Enzymolysis preparation process of albumin oligopeptide

Abstract

The invention relates to the technical field of albumin oligopeptide enzymolysis, in particular to an albumin oligopeptide enzymolysis preparation process which comprises the following steps: (1) constructing and expressing recombinant albumin containing a KRPAGY repetitive sequence; (2) purifying the recombinant albumin; (3) carrying out enzymolysis on the purified recombinant albumin by using a multiple enzymolysis system; (4) purifying the enzymolysis product through a multi-stage membrane separation system; (5) carrying out structural modification on the purified peptide fragment; and (6) evaluating the immunocompetence of the modified peptide fragment, through the design of a multiple enzymolysis system, accurate hydrolysis of albumin is realized, and the yield and specificity of the target peptide fragment are remarkably improved. The innovative membrane separation technology greatly improves the separation and purification efficiency; through N-terminal cysteine modification and C-terminal glycyl-prolyl modification, the stability and membrane permeability of the peptide fragment in the gastrointestinal tract environment are significantly improved.

Description

Technical Field

[0001] The invention relates to the technical field of albumin oligopeptide enzymatic hydrolysis, in particular to an albumin oligopeptide enzymatic hydrolysis preparation process. Background Art

[0002] In recent years, immunomodulatory peptides have shown great potential in the treatment of autoimmune diseases, inflammatory diseases and allergic diseases. Among them, albumin oligopeptides have become a research hotspot due to their natural origin, low immunogenicity and good biocompatibility. However, there are still many problems in the existing preparation technology and application of albumin oligopeptides:

[0003] Traditional albumin oligopeptide preparation mainly relies on single enzymatic hydrolysis or chemical hydrolysis, resulting in low peptide sequence diversity and difficulty in obtaining peptides with specific functions. In addition, it is difficult to accurately control the degree of hydrolysis with existing technologies, which often leads to over-hydrolysis or insufficient hydrolysis, affecting product quality and function.

[0004] Conventional chromatography separation technology is inefficient when dealing with complex peptide mixtures, making it difficult to achieve large-scale production. At the same time, existing technologies are difficult to effectively remove residual enzymes and other impurities, affecting the purity and safety of the product.

[0005] Existing albumin oligopeptides are often easily hydrolyzed by gastrointestinal enzymes and have low oral bioavailability. At the same time, many peptides have poor stability in the body and are difficult to maintain sustained therapeutic effects.

[0006] Most existing immunomodulatory peptides target only a single target or a single immune cell type, making it difficult to address complex immune disorders. This single mechanism of action limits its clinical application and therapeutic effect.

[0007] Existing immunomodulatory peptides often lack tissue specificity and are difficult to precisely regulate at specific inflammatory sites, which may lead to systemic immunosuppression and increase the risk of side effects such as infection.

[0008] Due to the complex preparation process and high cost, many potential albumin oligopeptides are difficult to achieve large-scale industrialization, which limits their clinical application and market promotion.

[0009] In view of the above problems, developing a novel, multifunctional and efficiently prepared albumin oligopeptide has important scientific significance and clinical value. The present invention is aimed at the deficiencies of the prior art and proposes an innovative solution. Summary of the invention

[0010] In view of the deficiencies in the prior art, the present invention provides an enzymatic preparation process for albumin oligopeptides.

[0011] To achieve the above object, the present invention provides the following technical solution: a process for preparing albumin oligopeptides by enzymatic hydrolysis, comprising the following steps:

[0012] (1) construct and express recombinant albumin containing KRPAGY repeat sequences;

[0013] (2) purifying the recombinant albumin;

[0014] (3) enzymatically hydrolyzing the purified recombinant albumin using a multiple enzymatic hydrolysis system;

[0015] (4) purifying the enzymatic hydrolysis product by a multi-stage membrane separation system;

[0016] (5) structural modification of the purified peptides;

[0017] (6) Evaluating the immune activity of the modified peptide.

[0018] Preferably, step (1) comprises:

[0019] a) designing and synthesizing a gene sequence encoding the recombinant albumin, wherein the gene sequence is shown in SEQ ID NO.1;

[0020] b) cloning the gene sequence into the pET28a vector to construct the pET28a-hKRPAGY-Albumin expression plasmid;

[0021] c) transforming the obtained plasmid into Escherichia coli BL21 (DE3) competent cells;

[0022] d) Incubate in LB medium containing 50 μg / mL kanamycin at 37°C with shaking at 200 rpm until OD600 reaches 0.6-0.8;

[0023] e) Cool down to 25°C, add IPTG to a final concentration of 0.5 mM, and continue culturing for 8-12 hours.

[0024] Preferably, step (2) comprises:

[0025] a) Collect the cells by centrifugation at 6000 g for 10 min at 4°C;

[0026] b) resuspending the bacterial cells in a lysis buffer, wherein the lysis buffer comprises 50 mM Tris-HCl (pH 8.0), 300 mM NaCl and 10 mM imidazole;

[0027] c) Ultrasonication in an ice bath: power 300 W, working for 3 seconds, interval 3 seconds, total time 10 minutes;

[0028] d) centrifugation at 12000 g for 30 min at 4°C and collecting the supernatant;

[0029] e) filtering the supernatant through a 0.22 μm filter membrane;

[0030] f) Purify the target protein using Ni-NTA affinity chromatography, wherein the elution buffer comprises 50 mM Tris-HCl (pH 8.0), 300 mM NaCl and 250 mM imidazole.

[0031] Preferably, the multiple enzymatic hydrolysis system in step (3) comprises trypsin, α-chymotrypsin, proteinase K and leucine aminopeptidase, wherein:

[0032] a) dissolving purified recombinant albumin (10 mg / mL) in 50 mM Tris-HCl buffer (pH 8.0);

[0033] b) Add the above enzymes in sequence, and their final concentrations are:

[0034] Trypsin 0.1-0.5 mg / mL; α-chymotrypsin 0.05-0.2 mg / mL; Proteinase K 0.01-0.05 mg / mL; Leucine aminopeptidase 0.02-0.1 mg / mL;

[0035] c) reacting at 37°C for 4 hours, taking samples for analysis every 30 minutes and adjusting the amount of each enzyme added according to the pH value and substrate concentration;

[0036] d) Heat in a 95°C water bath for 10 minutes to terminate the reaction.

[0037] Preferably, the multi-stage membrane separation system in step (4) comprises:

[0038] a) using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa, centrifuging at 4°C, 4000 g for 30 minutes, and collecting the filtrate;

[0039] b) using a nanofiltration column with a molecular weight cutoff of 500 Da, a column volume of 50 mL, a flow rate of 1 mL / min, and an elution buffer of 20 mM phosphate buffer (pH 7.4);

[0040] c) using a cation exchange column, the equilibration buffer was 20 mM phosphate buffer (pH 6.5), the elution buffer was 20 mM phosphate buffer (pH 6.5) containing a 0-1 M NaCl gradient, and the flow rate was 1 mL / min;

[0041] d) Pulse flow technique was applied in the above steps, with a pulse frequency of 0.5-2 Hz and a pulse amplitude of ±30% of the initial flow rate.

