aqueous solution of 40% methanol / 10% acetic acid), and gently shake and stain for 2 hours at room temperature.

[0034] 4. Decolorization: After dyeing, rinse with decolorizing solution (a 10% methanol / 10% acetic acid aqueous solution by volume) overnight.

[0035] 5. Mass Spectrometry Detection: The target band was cut and placed into a 1.5 mL EP tube in a clean bench, and then sent to Xuzhou Pubo Biotechnology Co., Ltd. for mass spectrometry detection. The detection results are as follows: Figure 2 As indicated by the A mark in the middle.

[0036] 6. Preparation of recombinant ACT domain protein: The gene sequence of the ACT domain protein (with a His tag at the C-terminus) from *F. rotavirus* was inserted into the pET28a plasmid.

[0037] Gene sequence of ACT domain protein in *Corynebacterium rota* (SEQ ID NO: 1): AAGGCCGTGATCACAGTTTGGGCGCCGACAGAACAGGCATTCTGGCTCTGGTGGCCTCTGAGTGCGCCGCCAGAAACTGCAATATCCTGGACGTGTCCCAGACCATCGTGGACGGCGTGTTCACCATGACCA TGATCGCCGACATGGACGGCCTGACCGAGAGCTTTGAAGCCTTCGCCGACCACATGGAAACCGTGGGAGCCGAGAATGACCTGGCCGTCAGAGTGATGAACCGGGCCATCTTCAAGGCCATGCACAGCATCTAG The amino acid sequence of the ACT domain protein of *Bacillus coccidia* (SEQ ID NO: 2): KAVITVVGADRTGILALVASECAARNCNILDVSQTIVDGVFTMTMIADMDGLTESFEAFADHMETVGAENDLAVRVMNRAIFKAMHSI Transformation was performed using a 42℃ heat shock method, with the competent strain being TOP10. Single colonies were selected for growth, and positive clones were screened by PCR amplification using bacterial detection primers. After confirming the positive plasmid's correctness through sequencing, ACT domain protein expression was induced. The procedure was briefly described as follows: The ACT-His-pET28a plasmid was transformed into the expression host bacterium Rosetta(DE3) (volume ratio 1:100, i.e., 1 mL of plasmid added to 100 mL of Rosetta(DE3) competent bacteria), and placed on ice for 20 min; a 42℃ heat shock was performed for 90 s, followed by immediate placement on ice for 3 min; 500 mL of LB medium was added; the culture was incubated at 37℃ for 2 h; 200 mL of the bacterial culture was plated onto LB agar plates containing 50 mg / mL kanamycin and incubated upside down at 37℃ overnight; single colonies were picked from the plates and inoculated into fresh LB liquid medium (containing 50 mg / mL kanamycin) and incubated at 37℃ for approximately 6 hours. OD... 600The pH was approximately 0.6. IPTG was added to a final concentration of 0.5 mM for induction for 16 hours. Cells were collected by centrifugation (6000 rpm, 5 min), and the supernatant was discarded. The cell pellet was resuspended in 100 mL of ice-cold Buffer D (20 mM Tris-HCl pH 8.0, 50 mM NaCl, 0.1% Triton-100), and cells were lysed by sonication (600 W-800 W, 30 min). After centrifugation (10000 rpm, 15 min), the supernatant was collected and passed through a Ni-IDA column. Wash 1: 200 mL Buffer E (20 mM Tris-HCl pH 8.0, 500 mM NaCl, 0.1% Triton X-100) was used to wash the Ni-IDA medium. Wash 2: 20 mL Buffer D1 (20 mM Tris-HCl pH 8.0, 50 mM NaCl, 0.1% Triton-100) was used to wash the Ni-IDA medium. Wash 3: 20 mL Buffer D2 (20 mM Tris-HCl pH 8.0, 50 mM NaCl, 0.1% Triton-100) was used to wash the Ni-IDA medium. Wash the Ni-IDA medium with Tris-HCl (pH 8.0), 50 mM NaCl, 0.1% Triton X-100, and 20 mM imidazole. Elute the Ni-IDA medium with an appropriate amount of Buffer C (20 mM Tris-HCl (pH 8.0), 50 mM NaCl, 0.1% Triton X-100, and 250 mM imidazole). The supernatant can be purified to the target protein. Dialyze the eluted target protein to PBS three times. After dialysis, centrifuge and collect the supernatant. Measure the protein concentration (1 mg / mL) using a BCA protein assay kit and aliquot for later use.

