Collagen peptide composition as well as effect and application thereof in improving immunity

Abstract

The invention discloses a composition with immunoregulation and anti-aging functions and a preparation method of the composition. The composition comprises the following core components: marine collagen peptide, fusion polypeptide CPF-1, a monoclonal antibody mAb Senolix targeting aging cells, pentagalloylglucose (PGG) and hericium erinaceus polysaccharide. The fusion polypeptide CPF-1 is designed through a structural domain fusion strategy, and has collagen binding, immune activation and antibacterial functions; the monoclonal antibody mAb Senolix is targeted to a senescent cell surface antigen p16 (INK4a), and senescent cells are cleared through an ADCC effect. Through the synergistic effect of multiple components, the composition significantly improves the activity of immune cells, promotes collagen synthesis and alleviates chronic inflammation, shows excellent immunoregulation and anti-aging effects in an in-vitro cell model and an immunosuppressive mouse model, and can be used for developing immunopotentiators, anti-aging drugs or functional foods.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a collagen peptide composition and its efficacy and use in improving immunity. Background Art

[0002] In the fields of biomedicine and functional products, the application research of collagen peptides continues to deepen. With the exploration of the relationship between its structure and function, researchers have found that low molecular weight (<2000Da) collagen peptides show unique advantages in transmembrane transport and biological activity expression. This type of peptide segment can not only activate the proliferation and differentiation signaling pathways of fibroblasts and promote the synthesis of extracellular matrix to improve skin structure, but also participate in the immune regulation process through interaction with surface receptors of immune cells. In the prior art, a variety of composite formulas based on collagen peptides have appeared: such as the combination of collagen peptides and pentagalloyl glucose disclosed in CN117653562A, which intervenes in the cell aging process by regulating oxidative stress-related signaling pathways and enhances the immune defense function of macrophages; the multi-component composite formula proposed in CN116636619A utilizes the synergistic effect of whey protein, Haematococcus pluvialis and collagen peptides to achieve certain effects in beauty and immune regulation.

[0003] However, this field still faces many technical bottlenecks: First, the immunomodulatory activity of most collagen peptide products relies on non-specific nutritional support and lacks precise design for regulating immune cell function, resulting in limited intensity and duration of their effects; second, existing technologies have not yet established an effective targeted delivery mechanism, making it difficult to achieve selective intervention in specific immune cell subsets or aging cells; third, due to the special structure of polypeptides, they are susceptible to protease degradation in the body and have low transmembrane efficiency, which greatly limits their bioavailability and clinical application potential.

[0004] In recent years, fusion peptide design strategies based on structural biology and bioinformatics have gradually become a research hotspot. Through computational simulation and machine learning algorithms, the spatial conformation and biological activity changes after the fusion of different functional peptide segments can be predicted. For example, the technology disclosed in CN119068997A achieves accurate prediction of the immune activity of fusion peptides by constructing a multidimensional characteristic parameter model, providing theoretical support for the design of new peptides. However, there are still gaps in the existing fusion peptide technology in the study of the synergistic mechanism of immune regulation and anti-aging, and a systematic solution has not yet been formed. Therefore, there is an urgent need for a new collagen peptide composition with significant immune enhancement and anti-aging effects developed by new technologies to fill the gap in the relevant technical field. Summary of the Invention

[0005] Based on the above problems, the purpose of the present invention is to provide a collagen peptide composition with a clear molecular structure and mechanism of action to solve the problems of weak effects and unclear mechanisms of existing products.

[0006] Therefore, in one aspect, the present invention provides a fusion polypeptide CPF-1, the amino acid sequence of the fusion polypeptide CPF-1 is shown in SEQ ID NO.1, and the codon-optimized nucleotide sequence of the fusion polypeptide CPF-1 is shown in SEQ ID NO.2.

[0007] On the one hand, the present invention also provides a collagen peptide composition for improving immunity, the core components of which are marine collagen peptide, fusion polypeptide CPF-1, monoclonal antibody mAb Senolix, pentagalloyl glucose (PGG) and Hericium erinaceus polysaccharide. Among them, the marine collagen peptide is derived from tilapia skin, and after enzymatic hydrolysis and ultrafiltration, the molecular weight is controlled at 800-1500Da, which can provide structural support and promote fibroblast proliferation; the fusion polypeptide CPF-1 is designed by domain fusion technology, containing collagen binding domain, immunomodulatory domain and antimicrobial peptide domain, which can activate NF-κB and ERK1 / 2 signaling pathways and enhance macrophage phagocytosis (phagocytic index increased by 51.6%); the monoclonal antibody mAb Senolix targets the aa89-103 epitope of the p16^ (INK4a) protein on the surface of senescent cells (the heavy chain and light chain variable region sequences are as shown in SEQ ID NO.3 and SEQ ID NO.4, respectively), with a KD value as low as 1.8×10 -9 M, efficiently clears senescent cells through ADCC effect (clearance rate 87.3%); PGG and collagen peptide form a complex with a mass ratio of 1:0.1-0.4 to enhance antioxidant capacity; Hericium erinaceus polysaccharide is prepared by water extraction and alcohol precipitation method, with a polysaccharide content of ≥85%, which can promote intestinal immune regulation.

[0008] Preferably, the composition of the present invention is composed of the following components in parts by weight:

[0009] (1) 50-70 parts of marine collagen peptide;

[0010] (2) fusion polypeptide CPF-15-15 copies;

[0011] (3) 1-5 copies of monoclonal antibody mAb Senolix;

[0012] (4) 3-8 parts of pentagalloylglucose, with a mass ratio of 1:0.1-0.4 to collagen peptide;

[0013] (5) 10-20 parts of Hericium erinaceus polysaccharide, with a polysaccharide content of ≥85%.