[0042] Preferably, the structural modification in step (5) includes N-terminal cysteine ​​modification and C-terminal glycyl-prolyl modification, wherein:

[0043] a) The steps of N-terminal cysteine ​​modification are:

[0044] i. Dissolve the purified peptide (5 mg / mL) in 0.1 M phosphate buffer (pH 7.4);

[0045] ii. Add N-succinimidyl-S-acetylthiocysteine ​​(SATA) at a molar ratio of 1:1.2;

[0046] iii. react at room temperature for 2 hours, stirring speed 200 rpm;

[0047] iv. Add 50 mM hydroxylamine and react at room temperature for 15 minutes;

[0048] b) The steps of C-terminal glycyl-prolyl modification are:

[0049] i. dissolving the above product in a DMF: water (1:1) mixed solvent;

[0050] ii. Add Fmoc-Gly-Pro-OH, molar ratio 1:1.5;

[0051] iii. Add 1.2 equivalents of EDC·HCl and HOBt;

[0052] iv. React at room temperature for 4 hours, stirring speed 200 rpm;

[0053] v. Add 20% piperidine / DMF solution and react at room temperature for 30 minutes.

[0054] Preferably, the method further comprises purifying the modified peptide using a semi-preparative HPLC system, wherein:

[0055] a) Use a C18 reverse phase column with a column size of 10 x 250 mm and a particle size of 5 μm;

[0056] b) mobile phase A is 0.1% TFA aqueous solution, and mobile phase B is 0.1% TFA acetonitrile solution;

[0057] c) using a gradient elution of 5-50% B for 30 minutes;

[0058] d) The flow rate was 3 mL / min and the detection wavelength was 220 nm;

[0059] e) Collect the target peak and freeze-dry it.

[0060] Preferably, the immune activity assessment in step (6) comprises:

[0061] a) T cell proliferation assay, in which human peripheral blood mononuclear cells (PBMCs) were co-cultured with modified peptides at different concentrations (0.1-100 μg / mL) for 48 hours, followed by BrdU incorporation assay;

[0062] b) Cytokine secretion detection, in which the levels of IL-2 and IFN-γ in the culture supernatant were detected using the ELISA method;

[0063] c) flow cytometry analysis of the activation status of CD4+ and CD8+ T cells using fluorescently labeled antibodies against CD3, CD4, CD8, and CD69;

[0064] d) Mouse autoimmune encephalomyelitis (EAE) model experiment, in which mice were given 1-5 mg / kg of the modified peptide for 21 consecutive days, and the clinical scores and histopathological changes were evaluated.

[0065] Preferably, the following quality control steps are also included:

[0066] a) Analyze the sequence and purity of the peptides using HPLC-MS;

[0067] b) Analyze the secondary structure of peptides using circular dichroism spectroscopy;

[0068] c) Dynamic light scattering was used to detect the aggregation state of peptides.

[0069] 10. The method according to claim 9, characterized in that the conditions of the HPLC-MS analysis include:

[0070] a) Use a C18 reverse phase column with a column size of 2.1 x 50 mm and a particle size of 1.8 μm;

[0071] b) mobile phase A is 0.1% formic acid in water, and mobile phase B is 0.1% formic acid in acetonitrile;

[0072] c) using a gradient elution of 5-95% B for 10 minutes;

[0073] d) The flow rate was 0.3 mL / min and the injection volume was 5 μL;

[0074] e) The mass spectrometer scan range is m / z 100-2000.

[0075] Compared with the prior art, the present invention has the following beneficial technical effects:

[0076] 1. Efficient and accurate preparation process: Through the design of multiple enzymatic hydrolysis systems, the present invention achieves accurate hydrolysis of albumin, significantly improving the yield and specificity of target peptides. At the same time, innovative membrane separation technology greatly improves separation and purification efficiency, laying the foundation for large-scale production.

[0077] 2. Excellent bioavailability: Through N-terminal cysteine ​​modification and C-terminal glycyl-prolyl modification, the present invention significantly improves the stability and membrane permeability of the peptide segment in the gastrointestinal environment. This not only improves oral bioavailability, but also provides the possibility for the development of long-acting preparations.

[0078] 3. Multi-target immune regulation: The optimized peptides of the present invention show regulatory effects on multiple T cell subsets and can also affect the function of dendritic cells. This multi-target regulatory mechanism is expected to play a wider therapeutic role in complex immune disorders.

[0079] 4. Epithelial barrier protection: In organoid and skin equivalent models, the peptides of the present invention showed significant epithelial barrier protection. This discovery provides new ideas for the treatment of diseases such as inflammatory bowel disease and atopic dermatitis.

[0080] 5. Tissue-specific regulation: In different tissue models, the peptides of the present invention all showed good effects, reflecting their tissue-specific regulatory ability. This characteristic helps to achieve precise treatment and reduce systemic side effects.

[0081] 6. Broad-spectrum anti-inflammatory effect: The peptides of the present invention show good inhibitory effects in inflammatory reactions mediated by Th1, Th2 and Th17, reflecting the potential of broad-spectrum anti-inflammatory. This property makes it possible to be used in the treatment of various autoimmune diseases.

[0082] 7. Industrialization potential: The preparation process of the present invention is simple and efficient, and is easy to scale up for production. At the same time, the multifunctional properties and broad-spectrum application prospects of the optimized peptide segment provide strong support for its industrialization and market promotion.

[0083] 8. Low toxicity and high efficacy: Compared with traditional immunosuppressants, the peptides of the present invention may have lower toxicity and side effects while achieving similar effects, which provides a new option for long-term medication and chronic disease management.

[0084] 9. Tissue repair potential: The peptides of the present invention show the ability to promote extracellular matrix remodeling, which not only helps resolve inflammation but also may promote tissue repair and regeneration.

[0085] In summary, the present invention has successfully developed a multifunctional and highly efficient new albumin oligopeptide through innovative preparation technology and sophisticated structural design. This peptide segment not only overcomes many limitations of the prior art, but also shows broad application prospects. Its comprehensive advantages in immunomodulation, inflammation control, tissue protection, etc., provide new hope for the treatment of a variety of intractable diseases, and have important scientific significance and significant clinical transformation value. DETAILED DESCRIPTION

[0086] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the specific embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0087] The present invention relates to an enzymatic preparation process of albumin oligopeptide, which comprises the following steps:

[0088] 1. Construct and express recombinant albumin containing KRPAGY repeat sequence.

[0089] This step includes the following detailed processes:

[0090] a) Designing and synthesizing a gene sequence encoding recombinant albumin, wherein the gene sequence is shown in SEQ ID NO. 1. The sequence of SEQ ID NO. 1 is as follows:

[0091] .

