A collagen peptide composition and its efficacy and use in improving immunity

Abstract

The application discloses a kind of composition with immunoregulation and anti-aging function and preparation method thereof, and core component is marine collagen peptide, fusion polypeptide CPF-1, targeting senescent cell monoclonal antibody mAb Senolix, five gallate acyl glucose (PGG) and Hericium erinaceus polysaccharide.Fusion polypeptide CPF-1 is designed by domain fusion strategy, with collagen binding, immune activation and antibacterial function;Monoclonal antibody mAb Senolix targets the surface antigen p16^(INK4a) of senescent cell, and removes senescent cell by ADCC effect.The composition significantly improves immune cell activity, promotes collagen synthesis and reduces chronic inflammation through the synergistic effect of multiple components, and shows excellent immunoregulation and anti-aging effect in in vitro cell model and immunosuppressed mouse model, and can be used for developing immune enhancer, anti-aging drug or functional food.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to a collagen peptide composition and its efficacy and use in improving immunity. BACKGROUND

[0002] In the field of biological medicine 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 (<2000 Da) collagen peptides have unique advantages in transmembrane transport and biological activity expression. This kind of peptide segment not only can activate the proliferation and differentiation signal pathway of fibroblasts, promote the synthesis of extracellular matrix to improve the structure of the skin, but also can participate in the immune regulation process through the interaction with the surface receptors of immune cells. In the prior art, there are many complex formulations based on collagen peptides: such as CN117653562A discloses the combination of collagen peptide and pentagalloyl glucose, which realizes the intervention on the cell aging process and enhances the immune defense function of macrophages by regulating the oxidative stress related signal pathway; CN116636619A proposes a multi-component complex formula, which utilizes the synergistic effect of whey protein, haematococcus pluvialis and collagen peptide to achieve certain effect in beauty and immune regulation.

[0003] However, there are still many technical bottlenecks in this field: first, the immune regulation activity of most collagen peptide products depends on non-specific nutritional support, and lacks precise design for the functional regulation of immune cells, resulting in limited action strength and duration; second, the existing technology has not established an effective targeted delivery mechanism, making it difficult to achieve selective intervention on specific immune cell subgroups or senescent cells; third, due to the special structure of polypeptides, they are easily degraded by proteases in vivo, and have low transmembrane efficiency, which greatly limits their bioavailability and clinical application potential.

[0004] In recent years, the design strategy of fusion polypeptides based on structural biology and bioinformatics has 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, CN119068997A discloses a technology that realizes the precise prediction of the immune activity of fusion polypeptides by constructing a multi-dimensional feature parameter model, providing theoretical support for the design of new polypeptides. However, the existing fusion polypeptide technology still has gaps in the research of the synergistic mechanism of immune regulation and anti-aging, and has not formed a systematic solution. Therefore, there is an urgent need for a new type of collagen peptide composition with significant immune enhancement and anti-aging efficacy developed by new technology, which fills the gap in the related technical field. SUMMARY

[0005] Based on the above problems, the purpose of the present application is to provide a collagen peptide composition with clear molecular structure and mechanism of action, which solves the problem of weak effect and unclear mechanism of existing products.

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

[0007] In one aspect, the present application 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. The marine collagen peptide is derived from tilapia skin, and its molecular weight is controlled at 800-1500 Da after enzymatic hydrolysis and ultrafiltration, which can provide structural support and promote fibroblast proliferation. The fusion polypeptide CPF-1 is designed by domain fusion technology, which contains collagen binding domain, immune regulation domain, and antibacterial peptide domain, can activate NF-κB and ERK1 / 2 signaling pathways, and enhance the phagocytic function of macrophages (the phagocytic index is increased by 51.6%). The monoclonal antibody mAb Senolix targets the aa89-103 epitope of p16^(INK4a) protein on the surface of senescent cells (the heavy chain and light chain variable region sequences are shown in SEQ ID NO. 3 and SEQ ID NO. 4, respectively), and the KD value is as low as 1.8×10 -9 M, which can efficiently remove senescent cells through ADCC effect (the removal rate is 87.3%); PGG forms a complex with collagen peptide at a mass ratio of 1:0.1-0.4, which enhances the antioxidant capacity; Hericium erinaceus polysaccharide is prepared by water extraction and alcohol precipitation, and the polysaccharide content is ≥85%, which can promote intestinal immune regulation.