[0038] 7. Western blot verification: The purified protein was subjected to Western blot detection, which is briefly described as follows: PMSF and PI (volume ratio 1:1000) were added to 10 μL of protein solution from step 6 and mixed thoroughly. Then add 2 μL of 5× loading buffer (Yisheng Biotechnology (Shanghai) Co., Ltd.), vortex to mix, briefly centrifuge, heat at 95℃ for 3 min, and centrifuge at 4℃ (13200 rpm) for 15 s to ensure no residual protein lysis buffer remains on the tube wall; fix the precast gel (GenScript Biotechnology Co., Ltd.) in the electrophoresis tank (Bio-Ray Biotech Co., Ltd.), pour in sufficient MOPS electrophoresis buffer (GenScript Biotechnology Co., Ltd.), and gently pull out the gel comb; add 15 μL of protein marker (Novozymes) or protein sample to the gel wells, and perform electrophoresis at 80 V; after electrophoresis, remove the gel plate and place the separating gel into the transfer buffer, while soaking the prepared nitrocellulose membrane (Zhenjiang Aibimeng Biotechnology Co., Ltd.) and thick filter paper in the transfer buffer for 15 min; then place the thick filter paper, NC membrane, separating gel, and another piece of thick filter paper in a semi-dry transfer instrument (Bio-Ray Biotech Co., Ltd.), and transfer at a constant current of 180 mA for 60 min; after transfer, wash the membrane with PBST, 10 After 1 minute, add 5 mL of 5% skim milk and 2.5 μL of primary antibody to the incubation box containing the nitrocellulose membrane, and incubate overnight at 4°C with gentle shaking. The next day, wash the NC membrane with PBST for 5-10 minutes, repeating three times. Discard the supernatant, add 5 mL of 5% skim milk and 1 μL of secondary antibody to the incubation box containing the nitrocellulose membrane, and incubate at room temperature for 2 hours. Wash the NC membrane with PBST for 10 minutes, repeating three times. Add freshly prepared chromogenic buffer (Suzhou Xinsaimei Biotechnology Co., Ltd.), and develop the image using a Bio-Rad chemiluminescence imaging system (Bio-Rad Life Sciences Co., Ltd.). Store the image for subsequent analysis. The detection results are as follows: Figure 2 As shown in B.

[0039] from Figure 2 As can be seen, the supernatant of the rodent bacteria culture medium contains ACT domain protein; Western blotting confirmed the presence of recombinant ACT domain protein.

[0040] 8. Antitumor effect of recombinant ACT domain protein: An MC-38 xenograft mouse model was established according to step 1 of Example 1. The tumor volume reached 50 mm² in step 1. 3 During the experiment, model mice were randomly divided into two groups. Every one day, tumor-bearing mice were injected via tail vein with either 100 μL of PBS or 100 μL of recombinant ACT domain protein (5 mg / kg). The long axis, short axis, and height of the tumor were measured using calipers and recorded as a, b, and c, respectively. The tumor volume was calculated using the formula: V = a × b × c. After the experiment, the mice were euthanized, and the tumor tissue was separated and weighed.

[0041] Depend on Figure 3 It can be seen that the recombinant ACT domain protein effectively inhibits the growth of MC-38 tumors ( Figure 3 AB) and tumor weight ( Figure 3 C). In summary: Recombinant ACT domain proteins can effectively inhibit the growth of CRC tumors.

[0042] Example 3: Identification and analysis of receptors that bind to the ACT domain protein and their effects on downstream pathways of the receptors.

[0043] 1. Overexpression of pcDNA3.1-ACT domain protein in HEK293T cells: Approximately 5 × 10⁻⁶ cells were overexpressed. 5 HEK293T cells were seeded into six-well plates, ensuring even distribution, and incubated overnight. The next day, cells were harvested and observed under a microscope for growth and distribution. When the cell density reached approximately 75%, transfection was performed. 2 μg of the empty and ACT domain protein particles were added to 1.5 mL centrifuge tubes and mixed thoroughly. Then, 200 μL of jetPRIME buffer was added to the mixed plasmid. 4 μL of jetPRIME Transfection reagent was added, gently mixed, and incubated at room temperature for 10 min. The mixture was then gently added dropwise to the HEK293T cell culture medium. On the third day, cell growth was observed under a microscope. Once good cell growth was confirmed, the culture medium was discarded, and the cells were collected.