[0092] The sequence encodes a modified albumin containing three KRPAGY repeating units while retaining the main structural domain of albumin.

[0093] b) The above gene sequence was cloned into the pET28a vector to construct the pET28a-hKRPAGY-Albumin expression plasmid. The specific steps are as follows:

[0094] i. Design primers:

[0095] Forward primer: 5'-GGAATTCCATATGAAGCACGGCATTCCCTTATGC-3' (containing NdeI restriction site);

[0096] Reverse primer: 5'-CCGCTCGAGTTAAGCAGGTCGCCTGTTCAAGC-3' (containing XhoI restriction site);

[0097] ii. Perform PCR amplification, the reaction conditions are:

[0098] Initial denaturation: 95°C, 5 minutes;

[0099] 35 cycles: 95°C for 30 sec, 58°C for 30 sec, 72°C for 2 min;

[0100] Final extension: 72°C, 10 min;

[0101] iii. Enzyme digestion reaction: 37°C, double digestion with NdeI and XhoI for 4 hours;

[0102] iv. Ligation reaction: 16°C, use T4 DNA ligase, react overnight;

[0103] c) Transform the constructed pET28a-hKRPAGY-Albumin plasmid into E. coli BL21 (DE3) competent cells. The transformation steps are as follows:

[0104] i. Mix the plasmid DNA with competent cells and place on ice for 30 minutes;

[0105] ii. Heat shock at 42°C for 90 seconds;

[0106] iii. Immediately place in ice bath for 2 minutes;

[0107] iv. Add 500 μL LB medium and culture at 37°C with shaking for 1 hour;

[0108] v. Spread on LB solid medium containing 50 μg / mL kanamycin and culture at 37°C overnight.

[0109] d) A single clone was selected and inoculated into 5 mL of LB liquid medium containing 50 μg / mL kanamycin, and cultured overnight at 37°C with shaking at 200 rpm.

[0110] e) Take 1 mL of the overnight culture and inoculate it into 100 mL of fresh LB medium (containing 50 μg / mL kanamycin) and culture at 37°C with shaking at 200 rpm until the OD600 reaches 0.6-0.8.

[0111] f) Cool down to 25°C, add IPTG to a final concentration of 0.5 mM, and continue culturing for 8-12 hours. At this stage, recombinant albumin begins to be expressed in large quantities.

[0112] 2. Purification of recombinant albumin.

[0113] This step includes the following detailed processes:

[0114] a) Centrifuge at 4°C, 6000 g for 10 minutes to collect the cells. This step is to separate the culture medium and the cells.

[0115] b) Resuspend the collected cells in 20 mL of lysis buffer. The lysis buffer consists of 50 mM Tris-HCl (pH 8.0), 300 mM NaCl and 10 mM imidazole. The purpose of this step is to break the cell wall and release intracellular proteins.

[0116] c) Perform ultrasonic disruption in an ice bath. The specific parameters are: power 300W, working 3 seconds, interval 3 seconds, total time 10 minutes. This step can effectively disrupt cells and release target proteins.

[0117] d) Centrifuge at 12,000 g for 30 minutes at 4°C and collect the supernatant. This step is to separate soluble proteins and cell debris.

[0118] e) The collected supernatant was filtered through a 0.22 μm filter membrane to remove residual cell debris and macromolecular aggregates.

[0119] f) Purify the target protein using Ni-NTA affinity chromatography. The specific steps are as follows:

[0120] i. Load the filtered supernatant onto a pre-equilibrated 1 mL Ni-NTA resin column;

[0121] ii. Wash with 20 mL of washing buffer (50 mM Tris-HCl, pH 8.0, 300 mM NaCl, 20 mM imidazole);

[0122] iii. Elute the target protein with 10 mL elution buffer (50 mM Tris-HCl, pH 8.0, 300 mM NaCl, 250 mM imidazole);

[0123] iv. Collect 1 mL of elution fraction and perform SDS-PAGE and Western blot analysis to confirm the purity and integrity of the target protein.

[0124] 3. Use a multiple enzymatic hydrolysis system to enzymatically hydrolyze the purified recombinant albumin.

[0125] This step uses a unique multi-enzyme digestion system, including the following proteases:

[0126] a) Trypsin (EC 3.4.21.4): specifically cleaves peptide bonds at the C-terminus of lysine and arginine;

[0127] b) α-chymotrypsin (EC 3.4.21.1): preferentially cleaves the peptide bond at the C-terminus of aromatic amino acids (such as tyrosine);

[0128] c) Proteinase K (EC 3.4.21.64): a broad-spectrum protease that can cleave a variety of hydrophobic amino acids;

[0129] d) Leucine aminopeptidase (EC 3.4.11.1): specifically removes the N-terminal leucine.

[0130] The specific enzymatic hydrolysis steps are as follows:

[0131] a) Purified recombinant hKRPAGY-Albumin (10 mg / mL) was dissolved in 50 mM Tris-HCl buffer (pH 8.0).

[0132] b) Add the above enzymes in sequence to the following final concentrations:

[0133] Trypsin: 0.1-0.5 mg / mL;

[0134] α-chymotrypsin: 0.05-0.2 mg / mL;

[0135] Proteinase K: 0.01-0.05 mg / mL;

[0136] Leucine aminopeptidase: 0.02-0.1 mg / mL;

[0137] c) react at 37°C for 4 hours. During the reaction, samples were taken and analyzed every 30 minutes, and the amount of each enzyme added was adjusted according to the real-time monitored pH value and substrate concentration. The pH value was monitored using a Mettler Toledo InLab Micro pH electrode, and the substrate concentration was monitored using a Nanodrop 2000 spectrophotometer at a wavelength of 280 nm. Based on the monitoring data, the amount of each enzyme added was adjusted every 30 minutes within a range of ±20% of the initial concentration.

[0138] d) After the reaction is completed, the reaction solution is placed in a 95°C water bath and heated for 10 minutes to terminate the enzymatic hydrolysis reaction.

[0139] 4. Purify the enzymatic hydrolysis products through a multi-stage membrane separation system.

[0140] This step uses a new type of multi-stage membrane separation system, including ultrafiltration, nanofiltration and ion exchange. The specific steps are as follows:

[0141] a) Ultrafiltration:

[0142] i. Use MilliporeAmiconUltra-15 centrifugal filter with a molecular weight cutoff of 10kDa;

[0143] ii. Centrifuge at 4000 g for 30 min at 4°C;

[0144] iii. Collect the filtrate and discard the retentate.

[0145] b) Nanofiltration:

[0146] i. Use GE Healthcare Sephadex G-25 nanofiltration column with a molecular weight cutoff of 500Da;

[0147] ii. The column volume was set to 50 mL and the flow rate was controlled at 1 mL / min;

[0148] iii. Use 20 mM phosphate buffer (pH 7.4) as elution buffer;

[0149] iv. Collect 1 mL of elution fraction and perform HPLC analysis.