[0014] Preferably, the composition of the present invention is composed of the following components in parts by weight:

[0015] (1) 60 parts of marine collagen peptide;

[0016] (2) 10 copies of the fusion polypeptide CPF-1;

[0017] (3) 3 copies of monoclonal antibody mAb Senolix;

[0018] (4) 5 parts of pentagalloylglucose, with a mass ratio of 1:0.2 to collagen peptide;

[0019] (5) 15 parts of Hericium erinaceus polysaccharide.

[0020] In one aspect, the present invention also provides a use of the fusion polypeptide CPF-1 in preparing a collagen peptide composition for improving immunity.

[0021] In one aspect, the present invention also provides a use of the monoclonal antibody mAb Senolix in preparing a collagen peptide composition for improving immunity.

[0022] The composition of the present invention exhibits significant beneficial effects through the synergistic action of multiple components: the fusion of multiple CPF-1 can activate the NF-κB and ERK1 / 2 signaling pathways, enhance the phagocytic function of macrophages, increase the phagocytic index by 51.6%, and promote fibroblast proliferation and collagen synthesis. In the H2O2-induced senescent cell model, the secretion of type I collagen is increased by 189% compared with the model group; the monoclonal antibody mAb Senolix targets and binds to the p16^(INK4a) antigen epitope on the surface of senescent cells, and efficiently eliminates senescent cells through the ADCC effect, with a clearance rate of 87.3%, which is 22.1% higher than that of similar antibodies; pentagalloyl glucose (PGG) and Hericium erinaceus polysaccharide synergistically alleviate chronic inflammation by enhancing antioxidant capacity (SOD activity increased by 2.3 times) and promoting intestinal immune regulation (IgA secretion increased by 102%), respectively.

[0023] In the in vivo immunosuppression model validation, the high-dose group (300 mg / kg·d) of the composition can restore the DTH footpad swelling of cyclophosphamide-induced immunosuppressive mice to 0.42 mm (0.18 mm in the model group), the serum hemolysin HC50 value reaches 98.7 (close to the normal level), and the CD4 + / CD8 + The ratio was restored to 2.15 (1.52 in the model group), and significantly reversed the decline in learning and memory ability of aging model mice (escape latency was shortened to 28.4s), and reduced the proportion of senescent cells in the liver to 8.2% (21.7% in the model group).

[0024] With its multi-target mechanism of action, this composition can be used in the medical field to develop drugs for treating immune aging-related diseases (such as chronic inflammation and immune dysfunction); in the field of health care products, it is suitable for making powders, capsules, functional beverages, etc. to meet the needs of anti-aging and immune enhancement; in the field of cosmetics, its properties of promoting skin repair and anti-wrinkle can be used in the development of topical preparations, and it has broad prospects for clinical transformation and market application. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 SDS-PAGE detection results of fusion polypeptide CPF-1. DETAILED DESCRIPTION

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains. The terms used herein in the specification of the present invention are for the purpose of describing specific embodiments only and are not intended to limit the present invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0027] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0028] Example 1: Design and preparation of fusion polypeptide CPF-1

[0029] 1. Design of fusion peptide CPF-1

[0030] To address the shortcomings of existing collagen peptide products, this invention designed a novel dual-functional fusion peptide, CPF-1, based on protein engineering and immunological principles. This 58-amino acid peptide employs a domain fusion strategy, combining an active collagen fragment, an immunomodulatory peptide, and an antimicrobial peptide via a flexible linker. Specifically, the peptide is structured as follows: N-terminal collagen domain "GPGPAGPPGPPGPPG"; flexible linker 1 "GGGGSGGGGSGGGGS"; central immunomodulatory domain "VGVGIGAGHKLGQGPGGPQGP"; flexible linker 2 "GGGGSGGGGS"; and C-terminal antimicrobial peptide domain "ERKRLVGGR." The amino acid sequence of the designed fusion peptide, CPF-1, is shown in SEQ ID NO. 1.

[0031] The N-terminal collagen domain is derived from the repetitive sequence of type I collagen (GXX' motif), where X and X' are primarily proline (Pro) and hydroxyproline (Hyp). This domain retains the characteristic triple-helical structure of collagen and can bind to cell surface collagen receptors (such as DDR2), activating downstream signaling pathways and promoting fibroblast proliferation and collagen synthesis. 1 The central immunomodulatory domain is derived from a functional fragment (VGVGIGAGHKLGQGP) of the human immunoglobulin heavy chain constant region. This domain has been modified to enhance its affinity for Fc receptors on the surface of macrophages, thereby enhancing immune cell activity by regulating phosphorylation levels of the NF-κB and ERK1 / 2 signaling pathways. The C-terminal antimicrobial peptide domain is derived from the functional core of human defensins (ERKRLVGGR) and possesses broad-spectrum antimicrobial activity and immunomodulatory functions. Experimental studies have shown that this peptide can enhance the phagocytic ability of macrophages against pathogens. A flexible linker (GGGGS)n is used to connect the functional domains, providing structural flexibility and ensuring that each functional domain folds correctly and performs independent functions, avoiding steric interference.

[0032] 2. Preparation of Fusion Peptide CPF-1

[0033] 1. Gene Synthesis and Vector Construction

[0034] (1) Codon optimization: The CPF-1 amino acid sequence was codon-optimized based on the E. coli codon usage frequency database to avoid the use of rare codons. The optimized DNA sequence is as shown in SEQ ID NO. 2, with an NdeI cleavage site added to the 5' end, an XhoI restriction site added to the 3' end, and a 6×His tag encoding sequence.

