Mitochondrial regulatory factor targeting TGF-B and TNF pathways and immunoregulation method of mitochondrial regulatory factor

Abstract

The invention discloses a mitochondrial regulatory factor targeting TGF-B and TNF pathways and an immunoregulation method thereof, and relates to the technical field of biologication.The immunoregulation method includes the steps of S1, molecular design and gene synthesis, S2, recombinant plasmid construction and verification, S3, mammalian cell expression and purification, S4, preparation freeze-drying and redissolving, and S5, preparation freeze-drying and redissolving. According to the mitochondrial targeting fusion protein disclosed by the invention, two inflammatory pathways of TGF-beta and TNF-alpha are targeted at the same time, so that collaborative regulation and control on immunoreaction are realized, and the structural domain A blocks excessive activation of a downstream SMAD signal pathway by inhibiting kinase activity of a TGF-beta receptor I, so that abnormal amplification of fibrosis-promoting and immunosuppressive cells is reduced, and the immunosuppressive effect of the mitochondrial targeting fusion protein is improved. The structural domain B simulates a soluble TNF receptor, neutralizes free TNF-alpha and inhibits an NF-kappa B inflammation cascade reaction triggered after the structural domain B is combined with a membrane receptor, the synergistic effect of the structural domain B and the soluble TNF-alpha can intervene in unbalance of proinflammatory and anti-inflammatory signals at the same time, and compensatory signal rebound possibly caused by single-channel inhibition is avoided.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to mitochondrial regulators targeting TGF-β and TNF pathways and their immunomodulatory methods. Background Art

[0002] In the treatment of immune-related diseases (such as autoimmune diseases, fibrotic diseases, and chronic inflammation), the abnormal activation of TGF-β and TNF-α signaling pathways is the core pathological mechanism. The TGF-β pathway drives the fibrotic process by activating SMAD proteins and induces an immunosuppressive microenvironment, while TNF-α amplifies the inflammatory response through pathways such as NF-κB. The two together lead to the imbalance of immune homeostasis. Although single-pathway inhibitors currently used clinically (such as TGF-β receptor kinase inhibitors or TNF-α antibodies) can partially relieve symptoms, they have limitations: the blockade of a single target is likely to trigger the reverse activation of compensatory signaling pathways. For example, inhibiting TGF-β may exacerbate TNF-α-mediated inflammatory damage, while neutralizing TNF-α may enhance TGF-β-driven fibrotic progression. This signal compensation effect not only reduces the therapeutic effect but also may lead to disease recurrence or exacerbation. In view of the above problems, the following solutions are proposed. Summary of the Invention

[0003] The purpose of the present invention is to provide mitochondrial regulators targeting TGF-β and TNF pathways and their immunomodulatory methods. By simultaneously targeting the two major inflammatory pathways of TGF-β and TNF-α through a mitochondrial-targeted fusion protein, the coordinated regulation of immune responses is achieved, and the problem that the blockade of a single target in the existing methods is likely to trigger the reverse activation of compensatory signaling pathways is solved.

[0004] To solve the above technical problems, the present invention is achieved through the following technical solutions:

[0005] The present invention provides a preparation method for a mitochondrial regulator preparation targeting TGF-β and TNF pathways, including:

[0006] Step S1, molecular design and gene synthesis: Design a fusion protein containing the TGF-β receptor type I (TβRI) inhibitory domain, the soluble TNF receptor (sTNFR) neutralizing domain, and the mitochondrial localization signal peptide COX8 MLS, optimize the gene sequence and chemically synthesize it;

[0007] Step S2, recombinant plasmid construction and verification: Clone the synthesized gene into the pcDNA3.1(+) vector, and obtain the correct recombinant plasmid through enzyme digestion ligation, transformation screening, and sequencing verification;

[0008] Step S3, mammalian cell expression and purification: Express the fusion protein in Expi293F cells, purify it by Flag-tag affinity chromatography, and dialyze and concentrate to obtain a high-purity protein;

[0009] Step S4, freeze-drying and reconstitution of the preparation: the purified protein is mixed with the protective agent, freeze-dried to prepare a freeze-dried powder, and reconstituted with sterile water to prepare an injection preparation when used.

