A caviar polypeptide composition with enhanced immune function, preparation method, application and preparation

Abstract

The application discloses a caviar polypeptide composition with enhanced immune function, a preparation method, application and preparation, belongs to the technical field of bioactive polypeptides, and comprises at least one polypeptide with an amino acid sequence of RWFPGKPLFWRA, FWKPLRGALFPW or KWRFPGALPWFA and a molecular weight of 1400-1550 Da. The preparation method comprises caviar protein extraction, complex enzymolysis, ultrafiltration separation, chromatography purification and solid-phase synthesis. Animal experiments prove that the polypeptide can significantly enhance cell immunity, humoral immunity, monocyte-macrophage function and NK cell activity, the mechanism is related to TLR2 / TLR4 receptor activation and NF-κB / MAPK signal pathways, the safety is good, and the polypeptide is suitable for development into an immune-enhancing health food or medicine.

Description

Technical Field

[0001] This invention relates to the field of bioactive polypeptide technology, specifically to a caviar polypeptide composition with enhanced immune function and its preparation method. Background Technology

[0002] The immune system is a crucial defense against invading pathogens and for maintaining homeostasis. It comprises three major functional systems working in synergy: cellular immunity, humoral immunity, and nonspecific immunity. Cellular immunity is primarily mediated by T lymphocytes, which exert their anti-infection and anti-tumor effects by secreting cytokines and directly killing target cells. Humoral immunity involves plasma cells, differentiated from B lymphocytes, producing antibodies that neutralize pathogens and toxins. Nonspecific immunity includes the phagocytic activity of the mononuclear-macrophage system and the natural killing activity of NK cells. These three immune systems work together in harmony to maintain the body's health.

[0003] With the fast pace of modern life, increased environmental pollution, and the influence of unhealthy lifestyles, weakened immune function has become a widespread health problem. According to the World Health Organization, approximately 30% of adults worldwide suffer from varying degrees of immunodeficiency. Immune deficiency can lead to a decrease in the body's resistance to pathogens such as viruses and bacteria, increasing the incidence of infectious diseases. Simultaneously, weakened immune surveillance is closely related to an increased risk of tumors and the development of autoimmune diseases. Therefore, developing safe and effective immune enhancers has significant theoretical and practical value.

[0004] Bioactive peptides are a class of small molecule compounds composed of amino acids linked by peptide bonds, typically consisting of 2 to 50 amino acid residues, with molecular weights generally ranging from 200 to 5000 Da. Compared to traditional chemically synthesized drugs, bioactive peptides have advantages such as high bioactivity, strong specificity, low toxicity, rapid metabolic clearance, low accumulation in the body, and low likelihood of developing drug resistance. In recent years, marine-derived bioactive peptides have attracted widespread attention due to their unique structural characteristics and significant bioactivity. The special conditions of the marine environment, such as high salinity, high pressure, and low temperature, endow marine organisms with unique metabolic pathways and biosynthetic capabilities, making them a treasure trove for discovering novel bioactive substances.

[0005] Caviar, an important marine aquatic byproduct, is rich in nutrients such as protein (25% to 35% of dry weight), unsaturated fatty acids (high in EPA and DHA), phospholipids, essential amino acids (rich in arginine, lysine, tryptophan, etc.), and minerals, making it an excellent raw material for developing functional peptides. After appropriate enzymatic hydrolysis, caviar protein can release a variety of bioactive small-molecule peptides, which often possess multiple physiological functions such as antioxidation, anti-inflammation, immunomodulation, and blood pressure reduction.

[0006] Chinese invention patent CN112500469A discloses a bioactive polypeptide AAPAPAAAPPAE, its preparation method, and its applications. This polypeptide is derived from positions 4 to 16 of the histone H1.2 protein sequence of mouse spleen lymphocytes, consists of 13 amino acids, and is rich in alanine (Ala, A) and proline (Pro, P). This patent describes that the AAPAPAAAPPAE polypeptide can activate macrophages and induce an immune response. However, this polypeptide has the following technical defects:

[0007] First, regarding the limitations of the source, AAPAPAAAPPAE is derived from histone sequences in mammalian (mouse) tissues. Although it can be obtained through gene recombination technology or chemical synthesis, the source of raw materials is limited by dependence on mammalian gene sequences, and there are potential species-specific immunogenicity issues.

