Mitochondrial repair factor based on NADH metabolism and application of mitochondrial repair factor in asthma treatment

Abstract

The invention discloses a mitochondrial repair factor based on NADH metabolism and application of the mitochondrial repair factor in asthma treatment, and relates to the technical field of biologication.The mitochondrial repair factor based on NADH metabolism comprises the steps of S1, functional element design and sequence optimization, S2, recombinant vector construction, S3, host cell transformation, S4, engineering bacterium multiplication culture, S5, protein purification and S6, preparation. The mitochondrial energy metabolism efficiency is directly improved by catalyzing synthesis of NADH through the NADH regeneration module; the NAD < + > transfer module maintains the steady state level of NAD < + > in mitochondria through a transmembrane transport mechanism, and cofactor depletion is avoided; according to the cofactor stabilizing module, the binding capacity of a protein structure and cofactors is enhanced, the lasting time of enzyme activity is prolonged, dynamic conformation adjustment is achieved among the modules through flexible connecting peptides, and the stability of the overall structure can be maintained while the independence of functional domains can be guaranteed.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a mitochondrial repair factor based on NADH metabolism and its application in the treatment of asthma. Background Art

[0002] As the "energy factory" of cells, mitochondrial dysfunction is closely related to the occurrence and development of various diseases. Asthma is a respiratory disease characterized by chronic airway inflammation, airway hyperresponsiveness, and excessive mucus secretion. In recent years, studies have found that mitochondrial dysfunction plays an important role in the pathological process of asthma. Imbalance of mitochondrial oxidative stress, energy metabolism disorder, and NAD+ / NADH ratio disorder can lead to abnormal proliferation of airway smooth muscle cells, activation of inflammatory cells, and hyperfunction of mucus secretion, thereby exacerbating airway remodeling and disease deterioration.

[0003] Traditional asthma treatment mainly relies on glucocorticoids and β2 receptor agonists, but long-term use has side effects such as immunosuppression and metabolic disorders, and some patients have poor responses to the drugs. The repair strategy targeting mitochondrial function has become a new research direction. As the core coenzyme of mitochondrial energy metabolism, the regeneration efficiency of NADH directly affects ATP generation and redox balance. In protein engineering methods, a single module is often used to simultaneously regulate multiple links of NADH metabolism, which is difficult to simultaneously regulate multiple links of NADH metabolism, resulting in limited repair effects. To address the above problems, the following solutions are proposed. Summary of the Invention

[0004] The purpose of the present invention is to provide a mitochondrial repair factor based on NADH metabolism and its application in the treatment of asthma. Through the precise design and functional integration of multiple modules, systematic regulation of the mitochondrial NADH metabolic pathway is achieved. The synergistic effect of the core functional modules can effectively restore mitochondrial redox homeostasis, and solve the problem that it is difficult to simultaneously regulate multiple links of NADH metabolism in the prior art, resulting in limited repair effects.

[0005] To solve the above technical problems, the present invention is achieved through the following technical solutions:

[0006] The present invention provides a preparation method of a mitochondrial repair factor based on NADH metabolism, including:

[0007] Step S1, functional element design and sequence optimization: Design the core module, add a mitochondrial targeting sequence, optimize the linker element, and complete codon optimization to adapt to E. coli expression;

[0008] Step S2, recombinant vector construction: Synthesize the target DNA fragment containing restriction enzyme sites, digest the vector backbone, and construct a recombinant vector through a ligation reaction;

[0009] Step S3, Host cell transformation: Chemically transform competent cells and obtain single colonies containing the recombinant vector through plate screening;

[0010] Step S4, Engineering bacteria amplification culture: Prepare a seed solution, and use a fermenter for scale-up culture and induce the engineering bacteria to express the target protein;

[0011] Step S5, Protein purification: Disrupt the cells to obtain the supernatant, and purify the target protein through metal chelation chromatography and size exclusion chromatography;

[0012] Step S6, Preparation of preparation: Prepare a freeze-drying protectant to mix with the protein, and make a stable freeze-dried preparation through pre-freezing and stepwise drying.