[0010] Furthermore, the step S1, molecular design and gene synthesis specifically includes the following steps:

[0011] Step S11, domain A design: select the intracellular kinase inhibitory fragment of TβRI: amino acid residues 198-220, eliminate ATP binding ability by site-directed mutagenesis (D203A); add a flexible linker peptide (GGGGS) × 3 at the N-terminus;

[0012] Step S12, domain B design: extract the extracellular domain of human sTNFR2: residues 29-211, retain the TNF-α binding site, delete the transmembrane region; insert a Flag tag (DYKDDDDK) at the C-terminus for subsequent purification and detection;

[0013] Step S13, selection of mitochondrial localization signal peptide: using the N-terminal mitochondrial targeting sequence (MLS, amino acids 1-25) of human cytochrome c oxidase subunit 8 (COX8), and inserting it into the N-terminus of the fusion protein;

[0014] Step S14, overall assembly: the assembly order is: MLS (1-25), domain A (198-220), connecting peptide, domain B (29-211), Flag tag; the coding gene sequence is synthesized after codon optimization;

[0015] The core functional modules of the fusion protein (TβRI inhibition, TNF-α neutralization and mitochondrial targeting) were determined, and the functions were ensured to be non-interfering through domain optimization and connecting peptide design. The codon-optimized gene sequence was synthesized to provide an accurate template for subsequent expression, laying the foundation for targeted therapy.

[0016] Furthermore, the step S2, recombinant plasmid construction and verification specifically includes the following steps:

[0017] Step S21, vector selection: using pcDNA3.1(+) eukaryotic expression vector containing CMV promoter and SV40 polyA signal;

[0018] Step S22, restriction digestion and ligation: the vector and the synthetic gene fragment were double-digested with XhoI and EcoRI, and after gel recovery and purification, they were ligated with T4 DNA ligase (NEB, M0202) at 16°C for 12 hours;

[0019] Step S23, transformation and screening: the ligation product was transformed into TOP10 competent cells (Thermo Fisher), spread on LB plates containing ampicillin, and cultured at 37° C. for 16 hours;

[0020] Pick a monoclonal colony, culture it with shaking to amplify, and then extract the plasmid (QIAprep Miniprep Kit). Verify by Sanger sequencing. The sequencing primer sequences are: CMV-F: 5’-CGCAAATGGGCGGTAGGCGTG-3’;

[0021] Insert the synthetic gene into the eukaryotic expression vector to construct a plasmid system that can be stably transcribed. Ensure the correctness of the gene sequence and reading frame through restriction enzyme digestion, ligation, and sequencing screening, providing a reliable genetic tool for efficient protein expression and avoiding translation errors or expression failures.

[0022] Further, in step S22, for the double restriction enzyme digestion in restriction enzyme digestion and ligation, the reaction temperature is set at 37°C and the duration is 2 hours;

[0023] The concentration of ampicillin in the LB plate is 100 μg / mL.

[0024] Further, step S3, mammalian cell expression and purification specifically includes the following steps:

[0025] Step S31, mammalian cell transfection: Use the Expi293F cell line (Thermo Fisher), adjust the density to 2×10 5 cells / mL, transfect the plasmid, and culture the transfected cells for 5 days at 37°C with a CO2 volume percentage of 8%;

[0026] Step S32, collection of secreted protein: Centrifuge to remove cell debris, and filter the supernatant through a 0.22 μm filter membrane;

[0027] Step S33, affinity chromatography purification: Use Anti-Flag M2 affinity gel (Sigma), pass the supernatant through the column at a flow rate of 1 mL / min, wash with PBS, and then elute with 0.1 M glycine, and immediately neutralize with 1 M Tris-HCl;