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

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

[0010] (2) fusion polypeptide CPF-1 5-15 parts;

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

[0012] (4) pentagalloyl glucose 3-8 parts, the mass ratio of which to collagen peptide is 1:0.1-0.4;

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

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

[0015] (1) Marine collagen peptide 60 parts;

[0016] (2) Fusion polypeptide CPF-1 110 parts;

[0017] (3) Monoclonal antibody mAb Senolix 3 parts;

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

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

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

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

[0022] The composition of the present application exhibits significant beneficial effects through multi-component synergistic effect: the fusion polypeptide 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 the proliferation of fibroblasts 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 the p16^(INK4a) antigen epitope on the surface of senescent cells, effectively removes senescent cells through the ADCC effect, with a removal rate of 87.3%, which is 22.1% higher than that of similar antibodies; five galloyl glucose (PGG) and hericium erinaceus polysaccharide can respectively enhance the antioxidant capacity (SOD activity is increased by 2.3 times) and promote intestinal immune regulation (IgA secretion is increased by 102%), and synergistically reduce chronic inflammation.

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

[0024] The composition can be used for developing drugs for treating immune aging related diseases (such as chronic inflammation and low immune function) in the medical field by virtue of the multi-target mechanism; in the health care product field, it is suitable for being made into powders, capsules, functional beverages and the like to meet the anti-aging and immune enhancement needs; in the cosmetic field, its characteristics of promoting skin repair and anti-wrinkle can be used for the development of external preparations, and has a broad clinical transformation and market application prospect. BRIEF DESCRIPTION OF DRAWINGS

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

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0027] Unless specifically noted, the reagents, methods, and equipment employed in the present application are the conventional reagents, methods, and equipment in the art. Unless specifically noted, the reagents and materials used in the following examples are commercially available.

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

[0029] I. Design of fusion polypeptide CPF-1

[0030] The present application aims at the deficiencies of existing collagen peptide products, and designs a new fusion polypeptide CPF-1 with double functions based on the principles of protein engineering and immunology. The full-length of the polypeptide is 58 amino acids, and the sequence design adopts a domain fusion strategy to reasonably splice the collagen active fragment, the immunomodulatory peptide and the antibacterial peptide through a flexible linker, as shown below: N-terminal collagen domain "GPGPAGPPGPPGPPG"; flexible linker 1 "GGGGSGGGGSGGGGS"; central immunomodulatory domain "VGVGIGAGHKLGQGPGGPQGP"; flexible linker 2 "GGGGSGGGGS"; C-terminal antibacterial peptide domain "ERKRLVGGR". The amino acid sequence of the fusion polypeptide CPF-1 designed above is shown as SEQ ID NO. 1.

[0031] The N-terminal collagen domain is derived from the repeating sequence (GXX'motif) of type I collagen, in which X and X' are mainly proline (Pro) and hydroxyproline (Hyp). This domain retains the characteristic triple-helical structure of collagen, can bind to the cell surface collagen receptor (such as DDR2), activate downstream signaling pathways, and promote fibroblast proliferation and collagen synthesis1. The central immunomodulatory domain is derived from a functional fragment of the human immunoglobulin heavy chain constant region (VGVGIGAGHKLGQGP). This domain is modified to enhance its affinity for the Fc receptor on the surface of macrophages, and by regulating the phosphorylation level of the NF-κB and ERK1 / 2 signaling pathways, it enhances the activity of immune cells. The C-terminal antibacterial peptide domain is derived from the functional core of human defensin (ERKRLVGGR), which has broad-spectrum antibacterial activity and immunomodulatory function. Experiments have shown that this peptide segment can enhance the phagocytic ability of macrophages against pathogenic bacteria. A flexible linker (GGGGS)n is used to connect the functional domains, providing structural flexibility to ensure that each functional domain folds correctly and functions independently, avoiding spatial steric hindrance interference.

[0032] II. Preparation of the fusion polypeptide CPF-1

[0033] 1. Gene synthesis and vector construction

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

[0035] (2) Gene synthesis and cloning: The optimized DNA fragment is synthesized by Biosune, and is cloned into the pET-28a(+) vector by NdeI / XhoI double digestion. The ligation product is transformed into DH5α competent cells, which are spread on LB plates containing 50 μg / mL kanamycin and incubated at 37°C for 16 hours.