[0150] c) Ion exchange:

[0151] i. Use GE Healthcare HiTrap SP HP cation exchange column with a column volume of 1 mL;

[0152] ii. Use 20 mM phosphate buffer (pH 6.5) as the equilibration buffer;

[0153] iii. The elution buffer was 20 mM phosphate buffer (pH 6.5) containing a 0-1 M NaCl gradient;

[0154] iv. Control the flow rate to 1 mL / min and collect 0.5 mL of elution fraction.

[0155] During the entire membrane separation process, pulse flow technology is used to improve separation efficiency. The specific parameters are as follows:

[0156] use pure protein purification system, equipped with a pulse flow module; the pulse frequency was set to 0.5-2 Hz; the pulse amplitude was controlled at ±30% of the initial flow rate.

[0157] 5. Structural modification of the purified peptide fragments.

[0158] This step includes two parts: N-terminal cysteine ​​modification and C-terminal glycyl-prolyl modification.

[0159] a) N-terminal cysteine ​​modification:

[0160] i. Dissolve the purified KRPAGY peptide (5 mg / mL) in 0.1 M phosphate buffer (pH 7.4);

[0161] ii. Add N-succinimidyl-S-acetylthiocysteine ​​(SATA) at a molar ratio of 1:1.2;

[0162] iii. react at room temperature for 2 hours with a stirring speed of 200 rpm;

[0163] iv. Add hydroxylamine to a final concentration of 50 mM and continue the reaction at room temperature for 15 minutes to remove the acetyl group.

[0164] b) C-terminal glycyl-prolyl modification:

[0165] i. dissolving the above product in a DMF: water (1:1) mixed solvent;

[0166] ii. Add Fmoc-Gly-Pro-OH, the molar ratio is controlled at 1:1.5;

[0167] iii. Add 1.2 equivalents of EDC·HCl and HOBt;

[0168] iv. react at room temperature for 4 hours with stirring speed maintained at 200 rpm;

[0169] v. Add 20% piperidine / DMF solution and react at room temperature for 30 minutes to remove the Fmoc protecting group.

[0170] The modified peptides were purified using a semi-preparative HPLC system, with the following specific steps:

[0171] a) Using Shimadzu LC-20AT semi-preparative HPLC system;

[0172] b) The chromatographic column used was Waters XBridge Peptide BEH C18 column (10x 250mm, 5μm);

[0173] c) Mobile phase A is 0.1% TFA aqueous solution; Mobile phase B is 0.1% TFA acetonitrile solution;

[0174] d) using a gradient elution of 5-50% B over 30 minutes;

[0175] e) The flow rate was set to 3 mL / min and the detection wavelength was 220 nm;

[0176] f) Collect the target peak and perform freeze-drying.

[0177] 6. Evaluate the immunological activity of the modified peptides.

[0178] This step includes an assessment of the following aspects:

[0179] a) T cell proliferation assay:

[0180] i. PBMCs were isolated from the peripheral blood of healthy volunteers using Ficoll-Paque density gradient centrifugation;

[0181] ii. PBMCs (1x 10^6 cells / mL) were co-cultured with modified peptides at different concentrations (0.1-100 μg / mL);

[0182] iii. The culture medium used was RPMI 1640 supplemented with 10% FBS and 1% penicillin / streptomycin;

[0183] iv. Incubate at 37°C, 5% CO2 for 48 hours;

[0184] v. Add BrdU (10 μM) and continue culturing for 12 hours;

[0185] vi. Cell proliferation was detected using BrdU cell proliferation ELISA kit (Roche).

[0186] b) Cytokine secretion detection:

[0187] i. Collect the supernatant of the above culture

[0188] ii. Human IL-2 and IFN-γ ELISA kits (R&D Systems) were used to detect cytokine levels. iii. The OD450 value was read using a SpectraMax M5 multi-function microplate reader according to the instructions of the kit.

[0189] c) Flow cytometry analysis:

[0190] i. Harvest the cultured cells and wash twice with PBS;

[0191] ii. Stain with the following antibody mixture for 30 minutes at 4°C in the dark:

[0192] Anti-CD3-FITC (BD Biosciences, 1:100 dilution);

[0193] Anti-CD4-PE (BD Biosciences, 1:100 dilution);

[0194] Anti-CD8-APC (BD Biosciences, 1:100 dilution);

[0195] Anti-CD69-PE-Cy7 (BD Biosciences, 1:50 dilution);

[0196] iii. Wash twice with PBS and resuspend in 200 μL PBS;

[0197] iv. Analyze using BD FACSCanto II flow cytometer, collecting at least 10,000 events per sample;

[0198] v. Analyze data using FlowJo software to assess the activation status of CD4+ and CD8+ T cells;

[0199] d) Mouse autoimmune encephalomyelitis (EAE) model experiment:

[0200] i. Select 6-8 week old female C57BL / 6 mice, 10 per group;

[0201] ii.EAE induction:

[0202] Day 0: subcutaneous injection of MOG35-55 peptide (200 μg / mouse) + complete Freund's adjuvant;

[0203] Day 0 and day 2: intraperitoneal injection of pertussis toxin (200 ng / mouse);

[0204] iii. Dosage regimen:

[0205] Control group: normal saline, intraperitoneal injection every day;

[0206] Low-dose group: modified peptide 1 mg / kg, intraperitoneal injection every day;

[0207] High-dose group: 5 mg / kg of modified peptide, intraperitoneal injection per day;

[0208] iv. Starting from day 0, clinical scores (0-5 points) were assessed every day;

[0209] v. On day 21, the mice were euthanized and the spinal cord tissues were collected for HE staining and immunohistochemical analysis.

[0210] 7. Quality control.

[0211] To ensure the quality of the prepared highly immunoreactive albumin oligopeptides, the following quality control steps were performed:

[0212] a) HPLC-MS analysis:

[0213] i. Using an Agilent 1290 Infinity II HPLC system coupled to an Agilent 6545Q-TOF mass spectrometer;

[0214] ii. Chromatographic column: Agilent ZORBAX RRHD Eclipse Plus C18 (2.1x 50mm, 1.8μm);

[0215] iii. Mobile phase A: 0.1% formic acid in water; Mobile phase B: 0.1% formic acid in acetonitrile;

[0216] iv. Gradient elution: 5-95% B, 10 minutes;

[0217] v. Flow rate: 0.3 mL / min, injection volume: 5 μL;

[0218] vi. ESI source parameters: drying gas temperature 350°C, flow rate 10 L / min, nebulizer pressure 35 psig;

[0219] vii. Mass spectrometry scanning range: m / z 100-2000;

[0220] viii. Data analysis: Agilent MassHunter software was used to confirm the peptide sequence and purity.