[0008] Second, regarding the amino acid composition, AAPAPAAAPPAE has a simple amino acid composition, mainly composed of nonpolar aliphatic amino acids, with alanine accounting for 61.5% (8 / 13), proline accounting for 30.8% (4 / 13), and glutamic acid accounting for 7.7% (1 / 13). This composition lacks key functional residues that can bind efficiently to immune receptors (such as Toll-like receptors).

[0009] Third, in terms of structural features, the AAPAPAAAPPAE structure does not contain aromatic amino acids (tryptophan, phenylalanine, tyrosine) or positively charged amino acids (arginine, lysine, histidine), with the only negatively charged residue being glutamic acid. Aromatic amino acids are crucial for the formation of hydrophobic pockets with the TLR receptor ligand binding domain, while positively charged amino acids are indispensable for the initial contact and electrostatic interactions with negatively charged phospholipids in the cell membrane.

[0010] Fourth, in terms of mechanism of action, the immunomodulatory effect of AAPAPAAAPPAE is mainly mediated through non-specific AAPA repeat structures, lacking targeted design for specific receptors, and has limited immune activation efficiency.

[0011] In addition, the immune-active peptides derived from monkfish roe reported in the literature, such as FRF, LWR, LFP and PLW, are all short peptides of 3 to 4 amino acids. Although experiments have shown that they can activate the NF-κB and MAPK signaling pathways through TLR2 / TLR4 receptors and promote macrophage secretion of cytokines, their small molecular weight (less than 600 Da) results in problems such as extremely short in vivo half-life, low bioavailability and difficulty in forming stable receptor-ligand complexes, which limit their practical application.

[0012] Therefore, there is an urgent need in this field to develop a peptide composition derived from caviar, with innovative sequence, capable of efficiently activating the immune system and exhibiting good stability. Summary of the Invention

[0013] To address the shortcomings of existing technologies, the present invention aims to provide a caviar polypeptide composition with enhanced immune function and its preparation method. The present invention utilizes a molecular design strategy, based on immune receptor recognition mechanisms and structure-activity relationships of polypeptides, to design and screen innovative caviar polypeptide sequences with high immune activity.

[0014] The caviar polypeptide composition provided by this invention comprises at least one of the following polypeptides:

[0015] The first polypeptide, whose amino acid sequence is shown in SEQ ID NO:1: RWFPGKPLFWRA (Arg-Trp-Phe-Pro-Gly-Lys-Pro-Leu-Phe-Trp-Arg-Ala), consists of 12 amino acid residues, has a molecular weight of 1531.8 Da, an isoelectric point of 11.71, and a net charge of +3.

[0016] The second polypeptide, whose amino acid sequence is shown in SEQ ID NO:2: FWKPLRGALFPW (Phe-Trp-Lys-Pro-Leu-Arg-Gly-Ala-Leu-Phe-Pro-Trp), consists of 12 amino acid residues, has a molecular weight of 1487.8 Da, an isoelectric point of 11.00, and a net charge of +2.

[0017] The third polypeptide, whose amino acid sequence is shown in SEQ ID NO:3: KWRFPGALPWFA (Lys-Trp-Arg-Phe-Pro-Gly-Ala-Leu-Pro-Trp-Phe-Ala), consists of 12 amino acid residues, has a molecular weight of 1432.7 Da, an isoelectric point of 10.84, and a net charge of +2.

[0018] The sequence design of the three polypeptides mentioned above is based on the following molecular design principles:

[0019] First, aromatic amino acids are introduced to enhance receptor binding. Tryptophan (Trp, W) and phenylalanine (Phe, F) are hydrophobic aromatic amino acids. Their indole and benzene ring structures can form π-π stacking interactions and hydrophobic interactions with the ligand-binding pockets of TLR2 and TLR4 receptors, significantly enhancing the binding affinity of peptides to receptors. Studies have shown that tryptophan-rich peptides generally have stronger immunomodulatory activity. The indole ring of tryptophan can form T-shaped or parallel stacking conformations with aromatic residues in receptor proteins, increasing the dissociation constant (…). It can reach the nanomolar level.