[0036] (3) Positive clone screening and sequencing verification: 10 single colonies are randomly picked and inoculated in 5 mL LB medium containing kanamycin and incubated at 37°C with shaking overnight. The plasmid is extracted and subjected to double digestion verification and Sanger sequencing. The recombinant plasmid with correct sequencing is named pET-28a-CPF-1.

[0037] 2. Fermentation expression

[0038] (1) Seed preparation: A single colony of BL21(DE3) / pET-28a-CPF-1 with correct sequencing is inoculated in 10 mL LB medium containing 50 μg / mL kanamycin and incubated at 37°C with shaking at 220 rpm for 12 hours as seed liquid.

[0039] (2) Batch fermentation: Seed culture was inoculated into 2L TB medium (containing 50 pg / mL kanamycin) at a ratio of 1:100, and incubated at 37°C, 220 rpm until OD 600 = 0.6. The temperature was reduced to 25°C, and IPTG was added to a final concentration of 0.5 mM, and expression was induced for 16 hours.

[0040] (3) Fermentation process monitoring: OD 600 values were measured every 2 hours, and after induction, the bacteria were collected by centrifugation at 4°C, 8000 rpm for 20 minutes, weighed, and stored at -80°C.

[0041] 3. Isolation and purification

[0042] (1) Cell disruption and inclusion body collection: The frozen bacteria were resuspended in lysis buffer (50 mM Tris-HCl, 300 mM NaCl, 10 mM imidazole, pH 8.0) (10 mL / g wet bacteria), and 1 mM PMSF and 1 mg / mL lysozyme were added, and incubated in an ice bath for 30 minutes. Ultrasonic disruption (power 300 W, work 3 s, interval 5 s, total time 15 minutes), and the inclusion body precipitate was collected by centrifugation at 4°C, 12000 rpm for 30 minutes.

[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, and the supernatant was collected by centrifugation at 4°C, 12000 rpm for 30 minutes. The supernatant was loaded onto a HisTrap HP 5 mL nickel column, and the impurities and endotoxins were washed away with equilibration buffer (8 M urea, 50 mM Tris-HCl, 300 mM NaCl, 20 mM imidazole, 2% Triton 114, pH 8.0), and then linear gradient elution was performed with elution buffer (8 M urea, 50 mM Tris-HCl, 300 mM NaCl, 500 mM imidazole, pH 8.0), and the target protein peak was collected.

[0044] (3) Gradient dialysis renaturation: The collected target protein solution was placed in a dialysis bag with a molecular weight cutoff of 3.5 kDa, and was sequentially placed in renaturation 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, and dialyzed for 4 hours each time, and finally dialyzed overnight in renaturation buffer without urea.

[0045] (4) Size Exclusion Chromatography (SEC) purification: The refolded protein solution was filtered through 0.22 μm filter membrane, and then loaded onto a Superdex 75 10 / 300 GL column, and 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 as the purified fusion polypeptide CPF-1.

[0046] 4. Detection

[0047] (1) SDS-PAGE purity analysis: 15% separating gel was prepared, and 10 μg of purified protein was loaded and electrophoresed at 120 V for 90 min. Coomassie Brilliant Blue R-250 staining was performed for 3 h, and the background was cleared by decolorizing solution (methanol:acetic acid:water = 4:1:5). The band purity was analyzed by ImageJ software. The results showed that a single main band appeared at about 9 kDa, and the purity was 98.2% by ImageJ analysis Figure 1

[0048] (2) Concentration determination: HPLC method was used with bovine serum albumin (BSA) as the standard. Column: TSKgel G2000SWXL (7.8 mm x 300 mm); mobile phase: 0.1 M sodium phosphate buffer (pH 7.0) containing 0.1 M sodium sulfate; flow rate: 0.5 mL / min; detection wavelength: 280 nm. 20 μL was injected, and the concentration was calculated by external standard method. The results showed that the fusion polypeptide CPF-1 had a peak at 12.5 min, and the peak area had a good linear relationship with the concentration (R 2 = 0.9998). The measured sample concentration was 9.25 mg / mL.