[0035] (2) Gene synthesis and cloning: We commissioned Sangon Biotechnology to synthesize the optimized DNA fragment, which was then cloned into the pET-28a(+) vector via NdeI / XhoI double digestion. The ligation product was transformed into DH5α competent cells, plated on LB plates containing 50 μg / mL kanamycin, and cultured at 37°C for 16 hours.

[0036] (3) Positive clone screening and sequencing verification: Ten single colonies were randomly selected and inoculated into 5 mL of LB medium containing kanamycin. The culture was shaken at 37°C overnight. The plasmid was extracted, double enzyme digestion verification, and Sanger sequencing was performed. The recombinant plasmid that was sequenced correctly was named pET-28a-CPF-1.

[0037] 2. Fermentation Expression

[0038] (1) Seed solution preparation: A single BL21(DE3) / pET-28a-CPF-1 colony with correct sequencing was selected and inoculated into 10 mL of LB medium containing 50 μg / mL kanamycin. The culture was shaken at 37°C and 220 rpm for 12 h to serve as the seed solution.

[0039] (2) Batch fermentation: The seed solution was inoculated into 2 L TB medium (containing 50 μg / mL kanamycin) at a ratio of 1:100, and cultured at 37°C and 220 rpm until the OD 600 The temperature was lowered to 25°C, IPTG was added to a final concentration of 0.5 mM, and expression was induced for 16 hours.

[0040] (3) Fermentation process monitoring: sampling and measuring OD every 2 hours 600 After induction, the cells were collected by centrifugation at 8000 rpm for 20 min at 4°C, weighed, and stored at -80°C.

[0041] 3. Separation and purification

[0042] (1) Cell disruption and inclusion body collection: Frozen cells were resuspended in lysis buffer (50 mM Tris-HCl, 300 mM NaCl, 10 mM imidazole, pH 8.0) (10 mL / g wet bacteria), 1 mM PMSF and 1 mg / mL lysozyme were added, and the cells were incubated on ice for 30 min. Ultrasonic disruption was performed (power 300 W, 3 s operation, 5 s interval, total time 15 min), and the inclusion body precipitate was collected by centrifugation at 12,000 rpm at 4°C for 30 min.

[0043] (2) Denaturation and nickel column affinity chromatography: The inclusion bodies were dissolved in denaturation buffer (8 M urea, 50 mM Tris-HCl, 300 mM NaCl, 10 mM imidazole, pH 8.0) at room temperature for 2 hours, centrifuged at 4°C, 12,000 rpm for 30 minutes, and the supernatant was collected. The supernatant was loaded onto a HisTrap HP 5 mL nickel column and washed with equilibration buffer (8 M urea, 50 mM Tris-HCl, 300 mM NaCl, 20 mM imidazole, 2% Triton 114, pH 8.0) to remove contaminants and endotoxins. The column was then eluted with elution buffer (8 M urea, 50 mM Tris-HCl, 300 mM NaCl, 500 mM imidazole, pH 8.0) for a linear gradient elution to collect the target protein peak.

[0044] (3) Gradient dialysis refolding: The collected target protein solution was placed in a dialysis bag with a molecular weight cutoff of 3.5 kDa and placed in refolding buffer (50 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA, 1 mM GSH, 0.1 mM GSSG, pH 8.0) containing 6 M, 4 M, 2 M, and 1 M urea, respectively. Each step was dialyzed for 4 hours, and finally dialyzed overnight with refolding buffer without urea.

[0045] (4) Size exclusion chromatography (SEC) purification: The renatured protein solution was filtered through a 0.22 μm filter membrane, loaded onto a Superdex 7510 / 300GL column, and isocratically eluted with SEC buffer (20 mM Tris-HCl, 150 mM NaCl, pH 7.4) at a flow rate of 0.5 mL / min. The main peak was collected, which was the purified fusion polypeptide CPF-1.

[0046] 4. Detection

[0047] (1) SDS-PAGE purity analysis: Prepare 15% separation gel, load 10 μg of purified protein, and electrophoresis at a constant voltage of 120 V for 90 minutes. Stain with Coomassie Brilliant Blue R-250 for 3 hours, decolorize with destaining solution (methanol: acetic acid: water = 4:1:5) until the background is clear, and analyze the purity of the bands using ImageJ software. The results show ( Figure 1 ), a single major band appeared at about 9 kDa, and the purity was 98.2% as analyzed by ImageJ.

[0048] (2) Concentration determination: HPLC method was used, with bovine serum albumin (BSA) as the standard. Chromatographic column: TSKgelG2000SWXL (7.8mm×300mm); mobile phase: 0.1M sodium phosphate buffer (pH7.0) containing 0.1M sodium sulfate; flow rate: 0.5mL / min; detection wavelength: 280nm. 20μL was injected, and the concentration was calculated by external standard method. The results showed that the fusion peptide CPF-1 peaked at 12.5 minutes, and the peak area showed a good linear relationship with the concentration (R 2 =0.9998), and the sample concentration was measured to be 9.25 mg / mL.

[0049] (3) Endotoxin detection: The Limulus Amebocyte Lysate (LAL) method was used, and the detection was performed based on the sensitivity of the LAL of 0.025 EU / mL. The sample was diluted to 1 mg / mL, and LAL was added. The sample was incubated at 37°C for 60 minutes. The gel formation was observed and the endotoxin was compared with the standard endotoxin curve. The results showed that the detection value of the sample after 10-fold dilution was 0.08 EU / mL, which was converted to 0.08 EU / mg, meeting the standard of <0.1 EU / mg.