[0028] Step S34, dialysis and concentration: Place the eluate into a Slide-A-Lyzer dialysis cassette (10 kDa MWCO, ThermoFisher), dialyze to PBS at 4°C, and ultrafiltrate and concentrate to 2 mg / mL (Amicon Ultra) to obtain highly pure protein;

[0029] Use mammalian cells (Expi293F) to complete the post-translational modification and secretory expression of the fusion protein, specifically capture the target protein through Flag-tag affinity chromatography, remove host cell impurities, and obtain a highly active and highly pure therapeutic protein after dialysis and concentration to meet the formulation requirements.

[0030] Further, in step S31, when transfecting mammalian cells, for every 1 μg of recombinant plasmid DNA used, 3 μL of Expifectamine transfection reagent is added;

[0031] In step S32, during the centrifugation operation for collecting the secreted protein, the centrifugal force intensity is 3000×g and it lasts for 10 minutes;

[0032] In step S33, in affinity chromatography purification, the pH of glycine is 3.0 and the pH of Tris-HCl is 8.0;

[0033] In step S34, in dialysis and concentration, the molecular weight cut-off value of the dialysis cassette is 10 kDa and the pH of the PBS buffer is 7.4.

[0034] Further, step S4, the freeze-drying and reconstitution of the preparation specifically includes the following steps:

[0035] Step S41, freeze-drying preparation formula: Mix the purified protein with the freeze-drying protectant, dispense into vials, pre-freeze at -80°C for 2 hours, and then transfer to a freeze-dryer;

[0036] Step S42, reconstitution method: Reconstitute with sterile injection water before use, with a final concentration of 1 mg / mL, and store at 4°C for no more than 72 hours;

[0037] Through the freeze-drying process, the liquid protein is transformed into a solid powder. Combining protectants such as trehalose maintains the long-term stability of the protein and reduces transportation and storage costs; after reconstitution, the biological activity is retained, ensuring accurate dosing and easy delivery during clinical use.

[0038] Further, the freeze-drying protectant contains 5% trehalose, 1% mannitol, 0.01% Tween-80. When the freeze-dryer is working, the freeze-drying temperature is -50°C, the vacuum pressure is 0.1 mbar, and the freeze-drying duration is 48 hours;

[0039] Further, the reconstituted preparation is administered by intravenous injection or local lesion injection (such as joint cavity), with a dose of 0.1 - 10 mg / kg, twice a week;

[0040] After the fusion protein penetrates the cell membrane through the blood circulation, the MLS guides its localization to the mitochondria. Domains A / B respectively block TGF-β and TNF signals, and at the same time reduce ROS by stabilizing MMP, reversing the imbalance of immune cells (such as Treg / Th17).

[0041] The present invention has the following beneficial effects:

[0042] 1. The mitochondrial targeted fusion protein of the present invention achieves coordinated regulation of immune response by simultaneously targeting two major inflammatory pathways, TGF-β and TNF-α. Domain A inhibits the kinase activity of TGF-β receptor I and blocks the excessive activation of the downstream SMAD signaling pathway, thereby reducing the abnormal proliferation of pro-fibrotic and immunosuppressive cells. Domain B simulates soluble TNF receptors, neutralizes free TNF-α, and inhibits the NF-κB inflammatory cascade reaction triggered by its binding to membrane receptors. The synergistic effect of the two can simultaneously intervene in the imbalance of pro-inflammatory and anti-inflammatory signals, avoiding compensatory signal rebound that may be caused by single pathway inhibition.

[0043] 2. The present invention introduces the mitochondrial localization signal peptide of human cytochrome c oxidase subunit 8, so that the fusion protein can be accurately enriched in the mitochondrial inner membrane and directly intervene in the immune abnormalities related to mitochondrial dysfunction. MTFP is designed to target mitochondria and selectively act on the mitochondria of diseased cells at a lower dose. It can repair the energy metabolism defects of immune cells by stabilizing the mitochondrial membrane potential and reducing the accumulation of reactive oxygen species, thereby reversing their pro-inflammatory or over-activated phenotypes.