[0044] 2. SDS-PAGE electrophoresis: Add protein lysis buffer containing 1% PMSF, 1% PI, and 1% SDS to the cells scraped and collected in step 1, and mix thoroughly. Take 12 μL of protein lysis buffer from each group, add 3 μL of 5× loading buffer (Yisheng Biotechnology (Shanghai) Co., Ltd.), vortex to mix, briefly centrifuge, heat at 95℃ for 3 min, and centrifuge at low temperature (4℃, 13200 rpm) for 15 s to ensure no protein lysis buffer residue remains on the tube wall; fix the precast gel (GenScript Biotechnology Co., Ltd.) in the electrophoresis tank (Bio-Ray Biotech Co., Ltd.), pour in sufficient MOPS electrophoresis buffer (GenScript Biotechnology Co., Ltd.), and gently pull out the gel comb; add 15 μL of protein marker (Novozymes) or protein sample to the gel wells, and perform electrophoresis at 80 V for 30 min.

[0045] 3. Gel fixation: After electrophoresis, place the gel in 200 mL of fixative (40% methanol / 10% acetic acid by volume) and gently shake at room temperature for 1 hour.

[0046] 4. Staining: Discard the fixative, add 200 mL of 0.1% Coomassie Brilliant Blue R-250 staining solution (R-250 dissolved in an aqueous solution of 40% methanol / 10% acetic acid), and gently shake and stain for 2 hours at room temperature.

[0047] 5. Decolorization: After dyeing, rinse with decolorizing solution (a 10% methanol / 10% acetic acid aqueous solution by volume) overnight.

[0048] 6. Mass spectrometry detection: The target band was cut in a clean bench and placed into a 1.5 mL EP tube, and then sent to Xuzhou Pubo Biotechnology Co., Ltd. for mass spectrometry detection.

[0049] 7. Bioinformatics Analysis: The mass spectrometry sequences were uploaded to the online platform of Shanghai Meiji Biomedical Technology Co., Ltd. for bioinformatics analysis. Results are as follows: Figure 4 As shown in Figure A: 63 membrane proteins were identified from the ACT domain proteome. Among the 63 membrane proteins, GO enrichment analysis suggested that the mitochondrial glutamate transporter SLC25A22 was the main candidate. Figure 4 B). Using GRAMM to predict the likelihood of interaction between the ACT domain protein and the SLC25A22 protein (B). Figure 5 A).

[0050] 8. Packaging lentiviruses and establishment of stable transgenic strains (completed in a p2 laboratory): Approximately 5 × 10 5 HEK293T cells were seeded into six-well plates, ensuring even distribution, and incubated overnight. The next day, cells were harvested and observed under a microscope for growth and distribution. When the cell density reached approximately 75%, transfection was performed. Tet-on-ACT domain protein particles and packaging plasmids PSPAX2 and PMD.2G were added to a 1.5 mL centrifuge tube at a ratio of 4:3:1 (total 4 μg) and mixed thoroughly. Then, 200 μL of jetPRIME buffer was added to the mixed plasmids, followed by 4 μL of jetPRIME Transfection reagent. The mixture was gently mixed and incubated at room temperature for 10 min. The mixture was then gently added dropwise to the HEK293T cell culture medium. On the third day, cell growth was observed under a microscope. After adding 1 mL of fresh culture medium, the cells were incubated in a cell culture incubator. On the fourth day, all supernatant was collected and centrifuged at low speed to remove residual cells. Centrifuge the virus-containing supernatant at 4°C for 3 hours. Discard the upper 80% of the supernatant from the centrifuge tube. Aliquot the remaining virus-containing supernatant and freeze it at -80°C for later use. Approximately 5 × 10⁻⁵ cells in good condition... 5 / mL of human colon cancer cell line HCT-8 (Shanghai Fuheng) was seeded into six-well plates, with 2 mL of culture medium added to each well. The next day, the cell culture medium was discarded, and after washing with PBS, the infection complex was added to the cell wells (the infection complex was prepared by mixing 20 μL of lentivirus with the lentivirus containing the lentivirus of ...