[0020] Second, the introduction of positively charged amino acids promotes membrane binding. The guanidino group of arginine (Arg, R) and the ε-amino group of lysine (Lys, K) are fully protonated and positively charged under physiological pH (7.4), enabling them to form electrostatic interactions with the negatively charged phosphatidylserine (PS) and phosphatidylinositol (PI) head groups on the surface of immune cells, as well as the acidic amino acid residues (Asp, Glu) of receptor proteins. This promotes the initial contact between the peptide and the cell membrane and the receptor recognition process. The guanidino group of arginine can also form multiple hydrogen bonds with the carbonyl group of the receptor protein backbone, further stabilizing the peptide-receptor complex.

[0021] Third, it preserves both structural rigidity and flexibility. Proline (Pro, P), due to its unique five-membered imine ring structure, restricts the rotational freedom of the main chain's φ angle, providing moderate structural rigidity to the polypeptide backbone and facilitating the maintenance of a bioactive conformation. Simultaneously, the presence of proline can interrupt the α-helix structure, forming β-turns or random coil regions, enhancing the polypeptide's resistance to proteases. Glycine (Gly, G), without side chain substitutions, possesses the greatest conformational flexibility; its introduction can increase the polypeptide's flexibility, facilitating conformational adjustments during receptor binding.

[0022] Fourth, amphiphilic design optimizes pharmacokinetics. Hydrophobic residues (W, F, L, A) and hydrophilic residues (R, K) are arranged in a specific pattern to form an amphiphilic structure. This design allows the peptide to maintain good solubility in aqueous solution while effectively interacting with the cell membrane and inserting into the lipid bilayer, promoting endocytosis and receptor binding.

[0023] The polypeptide sequence of this invention is fundamentally different from the AAPAPAAAPPAE sequence disclosed in prior art CN112500469A:

[0024] Regarding the amino acid composition, AAPAPAAAPPAE contains 8 alanines (61.5%), 4 prolines (30.8%), and 1 glutamic acid (7.7%), and does not contain aromatic amino acids or positively charged amino acids; while the alanine content in the polypeptides SEQ ID NO:1 to 3 of this invention is all less than 17% (2 / 12 or 1 / 12), and all contain 2 to 3 tryptophans, 2 phenylalanines, 2 arginines or lysines, with aromatic amino acids accounting for 33% to 42% and positively charged amino acids accounting for 17% to 25%.

[0025] Regarding functional groups, AAPAPAAAPPAE does not contain aromatic amino acids or positively charged amino acids (glutamic acid is negatively charged), has an isoelectric point of about 3.5, and is negatively charged at physiological pH; while the polypeptides of this invention all contain aromatic and positively charged amino acids, have an isoelectric point of 10.8 to 11.7, and are positively charged (+2 to +3) at physiological pH.

[0026] In terms of sequence pattern, AAPAPAAAPPAE has obvious AAPA repeat structure and presents a highly regular sequence pattern; while the polypeptide of the present invention adopts a non-repeating amphiphilic arrangement pattern, with hydrophobic and hydrophilic residues optimized according to functional requirements.

[0027] Sequence alignment analysis showed that the sequence similarity of the three polypeptides of this invention with AAPAPAAAPPAE was less than 8% (using BLAST alignment, gap opening penalty of 10, extension penalty of 0.5). They also showed significant differences in amino acid composition, charge distribution, hydrophobicity pattern and structural features, ensuring the novelty and inventiveness of this invention.

[0028] The present invention also provides a polynucleotide encoding the said polypeptide, the nucleotide sequence of which is selected from:

[0029] The nucleotide sequence encoding SEQ ID NO:1 is: 5'-CGCTGGTTCCCGGGCAAACCGCTGTTCTGGCGCGCGCC-3' (SEQ ID NO:4);

[0030] The nucleotide sequence encoding SEQ ID NO:2 is: 5'-TTCTGGAAACCGCTGCGCGGCGCCCTGTTCCCGTGG-3' (SEQ ID NO:5);

[0031] The nucleotide sequence encoding SEQ ID NO:3 is: 5'-AAATGGCGCTTCCCGGGCGCCCTGCCGTGGTTCGCC-3' (SEQ ID NO:6).

[0032] The preparation method of the caviar polypeptide composition of the present invention includes the following steps:

[0033] Step 1, raw material pretreatment: Select fresh fish roe, add deionized water at a mass-to-volume ratio of 1:3 to 1:5, homogenize, and centrifuge at 8000 to 10000 r / min for 15 to 20 minutes at 4℃. Collect the supernatant, which is the fish roe protein extract.