[0044] 3. The molecular design of the mitochondrial targeting fusion protein of the present invention improves its drug potential through multi-dimensional optimization. First, the site-directed mutagenesis of domain A eliminates cross-reactivity with non-target proteins, ensures specific inhibition of TβRI, and avoids off-target effects. Secondly, the introduction of flexible connecting peptides alleviates the steric hindrance between domains, maintains the conformational integrity of each functional region, and ensures the simultaneous activity of dual targets. In addition, the integration of the Flag tag simplifies the protein purification process. Combined with the mammalian cell expression system, it can efficiently secrete and express fusion proteins with correct folding modifications, thereby improving yield and batch consistency.

[0045] Of course, any product implementing the present invention does not necessarily need to achieve all of the advantages described above at the same time. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for describing the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative work.

[0047] Figure 1 The present invention is a schematic diagram of the process for preparing a mitochondrial regulatory factor preparation targeting TGF-B and TNF pathways. DETAILED DESCRIPTION

[0048] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without making creative efforts shall fall within the protection scope of the present invention.

[0049] Please refer to Figure 1 As shown, the present invention is a preparation method of a mitochondrial regulatory factor preparation targeting the TGF-β and TNF pathways, including:

[0050] Step S1, molecular design and gene synthesis:

[0051] Step S11, domain A design: Select the intracellular kinase inhibitory fragment of TβRI: amino acid residues 198-220, and eliminate the ATP binding ability (D203A) through site-directed mutagenesis; add a flexible linker peptide (GGGGS)×3 at the N-terminus;

[0052] Step S12, domain B design: Intercept the extracellular domain of human sTNFR2: residues 29-211, retain the TNF-α binding site, and delete the transmembrane region; insert a Flag tag (DYKDDDDK) at the C-terminus for subsequent purification and detection;

[0053] Step S13, selection of mitochondrial localization signal peptide: Adopt the N-terminal mitochondrial targeting sequence (MLS, amino acids 1-25) of human cytochrome c oxidase subunit 8 (COX8), and insert it at the N-terminus of the fusion protein;

[0054] Step S14, overall assembly: The assembly sequence is: MLS (1-25), domain A (198-220), linker peptide, domain B (29-211), Flag tag; the coding gene sequence is synthesized after codon optimization;

[0055] Determine the core functional modules of the fusion protein (TβRI inhibition, TNF-α neutralization, and mitochondrial targeting), ensure that the functions do not interfere with each other through domain optimization and linker design, synthesize the codon-optimized gene sequence, provide an accurate template for subsequent expression, and lay the foundation for targeted therapy;

[0056] Step S2, recombinant plasmid construction and verification: Step S21, vector selection: Use the pcDNA3.1(+) eukaryotic expression vector, containing the CMV promoter and the SV40 polyA signal;

[0057] Step S22, digestion and ligation: Double-digest the vector and the synthesized gene fragment with XhoI and EcoRI, recover and purify by gel electrophoresis, and then ligate with T4 DNA ligase (NEB, M0202) at 16°C for 12 hours;

[0058] Step S23, Transformation and Screening: Transform the ligation product into TOP10 competent cells (Thermo Fisher), spread it on an LB plate containing ampicillin, and culture at 37 °C for 16 hours.

[0059] Pick monoclonal colonies, culture with shaking and then extract plasmids (QIAprep Miniprep Kit), verify by Sanger sequencing. The sequencing primer sequences are: CMV-F: 5’-CGCAAATGGGCGGTAGGCGTG-3’.

[0060] Insert the synthetic gene into the eukaryotic expression vector to construct a plasmid system for stable transcription. Ensure the correctness of the gene sequence and reading frame through restriction digestion, ligation and sequencing screening, providing a reliable genetic tool for efficient protein expression and avoiding translation errors or expression failures.