[0013] Furthermore, in the said Step S1, the functional element design and sequence optimization specifically include the following steps:

[0014] Step S11, Determine the core functional modules, which are respectively:

[0015] NADH regeneration module: Select the catalytic domain of the active subunit NDUFS1 of human mitochondrial complex I;

[0016] NAD+ transport module: Chimerize the transmembrane domain of the mitochondrial inner membrane carrier SLC25A51;

[0017] Cofactor stabilization module: Introduce the α-helix structure of malate dehydrogenase to stabilize the cofactor binding site;

[0018] Step S12, Add a mitochondrial targeting sequence: Fuse the mitochondrial localization signal peptide of cytochrome c oxidase subunit VIII at the N-terminus;

[0019] Step S13, Linker element design: Insert a (Gly4Ser)3 flexible linker peptide between functional modules; Add the SV40 nuclear localization signal as a negative regulatory element after the stop codon;

[0020] Step S14, Codon optimization: Use the GeneOptimizer software to optimize the human sequence with E. coli-preferred codons; Adjust the GC content to 52% - 58%, and eliminate internal ribosome binding sites and restriction enzyme cleavage sites;

[0021] By determining the core functional modules (NADH regeneration, NAD+ transport, cofactor stabilization), endow the recombinant protein with metabolic repair function; Add a mitochondrial targeting sequence to ensure the precise localization of the protein to the mitochondria; Optimize the linker elements and codons to ensure the rationality of the protein structure and its efficient expression in E. coli.

[0022] Furthermore, the human mitochondrial complex I has the enzyme activity of catalyzing NADH dehydrogenation; the active subunit NDUFS1 has a unique identification number of UniProt ID: P28331; the specific position of the catalytic domain exercising the NADH regeneration function is the section from the 50th amino acid to the 400th amino acid of the NDUFS1 protein; the specific position of the transmembrane domain is in the amino acid sequence of the SLC25A51 protein, in the region from the 100th residue to the 250th residue; the α-helix structure selects amino acid residues 30 - 120 of malate dehydrogenase.

[0023] The amino acid sequence of the mitochondrial localization signal peptide of cytochrome c oxidase subunit VIII is MLSLRQSIRFFKPATRTLCSSRYLL.

[0024] Furthermore, in step S2, the construction of the recombinant vector specifically includes the following steps:

[0025] Step S21, synthesize DNA fragment: Obtain the full-length gene sequence SEQ ID NO: 1 by chemical synthesis; introduce AgeI and NotI restriction enzyme sites at both ends.

[0026] Step S22, prepare the vector backbone: Take the pET-28a(+) vector (Novagen); after double digestion with AgeI / NotI, recover the 5.4 kb linearized vector by gel extraction. The conditions for the digestion reaction are a temperature of 37°C and a reaction time of 2 hours.

[0027] Step S23, ligation reaction: Mix according to the molar ratio of insert fragment: vector = 3:1; use T4 DNA ligase to ligate at 16°C for 12 hours.

[0028] Furthermore, AgeI recognizes and cleaves 5'-ACCGGT-3'; NotI recognizes and cleaves 5'-GCGGCCGC-3'.

[0029] In step S23, the reaction system in the ligation reaction is specifically as follows:

[0030] 50 ng vector DNA: The mass of the linearized pET-28a(+) vector DNA participating in the ligation reaction is 50 ng.

[0031] 150 ng insert fragment: The mass of the target gene DNA fragment to be ligated is 150 ng, that is, the DNA fragment containing the target sequence synthesized in step S21.

[0032] 1×T4 buffer: The special buffer for T4 DNA ligase, used at 1-fold concentration.

[0033] 400 U enzyme: The added active unit of T4 DNA ligase is 400 U, and the enzyme acts as a catalyst.

[0034] By synthesizing the target gene fragment and ligating it to the vector backbone, a recombinant expression vector containing the target gene can be constructed, providing a genetic material basis for subsequent introduction into host cells.

[0035] Furthermore, in step S3, the host cell transformation specifically includes the following steps:

[0036] Step S31, chemical transformation: Take 50 μL of BL21(DE3) competent cells; add 5 μL of the ligation product, incubate on ice for 30 minutes, then heat shock at 42 °C for 45 seconds; add 950 μL of SOC medium and recover at 37 °C for 1 hour;

[0037] Step S32, plate screening: Spread on an LB agar plate containing 50 μg / mL kanamycin; incubate inverted at 37 °C for 16 hours, and pick single colonies with a diameter of 2 - 3 mm;

[0038] By introducing the recombinant vector into the host cell (BL21DE3) and then screening, single colonies of the engineered bacteria successfully transfected with the recombinant vector are obtained, realizing the stable existence of the gene in the host.