[0051] 9. Western blot verification of ACT domain protein expression in stable transfectants: Approximately 5 × 10⁻⁶ protein molecules were used. 5HCT-8-ACT cells were seeded into six-well plates, ensuring even distribution, and incubated overnight. The next day, 1 μg / mL doxycycline (DOX) was added for induction for 48 h. 48 hours later, cells were scraped and collected using a protein lysis buffer containing 1% PMSF, 1% PI, and 1% SDS, and thoroughly mixed. Western blotting was then performed, briefly as follows: 2 μL of 5× loading buffer (Yisheng Biotechnology (Shanghai) Co., Ltd.) was added to 10 μL of the above protein solution, vortexed, briefly centrifuged, heated at 95℃ for 3 min, and centrifuged at 4℃, 13200 rpm for 15 s to ensure no residual protein lysis buffer remained on the tube wall; the pre-cast gel (GenScript Biotechnology Co., Ltd.) was fixed in the electrophoresis tank (Bio-Live Biomedical Products Co., Ltd.), and sufficient MOPS electrophoresis buffer (GenScript Biotechnology Co., Ltd.) was poured in. The gel comb was gently removed; 15 μL of protein marker (Novozymes) or protein sample was added to the wells, and electrophoresis was performed at 80 V; after electrophoresis, the gel plate was removed, and the separating gel was placed in the transfer buffer. Simultaneously, a pre-prepared nitrocellulose membrane (Zhenjiang Aibimeng Biotechnology Co., Ltd.) and thick filter paper were soaked in the transfer buffer for 15 minutes. min; sequentially lay thick filter paper, NC membrane, separating gel, and another piece of thick filter paper in a semi-dry transfer instrument (Bio-Ray Biotech Co., Ltd.), transfer at a constant current of 180 mA for 60 min; after transfer, wash the membrane with PBST, and after 10 min, add 5 mL of 5% skim milk and 2.5 μL of primary antibody to the incubation box containing the nitrocellulose membrane, and incubate overnight at 4°C with gentle shaking; the next day, take the NC membrane and wash it with PBST for 5-10 min, repeat three times; discard the supernatant, add 5 mL of 5% skim milk and 1 μL of secondary antibody to the incubation box containing the nitrocellulose membrane, and incubate at room temperature for 2 h; take the NC membrane and wash it with PBST for 10 min, repeat three times; add freshly prepared chromogenic solution (Suzhou Xinsaimei Biotechnology Co., Ltd.), develop using a Bio-Ray chemiluminescence imaging system (Bio-Ray Biotech Co., Ltd.), and store the image for subsequent analysis.

[0052] 10. Protein immunoprecipitation verification: approximately 3 × 10 5HCT-8-ACT cells were seeded at 1 / mL in p100 culture dishes, ensuring even distribution, and incubated overnight. The next day, different concentrations (0 μg / mL, 1 μg / mL) of doxycycline were added for induction for 48 h. After 48 h, cells were scraped and collected using RIPA protein lysis buffer (weak) containing 1% PMSF and 1% PI, and thoroughly mixed. After incubation at 4°C on a shaker for 30 min, the cells were centrifuged at low temperature (4°C, 13200 rpm) for 10 min, and the supernatant was collected. After determining protein concentration using the Bradford kit, add 5×SDS loading buffer to 50 mg (approximately 1% v / v) of protein sample and label it "Input". Add anti-Flag antibody to the remaining approximately 1 mg of protein sample at a ratio of 1 µg / mg and incubate overnight at 4°C with shaking. The next day, add 20 µL of protein A / G to each tube, incubate at 4°C with shaking for 3 h, and centrifuge at 2000 rpm for 2 min at 4°C. Discard the supernatant, add 1 mL of 0.1% NP-40 lysis buffer (containing 1 mM PMSF), and gently wash 3 times, centrifuging at 2000 rpm for 3 min each time. After the last wash, completely remove the washing buffer, add 20 µL of 1×SDS loading buffer, heat at 95°C for 3 min, then centrifuge at low temperature (4°C, 13200 rpm) for 15 s. Then, perform Western blotting according to step 8. The detection results are as follows: Figure 5 As shown in Figure B: The ACT domain protein binds to the glutamate transporter SLC25A22.