[0050] Example 2: Design and preparation of monoclonal antibody mAb Senolix

[0051] 1. Antibody Design and Screening

[0052] 1. Epitope Analysis: The three-dimensional structure of p16^(INK4a) was predicted using homology modeling. Immunoinformatics tools were used to analyze its surface-exposed regions, and aa89-103 (FLDTLVVLHRAGAR) of P42771 was selected as the target epitope. This peptide was synthesized and conjugated to the KLH carrier protein. Its binding affinity to native p16^(INK4a) was determined using Biacore T200 (KD = 3.2 × 10^(-7) M).

[0053] 2. Phage library screening: construction capacity is 2.5×10 10 Human antibody Fab phage library was screened for 4 rounds to enrich specific clones:

[0054] Round 1: coating antigen concentration 100 nM, elution condition pH 2.2 (glycine-HCl buffer);

[0055] Rounds 2-4: The antigen concentration was gradually decreased (50 nM→20 nM→10 nM), and competitive elution was introduced (10 μM of non-related peptide was added); finally, 12 positive clones were screened and their binding activity was verified by ELISA.

[0056] 3. Affinity maturation: Saturation mutation was performed on the CDR-H3 region to construct a secondary library (capacity 1.8×10 9 High-affinity variants were identified through SPR screening, with the mAb Senolix achieving a KD value of 1.8×10^(-9)M, a 42-fold increase compared to the original clone and also superior to the commercial monoclonal antibody (ab270058) (KD value of 5.2×10^(-9)M).

[0057] 2. Cell Line Construction and Reactor Culture

[0058] 1. Expression vector construction: The genes of the heavy chain variable region (amino acid sequence as shown in SEQ ID NO.3) and light chain variable region (amino acid sequence as shown in SEQ ID NO.4) of the best monoclonal antibody (mAb Senolix) screened above were cloned into pOptiVEC TM -TOPO vector, with human IgG1 constant region and κ chain constant region sequences connected downstream. The vector was linearized with PacI and transfected into CHO-K1 cells by electroporation (150V, 10ms, 3 pulses).

[0059] 2. Pressure screening and cloning: 25 μM MSX was added 48 hours after transfection, and the medium was changed every 3 days. On the 14th day, single clones were picked and expanded to 24-well plates. High-yielding clones were screened by ELISA and the pressure was further increased to 100 μM MSX. Finally, the stable cell line SL-23 was obtained, with an expression level of 680 mg / L.

[0060] 3. Reactor culture optimization: Batch fed-batch culture was performed in a 5 L bioreactor, and parameter control was:

[0061] Temperature: 37°C from 0 to 72 hours, then drop to 33°C after 72 hours;

[0062] pH: maintained at 7.0 ± 0.1 by CO2 and NaOH;

[0063] DO: Maintain 30% saturation with air / oxygen mixture;

[0064] Feeding strategy: Feed A (containing glucose and amino acids) was added starting on the 3rd day, and Feed B (containing vitamins and trace elements) was added starting on the 5th day. The total feeding amount was 40% of the initial volume.

[0065] 4. Purification process development

[0066] (1) Protein A affinity chromatography

[0067] Equilibration buffer: 20 mM Tris-HCl, 150 mM NaCl, pH 7.4;

[0068] Sample load: 10 mg antibody / mL resin;

[0069] Elution conditions: 25 mM citric acid, pH 3.5, linear gradient elution;

[0070] The main peak was collected and immediately neutralized with 1 M Tris to pH 7.0.

[0071] (2) Virus inactivation and anion exchange

[0072] Virus inactivation: pH 3.8, incubation at 25°C for 60 min;

[0073] Anion exchange: Capto Q column, flow rate 3CV / h;

[0074] Buffer A: 20 mM Tris-HCl, pH 8.0;

[0075] Buffer B: 20 ​​mM Tris-HCl, 1 M NaCl, pH 8.0;

[0076] Gradient elution: 0-30% B, 30CV.

[0077] (3) Ultrafiltration and sterilizing filtration

[0078] Ultrafiltration: Use a 10kDa MWCO membrane to replace the buffer with PBS.

[0079] Sterile filtration: 0.22μm PVDF filter membrane, operating pressure ≤0.2MPa.

[0080] (4) The purification data are summarized in Table 1. The concentration of the qualified mAb Senolix was adjusted to 1.0 mg / mL and then lyophilized. After lyophilization, it was stored at -80°C for future use.

[0081] Table 1 Summary of purification data

[0082]

[0083] 5. Functional Verification

[0084] (1) In vitro ADCC activity assay (Jurkat-NFAT reporter gene system)

[0085] Cell preparation: Target cells were cultured senescent cell lines expressing p16^(INK4a) (H2O2-induced HSF cells), and effector cells were cultured Jurkat cells stably transfected with the NFAT reporter gene (expressing human FcγRIIIa).

[0086] Antibody treatment: mAb Senolix, commercial monoclonal antibody (positive control) and human IgG1 (negative control) were diluted to three concentration gradients of 0.1 nM, 1 nM and 10 nM, respectively.

[0087] Co-culture reaction: Effector cells and target cells were seeded into 96-well plates at an effector:target ratio of 20:1, and different concentrations of antibodies were added. The cells were incubated at 37°C for 6 hours.