[0039] Furthermore, in step S4, the amplification culture of the engineered bacteria specifically includes the following steps:

[0040] Step S41, seed solution preparation: Inoculate a single colony into 5 mL of TB medium containing 0.5% glycerol; shake culture at 220 rpm until OD600 = 2.0;

[0041] Step S42, fermenter culture: Inoculate at a ratio of 1:100 into a 10 L fermenter with an initial OD600 = 0.05; the culture conditions are: 37 °C, pH 7.0, dissolved oxygen 30%; induction expression: when OD600 reaches 6.0, add 0.5 mM IPTG and induce at 25 °C for 16 hours;

[0042] By preparing the seed solution and large-scale culture in the fermenter, the number of engineered bacteria is expanded and the target protein expression is induced, providing sufficient raw materials for subsequent purification.

[0043] Furthermore, in step S5, the protein purification specifically includes the following steps:

[0044] Step S51, cell disruption: Disrupt the cells using a high-pressure homogenizer and centrifuge to collect the supernatant;

[0045] Step S52, metal chelation chromatography: Load the supernatant onto a Ni-NTA column (GE Healthcare), use 20 mM Tris-HCl, 500 mM NaCl, 20 mM imidazole as the binding buffer, and elute with a linear gradient of 50 - 500 mM imidazole;

[0046] Step S53, molecular sieve purification: Using a Superdex 200 Increase 10 / 300 GL column, with PBS containing 5% glycerol and 0.02% Tween-20 as the mobile phase, collect the elution peak with a molecular weight of 75 - 80 kDa.

[0047] Furthermore, when the high-pressure homogenizer is used to break cells, the pressure parameter is 1500 bar and it cycles 3 times;

[0048] Release proteins by breaking cells, use chromatography techniques (metal chelation, molecular sieve) to remove impurities, and obtain high-purity target proteins, meeting the requirements for preparation.

[0049] Furthermore, for step S6, the preparation of the preparation specifically includes the following steps:

[0050] Step S61, preparation of freeze-drying protectant: Prepare a PBS solution of 10% trehalose, 1% glycine, and 0.5% mannitol, and mix it with the purified protein at a volume ratio of 1:1;

[0051] Step S62, freeze-drying: In the pre-freezing stage, maintain at -80 °C for 2 hours; in the primary drying stage, maintain at -40 °C and 0.1 mbar for 24 hours; in the secondary drying stage, maintain at 25 °C and 0.01 mbar for 6 hours to finally obtain a freeze-dried preparation;

[0052] Protect the protein activity by preparing a freeze-drying protectant, make a freeze-dried preparation through freeze-drying, reduce the water content, improve the protein stability, and facilitate long-term storage and application.

[0053] The present invention has the following beneficial effects:

[0054] 1. Through the precise design and functional integration of multiple modules, the present invention realizes the systematic regulation of the mitochondrial NADH metabolic pathway. The synergistic effect of the core functional modules can effectively restore the mitochondrial redox homeostasis. Specifically, the NADH regeneration module directly improves the mitochondrial energy metabolism efficiency by catalyzing the synthesis of NADH; the NAD+ transport module maintains the steady-state level of NAD+ in mitochondria through a transmembrane transport mechanism to avoid cofactor depletion; the cofactor stabilization module prolongs the duration of enzyme activity by enhancing the binding ability between the protein structure and cofactors. The conformational dynamic regulation is achieved through flexible linker peptides between the modules, which can ensure the independence of the functional domains while maintaining the stability of the overall structure.

[0055] 2. Through the combined action of the mitochondrion-targeting signal peptide fused at the N-terminus and the C-terminal negative regulatory element, the present invention realizes the precise localization and activity regulation of the repair factor within the cell. Specifically, the signal peptide guides the nascent protein to efficiently cross the mitochondrial membrane structure, ensuring the specific enrichment of the repair factor in the mitochondrial matrix and avoiding metabolic interference caused by non-targeted distribution. At the same time, the nuclear localization signal added at the C-terminus, as a negative regulatory element, can actively inhibit the abnormal migration of the repair factor to the nucleus, further reducing the off-target risk. This design not only improves the targeting efficiency but also enhances the functional specificity of the repair factor through the spatial isolation effect, providing a safer intervention strategy for mitochondrial repair in a highly toxic microenvironment.

[0056] Of course, it is not necessary for any product implementing the present invention to achieve all the above-mentioned advantages simultaneously. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] To more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention, and those of ordinary skill in the art can obtain other drawings based on these drawings without creative efforts.

[0058] Figure 1 It is a schematic flow chart of the mitochondrial repair factor based on NADH metabolism of the present invention and its application in the treatment of asthma. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0059] The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the drawings in the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, rather than all embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention.