[0061] In step S22, the temperature of the double restriction digestion reaction in restriction digestion and ligation is set at 37 °C and the duration is 2 hours.

[0062] The concentration of ampicillin in the LB plate is 100 μg / mL.

[0063] Step S3, Mammalian Cell Expression and Purification:

[0064] Step S31, Mammalian Cell Transfection: Use the Expi293F cell line (Thermo Fisher), adjust the density to 2×10 5 cells / mL, transfect the plasmid, and culture the transfected cells for 5 days at 37 °C with a CO2 volume percentage of 8%.

[0065] Step S32, Secreted Protein Collection: Centrifuge to remove cell debris, and filter the supernatant through a 0.22 μm filter membrane.

[0066] Step S33, Affinity Chromatography Purification: Use Anti-Flag M2 affinity gel (Sigma), pass the supernatant through the column at a flow rate of 1 mL / min, wash with PBS, elute with 0.1 M glycine, and immediately neutralize with 1 M Tris-HCl.

[0067] Step S34, Dialysis and Concentration: Place the eluate into a Slide-A-Lyzer dialysis cassette (10 kDa MWCO, ThermoFisher), dialyze against PBS at 4 °C, and ultrafiltrate and concentrate to 2 mg / mL (Amicon Ultra) to obtain high-purity protein.

[0068] The post-translational modification and secretory expression of the fusion protein are completed using mammalian cells (Expi293F). The target protein is specifically captured by Flag-tag affinity chromatography to remove host cell impurities. After dialysis and concentration, a highly active and highly pure therapeutic protein is obtained to meet the formulation requirements;

[0069] In step S31, when transfecting mammalian cells, for every 1 μg of recombinant plasmid DNA used, 3 μL of Expifectamine transfection reagent is used;

[0070] In step S32, during the centrifugation operation for collecting secreted proteins, the centrifugal force intensity is 3000×g for 10 minutes;

[0071] In step S33, for affinity chromatography purification, the pH of glycine is 3.0 and the pH of Tris-HCl is 8.0;

[0072] In step S34, for dialysis and concentration, the molecular weight cut-off value of the dialysis cassette is 10 kDa, and the pH of the PBS buffer is 7.4;

[0073] Step S4, formulation lyophilization and reconstitution:

[0074] Step S41, lyophilized formulation recipe: Mix the purified protein with a lyoprotectant, dispense into vials, pre-freeze at -80°C for 2 hours, and then transfer to a freeze dryer;

[0075] Step S42, reconstitution method: Reconstitute with sterile water for injection before use, with a final concentration of 1 mg / mL, and store at 4°C for no more than 72 hours;

[0076] The liquid protein is converted into a solid powder through the lyophilization process. Combining protectants such as trehalose maintains the long-term stability of the protein and reduces transportation and storage costs; after reconstitution, the biological activity is retained, ensuring accurate dosing and easy delivery during clinical use;

[0077] The lyoprotectant contains 5% trehalose, 1% mannitol, and 0.01% Tween-80. When the dryer is working, the lyophilization temperature is -50°C, the vacuum pressure is 0.1 mbar, and the lyophilization duration is 48 hours;

[0078] The reconstituted formulation is administered by intravenous injection or local lesion injection (such as joint cavity), with a dose of 0.1 - 10 mg / kg, twice a week;

[0079] After the fusion protein penetrates the cell membrane through the blood circulation, MLS guides its localization to the mitochondria. Domains A / B block TGF-β and TNF signals respectively, and at the same time reduce ROS by stabilizing MMP, reversing the imbalance of immune cells (such as Treg / Th17).