[0088] Detect fluorescence signal: add fluorescein substrate and use a microplate reader to detect the fluorescence intensity at 485 / 520 nm. Calculate the ADCC activity percentage = (experimental group signal - negative control signal) / (positive control signal - negative control signal) × 100%.

[0089] The results showed (Table 2) that the ADCC activity of mAb Senolix was significantly higher than that of similar antibodies, inducing 82.4% cytotoxicity at a concentration of 10 nM.

[0090] Table 2 In vitro ADCC activity assay results

[0091]

[0092] (2) Senescent cell clearance experiment (β-galactosidase staining)

[0093] Establishment of senescent cell model: HSF cells were treated with 100 μM H2O2 for 48 h to induce senescence, and the proportion of senescent cells (≥60%) was verified by SA-β-gal staining.

[0094] Antibody treatment: Senescent cells were seeded in 24-well plates and 10 nM of mAb Senolix, commercial monoclonal antibodies, and human IgG1 were added, respectively, and incubated at 37°C for 72 hours.

[0095] Staining and detection: discard the supernatant, wash twice with PBS, add SA-β-gal staining solution (pH 6.0), incubate at 37°C for 16 hours, randomly select 5 fields under a microscope, count the proportion of blue-positive cells, and calculate the clearance rate: clearance rate (%) = (positive rate of untreated group - positive rate of treated group) / positive rate of untreated group × 100%.

[0096] The results showed (Table 3) that the in vitro senescent cell clearance experiment of mAb Senolix showed that its clearance rate reached 87.3%, which was 22.1% higher than that of similar antibodies.

[0097] Table 3 Results of senescent cell clearance experiment

[0098]

[0099] Example 3: Composition formula and preparation method

[0100] 1. The composition of the present invention is composed of the following components in specific proportions (Table 4):

[0101] Table 4 Composition formula

[0102]

[0103] 2. Preparation process

[0104] 1. Key pre-processing steps

[0105] (1) Preparation of marine collagen peptide

[0106] Raw material treatment: Tilapia skin was soaked in 0.1 M NaOH for 12 h to remove impurities and then rinsed with clean water until neutral;

[0107] Enzymatic hydrolysis process: compound protease (1500U / g) + flavor protease (800U / g), enzymatic hydrolysis at 50℃ for 4h;

[0108] Ultrafiltration purification: pass through 10kDa and 1kDa ultrafiltration membranes in sequence, retaining 800-1500Da components;

[0109] Spray drying: inlet air temperature 180℃, outlet air temperature 85℃, yield ≥65%.

[0110] (2) Extraction of Hericium erinaceus polysaccharides

[0111] Water extraction conditions: solid-liquid ratio 1:20, 95℃ extraction for 3h, repeated 2 times;

[0112] Alcohol precipitation process: add 4 times the volume of 95% ethanol to the concentrate and let it stand at 4℃ for 12h;

[0113] Purification: deproteinization by sevage method and desalting by dialysis (molecular weight cut-off 3.5 kDa);

[0114] Vacuum drying: Dry at 60℃ under reduced pressure until the moisture content is ≤5%.

[0115] 2. Composition Preparation Process

[0116] (1) Complex formation: Collagen peptide and PGG were placed in a jacketed reactor at a mass ratio of 5:1, and water for injection (solid-liquid ratio 1:10) was added. The mixture was stirred at a constant temperature of 45°C for 30 min (speed 200 rpm) to form a stable complex (detected by dynamic light scattering, particle size 100-300 nm).

[0117] (2) Polypeptide addition and homogenization: Cool to 30°C, slowly add CPF-1 solution (10 mg / mL), stir at 150 rpm for 60 min to ensure complete dissolution, add Hericium erinaceus polysaccharide solution (20 mg / mL), and homogenize at 5000 rpm for 10 min.

[0118] (3) Antibody compounding and lyophilization: Cool to 4°C, add mAb Senolix lyophilized powder, shake gently at 100 rpm for 30 min, divide into lyophilization bottles, pre-freeze at -45°C for 4 h, vacuum degree 0.1 mbar, heat to 25°C, lyophilize for 24 h, fill with nitrogen and seal, moisture content ≤3%.

[0119] 3. The components of this composition work synergistically: collagen peptides provide basic nutritional support; CPF-1 directly activates immune cells; mAb Senolix eliminates senescent cells that cause immune senescence; PGG enhances antioxidant capacity; and Hericium erinaceus polysaccharides promote intestinal immune regulation.

[0120] 3. Composition efficacy verification experiment

[0121] 1. In vitro immune activity assay

[0122] 1.1 Cell culture and model establishment

[0123] (1) Cell recovery and passage: After recovery, human skin fibroblasts (HSF) were cultured in DMEM medium containing 10% FBS. The medium was changed every 2 days. When the cell confluence reached 80%, the cells were passaged at a ratio of 1:3. Cells in the logarithmic growth phase were used for experiments.

[0124] (2) Experimental groups and treatments are shown in Table 5:

[0125] Table 5 HSF cell repair experimental design

[0126]

[0127]

[0128] (3) H2O2-induced aging model: cells were seeded in 96-well plates (5×10 3 cells / well), cultured for 24 h, discarded the original culture medium, washed twice with PBS, added serum-free culture medium containing 200 μM H2O2, incubated at 37°C for 2 h, aspirated and discarded the H2O2 solution, and washed three times with PBS to remove residual oxidants.

[0129] 1.2 Drug handling and testing

[0130] (1) Composition treatment: The culture medium for each group was prepared according to Table 5, sterilized by filtration with a 0.22 μm filter membrane, 100 μL of the corresponding culture medium was added to each well, 6 replicate wells were set up for each group, and cultured at 37°C, 5% CO2 for 48 hours.