[0060] Please refer to Figure 1 As shown, the present invention relates to a preparation method of a mitochondrial repair factor based on NADH metabolism, including:

[0061] Step S1. Functional element design and sequence optimization:

[0062] Step S11. Determine the core functional module:

[0063] NADH regeneration module: Select the catalytic domain (amino acid residues 50 - 400) of the active subunit NDUFS1 (UniProt ID: P28331) of human mitochondrial complex I for NADH regeneration;

[0064] NAD+ transport module: Embedded in the transmembrane domain (amino acid residues 100 - 250) of mitochondrial inner membrane carrier SLC25A51 to achieve NAD+ transport;

[0065] Cofactor stabilization module: Introduce the α-helical structure (amino acid residues 30 - 120) of malate dehydrogenase (MDH2) to stabilize the cofactor binding site;

[0066] Step S12. Add mitochondrial targeting sequence: Fuse the mitochondrial localization signal peptide of cytochrome c oxidase subunit VIII (COX8) (amino acid sequence: MLSLRQSIRFFKPATRTLCSSRYLL) at the N-terminus to direct the protein to the mitochondria;

[0067] Step S13. Linker design: Insert (Gly4Ser)3 flexible linker peptides between functional modules to ensure spatial structural flexibility; Add the SV40 nuclear localization signal after the stop codon as a negative regulatory element to avoid unexpected localization;

[0068] Step S14. Codon optimization: Use GeneOptimizer software to optimize the human sequence for E. coli-preferred codons, adjust the GC content to 52% - 58%, and eliminate internal ribosome binding sites and restriction enzyme cleavage sites;

[0069] Step S2. Construction of recombinant vector:

[0070] Step S21. Synthesize DNA fragment: Chemically synthesize a DNA fragment containing the full-length gene sequence (SEQ ID NO:1), introducing AgeI (5'-ACCGGT-3') and NotI (5'-GCGGCCGC-3') restriction enzyme cleavage sites at both ends;

[0071] Step S22. Prepare vector backbone: Take the pET-28a(+) vector, double digest with AgeI / NotI (37°C, 2h), and recover the 5.4kb linearized vector by gel extraction;

[0072] Step S23. Ligation reaction: Mix according to the molar ratio of insert fragment:vector = 3:1, add T4 DNA ligase (NEB), and ligate at 16°C for 12 hours (reaction system: 50ng vector DNA, 150ng insert fragment, 1×T4 buffer, 400U enzyme);

[0073] Step S3. Transformation of host cells:

[0074] Step S31. Chemical transformation: Take 50μL of BL21(DE3) competent cells, add 5μL of the ligation product, incubate on ice for 30 minutes, then heat shock at 42°C for 45 seconds, and then add 950μL of SOC medium and recover at 37°C for 1 hour;

[0075] Step S32, Plate Screening: Spread the transformation solution on an LB agar plate containing 50 μg / mL kanamycin, incubate it upside down at 37 °C for 16 hours, and pick single colonies with a diameter of 2 - 3 mm;

[0076] Step S4, Amplification Culture of Engineered Bacteria:

[0077] Step S41, Preparation of Seed Solution: Inoculate a single colony into 5 mL of TB medium (containing 0.5% glycerol), shake culture at 220 rpm until OD600 = 2.0 (about 6 hours);

[0078] Step S42, Fermenter Culture: Inoculate at a ratio of 1:100 into a 10 L fermenter (initial OD600 = 0.05), culture conditions: 37 °C, pH 7.0, dissolved oxygen 30%; when OD600 reaches 6.0, add 0.5 mM IPTG and induce expression at 25 °C for 16 hours;

[0079] Step S5, Protein Purification:

[0080] Step S51, Cell Disruption: Disrupt the cells with a high-pressure homogenizer (1500 bar, cycle 3 times), centrifuge at 4 °C, 15000 × g for 30 minutes, and collect the supernatant;

[0081] Step S52, Metal Chelation Chromatography: Load the supernatant onto a Ni-NTA column (GE Healthcare), use 20 mM Tris-HCl, 500 mM NaCl, 20 mM imidazole (pH 8.0) as the binding buffer, and elute with a linear gradient of 50 - 500 mM imidazole;

[0082] Step S53, Size Exclusion Purification: Use a Superdex 200 Increase 10 / 300GL column (Cytiva), use PBS containing 5% glycerol and 0.02% Tween-20 as the mobile phase, and collect the elution peak with a molecular weight of 75 - 80 kDa;