[0080] A specific application of this example is:

[0081] Immunomodulatory effect of MTFP preparation in collagen-induced arthritis (CIA) model

[0082] 1. Experimental design:

[0083] Animal model: Male DBA / 1 mice (8 weeks old, n = 30), divided into 3 groups:

[0084] Experimental group: MTFP preparation (5 mg / kg, intravenous injection, twice a week);

[0085] Positive control group: Anti-TNF-α monoclonal antibody (10 mg / kg, same frequency);

[0086] Negative control group: Normal saline (equal volume injection);

[0087] Disease induction: Bovine type II collagen and complete Freund's adjuvant (CFA) emulsion (1:1) were subcutaneously injected at the base of the tail (on day 0 and day 21);

[0088] 2. Experimental procedure:

[0089] Step 1. Model establishment and drug administration:

[0090] Primary immunization: Bovine type II collagen (2 mg / mL) was emulsified with CFA (sonicated on ice for 10 minutes to form a homogeneous emulsion); 100 μL of the emulsion was subcutaneously injected at the base of the tail of each mouse;

[0091] Secondary immunization (on day 21): Repeat the injection of the same dose of emulsion (boost the immune response);

[0092] Drug administration plan (starting from day 28): The experimental group and the control group were injected according to the preset dose for 4 weeks;

[0093] Step 2. Phenotype observation and sample collection:

[0094] Arthritis score: Record the swelling degree of the hind limb joints of mice every week (0 - 4 score standard: 0 = no redness and swelling, 4 = severe deformation); Measure the diameter of the ankle joint (vernier caliper, accurate to 0.1 mm);

[0095] Serum collection (on day 56): Blood was taken from the orbital cavity, allowed to stand for 30 minutes and then centrifuged (3000×g, 10 minutes), and the serum was separated and stored at -80 °C;

[0096] Tissue sample processing: After sacrificing the mice, the hind limb joints were fixed in 4% paraformaldehyde (for 24 hours), decalcified and then embedded in paraffin for sectioning; The spleen was ground through a 70 μm sieve to separate single cell suspensions for flow cytometry analysis;

[0097] Step 3. Analysis of immune cell subsets:

[0098] Detection of Treg / Th17 ratio: After stimulating splenocytes with PMA / ionomycin (for 5 h), fix and permeabilize the cells, and stain: Treg: CD4+CD25+FoxP3+; Th17: CD4+IL-17A+; Detect using a flow cytometer (BD FACS Canto II) and analyze the data with FlowJo;

[0099] Determination of cytokine levels: Concentrations of TNF-α, TGF-β1, and IL-6 in serum (ELISA kit, R&D Systems), operate according to the instructions;

[0100] Step 4, Analysis of mitochondrial function association:

[0101] Detection of mitochondrial membrane potential (MMP): Isolate splenic CD4+ T cells, incubate with JC-1 dye (5 μM, 37 °C for 20 minutes), and detect the red / green fluorescence ratio by flow cytometry (a decrease in the ratio indicates a decrease in MMP);

[0102] Determination of ROS levels: Incubate cells with DCFH-DA (10 μM, 37 °C for 30 minutes), and detect the fluorescence intensity by flow cytometry (excitation / emission: 488 / 525 nm);

[0103] 3. Data analysis:

[0104] Statistical method: Data are expressed as mean ± SEM, and the intergroup differences are tested by one-way ANOVA and Tukey's multiple comparison test (GraphPad Prism 9.0);

[0105] Comparison of key indicators:

[0106] Experimental group vs. positive control group: Treg / Th17 ratio, degree of decrease in joint score;

[0107] Experimental group vs. negative control group: Serum TNF-α / TGF-β1 levels, improvement rates of MMP / ROS.