[0131] (2) CCK-8 cell proliferation assay: 10 μL of CCK-8 reagent was added to each well and incubated for 2 hours. The absorbance at 450 nm was measured with a microplate reader and the proliferation rate was calculated. Proliferation rate (%) = (OD value of experimental group / OD value of negative control group) × 100%.

[0132] (3) SA-β-gal staining: discard the culture medium, wash twice with PBS, add 100 μL of fixative (2% formaldehyde + 0.2% glutaraldehyde) to each well, fix at room temperature for 15 minutes, discard the fixative, wash three times with PBS, add 100 μL of staining working solution (pH 6.0) to each well, incubate at 37°C in the dark for 16 hours, randomly select 5 fields of view under an optical microscope (200×), and count the proportion of blue-positive cells.

[0133] (4) Type I collagen ELISA assay: The cell supernatant was collected and centrifuged at 12,000 rpm for 10 minutes to remove impurities. The ELISA kit was operated according to the instructions and a standard curve was drawn to calculate the collagen content.

[0134] 1.3 Experimental Results

[0135] (1) The results of the cell proliferation experiment are shown in Table 6.

[0136] H2O2 model verification: Compared with the negative control group, H2O2 treatment significantly reduced the cell proliferation rate to 48.4% (p<0.001), indicating that the aging model was successfully established.

[0137] The combination significantly promoted proliferation: the proliferation rate of combination group 2 reached 88.3%, which was 82.4% higher than that of the model group (p<0.001), and was significantly better than that of each single component (p<0.05).

[0138] Synergistic effect: The proliferation rate of combination group 2 was 37.8% higher than that of the collagen peptide alone, indicating that the components synergistically promoted cell regeneration.

[0139] Table 6 Cell proliferation assay results

[0140]

[0141]

[0142] (2) The results of SA-β-gal staining are shown in Table 7.

[0143] SA-β-gal positive rate: H2O2 treatment increased the positive rate from 18.5% to 67.8%, while composition group 2 reduced it to 22.3%, close to the normal level.

[0144] Better than a single component: The positive rate of combination group 2 was 56.8% lower than that of the collagen peptide single component and 54.4% lower than that of the CPF-1 single component.

[0145] Dose dependence: The positive rate of composition group 1 was 32.7%, indicating that the anti-aging effect was enhanced with the increase of component concentration.

[0146] Table 7 SA-β-gal staining results

[0147]

[0148] (3) The results of type I collagen secretion are shown in Table 8.

[0149] Model injury: H2O2 treatment reduced the secretion of type I collagen from 42.5 ng / mL to 12.3 ng / mL, a decrease of 71%.

[0150] Composition repair: The collagen secretion of composition group 2 reached 35.6 ng / mL, recovered to 83.8% of the normal group, and increased by 189% compared with the model group.

[0151] Synergistic enhancement: The collagen secretion of combination group 2 was 92.4% higher than that of the collagen peptide single component, confirming that multiple components synergistically promoted the synthesis of extracellular matrix more effectively.

[0152] Table 8 Type I collagen secretion results

[0153]

[0154] 2. Macrophage phagocytic function experiment

[0155] 2.1 Cell inoculation: RAW264.7 cells in the logarithmic growth phase were obtained, digested with 0.25% trypsin, centrifuged at 1000 rpm for 5 min, discarded the supernatant, and resuspended in fresh culture medium. After counting, the cell concentration was adjusted to 2 × 10 6 Cells were cultured at 2×10 5 The cells were seeded into 24-well plates at a density of 1 mL per well and cultured in an incubator for 24 h until the cells adhered to the wall.

[0156] 2.2 Drug treatment:

[0157] (1) Group settings:

[0158] Control group: normal culture medium without the composition was added.

[0159] Collagen peptide alone group: culture medium containing 200 μg / mL collagen peptide was added.

[0160] Combination group: culture medium containing CPF-1 100 μg / mL + mAb Senolix 20 μg / mL was added.

[0161] (2) Set up 6 replicate wells for each group, replace the corresponding culture medium, and continue incubating in a 37°C, 5% CO2 incubator for 24 hours.

[0162] 2.3 Neutral red phagocytosis assay

[0163] (1) Add 100 μL of 0.1% neutral red solution to each well and incubate at 37°C for 1 hour to allow macrophages to phagocytize neutral red particles. Discard the liquid in the wells and gently wash the cells three times with PBS. Centrifuge at 1000 rpm for 5 minutes after each wash and discard the supernatant to remove as much unphagocytosed neutral red as possible. Add 500 μL of cell lysis buffer to each well and shake at room temperature for 10 minutes to fully lyse the cells and release the phagocytic neutral red into the lysis buffer.

[0164] (2) The lysed liquid was transferred to a 96-well plate, 200 μL per well, and the absorbance (OD) of each well was measured at a wavelength of 540 nm using a microplate reader. 540 ).

[0165] (3) Data calculation:

[0166] Phagocytic index = OD of experimental group 540 Mean / control group OD 540 Mean.

[0167] Synergy index (CI) was calculated by the Chou-Talalay method and analyzed with CompuSyn software. The half-maximal effect concentration (EC50 ) was used to calculate the CI value, and CI < 1 indicated a synergistic effect.

[0168] 2.4 The experimental results are shown in Tables 9 and 10.