[0221] b) Circular dichroism (CD) analysis:

[0222] i. Using Jasco J-815CD spectrometer;

[0223] ii. Sample concentration: 0.1 mg / mL, dissolved in 10 mM phosphate buffer (pH 7.4);

[0224] iii. Scanning range: 190-260nm, scanning speed: 50nm / min;

[0225] iv. Data acquisition interval: 0.1nm, bandwidth: 1nm;

[0226] v. Temperature: 25℃, accumulated 3 times;

[0227] vi. Data analysis: Use CDNN software to predict secondary structure;

[0228] c) Dynamic Light Scattering (DLS) Analysis:

[0229] i. Using the Malvern ZetasizerNano ZS instrument;

[0230] ii. Sample concentration: 1 mg / mL, dissolved in PBS buffer (pH 7.4);

[0231] iii. Temperature: 25°C;

[0232] iv. Equilibration time: 120 seconds;

[0233] v. Each sample was measured 3 times, and each measurement lasted 60 seconds;

[0234] vi. Data Analysis: Zetasizer software was used to evaluate the size distribution and polydispersity index of the peptides.

[0235] Through the above detailed steps, we can prepare albumin oligopeptides with high immunological activity. This method combines genetic engineering, protein expression and purification, multiple enzymatic hydrolysis, membrane separation technology, peptide modification, and comprehensive activity evaluation and quality control. Each step is carefully designed and optimized to ensure the quality and activity of the final product.

[0236] The innovations of this method are mainly reflected in the following aspects:

[0237] 1. A recombinant albumin rich in KRPAGY sequence was designed to increase the yield of the target peptide.

[0238] 2. Adopt multiple enzymatic hydrolysis system to precisely control the hydrolysis process through the synergistic action of different enzymes.

[0239] 3. The use of a multi-stage membrane separation system combined with pulse flow technology improves the purification efficiency of the target peptide.

[0240] 4. The stability and bioavailability of the peptide are enhanced through structural modification of the N-terminus and C-terminus.

[0241] 5. A comprehensive immune activity evaluation system has been established, including in vitro cell experiments and in vivo animal models.

[0242] This method can not only be used to prepare highly immunoactive KRPAGY albumin oligopeptides, but can also be used as a general platform for the design, preparation and evaluation of other functional peptides. By adjusting gene design, enzymatic conditions and modification strategies, various bioactive peptides with specific functions can be prepared, providing important technical support for the development of new immunomodulators, functional food additives, etc.

[0243] In order to screen out the optimal process of the present invention, we designed the following experimental groups:

[0244] Experimental group 1: standard process group (control group);

[0245] Experimental group 2: optimized enzymatic hydrolysis group (adjusting enzyme ratio);

[0246] Experimental group 3: membrane separation optimization group (adjustment of pulse flow parameters);

[0247] Experimental group 4: peptide modification optimization group (adjustment of modification ratio).

[0248] The specific parameters of each experimental group are as follows:

[0249] 1. Standard process group: Follow the standard process described above.

[0250] 2. Optimized enzymatic hydrolysis group: a) trypsin: 0.3 mg / mL; b) α-chymotrypsin: 0.15 mg / mL; c) proteinase K: 0.03 mg / mL; d) leucine aminopeptidase: 0.06 mg / mL.

[0251] 3. Membrane separation optimization group: Pulse flow parameters: frequency 1.5 Hz, amplitude ±25% of the initial flow rate.

[0252] 4. Peptide modification optimization group: a) N-terminal cysteine ​​modification: SATA to peptide molar ratio 1:1.5; b) C-terminal modification: Fmoc-Gly-Pro-OH to peptide molar ratio 1:2.

[0253] The test results are shown in the following table:

[0254] Table 1. Product yield and purity of different experimental groups

[0255]

[0256]

[0257] Table 2. Evaluation of the immune activity of products in different experimental groups

[0258]

[0259] Table 3. Stability evaluation of products in different experimental groups

[0260]

[0261] Table 4. Therapeutic effects in EAE mouse model (score after 21 days)

[0262]

[0263]

[0264] Based on the above data, we can draw the following conclusions:

[0265] 1. The optimized enzymatic hydrolysis group significantly improved the product yield and purity, which may be due to the fact that the optimized enzyme ratio better adapted to the structural characteristics of recombinant albumin and improved the enzymatic hydrolysis efficiency and specificity.

[0266] 2. The membrane separation optimization group slightly improved the product purity, indicating that the optimized pulse flow parameters can help reduce membrane fouling and improve separation efficiency.

[0267] 3. The peptide modification optimization group performed best in terms of immune activity and stability, which may be due to the fact that a higher proportion of modifications improved the bioavailability and stability of the peptide.

[0268] 4. In the EAE mouse model, the peptide modification optimization group showed the best therapeutic effect, significantly reducing the average clinical score, incidence rate and spinal cord inflammation index.

[0269] Based on these results, we can conclude that the present invention has the following unexpected technical effects:

[0270] 1. Synergistic effect: The optimized multiple enzymatic hydrolysis system not only improves the product yield and purity, but also indirectly enhances the immunological activity of the product. This may be because the more precise enzymatic hydrolysis process retains more key immunologically active epitopes.

[0271] 2. Optimization of structure-function relationship: By adjusting the ratio of N-terminal and C-terminal modifications, we not only improved the stability of the peptide, but also significantly enhanced its immunomodulatory activity. This suggests that the modification not only plays a protective role, but also may optimize the interaction between the peptide and the immune cell receptor.

[0272] 3. Broad-spectrum immunomodulation: From T cell proliferation, cytokine secretion to CD4+ and CD8+ T cell activation, the optimized peptides showed comprehensive immunomodulatory capabilities. This broad spectrum suggests that the peptides may work synergistically through multiple mechanisms to achieve more effective immunomodulation.

[0273] 4. Excellent in vivo effect: In the EAE model, the optimized peptide showed a therapeutic effect far beyond expectations. The 60% reduction in incidence and the spinal cord inflammation index of 1.1 showed that the peptide could not only effectively inhibit the occurrence of the disease, but also significantly reduce the severity of the disease that had already occurred.

[0274] 5. Balance between stability and activity: Usually, improving the stability of proteins or peptides may sacrifice their biological activity. However, the present invention significantly improves the stability and enhances the immunomodulatory activity through precise structural modification. The realization of this balance is very rare and provides a new idea for the development of long-acting and highly active immunomodulators.

[0275] 6. Potential oral administration: The optimized peptide segment showed excellent stability under both acidic and enzymatic conditions, which provides the possibility for the development of oral preparations. If oral administration can be achieved, it will greatly improve the patient's medication compliance and quality of life.

[0276] In summary, the present invention successfully prepared an albumin oligopeptide with high immune activity, excellent stability and significant in vivo effects through multiple enzymatic hydrolysis systems, sophisticated membrane separation technology and ingenious peptide modification strategies. This peptide not only performed well in in vitro experiments, but also showed strong therapeutic potential in complex in vivo environments. These findings have opened up new directions for the development of a new generation of immunomodulators and drugs for the treatment of autoimmune diseases, and have important scientific significance and application prospects.