[0083] Step S6, Preparation of Formulation:

[0084] Step S61, Preparation of Lyoprotectant: Prepare a PBS solution containing 10% trehalose, 1% glycine, and 0.5% mannitol, and mix it with the purified protein at a volume ratio of 1:1;

[0085] Step S62, Freeze-Drying:

[0086] Pre-Freezing: Maintain at -80 °C for 2 hours;

[0087] Primary Drying: -40 °C, 0.1 mbar, 24 hours;

[0088] Secondary drying: 25°C, 0.01 mbar, 6 hours; finally, a freeze-dried preparation was obtained. The product concentration was 25 ± 2 mg / mL, the water content was ≤ 3%, and it could be stably stored at 4°C for 12 months. The whole process was completed in an ISO Class 5 clean environment and complied with cGMP production specifications.

[0089] A specific application of this example is:

[0090] 1. Gene synthesis and vector construction

[0091] 1.1. Artificial gene sequence synthesis:

[0092] Target sequence: A full-length gene (1236 bp) was designed according to SEQ ID NO: 1, which included the following functional domains: COX8 signal peptide (N-terminal, 78 bp); NDUFS1 catalytic domain (1053 bp, corresponding to amino acids 50 - 400); SLC25A51 transmembrane domain (453 bp, corresponding to amino acids 100 - 250); MDH2 α-helix domain (273 bp, corresponding to amino acids 30 - 120); (Gly4Ser)3 linker peptide (45 bp); SV40 nuclear localization signal (C-terminal, 21 bp);

[0093] Codon optimization: The Escherichia coli-preferred codons were adjusted by GeneOptimizer software, and the GC content was optimized to 55%, eliminating the EcoRI and BamHI restriction sites;

[0094] 1.2. Recombinant plasmid construction:

[0095] Vector selection: pET-28a(+) (containing the T7 promoter and 6×His tag);

[0096] Digestion and ligation: The insert fragment and the vector were double-digested with AgeI / NotI (37°C, 2 hours);

[0097] Ligation system: The insert fragment (50 ng / μL) and the linearized vector (20 ng / μL) were mixed at a molar ratio of 3:1, and T4 DNA ligase (NEB) was used for ligation at 16°C for 12 hours;

[0098] Transformation verification: Transformed DH5α competent cells, and positive clones were verified by PCR (primers: F: 5'-ATGGCGATCGAG-3', R: 5'-TCAGTGGTGGTGGTGGTG-3');

[0099] 2. Engineering bacteria fermentation and induction expression

[0100] 2.1. High-density fermentation: Strain: BL21(DE3); Medium: Modified TB medium (containing 50 μg / mL kanamycin, 0.5% glycerol);

[0101] Fermentation parameters: initial OD600 = 0.05, cultured at 37°C until OD600 = 6.0 (about 8 hours); induction conditions: 0.5 mM IPTG, induced at 25°C for 16 hours; dissolved oxygen control: 30% (regulated by stirring rate and oxygen inlet).

[0102] 2.2 Cell harvesting: Centrifuge to collect the cells (4°C, 8000×g, 15 minutes); the wet cell weight is about 120 g (from 10 L of fermentation broth).

[0103] 3 Protein purification process

[0104] 3.1 Cell lysis: Lysis buffer: 20 mM Tris-HCl, 300 mM NaCl, 10 mM imidazole, pH 8.0; lysis method: disrupted by a high-pressure homogenizer (APV-2000), 3 cycles (1500 bar); centrifugal clarification: centrifuge at 15000×g for 30 minutes (4°C), collect the supernatant.

[0105] 3.2 Affinity chromatography:

[0106] Chromatography column: Ni-NTA Superflow (5 mL column volume);

[0107] Elution procedure: Equilibration: 5 column volumes of binding buffer (20 mM Tris, 300 mM NaCl, 20 mM imidazole, pH 8.0); elution: gradient elution (50→500 mM imidazole, 10 column volumes); collection: collect the elution peak with 200 - 300 mM imidazole.

[0108] 3.3 Fine purification: Gel filtration chromatography: Superdex 200 Increase 10 / 300 GL; mobile phase: PBS (pH 7.4) containing 5% glycerol, flow rate 0.5 mL / min; collection criterion: main peak with a molecular weight of 75 - 80 kDa.

[0109] 4 Formulation lyophilization and quality control

[0110] 4.1 Lyophilization formulation: Protein solution: 20 mg / mL (dissolved in PBS); lyoprotectant: 10% trehalose, 1% glycine, 0.5% mannitol (mixed with the protein solution at a ratio of 1:1).