[0049] (3) Endotoxin detection: Limulus amebocyte lysate (LAL) method was used for detection with a limulus amebocyte lysate sensitivity of 0.025 EU / mL. The sample was diluted to 1 mg / mL, and limulus amebocyte lysate was added. After incubation at 37°C for 60 min, the gel formation was observed, and the quantitative comparison with the standard endotoxin curve was made. The results showed that the detection value was 0.08 EU / mL after 10-fold dilution, which was equivalent 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] I. Antibody design and screening

[0052] ​1. Epitope analysis: The three-dimensional structure of p16^(INK4a) was predicted by homology modeling, and its surface-exposed regions were analyzed by immunoinformatics tools. The aa89-103 (FLDTLVVLHRAGAR) of P42771 was selected as the target epitope. The peptide was synthesized and coupled to the KLH carrier protein. The binding affinity of the peptide to the native p16^(INK4a) protein was determined by Biacore T200 (KD = 3.2 x 10^(-7) M).

[0053] 2. Phage library screening: A human Fab phage library with a capacity of 2.5 x 10 10 was constructed, and specific clones were enriched through 4 rounds of panning:

[0054] Round 1: The antigen was coated at a concentration of 100 nM, and the elution condition was 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 an unrelated peptide was added); finally, 12 positive clones were screened out, and the binding activity was verified by ELISA.

[0056] 3. Affinity maturation: The CDR-H3 region was subjected to saturation mutation, and a secondary library (capacity 1.8 x 10 9 ) was constructed. High-affinity variants were obtained by SPR screening, in which the KD value of mAb Senolix reached 1.8 x 10^(-9) M, which was 42 times higher than that of the original clone and better than that of the commercial monoclonal antibody (ab270058) (KD value of 5.2 x 10^(-9) M).

[0057] II. 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 the 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 the pOptiVEC TM -TOPO vector, and the human IgG1 constant region and kappa chain constant region sequences were connected downstream. After the vector was linearized by PacI, CHO-K1 cells were transfected by electroporation (150 V, 10 ms, 3 pulses).

[0059] 2. Pressure screening and cloning: 48 h after transfection, 25 μM MSX was added, and the medium was changed every 3 days; on day 14, single clones were picked and cultured to a 24-well plate; high-yield clones were screened by ELISA, and further pressure was applied to 100 μM MSX; finally, the stable cell line SL-23 was obtained, and the expression amount reached 680 mg / L.

[0060] 3. Reactor cultivation optimization: fed-batch cultivation was performed in a 5L bioreactor with the following parameters control:

[0061] Temperature: 37°C for 0-72h, then decreased to 33°C after 72h;

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

[0063] DO: maintained at 30% saturation by air / oxygen mixture;

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

[0065] 4. Purification process development

[0066] (1) Protein A affinity chromatography

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

[0068] Loading amount: 10mg antibody / mL resin;

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

[0070] Collect the main peak and immediately neutralize to pH 7.0 with 1M Tris.

[0071] (2) Virus inactivation and anion exchange

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

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

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

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

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

[0077] (3) Ultrafiltration and sterilization filtration

[0078] Ultrafiltration replacement: using 10kDa MWCO membrane package, replaced to PBS buffer;

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

[0080] (4) The purification data summary is shown in Table 1. The qualified mAb Senolix was adjusted to 1.0 mg / mL before lyophilization, and then stored at -80 °C for standby after lyophilization.

[0081] Table 1 Purification data summary

[0082]

[0083] 5. Function verification

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

[0085] Cell preparation: Target cells were cultured senescent cell line expressing pl6^(INK4a) (H2O2-induced HSF cells), and effector cells were Jurkat cells stably transfected with NFAT reporter (expressing human FcyRIIIa).

[0086] Antibody treatment: mAb Senolix, commercialized mAb (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: Effect cells and target cells were inoculated in a 20:1 effector-to-target ratio in a 96-well plate, different concentrations of antibodies were added, and incubated at 37 °C for 6 hours.

[0088] Detection of fluorescence signal: Add luciferin substrate, use a microplate reader to detect 485 / 520 nm fluorescence intensity, and calculate the percentage of ADCC activity = (experimental group signal-negative control signal) / (positive control signal-negative control signal) x 100%.

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

[0090] Table 2 Results of in vitro ADCC activity assay

[0091]

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

[0093] Establishment of senescent cell model: HSF cells were treated with 100 mM H2O2 for 48 hours 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, commercialized mAb and human IgG1 were added respectively, and incubated at 37℃ for 72 hours.

[0095] Staining and detection: the supernatant was discarded, and the cells were washed with PBS twice, SA-β-gal staining solution (pH 6.0) was added, and incubated at 37℃ for 16 hours, 5 fields of view were randomly selected under a microscope, the proportion of blue positive cells was counted, and the clearance rate was calculated: clearance rate (%) = (positive rate of untreated group - positive rate of treated group) / positive rate of untreated group x 100%.