[0277] In addition, the applicant also established multiple models to conduct performance tests to evaluate the effectiveness of the solution of the present invention and provided detailed experimental design, test results and analysis.

[0278] Model 1: Humanized immune cell in vitro model

[0279] Experimental conditions:

[0280] Cells: Human peripheral blood mononuclear cells (PBMCs);

[0281] Culture conditions: RPMI 1640 + 10% FBS, 37°C, 5% CO 2 ;

[0282] Experimental period: 72 hours.

[0283] Experimental method steps:

[0284] 1. Isolate PBMCs from the peripheral blood of healthy volunteers;

[0285] 2. PBMCs (1×10 6 cells / mL) were co-cultured with different concentrations of peptides (0.1, 1, 10, 100 μg / mL);

[0286] 3. Collect the supernatant and cells after 72 hours;

[0287] 4. Detect T cell proliferation, cytokine secretion and T cell subset activation.

[0288] Model 2: Autoimmune encephalomyelitis (EAE) mouse model

[0289] Experimental conditions:

[0290] Animals: 6-8 week old female C57BL / 6 mice;

[0291] Experimental period: 28 days.

[0292] Experimental method steps:

[0293] 1. EAE induction: subcutaneous injection of MOG35-55 peptide + complete Freund's adjuvant, intraperitoneal injection of pertussis toxin;

[0294] 2. Randomly grouped, 10 in each group;

[0295] 3. Start medication from day 0 for 28 consecutive days;

[0296] 4. Evaluate clinical scores every day;

[0297] 5. On day 28, the mice were euthanized and the spinal cord and lymph node tissues were collected.

[0298] Model 3: Artificial gastrointestinal digestion model

[0299] Experimental conditions:

[0300] Simulated gastric fluid: 0.1M HCl + 3.2mg / mL pepsin;

[0301] Simulated intestinal fluid: 0.1M NaHCO3+1mg / mL pancreatin;

[0302] Experimental period: 4 hours.

[0303] Experimental method steps:

[0304] 1. Dissolve the peptide in simulated gastric fluid and incubate at 37°C for 2 hours;

[0305] 2. Adjust the pH to 6.8, add simulated intestinal fluid, and continue incubation for 2 hours;

[0306] 3. Take samples regularly and analyze the integrity and degradation products of peptides by HPLC-MS.

[0307] Experimental group design:

[0308] 1. Standard process group (control group);

[0309] 2. Optimize the enzymatic hydrolysis group: adjust the ratio of each enzyme in the multiple enzymatic hydrolysis system;

[0310] 3. Membrane separation optimization group: optimize pulse flow parameters;

[0311] 4. Peptide modification optimization group A: increase the proportion of N-terminal cysteine ​​modification;

[0312] 5. Peptide modification optimization group B: increase the C-terminal glycyl-prolyl modification ratio;

[0313] 6. Comprehensive optimization group: Combined optimization of the optimal parameters of enzymatic hydrolysis, membrane separation and peptide modification. Test results:

[0314] Table 5. Results of humanized immune cell in vitro model (100 μg / mL peptide concentration)

[0315]

[0316] Table 6. EAE mouse model results (evaluated after 28 days)

[0317]

[0318] Table 7. Results of artificial gastrointestinal digestion model

[0319]

[0320] Based on the above experimental results, we can draw the following conclusions:

[0321] 1. The comprehensive optimization group showed the best effect in all models, which confirmed the synergistic effect of various innovative points of the present invention.

[0322] 2. In the humanized immune cell in vitro model, the comprehensive optimization group significantly enhanced T cell proliferation, the secretion of pro-inflammatory cytokines (IL-2, IFN-γ) and anti-inflammatory cytokines (IL-10), and the activation of T cell subsets. In particular, the increase in the proportion of Treg cells suggests that the peptide may have the effect of regulating immune balance.

[0323] 3. In the EAE mouse model, the comprehensive optimization group significantly reduced the severity and incidence of the disease. The reduction of pro-inflammatory T cells (IL-17A+ and IFN-γ+) in the spinal cord and the increase of Treg cells in the lymph nodes further confirmed the immunomodulatory effect of this peptide.

[0324] 4. The results of the artificial gastrointestinal digestion model showed that the comprehensive optimization group had the best digestive tract stability, which provided the possibility for the development of oral preparations.

[0325] The present invention has the following unexpected technical effects:

[0326] 1. Multiple immune regulation mechanisms: The present invention can not only inhibit pro-inflammatory T cell subsets, but also promote the production of regulatory T cells. This bidirectional regulation effect is not available in many existing immunomodulators, and is expected to reduce the risk of excessive immunosuppression while treating autoimmune diseases.

[0327] 2. Potential for oral administration: Through structural optimization, the present invention significantly improves the stability of the peptide segment in a simulated gastrointestinal environment. This discovery provides the possibility for the development of oral preparations, which is expected to greatly improve patients' medication compliance and quality of life.

[0328] 3. Improvement in dose-effect relationship: The comprehensive optimization group can achieve significant immunomodulatory effects at lower concentrations, which means that the clinical drug dose may be reduced, thereby reducing potential side effects.

[0329] 4. Broad-spectrum immunomodulation: The present invention not only regulates T cell subsets, but also affects the cytokine network. This broad-spectrum regulation may make it play a role in a variety of autoimmune diseases.

[0330] 5. Central nervous system protective effect: In the EAE model, the present invention significantly reduced the spinal cord inflammation index, which suggests that in addition to the immunomodulatory effect, the peptide may also have a direct neuroprotective effect.

[0331] 6. Potential long-term therapeutic effects: By regulating the proportion of Treg cells, the present invention may help to reestablish immune tolerance and thus achieve long-term disease remission.

[0332] 7. Potential for personalized treatment: Different optimization groups have different effects on different immune indicators, which makes it possible to select the most suitable peptide variant according to the individual patient's condition.

[0333] Summary: This invention successfully prepared an albumin oligopeptide with multiple immunomodulatory effects, excellent stability and significant in vivo effects through multiple enzymatic hydrolysis systems, fine membrane separation technology and ingenious peptide modification strategies. This peptide not only performed well in in vitro experiments, but also showed strong therapeutic potential in complex in vivo environments. Its multiple mechanisms of action, oral administration potential and personalized treatment prospects have opened up a new direction for the development of a new generation of drugs for the treatment of autoimmune diseases, and have important scientific significance and great application prospects.

[0334] Model 4: Humanized dendritic cell-T cell co-culture model

[0335] Experimental conditions:

[0336] Cells: Dendritic cells (DCs) and naive T cells isolated from human peripheral blood mononuclear cells (PBMCs);

[0337] Culture conditions: RPMI 1640 + 10% FBS, 37°C, 5% CO 2 .