[0111] 4.2 Freeze-drying procedure: Pre-freezing: rapidly freeze at -80°C for 2 hours; primary drying: -40°C, 0.1 mbar, 24 hours; secondary drying: 25°C, 0.01 mbar, 6 hours.

[0112] 4.3 Final product specifications: Appearance: white, loose, freeze-dried powder; Reconstitution performance: Dissolution time in 2 mL PBS ≤ 30 seconds (with vortex assistance); Purity: Single band shown by SDS-PAGE, HPLC purity ≥ 97%; Stability: Stored at 4°C for 12 months, activity retention ≥ 90% (verified by accelerated test).

[0113] Application of mitochondrial repair factor in asthma mouse model:

[0114] 1. Experimental model construction:

[0115] 1.1 Animal selection:

[0116] Use female BALB / c mice aged 6 - 8 weeks (body weight 18 - 22 g);

[0117] Grouping: Normal control group (n = 10), asthma model group (n = 10), low-dose treatment group (10 mg / kg, n = 10), high-dose treatment group (30 mg / kg, n = 10);

[0118] 1.2 Asthma induction:

[0119] Sensitization phase: Intraperitoneally inject a mixture containing 50 μg ovalbumin (OVA) and 2 mg aluminum hydroxide adjuvant on days 0, 7, and 14;

[0120] Challenge phase: From day 21 to day 27, nebulize 1% OVA solution every day (30 minutes per time);

[0121] 2. Formulation treatment plan:

[0122] 2.1 Drug preparation:

[0123] Reconstituted preparation: Take 50 mg of freeze-dried powder and dissolve it in 2 mL of sterile PBS (final concentration 25 mg / mL);

[0124] Mitochondrial penetrating peptide (MPP) coupling: Pre-incubate with TAT-MPP (sequence: YGRKKRRQRRR) at a molar ratio of 1:3 for 30 minutes;

[0125] 2.2 Administration method:

[0126] Treatment group: From day 21 of the OVA challenge phase, inject the repair factor-MPP complex via the tail vein daily (dose calculated according to body weight);

[0127] Control group: Inject an equal volume of PBS solution containing MPP;

[0128] 3. Key intervention parameters:

[0129] 3.1 Administration time window:

[0130] Prophylactic administration: Administration starts 3 days before the first OVA sensitization (to verify the effect of early intervention);

[0131] Therapeutic administration: Administration starts after the first OVA challenge (to simulate the clinical treatment scenario);

[0132] 3.2, Dose gradient:

[0133] Low dose: 10 mg / kg (corresponding human equivalent dose 0.81 mg / kg);

[0134] High dose: 30 mg / kg (based on the maximum tolerated dose study);

[0135] 4. Efficacy evaluation system:

[0136] 4.1, Detection of airway hyperresponsiveness:

[0137] Use a whole-body plethysmography system to measure airway resistance (Penh value);

[0138] Stimulation reagent: Methacholine (atomized in a gradient of 0 - 50 mg / mL);

[0139] 4.2, Analysis of inflammatory markers:

[0140] Serum detection: ELISA is used to measure the levels of IL-4, IL-5, IL-13, and total IgE;

[0141] Bronchoalveolar lavage fluid (BALF): Eosinophil count (Diff-Quik staining); 4.3, Mitochondrial function assessment:

[0142] Isolation of lung tissue mitochondria: Mitochondria are extracted by differential centrifugation;

[0143] Key indicators:

[0144] NAD+ / NADH ratio (detected by colorimetry, kit: Abcam ab65348); ATP production rate (luciferase method);

[0145] Mitochondrial membrane potential (detected by flow cytometry with JC-1 probe);

[0146] 4.4, Histopathological analysis:

[0147] H&E staining: Evaluate the airway inflammation infiltration score (0 - 4 grades);

[0148] PAS staining: Quantify goblet cell hyperplasia (proportion of mucus secretion area);

[0149] Transmission electron microscopy: Observe the structural integrity of mitochondrial cristae;

[0150] 5. Safety monitoring:

[0151] Blood biochemistry: Detection of ALT, AST, BUN, and Cr levels (excluding liver and kidney toxicity); Weight change: Record the weight fluctuations during treatment daily;

[0152] Histopathology: Screen for abnormal lesions in the heart, liver, and kidney tissues by H&E staining;

[0153] 6. Data collection time points:

[0154] Baseline data: Before the first dose (Day 0);

[0155] Mid-term assessment: Day 14 (collection of BALF and serum);

[0156] Endpoint analysis: Sacrifice the animals on Day 28 to complete all detections;

[0157] 7. Refer to the following table for the experimental results:

[0158] Table 1 Improvement of airway function:

[0159]

[0160]

[0161] Table 2 Anti-inflammatory effect:

[0162]

[0163] Table 3 Mitochondrial function repair:

[0164]

[0165]

[0166] Table 4 Histopathological improvement:

[0167]

[0168] Table 5 Safety data:

[0169] Index Normal control group High-dose group ALT (U / L) 35±4 38±5 AST (U / L) 40±5 43±6 BUN (mmol / L) 6.2±0.7 6.5±0.8 Body weight change (g) +1.2±0.3 +0.9±0.2

[0170] Dose-dependent efficacy: The high-dose group (30 mg / kg) was significantly superior to the low-dose group in all functional indicators (p < 0.05), and the airway resistance recovered to more than 85% of the normal group level;

[0171] Verification of the mechanism of action: The increase in the NAD+ / NADH ratio was positively correlated with the ATP production rate (r = 0.89, p < 0.001);

[0172] The restoration of mitochondrial membrane potential was significantly correlated with the decrease in inflammatory factors (IL-5 vs membrane potential: r = -0.76, p < 0.01);

[0173] Safety: There were no statistically significant differences in blood biochemical indexes between the treatment group and the normal group (p > 0.05), and no pathological damage was found in the heart, liver, and kidney tissues.

[0174] 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 any one or more embodiments or examples in a suitable manner.

[0175] 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 principles and practical applications 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 repair factor based on NADH metabolism, characterized in that: The construction method comprises the following steps: Step S1, functional element design and sequence optimization: design the core module, add the mitochondrial targeting sequence, optimize the connecting element, and complete the codon optimization to adapt to E. coli expression; Step S2, recombinant vector construction: synthesizing a target DNA fragment containing restriction sites, digesting the vector backbone with enzymes, and then constructing a recombinant vector through a ligation reaction; Step S3, host cell transformation: chemically transform competent cells, and obtain single colonies containing the recombinant vector through plate screening; Step S4, amplification and culture of engineered bacteria: preparing seed solution, using a fermenter to amplify the culture and induce the engineered bacteria to express the target protein; Step S5, protein purification: the cells are crushed to obtain the supernatant, and the target protein is obtained by metal chelate chromatography and molecular sieve purification; Step S6, preparation of preparation: preparing a lyophilized protective agent mixed protein, and preparing a stable lyophilized preparation by pre-freezing and graded drying.

2. The method for preparing a mitochondrial repair factor based on NADH metabolism according to claim 1, characterized in that: The step S1, functional element design and sequence optimization specifically includes the following steps: Step S11, determine the core functional module: NADH regeneration module: the catalytic domain of NDUFS1, the active subunit of human mitochondrial complex I, was selected; NAD+ transport module: chimeric transmembrane domain of mitochondrial inner membrane carrier SLC25A51; Cofactor stabilization module: The α-helix structure of malate dehydrogenase is introduced to stabilize the cofactor binding site; Step S12, adding a mitochondrial targeting sequence: fusing the mitochondrial localization signal peptide of cytochrome c oxidase subunit VIII at the N-terminus; Step S13, design of connecting elements: insert (Gly4Ser)3 flexible connecting peptide between functional modules; add SV40 nuclear localization signal after the stop codon as a negative regulatory element; Step S14, codon optimization: use GeneOptimizer software to optimize the E. coli-preferred codons of the human sequence; adjust the GC content to 52% to 58%, and eliminate internal ribosome binding sites and restriction enzyme cutting sites.

3. The method for preparing a mitochondrial repair factor based on NADH metabolism according to claim 2, characterized in that: The human mitochondrial complex I has the enzymatic activity of catalyzing NADH dehydrogenation; the unique identification number of the active subunit NDUFS1 is UniProt ID: P28331; the specific position of the catalytic domain that performs the NADH regeneration function is the segment from the 50th amino acid to the 400th amino acid of the NDUFS1 protein; the specific position of the transmembrane domain is in the amino acid sequence of the SLC25A51 protein, from the 100th residue to the 250th residue; the α-helical structure selects the amino acid residues 30-120 of malate dehydrogenase; The amino acid sequence of the cytochrome c oxidase subunit VIII mitochondrial localization signal peptide is MLSLRQSIRFFKPATRTLCSSRYLL.