[0096] The results show (Table 3) that the clearance rate of mAb Senolix in the in vitro senescent cell clearance experiment is 87.3%, which is 22.1% higher than that of the same antibody.

[0097] Table 3: Results of senescent cell clearance experiment

[0098]

[0099] Example 3: Composition formula and preparation method

[0100] Firstly, the composition of the present application is composed of the following components in a specific ratio (Table 4):

[0101] Table 4: Composition formula

[0102]

[0103] Secondly, the preparation process

[0104] 1. Key pretreatment steps

[0105] (1) Preparation of marine collagen peptides

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

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

[0108] Ultrafiltration purification: sequentially pass through 10kDa and 1kDa ultrafiltration membranes to remove components with molecular weight of 800-1500Da;

[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℃ for 3h, repeated twice;

[0112] Alcohol precipitation process: add 4 times the volume of 95% ethanol to the concentrated solution, stand at 4°C for 12h;

[0113] Purification treatment: remove protein by sevage method, desalt by dialysis (molecular weight cutoff 3.5kDa);

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

[0115] 2. Composition preparation process

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

[0117] (2) Polypeptide addition and homogenization: the temperature was reduced to 30°C, and CPF-1 solution (10 mg / mL) was slowly added. The mixture was stirred at 150 rpm for 60 min to ensure complete dissolution, and Hericium erinaceus polysaccharide solution (20 mg / mL) was added and homogenized at 5000 rpm for 10 min.

[0118] (3) Antibody compounding and lyophilization: the temperature was cooled to 4°C, and mAb Senolix lyophilized powder was added. The mixture was gently shaken at 100 rpm for 30 min, then aliquoted into lyophilized bottles, pre-frozen at -45°C for 4 h, and then lyophilized at 25°C under a vacuum of 0.1 mbar for 24 h. The bottles were sealed with nitrogen and the moisture content was ≤3%.

[0119] 3. The components in the composition work synergistically: collagen peptide provides basic nutritional support; CPF-1 directly activates immune cells; mAb Senolix removes senescent cells that cause immune aging; PGG enhances antioxidant capacity; Hericium erinaceus polysaccharide promotes intestinal immune regulation.

[0120] III. Efficacy verification experiment of the composition

[0121] 1. In vitro immune activity experiment

[0122] 1.1 Cell culture and model establishment

[0123] (1) Cell recovery and subculture: after recovery, human skin fibroblasts (HSF) were cultured in DMEM medium containing 10% FBS, the medium was changed every 2 days, and the cells were subcultured at a ratio of 1:3 when the confluence reached 80%. Logarithmic growth phase cells were used for experiments.

[0124] (2) Experimental grouping and treatment as shown in Table 5:

[0125] Table 5 Design of HSF cell repair experiment

[0126]

[0127]

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

[0129] 1.2 Drug treatment and detection

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

[0131] (2) CCK-8 cell proliferation detection: 10 μL of CCK-8 reagent was added to each well, and the incubation was continued for 2 hours. The absorbance at 450 nm was detected by a microplate reader, and the proliferation rate was calculated. The proliferation rate (%) = (OD value of the experimental group / OD value of the negative control group) x 100%.

[0132] (3) SA-β-gal staining: The culture medium was discarded, and the cells were washed twice with PBS. Then, 100 μL of fixing solution (2% formaldehyde + 0.2% glutaraldehyde) was added to each well, and the cells were fixed at room temperature for 15 minutes. The fixing solution was removed, and the cells were washed three times with PBS. Then, 100 μL of staining working solution (pH 6.0) was added to each well, and the cells were incubated at 37°C for 16 hours in the dark. Under a light microscope, five fields of view (200x) were randomly selected, and the proportion of blue positive cells was counted.

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

[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] Composition significantly promotes proliferation: The proliferation rate of the composition group 2 was 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 composition group 2 was 37.8% higher than that of the collagen peptide single component, showing that the components had a synergistic effect on promoting cell regeneration.

[0139] Table 6 Cell proliferation experiment results

[0140]

[0141]

[0142] (2) SA-β-gal staining results 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 single component: The positive rate of composition 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-dependent: The positive rate of composition group 1 was 32.7%, indicating that the anti-aging effect was enhanced with increasing component concentration.

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

[0147]

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

[0149] Model injury: H2O2 treatment reduced the collagen type I secretion 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, which was 83.8% of the normal group and 189% higher than the model group.