[0338] Experimental period: 7 days

[0339] Experimental method steps:

[0340] 1. Isolate PBMCs from the peripheral blood of healthy volunteers;

[0341] 2. Use CD14 magnetic beads to sort monocytes and culture for 6 days to induce mature DCs;

[0342] 3. Use CD3 / CD28 magnetic beads to sort out naive T cells;

[0343] 4. Mature DCs were co-cultured with naive T cells at a ratio of 1:10, and peptides from different treatment groups were added (10 μg / mL);

[0344] 5. Collect the supernatant and cells after 7 days of culture;

[0345] 6. Detect T cell proliferation, cytokine secretion profile and T cell subset differentiation.

[0346] Model 5: Humanized organoid immune-inflammation model

[0347] Experimental conditions:

[0348] Cells: Human intestinal epithelial organoids + PBMCs;

[0349] Culture conditions: Advanced DMEM / F12 + growth factors, 37°C, 5% CO 2；

[0350] Experimental period: 14 days.

[0351] Experimental method steps:

[0352] 1. Cultivate human intestinal epithelial organoids to maturity (about 7 days);

[0353] 2. Add TNF-α and IFN-γ to induce inflammation (24 hours);

[0354] 3. Add PBMCs and peptides from different treatment groups (20 μg / mL);

[0355] 4. Cultivate for 7 days, and replace the medium containing peptide fragments every other day;

[0356] 5. Collect the supernatant and digest the organoids to collect cells;

[0357] 6. Detect inflammatory factor levels, epithelial integrity and immune cell infiltration.

[0358] Model 6: Humanized Skin Equivalent Model

[0359] Experimental conditions:

[0360] Cells: human keratinocytes + human skin fibroblasts + PBMCs;

[0361] Culture conditions: air-liquid interface culture, 37°C, 5% CO 2 ;

[0362] Experimental period: 21 days.

[0363] Experimental method steps:

[0364] 1. Construction of three-dimensional skin equivalents (14 days).

[0365] 2. Addition of IL-4 and IL-13 induced atopic dermatitis-like inflammation (24 hours).

[0366] 3. Add peptides (50 μg / mL) from different treatment groups to the air-liquid interface.

[0367] 4. Cultivate for 7 days and add peptides once a day.

[0368] 5. Collect the culture medium and cryosection the skin equivalents.

[0369] 6. Detect stratum corneum integrity, inflammatory factor expression and immune cell infiltration.

[0370] Experimental group design:

[0371] 1. Negative control group: PBS;

[0372] 2. Positive control group: dexamethasone (1 μM);

[0373] 3. Standard peptide group: unoptimized KRPAGY peptide;

[0374] 4. The optimized peptide group of the present invention: KRPAGY peptide segment optimized by multiple enzymatic hydrolysis, membrane separation and structural modification;

[0375] 5. Commercialized immunomodulatory peptide (thymopentin).

[0376] The test results are shown in the following table:

[0377] Table 8. Results of humanized dendritic cell-T cell co-culture model

[0378]

[0379]

[0380] Table 9. Results of humanized organoid immune inflammation model

[0381]

[0382] Table 10. Humanized skin equivalent model results

[0383]

[0384] Based on the above experimental results, we can draw the following conclusions:

[0385] 1. In all three models, the optimized peptide group of the present invention showed an effect superior to that of the standard peptide group and commercial immunomodulatory peptides, and was close to that of the positive control group (dexamethasone).

[0386] 2. In the dendritic cell-T cell co-culture model, the optimized peptide of the present invention significantly inhibited T cell proliferation and effectively regulated the balance of T cell subsets, especially increasing the proportion of Treg cells. This shows that the peptide has a strong immunomodulatory ability.

[0387] 3. In the organoid immune inflammation model, the optimized peptide of the present invention significantly improved epithelial integrity, reduced the level of inflammatory factors, and reduced T cell infiltration. This shows that the peptide not only has an immunomodulatory effect, but also may directly protect the epithelial barrier function.

[0388] 4. In the skin equivalent model, the optimized peptide of the present invention effectively increased the thickness of the stratum corneum, reduced transepidermal water loss (TEWL), inhibited the production of Th2-related cytokines, and reduced the infiltration of eosinophils. This indicates that the peptide may have good application prospects in allergic skin diseases.

[0389] The present invention has the following unexpected technical effects:

[0390] 1. Multi-target immunomodulation: The optimized peptide of the present invention can not only regulate the balance of T cell subsets, but also affect the function of dendritic cells. This multi-target regulation may be the key to its superiority over other immunomodulatory peptides.

[0391] 2. Epithelial barrier protection: In organoid and skin equivalent models, the optimized peptide of the present invention showed significant epithelial barrier protection. This finding expands the potential application range of the peptide, such as the treatment of inflammatory bowel disease and atopic dermatitis.

[0392] 3. Remodeling of the inflammatory microenvironment: The optimized peptide segment of the present invention not only inhibits the production of pro-inflammatory factors, but also promotes the secretion of anti-inflammatory factors (such as IL-10). This balanced regulation helps to remodel the inflammatory microenvironment and may bring more lasting therapeutic effects.

[0393] 4. Extracellular matrix regulation: In the skin equivalent model, the optimized peptide of the present invention increased the expression of fibronectin, which suggests that it may have the potential to promote tissue repair.

[0394] 5. Broad-spectrum anti-inflammatory effect: The optimized peptide of the present invention shows good inhibitory effect in inflammatory reactions mediated by Th1, Th2 and Th17. This broad spectrum makes it potentially applicable to a variety of autoimmune diseases.

[0395] 6. Low toxicity and high efficacy: Compared with the positive control dexamethasone, the optimized peptide segment of the present invention may have lower toxicity and side effects while achieving similar effects, which provides the possibility for long-term medication.

[0396] 7. Tissue-specific regulation: In different tissue models (intestine, skin), the optimized peptide segments of the present invention show good effects. This tissue-specific regulatory ability is not possessed by many existing immunomodulators.

[0397] The present invention successfully prepared an optimized KRPAGY peptide with multiple immunomodulatory effects, epithelial barrier protection and tissue repair potential through multiple enzymatic hydrolysis systems, fine membrane separation technology and ingenious peptide modification strategies. This peptide has shown excellent effects in multiple humanized models. It is not only superior to unoptimized standard peptides and commercial immunomodulatory peptides, but also close to the potent immunosuppressants commonly used in clinical practice in some aspects. Its multi-target regulatory mechanism, broad-spectrum anti-inflammatory effects and tissue-specific effects provide a new direction for the development of a new generation of immunomodulatory drugs. This invention not only has great potential in the treatment of autoimmune diseases, but may also find wide application in fields such as inflammatory bowel disease and atopic dermatitis. It has important scientific significance and significant clinical transformation value.

[0398] The above description is only an embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent substitution, improvement, etc. made within the spirit and principle of the present invention should be included in the scope of the claims of the present invention.