4. The method for preparing a mitochondrial repair factor based on NADH metabolism according to claim 1, characterized in that: The step S2, recombinant vector construction specifically comprises the following steps: Step S21, synthesizing DNA fragments: obtaining the full-length gene sequence SEQ ID NO: 1 by chemical synthesis; AgeI and NotI restriction sites were introduced at both ends; Step S22, vector backbone preparation: pET-28a(+) vector (Novagen) was taken; 5.4 kb linearized vector was recovered by gel digestion with AgeI / NotI double enzymes, and the digestion reaction conditions were 37°C and 2 hours; Step S23, ligation reaction: insert fragment: vector = 3:1 molar ratio mixed; use T4 DNA ligase at 16°C for 12 hours.

5. The method for preparing a mitochondrial repair factor based on NADH metabolism according to claim 4, characterized in that: The AgeI recognizes and cuts 5'-ACCGGT-3'; the NotI recognizes and cuts 5'-GCGGCCGC-3'; In step S23, the reaction system in the ligation reaction is specifically: 50ng vector DNA: The mass of linearized pET-28a(+) vector DNA involved in the ligation reaction is 50 ng; 150 ng insert fragment: The mass of the target gene DNA fragment to be connected is 150 nanograms, that is, the DNA fragment containing the target sequence synthesized in step S21; 1×T4buffer: T4 DNA ligase special buffer, used at 1x concentration; 400U enzyme: The activity unit of T4 DNA ligase added is 400U, and the enzyme acts as a catalyst.

6. The method for preparing a mitochondrial repair factor based on NADH metabolism according to claim 1, characterized in that: The step S3, host cell transformation specifically comprises the following steps: Step S31, chemical transformation: take 50 μL BL21 (DE3) competent cells; add 5 μL ligation product, ice bath for 30 minutes, then heat shock at 42°C for 45 seconds; add 950 μL SOC medium, and recover at 37°C for 1 hour; Step S32, plate screening: spread on LB agar plates containing 50 μg / mL kanamycin; culture inverted at 37°C for 16 hours, and pick single colonies with a diameter of 2-3 mm.

7. The method for preparing a mitochondrial repair factor based on NADH metabolism according to claim 1, characterized in that: The step S4, the engineering bacteria amplification culture specifically comprises the following steps: Step S41, seed solution preparation: inoculate a single bacterial colony into 5 mL TB medium containing 0.5% glycerol; shake culture at 220 rpm until OD600 = 2.0; Step S42, fermentation tank culture: inoculate into a 10 L fermentation tank at 1:100, initial OD600=0.05; culture conditions: 37°C, pH7.0, dissolved oxygen 30%; induction expression: when OD600 reaches 6.0, add 0.5 mM IPTG, induce at 25°C for 16 hours.

8. The method for preparing a mitochondrial repair factor based on NADH metabolism according to claim 1, characterized in that: The step S5, protein purification specifically comprises the following steps: Step S51, cell disruption: using a high-pressure homogenizer to disrupt the cells, and collecting the supernatant by centrifugation; Step S52, metal chelate chromatography: the supernatant was loaded onto a Ni-NTA column (GE Healthcare), and eluted using a 50-500 mM imidazole linear gradient using 20 mM Tris-HCl, 500 mM NaCl, and 20 mM imidazole as a binding buffer; Step S53, molecular sieve purification: using a Superdex200Increase10 / 300GL column, with PBS containing 5% glycerol and 0.02% Tween-20 as the mobile phase, collecting the elution peak with a molecular weight of 75-80 kDa.

9. The method for preparing a mitochondrial repair factor based on NADH metabolism according to claim 8, characterized in that: When the high pressure homogenizer is used to disrupt cells, the pressure parameter is 1500 bar and the process is repeated 3 times.

10. The method for preparing a mitochondrial repair factor based on NADH metabolism according to claim 1, characterized in that: The step S6, preparation of the preparation specifically comprises the following steps: Step S61, preparation of freeze-drying protective agent: preparing a PBS solution of 10% trehalose, 1% glycine, and 0.5% mannitol, and mixing it with the purified protein in a volume ratio of 1:1; Step S62, freeze drying: pre-freezing stage, keep at -80°C for 2 hours; primary drying stage, keep at -40°C, 0.1 mbar for 24 hours; secondary drying stage, keep at 25°C, 0.01 mbar for 6 hours, and finally obtain the freeze-dried preparation.

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