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

[0152] Table 8 Collagen type I secretion results

[0153]

[0154] 2. Macrophage phagocytosis experiment

[0155] 2.1 Cell inoculation: Take the logarithmic growth phase of RAW264.7 cells, digest with 0.25% trypsin, centrifuge at 1000 rpm for 5 min, discard the supernatant, resuspend the cells with fresh culture medium, count and adjust the cell concentration to 2x10 6 6 / mL. Cells were inoculated at a density of 2x10 5 4 / well in a 24-well plate, 1 mL per well, and incubated in an incubator for 24 h until the cells adhered.

[0156] 2.2 Drug treatment:

[0157] (1) Group setting:

[0158] Control group: Add normal culture medium without composition.

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

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

[0161] (2) Set 6 replicates for each group, replace the corresponding medium as above, and continue to incubate at 37°C, 5% CO2 incubator for 24 h.

[0162] 2.3 Neutral red phagocytosis experiment

[0163] (1) Add 100 μL of 0.1% neutral red solution to each well, incubate at 37°C for 1 h to allow macrophages to phagocytose neutral red particles. Discard the liquid in the well, gently wash the cells with PBS 3 times, centrifuge at 1000 rpm for 5 min after each washing, discard the supernatant, and try to remove the unphagocytosed neutral red. Add 500 μL of cell lysis solution to each well, shake at room temperature for 10 min to fully lyse the cells and release the phagocytosed neutral red into the lysis solution.

[0164] (2) Transfer the lysed liquid to a 96-well plate, 200 μL per well, and use a microplate reader to measure the absorbance (OD 540 ) of each well at 540 nm wavelength.

[0165] (3) Data calculation:

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

[0167] Synergy index (CI) calculation: Chou-Talalay method, analyzed using CompuSyn software, the half maximal effective concentration (EC50 ) Calculate CI value, CI <1 indicates synergistic effect.

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

[0169] (1) Enhanced phagocytic function: Compared with the control group, the phagocytic index of macrophages in the collagen peptide group and the composition group was significantly increased. Among them, the phagocytic index of the composition group reached 1.85±0.12, which was 51.6% higher than that of the collagen peptide group (1.22±0.08), 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 Chou-Talalay method is 0.78, which is less than 1 and within the 95% confidence interval, which clearly confirms that CPF-1 and mAb Senolix have a significant synergistic effect in promoting the phagocytic function of macrophages. This means that when the two components are used together, their effect on promoting macrophage phagocytosis is not simply additive, but produces a stronger synergistic effect, which may be through different mechanisms to regulate the function of macrophages, thereby enhancing their phagocytic ability to pathogens and other foreign substances.

[0171] Table 9 Phagocytic index results

[0172]

[0173] Table 10 Synergistic index calculation results

[0174]

[0175] The CompuSyn software calculates the synergistic index CI = 0.78 (95% confidence interval: 0.72-0.84).

[0176] 2.5 Summary

[0177] This experiment systematically studied the effect of collagen peptide composition on the phagocytic function of RAW264.7 macrophages by neutral red phagocytosis method. The results showed that the collagen peptide composition of the present application, especially the combination of CPF-1 and mAb Senolix, can significantly enhance the phagocytic activity of macrophages, and the two show good synergistic effect. This result provides experimental evidence at the cellular level 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, and has potential medicinal and health care value, 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, 10 in each group, and the specific treatments are shown in Table 11, wherein the composition is prepared by the method of the first part of this example.

[0180] Table 11 Experimental grouping and treatment

[0181]

[0182] 3.2 Detection index and method

[0183] (1) Delayed hypersensitivity reaction (DTH) determination: 24 h after the last administration, each mouse was subcutaneously injected with 2 x 10 9

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

[0185] After DTH detection, the eyeball was bled, and the serum was separated by centrifugation at 4°C and 3000 rpm for 10 min after standing at 37°C for 1 h.

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

[0187] Preparation of standard curve: SRBC hemolysin solutions with different concentrations (0, 50, 100, 200, 400, 800 U / mL) were prepared, and the OD 540 value was determined, and a standard curve was drawn to calculate the HC50 value of the sample.