[0169] (1) Enhanced phagocytic function: Compared with the control group, the macrophage phagocytic index in both the collagen peptide group and the combination group was significantly increased. The phagocytic index in the combination group reached 1.85±0.12, which was 51.6% higher than the 1.22±0.08 in the collagen peptide group alone, indicating that the combination of CPF-1 and mAb Senolix can more effectively promote the phagocytic function of macrophages.

[0170] (2) Verification of synergistic effect: The synergistic index (CI) calculated by the Chou-Talalay method was 0.78, which was less than 1 and within the 95% confidence interval, clearly confirming that CPF-1 and mAb Senolix have a significant synergistic effect in promoting macrophage phagocytosis. This means that when the two components are used together, their effect in promoting macrophage phagocytosis is not simply additive, but rather produces a stronger synergistic effect. This may be due to the fact that they jointly regulate the function of macrophages through different mechanisms of action, thereby enhancing their ability to phagocytose foreign bodies such as pathogens.

[0171] Table 9 Phagocytic index results

[0172]

[0173] Table 10 Synergy index calculation results

[0174]

[0175] Calculated by CompuSyn software, the synergy index CI = 0.78 (95% confidence interval: 0.72-0.84).

[0176] 2.5 Summary

[0177] This experiment systematically studied the effect of the collagen peptide composition on the phagocytic function of RAW264.7 macrophages by using the neutral red phagocytosis method. The results showed that the collagen peptide composition of the present invention, especially the combination of CPF-1 and mAb Senolix, can significantly enhance the phagocytic activity of macrophages, and the two show a good synergistic effect. This result provides a cellular-level experimental basis for the composition to enhance the body's immunity, suggesting that it may play an important role in the immune defense process by activating macrophages, a key immune cell. It has potential medicinal and health value and is worthy of further study of its mechanism of action and application scenarios.

[0178] 3. In vivo immunity enhancement experiment

[0179] 3.1 Animal grouping and treatment: 50 mice (6-week-old SPF BALB / c mice) were randomly divided into 5 groups, with 10 mice in each group. The specific treatments are shown in Table 11, where the composition was prepared by the method of the first part of this example.

[0180] Table 11 Experimental groups and treatments

[0181]

[0182] 3.2 Detection indicators and methods

[0183] (1) Delayed-type hypersensitivity (DTH) assay: 24 h after the last administration, each mouse was subcutaneously injected with 2×10 9 20 μL of SRBCs / mL was added; 24 h after injection, the thickness of the left and right hind footpads of the mice was measured with a digital vernier caliper, and each footpad was measured 3 times to obtain the average value; the footpad swelling was calculated as follows: footpad swelling (mm) = right hind footpad thickness - left hind footpad thickness.

[0184] (2) Serum hemolysin (HC50) determination

[0185] After DTH detection, blood was collected from the eyeball, kept at 37°C for 1 h, and centrifuged at 4°C and 3000 rpm for 10 min to separate the serum.

[0186] The HC50 method was used to determine the hemolysis value: 100 μL of serum was added with 50 μL of 2% SRBC and 50 μL of 10% complement (guinea pig serum), incubated in a 37°C water bath for 30 min, and centrifuged at 4°C and 3000 rpm for 10 min; 100 μL of the supernatant was placed in a 96-well plate, 100 μL of distilled water was added, and the OD was measured using a microplate reader. 540 value.

[0187] Preparation of standard curve: Prepare SRBC hemolysin solutions of different concentrations (0, 50, 100, 200, 400, 800 U / mL) and measure OD 540 The standard curve was drawn and the HC50 value of the samples was calculated.

[0188] (3) Calculation of immune organ index: After blood collection, mice were killed, and the spleen and thymus were aseptically separated, the surrounding fat and connective tissue were removed, and the tissues were rinsed with pre-cooled saline. The surface moisture of the tissues was absorbed with filter paper, and the weights of the spleen (W spleen) and thymus (W chest) were recorded using an electronic balance. The immune organ index was calculated as follows: spleen index (mg / g) = W spleen / mouse body weight (g) × 1000, thymus index (mg / g) = W chest / mouse body weight (g) × 1000.

[0189] (4) Flow cytometry analysis of T cell subsets: The spleen was removed and placed on a 200-mesh cell sieve. The cells were ground with a syringe core to prepare a single-cell suspension. Red blood cell lysis buffer was added and the cells were lysed at room temperature for 5 min. The cells were washed twice with PBS and centrifuged at 1000 rpm for 5 min. The cell concentration was adjusted to 1×10 6 100 μL of cell suspension was taken, 5 μL of anti-mouse CD4-PE and anti-mouse CD8-FITC antibodies were added, and the cells were incubated at 4°C in the dark for 30 min; the cells were washed twice with PBS, and the cells were resuspended in 500 μL of PBS. CD4 + 、CD8 + T cell ratio, calculate CD4 + / CD8 + ratio.

[0190] 3.3 Experimental data

[0191] (1) DTH reaction, as shown in Table 12. The footpad swelling of the cyclophosphamide model group was significantly lower than that of the control group (p < 0.001), indicating that the immunosuppression model was successfully established. The footpad swelling of the high-dose combination group (0.42 ± 0.05 mm) was significantly higher than that of the model group (p < 0.001), and there was no significant difference with the positive drug group (p > 0.05), suggesting that the high-dose combination can effectively restore the body's cellular immune function.

[0192] Table 12DTH reaction results

[0193]

[0194] (2) Serum hemolysin (HC50), as shown in Table 13. The HC50 value of the model group was significantly lower than that of the control group. The HC50 value of the high-dose group of the composition (98.7±6.3) was close to the level of the control group and significantly higher than that of the model group (p<0.001), indicating that the composition can effectively enhance the humoral immune response and enhance the antibody secretion capacity.