[0053] 11. rACT protein against -ketoglutaric acid ( The effect of -KG) level: approximately 5×10 5 HCT-8 cells were resuspended in low-glucose DMEM medium at a concentration of 0 μg / mL and seeded into six-well plates, ensuring even cell distribution. The plates were incubated overnight. The following day, 0 μg / mL or 1 μg / mL rACT protein was added, and the plates were incubated again. After 24 hours, the cells were analyzed using... - Ketoglutarate content assay kit (Jiangsu Aidisheng Biotechnology Co., Ltd.) for the detection of ketoglutarate content in cells -KG level. Test results as follows Figure 5 As shown in C: ACT domain proteins can reduce the cell's... -KG level.

[0054] Example 4: Effect of rACT protein on the number of immune cells infiltrating the CRC tumor microenvironment

[0055] 1. Preparation of single-cell suspension of tumor tissue: rACT protein was established and administered to MC-38 tumor-bearing mice according to step 8 of Example 2. At the end of the experiment, the mice were euthanized, the tumor tissue was isolated, necrotic and fatty areas were removed, and the tumor tissue was cut into 1-3 mm³ pieces with surgical scissors; 5 mL of DMEM complete medium containing 1×Collagenase II and 1×DNase I was added, and the mixture was vortexed at 37°C for 60 min; every 20 min, the cells were gently pipetted 20-30 times with a 10 mL pipette to aid dissociation; then, 2 mMEDTA-PBS was added to 10 mL to stop the enzyme digestion, and the cells were passed through 70 mm and 40 mm sieves to remove undigested fragments; the cells were collected by centrifugation at low temperature (4°C, 4000 rpm) for 5 min, the supernatant was discarded, and the cells were resuspended in PBS for later use.

[0056] 2. Antibody labeling: The cell suspension obtained in step 1 was labeled with different fluorescently labeled anti-mouse CD45, F4 / 80, CD8 and TCR antibodies. Monoclonal antibody (1:200 dilution, BD) was incubated at 4°C in the dark. After 20 min, the cells were washed with 1 mL of phosphate buffer (0.01 M, pH 7.2-7.4, Nanjing Shenghang Biotechnology Co., Ltd.), centrifuged at low speed (25°C, 1000 rpm, 5 min) to collect cell clusters, and then resuspended in 3 mL of phosphate buffer (0.01 M, pH 7.2-7.4, Nanjing Shenghang Biotechnology Co., Ltd.) for later use. 3. Flow cytometry analysis: Turn on the flow cytometer (FACSAria III, BD), open the instrument's built-in software (BDFACSDiva Software) and all lasers, and set the parameters to analyze the immune cells infiltrating the tumor tissue. The test results are as follows: Figure 6 As shown: Compared with the control group, CD45 infiltration in the tumor microenvironment was higher in the rACT treatment group. + Increased cell number ( Figure 6 A), CD45 + In cells, DC cells ( Figure 6 BE), tumor-associated macrophages (F4 / 80) + () Figure 6 FH), CD4 + and CD8 + The number of T cells increased ( Figure 6 IL).

[0057] Example 5: Effect of ACT domain protein on survival rate of wild-type mice after irradiation

[0058] 1. Collection of bone marrow cells: Wild-type mice were CO2 euthanized and soaked in 75% ethanol for 5 min. The mice were then removed and placed on a sterile operating table, ventral side up. The tibia and femur were separated and removed, and the surrounding tissues were further separated and removed. The tibia and femur were then placed in a cell culture dish containing 2 mL of RPMI-1640 medium containing penicillin-streptomycin (Gibco) and 10% FBS (Gibco). The ends of the tibia and femur were cut open with ophthalmic scissors. Then, 1 mL of complete culture medium was drawn up with a syringe and used to rinse the bone marrow cells from one end of the bone into a 15 mL sterile centrifuge tube. Collect single-cell suspensions, centrifuge at low speed (25℃, 800 rpm) for 5 min to obtain cell pellets, resuspend in erythrocyte lysis buffer (Formax Biotechnology Co., Ltd.), mix well and let stand for 1 min to allow erythrocytes to fully lyse, add an equal volume of phosphate buffer (0.01 M, pH 7.2~7.4, Nanjing Shenghang Biotechnology Co., Ltd.) to terminate lysis, centrifuge at low speed (25℃, 800 rpm) for 5 min, and resuspend in 2 mL of phosphate buffer (0.01 M, pH 7.2~7.4, Nanjing Shenghang Biotechnology Co., Ltd.) for later use.