[0188] (3) Calculation of immune organ index: After bleeding, the mice were sacrificed, and the spleen and thymus were aseptically isolated, and the surrounding fat and connective tissue was removed, and then washed with pre-cooled physiological saline; the surface moisture of the tissue was absorbed with filter paper, and the weight of the spleen (W

[0189] ​(4) Flow cytometry analysis of T cell subsets: the spleen was taken and placed on a 200-mesh cell sieve, ground with a syringe core to prepare a single cell suspension; add red blood cell lysate, lyse at room temperature for 5 min, wash with PBS for 2 times, centrifuge at 1000 rpm for 5 min; adjust the cell concentration to 1 x 10 6 + + + +

[0190] 3.3 Experimental data

[0191] (1) DTH reaction, as shown in Table 12. The footpad swelling degree of the cyclophosphamide model group was significantly lower than that of the control group (p<0.001), indicating that the immunosuppression model was successfully constructed. The footpad swelling degree of the high-dose composition 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), indicating that the high-dose composition can effectively restore the cellular immune function of the body.

[0192] Table 12 DTH 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 composition group (98.7±6.3) was close to the level of the control group, and was 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 index, as shown in Table 14. The spleen and thymus indices of the model group were significantly lower than those of the control group (p<0.001). The spleen index (3.89±0.32 mg / g) and thymus index (1.95±0.21 mg / g) of the high-dose composition group were significantly higher than those of the model group (p<0.01), indicating that the composition can reduce the damage of immunosuppression to immune organs and promote their growth and development.

[0198] Table 14 Immune organ index results

[0199] ​​​​​

[0200] (4) T cell subgroups, as shown in Table 15. The CD4 + / CD8 + ratio of the model group was significantly lower than that of the control group (p<0.01). The CD4 + / CD8 + ratio (2.15±0.18) of the high-dose composition group was significantly higher than that of the model group (p<0.01), close to the level of the control group, indicating that the composition can regulate the balance of T cell subgroups and enhance the immune regulation ability of the body.

[0201] Table 15 T cell subgroup results

[0202]

[0203] 3.4 Summary

[0204] In this experiment, the immune function of the body was systematically evaluated by using a cyclophosphamide-induced immunosuppressed mouse model. The results showed that the high-dose composition can significantly improve the cellular immunity (DTH reaction) and humoral immunity (serum hemolysin) function of immunosuppressed mice, promote the development of immune organs, and regulate the balance of T cell subgroups. The experimental data confirm that the composition has a significant effect on improving the body's immunity, and its effect is comparable to that of the positive drug levamisole, providing reliable in vivo experimental basis for the development of the composition as an immunoregulatory product. Subsequent studies can further investigate the mechanism of action and the optimal application dose, and promote its conversion and application in the field of medicine and health products.

[0205] Example 4: Composition of other formulations

[0206] I. Oral powder

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

[0208] Preparation: Screen each component through a 100-mesh sieve, mix uniformly in a three-dimensional mixer for 30 minutes, and pack into aluminum foil bags (5 g per bag).

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

[0210] II. Capsules

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

[0212] Preparation: Fill capsules after mixing (500 mg per capsule).

[0213] Usage: 3 capsules per day, twice a day.

[0214] Three, functional beverage

[0215] Formulation: 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: ingredient → homogenization (50 MPa) → UHT sterilization (137℃, 5s) → aseptic filling.

[0217] Four, soluble film (oral mucosal administration)

[0218] Formulation: CPF-1 (15 mg / cm 2 ), mAb Senolix (5 mg / cm 2 ), collagen peptide (50 mg / cm 2 ), hydroxypropyl methyl cellulose (matrix).

[0219] Preparation: cast film, cut into 2 cm × 2 cm tablets.

[0220] Advantages: avoid gastrointestinal degradation, improve bioavailability.

[0221] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods, all of which are included in the protection scope of the present application.

Claims

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

1.

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

3. The composition of claim 2, wherein, The composition is composed of the following components by weight: (1) marine collagen peptide 60 parts; (2) fusion polypeptide CPF-1 10 parts; (3) monoclonal antibody mAb Senolix 3 parts; (4) five galloyl glucose 5 parts, the mass ratio of collagen peptide is 1:0.2; (5) Hericium erinaceus polysaccharide 15 parts.

4. The composition of claim 2, wherein, The monoclonal antibody mAb Senolix targets p16INK4a protein aa89-103 epitope, and the amino acid sequence is FLDTLVVLHRAGAR.

5. The use of the fusion polypeptide CPF-1 of claim 1 in the preparation of collagen peptide composition for improving immunity.

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