[0195] Table 13 Serum hemolysin (HC50) results

[0196]

[0197] (3) Immune organ indexes, as shown in Table 14. The spleen and thymus indexes in the model group were significantly lower than those in the control group (p < 0.001). The spleen index (3.89 ± 0.32 mg / g) and thymus index (1.95 ± 0.21 mg / g) in the high-dose group of the composition were significantly higher than those in the model group (p < 0.01), indicating that the composition can reduce the damage to immune organs caused by immunosuppression and promote their growth and development.

[0198] Table 14 Immune organ index results

[0199]

[0200] (4) T cell subsets, as shown in Table 15. Model group CD4 + / CD8 + The ratio was significantly lower than that of the control group (p<0.01). + / CD8 + The ratio (2.15±0.18) was significantly higher than that of the model group (p<0.01) and close to the level of the control group, indicating that the composition can regulate the balance of T cell subsets and enhance the body's immune regulation ability.

[0201] Table 15 T cell subset results

[0202]

[0203] 3.4 Summary

[0204] This experiment systematically evaluated the effect of the composition of the present invention on the immune function of the body through a cyclophosphamide-induced immunosuppressive mouse model. The results showed that the high-dose composition can significantly improve the cellular immunity (DTH reaction) and humoral immunity (serum hemolysin) functions of immunosuppressed mice, promote the development of immune organs, and regulate the balance of T cell subsets. Experimental data confirmed that the composition has a significant effect in improving the body's immunity, and its effect is comparable to that of the positive drug levamisole, providing a reliable in vivo experimental basis for the development of the composition as an immunomodulatory product. Further research can be conducted on its mechanism of action and optimal application dose to promote its transformation and application in the fields of medicine and health products.

[0205] Example 4: Compositions of other formulations

[0206] 1. Oral powder

[0207] Formula: collagen peptide (60g), CPF-1 (10g), mAb Senolix lyophilized powder (3g), PGG (5g), Hericium erinaceus polysaccharide (15g), flavoring agent (7g).

[0208] Preparation: Each component was passed through a 100-mesh sieve, mixed in a three-dimensional mixer for 30 minutes, and packaged into aluminum foil bags (5 g per bag).

[0209] Usage: 1-2 times a day, take with warm water.

[0210] 2. Capsules

[0211] Formula: Collagen peptide (55g), CPF-1 (8g), mAb Senolix (2g), PGG (4g), Hericium erinaceus polysaccharide (20g), microcrystalline cellulose (11g).

[0212] Preparation: After mixing, fill capsules (500 mg each).

[0213] Usage: 2 times a day, 3 capsules each time.

[0214] 3. Functional Drinks

[0215] Formula: Collagen peptide (3%), CPF-1 (0.5%), mAb Senolix (0.2%), PGG (0.15%), Hericium erinaceus polysaccharide extract (10%), xylitol (5%), citric acid (0.1%).

[0216] Process: batching → homogenization (50MPa) → UHT sterilization (137℃, 5s) → aseptic filling.

[0217] 4. Soluble film (oral mucosal administration)

[0218] Formula: CPF-1 (15mg / cm 2 )、mAb Senolix(5mg / cm 2 ), collagen peptide (50mg / cm 2 ), Hydroxypropyl methylcellulose (base).

[0219] Preparation: Cast into film and cut into 2cm×2cm tablets.

[0220] Advantages: Avoid gastrointestinal degradation and improve bioavailability.

[0221] The above embodiments are preferred implementations of the present invention, but the implementations of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A fusion polypeptide, characterized in that: The fusion polypeptide is the fusion polypeptide CPF-1, and its amino acid sequence is shown in SEQ ID NO.

1.

2. The fusion polypeptide according to claim 1, characterized in that The nucleotide sequence of the fusion polypeptide CPF-1 after codon optimization is shown in SEQ ID NO.

2.

3. A collagen peptide composition for improving immunity, characterized in that: The composition is composed of the following components in parts by weight: (1) 50-70 parts of marine collagen peptide, molecular weight 800-1500 Da, derived from tilapia skin; (2) 15 copies of the fusion polypeptide CPF-15 according to claim 1; (3) monoclonal antibody mAb Senolix 1-5, whose heavy chain variable region and light chain variable region sequences are shown in SEQ ID NO. 3 and SEQ ID NO. 4, respectively; (4) 3-8 parts of pentagalloylglucose, with a mass ratio of 1:0.1-0.4 to collagen peptide; (5) 10-20 parts of Hericium erinaceus polysaccharide, with a polysaccharide content of ≥85%.

4. The composition according to claim 3, characterized in that The composition is composed of the following components in parts by weight: (1) 60 parts of marine collagen peptide; (2) 10 copies of the fusion polypeptide CPF-1; (3) 3 copies of monoclonal antibody mAb Senolix; (4) 5 parts of pentagalloylglucose, with a mass ratio of 1:0.2 to collagen peptide; (5) 15 parts of Hericium erinaceus polysaccharide.

5. The composition according to claim 3, characterized in that The monoclonal antibody mAb Senolix targets the p16^(INK4a) protein aa89-103 epitope, and its amino acid sequence is FLDTLVVLHRAGAR.

6. Use of the fusion polypeptide CPF-1 according to claim 1 in preparing a collagen peptide composition for improving immunity.

7. Use of the monoclonal antibody mAb Senolix according to claim 3 in preparing a collagen peptide composition for improving immunity.

Images