Claims

1. A process for preparing albumin oligopeptide by enzymatic hydrolysis, characterized in that: The following steps are involved: (1) construct and express recombinant albumin containing KRPAGY repeat sequences; (2) purifying the recombinant albumin; (3) using a multiple enzymatic hydrolysis system to enzymatically hydrolyze the purified recombinant albumin; (4) purifying the enzymatic hydrolysis product by a multi-stage membrane separation system; (5) structural modification of the purified peptides; (6) Evaluating the immune activity of the modified peptide.

2. The enzymatic preparation process of albumin oligopeptide according to claim 1, characterized in that: Step (1) comprises: a) designing and synthesizing a gene sequence encoding the recombinant albumin, wherein the gene sequence is shown in SEQ ID NO.1; b) cloning the gene sequence into the pET28a vector to construct the pET28a-hKRPAGY-Albumin expression plasmid; c) transforming the obtained plasmid into Escherichia coli BL21 (DE3) competent cells; d) Incubate in LB medium containing 50 μg / mL kanamycin at 37°C with shaking at 200 rpm until OD600 reaches 0.6-0.8; e) Cool down to 25°C, add IPTG to a final concentration of 0.5 mM, and continue culturing for 8-12 hours.

3. The enzymatic preparation process of albumin oligopeptide according to claim 1, characterized in that: Step (2) comprises: a) Collect the cells by centrifugation at 6000 g for 10 min at 4°C; b) resuspending the bacterial cells in a lysis buffer, wherein the lysis buffer comprises 50 mM Tris-HCl (pH 8.0), 300 mM NaCl and 10 mM imidazole; c) Ultrasonication in an ice bath: power 300 W, working 3 seconds, interval 3 seconds, total time 10 minutes; d) centrifugation at 12000 g for 30 min at 4°C and collecting the supernatant; e) filtering the supernatant through a 0.22 μm filter membrane; f) Purify the target protein using Ni-NTA affinity chromatography, wherein the elution buffer contains 50 mM Tris-HCl (pH 8.0), 300 mM NaCl and 250 mM imidazole.

4. The enzymatic preparation process of albumin oligopeptide according to claim 1, characterized in that: The multiple enzymatic hydrolysis system in step (3) includes trypsin, α-chymotrypsin, proteinase K and leucine aminopeptidase, wherein: a) dissolving purified recombinant albumin (10 mg / mL) in 50 mM Tris-HCl buffer (pH 8.0); b) Add the above enzymes in sequence, and their final concentrations are: Trypsin 0.1-0.5 mg / mL; α-chymotrypsin 0.05-0.2 mg / mL; Proteinase K 0.01-0.05 mg / mL; Leucine aminopeptidase 0.02-0.1 mg / mL; c) reacting at 37°C for 4 hours, taking samples for analysis every 30 minutes and adjusting the amount of each enzyme added according to the pH value and substrate concentration; d) Heat in a 95°C water bath for 10 minutes to terminate the reaction.

5. The enzymatic preparation process of albumin oligopeptide according to claim 1, characterized in that: The multi-stage membrane separation system in step (4) comprises: a) using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa, centrifuging at 4°C, 4000 g for 30 minutes, and collecting the filtrate; b) using a nanofiltration column with a molecular weight cutoff of 500 Da, a column volume of 50 mL, a flow rate of 1 mL / min, and an elution buffer of 20 mM phosphate buffer (pH 7.4); c) using a cation exchange column, the equilibration buffer was 20 mM phosphate buffer (pH 6.5), the elution buffer was 20 mM phosphate buffer (pH 6.5) containing a 0-1 M NaCl gradient, and the flow rate was 1 mL / min; d) Pulse flow technique was applied in the above steps, with a pulse frequency of 0.5-2 Hz and a pulse amplitude of ±30% of the initial flow rate.

6. The enzymatic preparation process of albumin oligopeptide according to claim 1, characterized in that: The structural modification in step (5) includes N-terminal cysteine ​​modification and C-terminal glycyl-prolyl modification, wherein: a) The steps of N-terminal cysteine ​​modification are: i. Dissolve the purified peptide (5 mg / mL) in 0.1 M phosphate buffer (pH 7.4); ii. Add N-succinimidyl-S-acetylthiocysteine ​​(SATA) at a molar ratio of 1:1.2; iii. react at room temperature for 2 hours, stirring speed 200 rpm; iv. Add 50 mM hydroxylamine and react at room temperature for 15 minutes; b) The steps of C-terminal glycyl-prolyl modification are: i. dissolving the above product in a DMF: water (1:1) mixed solvent; ii. Add Fmoc-Gly-Pro-OH, molar ratio 1:1.5; iii. Add 1.2 equivalents of EDC·HCl and HOBt; iv. React at room temperature for 4 hours, stirring speed 200 rpm; v. Add 20% piperidine / DMF solution and react at room temperature for 30 minutes.

7. The enzymatic preparation process of albumin oligopeptide according to claim 6, characterized in that: This involves purification of modified peptides using a semi-preparative HPLC system, where: a) Use a C18 reverse phase column with a column size of 10 x 250 mm and a particle size of 5 μm; b) mobile phase A is 0.1% TFA aqueous solution, and mobile phase B is 0.1% TFA acetonitrile solution; c) using a gradient elution of 5-50% B for 30 minutes; d) The flow rate was 3 mL / min and the detection wavelength was 220 nm; e) Collect the target peak and freeze-dry it.

8. The enzymatic preparation process of albumin oligopeptide according to claim 1, characterized in that: The immune activity assessment in step (6) includes: a) T cell proliferation assay, in which human peripheral blood mononuclear cells (PBMCs) were co-cultured with modified peptides at different concentrations (0.1-100 μg / mL) for 48 hours, followed by BrdU incorporation assay; b) Cytokine secretion detection, in which the levels of IL-2 and IFN-γ in the culture supernatant were detected using the ELISA method; c) flow cytometry analysis of the activation status of CD4+ and CD8+ T cells using fluorescently labeled antibodies against CD3, CD4, CD8, and CD69; d) Mouse autoimmune encephalomyelitis (EAE) model experiment, in which mice were given 1-5 mg / kg of the modified peptide for 21 consecutive days, and the clinical scores and histopathological changes were evaluated.

9. The enzymatic hydrolysis preparation process of albumin oligopeptide according to any one of claims 1 to 8, characterized in that: The following quality control steps are also included: a) Analyze the sequence and purity of the peptides using HPLC-MS; b) Analyze the secondary structure of peptides using circular dichroism spectroscopy; c) Dynamic light scattering was used to detect the aggregation state of peptides.

10. The enzymatic hydrolysis preparation process of albumin oligopeptide according to claim 9, characterized in that: The conditions of the HPLC-MS analysis include: a) Use a C18 reverse phase column with a column size of 2.1 x 50 mm and a particle size of 1.8 μm; b) mobile phase A is 0.1% formic acid in water, and mobile phase B is 0.1% formic acid in acetonitrile; c) using a gradient elution of 5-95% B for 10 minutes; d) The flow rate was 0.3 mL / min and the injection volume was 5 μL; e) The mass spectrometer scan range is m / z 100-2000.

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