[0108] 4. Experimental results:

[0109]

[0110] Table 1 Comparison of clinical indicators in CIA model

[0111] In Table 1: Data are expressed as mean ± SEM (n = 10), * indicates p < 0.05 compared with the negative control group, and # indicates p < 0.05 compared with the positive control group;

[0112] The disease incidence rate is defined as the proportion of mice with an ankle joint diameter ≥ 3.0 mm;

[0113]

[0114]

[0115] Table 2 Subsets of immune cells and cytokine levels

[0116] In Table 2: The Treg / Th17 ratio was calculated by flow cytometry (CD4+FoxP3+ / CD4+IL-17+);

[0117] Serum cytokines were detected by ELISA. * indicates p < 0.05 compared with the negative control group, and # indicates p < 0.05 compared with the positive control group;

[0118]

[0119] Table 3 Indicators related to mitochondrial function

[0120] In Table 3: MMP (mitochondrial membrane potential) was detected by JC-1 dye, and ROS was measured by DCFH-DA fluorescent probe;

[0121] ATP production was detected by luciferase method. * indicates p < 0.05 compared with the negative control group, and # indicates p < 0.05 compared with the positive control group;

[0122] The joint inflammation score of the experimental group mice was reduced by more than 24% compared with the positive control group, the Treg / Th17 ratio increased by 2-fold, and the mitochondrial ROS level decreased by 30%, indicating that MTFP improved immune imbalance through dual pathway inhibition and mitochondrial regulation in synergy.

[0123] In the description of this specification, the descriptions referring to the terms "one embodiment", "example", "specific example", etc. mean that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in a suitable manner in any one or more embodiments or examples.

[0124] The preferred embodiments of the present invention disclosed above are only used to help explain the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the invention to the specific embodiments described. Obviously, many modifications and variations can be made according to the content of this specification. These embodiments are selected and specifically described in this specification to better explain the principle and practical application of the present invention, so that those skilled in the art can understand and utilize the present invention well. The present invention is only limited by the claims and their full scope and equivalents.

Claims

1. A method for preparing a mitochondrial regulatory factor preparation targeting TGF-B and TNF pathways, characterized in that: The preparation method comprises the following steps: Step S1, molecular design and gene synthesis: design a fusion protein containing the TβRI inhibitory domain, the sTNFR neutralizing domain and the mitochondrial localization signal peptide COX8 MLS, optimize the gene sequence and chemically synthesize; Step S2, recombinant plasmid construction and verification: the synthetic gene was cloned into the pcDNA3.1(+) vector, and the correct recombinant plasmid was obtained through restriction ligation, transformation screening and sequencing verification; Step S3, mammalian cell expression and purification: expressing the fusion protein in Expi293F cells, purifying it by Flag tag affinity chromatography, and dialysis concentration to obtain high-purity protein; Step S4, freeze-drying and reconstitution of the preparation: the purified protein is mixed with the protective agent, freeze-dried to prepare a freeze-dried powder, and reconstituted with sterile water to prepare an injection preparation when used.

2. The method for preparing a mitochondrial regulatory factor preparation targeting TGF-B and TNF pathways according to claim 1, characterized in that: The step S1, molecular design and gene synthesis specifically includes the following steps: Step S11, domain A design: select the intracellular kinase inhibitory fragment of TβRI: amino acid residues 198-220, eliminate the ATP binding ability by site-directed mutagenesis; add a flexible linker peptide (GGGGS) × 3 at the N-terminus; Step S12, domain B design: extract the extracellular domain of human sTNFR2: residues 29-211, retain the TNF-α binding site, delete the transmembrane region; insert a Flag tag at the C-terminus for subsequent purification and detection; Step S13, selection of mitochondrial localization signal peptide: using the N-terminal mitochondrial targeting sequence of human cytochrome c oxidase subunit 8, and inserting it into the N-terminus of the fusion protein; Step S14, overall assembly: the assembly order is: MLS, domain A, connecting peptide, domain B, Flag tag; the coding gene sequence is synthesized after codon optimization.