[0059] 2. Antibody labeling: The cell suspension obtained in step 1 was mixed with mouse CD117 antibodies. + Monoclonal antibody (1:200 dilution, BD) was incubated at 4°C for 20 min. After that, the cells were washed with 1 mL of phosphate buffer (0.01 M, pH 7.2-7.4, Nanjing Shenghang Biotechnology Co., Ltd.), centrifuged at low speed (25°C, 1000 rpm, 5 min) to collect cell clusters, and then resuspended in 3 mL of phosphate buffer (0.01 M, pH 7.2-7.4, Nanjing Shenghang Biotechnology Co., Ltd.) for later use. 3. Flow cytometry sorting to collect CD117 cells rich in hematopoietic stem cells + Cells: Turn on the flow cytometer (FACSAriaIII, BD), open the instrument's built-in software (BD FACSDiva Software) and all lasers, adjust the flow rate and set the sorting parameters (Drop Delay: 26.53, left rack value: 58). Analysis will yield CD117. + Two groups of cells were sorted and CD117 were collected. + Transfer the cells to a 15 mL centrifuge tube, centrifuge at low speed (25℃, 800 rpm) for 5 min to collect the cell clusters and resuspend them in an appropriate amount of RPMI-1640 medium; 4. Lentiviral infection and screening: The infection complex was added to the cells collected in step 3 (preparation of infection complex: 20 μL Tet-on-ACT domain protein lentivirus, 480 μL complete medium, 500 μL Enhancer reagent and 1 μL 10 mg / mL polybrene, mixed thoroughly); after 18 h, 1 mL of RPMI complete medium was added to the transfection well for further culture; the cell status was observed under a microscope; after 48 h, the supernatant was discarded and RPMI complete medium was added; after 72 h, screening began: the medium was replaced with RPMI complete medium containing 2 μg / mL puromycin and cultured for 3 days.

[0060] 5. Bone marrow transplantation: Wild-type mice were subjected to whole-body irradiation of 5 Gy. Six hours after irradiation, the cells from step 4 were reinfused into the irradiated mice via the tail vein. The mice were randomly divided into two groups: one group received normal drinking water, and the other received drinking water containing 2 mg / mL doxycycline. The survival time of the mice was dynamically monitored. Results are as follows: Figure 7 As shown, compared with the control group, the induced ACT domain protein significantly prolonged the survival of irradiated mice.

[0061] As can be seen from the above embodiments, the present invention provides the application of bacterial ACT domain proteins in the preparation of anticancer drugs, preparations that target and inhibit mitochondrial glutamate transporter SLC25A22, preparations that enhance the host's antitumor immune response, and the application of bacterial ACT domain proteins in the preparation of research and / or treatment of tumors such as CRC, radiation diseases, and organ transplantation immunity.

[0062] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An ACT domain protein, characterized in that, Its amino acid sequence is shown in SEQ ID NO:

2.

2. A nucleic acid molecule encoding the ACT domain protein as described in claim 1, characterized in that, Its nucleotide sequence is shown in SEQ ID NO:

1.

3. The use of the ACT domain protein as described in claim 1 in the preparation of a medicament for treating cancer.

4. The application according to claim 3, characterized in that, The ACT domain protein functions by targeting and inhibiting the mitochondrial glutamate transporter SLC25A22.

5. The application according to claim 3, characterized in that, The cancer in question is rectal cancer.

6. The use of the ACT domain protein as described in claim 1 in the preparation of a medicament for enhancing the host's anti-tumor immune response.

7. The use of the ACT domain protein as described in claim 1 in the preparation of a drug for preventing and treating damage from ionizing radiation.

8. The use of the ACT domain protein as described in claim 1 in the preparation of a medicament for treating immune-related diseases in organ transplantation.

9. A pharmaceutical composition, characterized in that, It comprises the ACT domain protein of claim 1 and a pharmaceutically acceptable carrier.

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