3. The method for preparing a mitochondrial regulatory factor preparation targeting TGF-B and TNF pathways according to claim 1, characterized in that: The step S2, recombinant plasmid construction and verification, specifically comprises the following steps: Step S21, vector selection: using pcDNA3.1(+) eukaryotic expression vector containing CMV promoter and SV40 polyA signal; Step S22, restriction digestion and ligation: the vector and the synthetic gene fragment were double-digested with XhoI and EcoRI, and after gel recovery and purification, they were ligated with T4 DNA ligase at 16°C for 12 hours; Step S23, transformation and screening: transform the ligation product into TOP10 competent cells, spread on LB plates containing ampicillin, and culture at 37° C. for 16 hours; Single clone colonies were picked, and plasmids were extracted after amplification by shaking. The plasmids were verified by Sanger sequencing. The sequencing primer sequence was: CMV-F: 5'-CGCAAATGGGCGGTAGGCGTG-3'.

4. The method for preparing a mitochondrial regulatory factor preparation targeting TGF-B and TNF pathways according to claim 3, characterized in that: In step S22, the double enzyme digestion reaction temperature in the enzyme digestion and ligation is set to 37° C. and the duration is 2 hours; The concentration of ampicillin in the LB plate was 100 μg / mL.

5. The method for preparing a mitochondrial regulatory factor preparation targeting TGF-B and TNF pathways according to claim 1, characterized in that: The step S3, mammalian cell expression and purification specifically comprises the following steps: Step S31, mammalian cell transfection: using Expi293F cell line, the density was adjusted to 2×10 5 cells / mL, transfected plasmids, and cultured the cells transfected with plasmids at 37°C and 8% CO2 for 5 days; Step S32, secretory protein collection: centrifugation to remove cell debris, and filtering the supernatant through a 0.22 μm filter membrane; Step S33, affinity chromatography purification: using Anti-Flag M2 affinity gel, the supernatant was passed through the column at a flow rate of 1 mL / min, washed with PBS, eluted with 0.1 M glycine, and immediately neutralized with 1 M Tris-HCl; Step S34, dialysis and concentration: the eluate was placed in a Slide-A-Lyzer dialysis cassette, dialyzed into PBS at 4°C, and concentrated by ultrafiltration to 2 mg / mL to obtain a high-purity protein.

6. The method for preparing a mitochondrial regulatory factor preparation targeting TGF-B and TNF pathways according to claim 5, characterized in that: In step S31, when mammalian cells are transfected, 1 μg of recombinant plasmid DNA is used with 3 μL of Expifectamine transfection reagent; In step S32, the centrifugal force intensity during the centrifugation operation for collecting secretory proteins is 3000×g for 10 minutes; In the step S33, the pH of glycine in the affinity chromatography purification is 3.0, and the pH of Tris-HCl is 8.0; In the step S34, the molecular weight cutoff value of the dialysis box in the dialysis and concentration is 10 kDa, and the pH of the PBS buffer is 7.

4.

7. The method for preparing a mitochondrial regulatory factor preparation targeting TGF-B and TNF pathways according to claim 1, characterized in that: The step S4, freeze-drying and reconstitution of the preparation specifically comprises the following steps: Step S41, freeze-dried preparation formulation: the purified protein is mixed with a freeze-dried protective agent, divided into vials, pre-frozen to -80°C for 2 hours, and then transferred to a freeze dryer; Step S42, reconstitution method: reconstitute with sterile water for injection before use, the final concentration is 1 mg / mL, and store at 4°C for no more than 72 hours.

8. The method for preparing a mitochondrial regulatory factor preparation targeting TGF-B and TNF pathways according to claim 7, characterized in that: The freeze-drying protective agent contains 5% trehalose, 1% mannitol, and 0.01% Tween-80. When the dryer is working, the freeze-drying temperature is -50°C, the vacuum pressure is 0.1 mbar, and the freeze-drying duration is 48 hours.

9. The method for preparing a mitochondrial regulatory factor preparation targeting TGF-B and TNF pathways according to claim 7, characterized in that: The reconstituted preparation is injected intravenously or locally at a dosage of 0.1-10 mg / kg, twice a week.

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