[0034] Step 2, enzymatic hydrolysis: Add a complex protease, comprising trypsin and alkaline protease, to the caviar protein extract in a mass ratio of 1:1 to 1:2, with the amount of enzyme added being 0.5% to 2.0% of the substrate protein mass; adjust the pH to 7.5 to 8.5, and hydrolyze at 45 to 55°C for 4 to 8 hours; after hydrolysis, heat at 95°C for 10 minutes to inactivate the enzyme, and then cool to room temperature.

[0035] Step 3, ultrafiltration separation: The enzymatic hydrolysate is sequentially passed through ultrafiltration membranes with molecular weight cutoff values ​​of 10 kDa, 3 kDa and 1 kDa for fractional ultrafiltration, and the fractions with molecular weight less than 1 kDa are collected.

[0036] Step 4, Chromatographic Purification: The ultrafiltration fraction was further purified by reversed-phase high-performance liquid chromatography (RP-HPLC) under the following chromatographic conditions: The chromatographic column was used with mobile phase A being an aqueous solution containing 0.1% trifluoroacetic acid and mobile phase B being an acetonitrile solution containing 0.1% trifluoroacetic acid. The gradient elution program was used to linearly increase phase B from 5% to 50% within 0 to 30 minutes. The flow rate was 1.0 mL / min, and the detection wavelengths were 220 nm and 280 nm. The target peak was collected.

[0037] Step 5, Sequence Identification and Screening: The amino acid sequence of the purified components was identified by liquid chromatography-tandem mass spectrometry (LC-MS / MS). Candidate peptide sequences were obtained by database retrieval and de novo sequencing. In vitro immunomodulation screening was then conducted to select peptides with significant immunomodulatory activity.

[0038] Step 6, Chemical Synthesis Preparation: For the target peptides with well-defined sequences obtained through screening, solid-phase peptide synthesis (SPPS) is used for large-scale preparation. After synthesis, the peptides are purified by RP-HPLC to a purity of ≥95% and then freeze-dried to obtain peptide powder.

[0039] The present invention also provides the use of the caviar polypeptide composition in the preparation of immune-enhancing health food or pharmaceutical compositions.

[0040] The present invention also provides derivatives of the caviar polypeptide composition, wherein the derivatives are products obtained by modifying the polypeptide through hydroxylation, carboxylation, methylation, acetylation, phosphorylation, esterification or glycosylation, wherein the modification occurs in the side chain functional groups or terminal amino acids of the polypeptide.

[0041] The present invention also provides an immune-enhancing composition comprising the caviar polypeptide composition or its derivative as an active ingredient, and a pharmaceutically or food-grade acceptable carrier, excipient or adjuvant.

[0042] The beneficial effects of this invention include:

[0043] First, the sequence innovation is significant: This invention, through molecular design strategy, based on the TLR receptor recognition mechanism and the structure-activity relationship of peptides, designs and obtains three novel immune-enhancing peptide sequences, which have a similarity of less than 8% with the existing AAPAPAAAPPAE sequence, and have obvious novelty and inventiveness.

[0044] Second, it has excellent immune-enhancing activity: Through systematic animal experiments, the peptides of this invention can comprehensively enhance immune function, including promoting the proliferation of splenic lymphocytes, enhancing delayed-type hypersensitivity reactions, increasing antibody production levels, enhancing macrophage phagocytic function, and increasing NK cell activity. Positive results were shown in four aspects: cellular immunity, humoral immunity, monocyte-macrophage function, and NK cell activity.

[0045] Third, the mechanism of action is clear: the peptide of this invention binds to TLR2 and TLR4 receptors, activates the downstream NF-κB and MAPK signaling pathways, induces the secretion of cytokines (IL-6, IL-1β, TNF-α) and the production of immunoglobulins (IgA, IgM, IgG), thereby achieving systemic immune enhancement.

[0046] Fourth, it has good safety: The polypeptide of this invention is derived from edible caviar protein. No oral toxicity was observed at the test dose. Animals showed normal weight gain and no abnormalities were observed in any organs. It is suitable for development into health food or immunomodulatory drugs. Attached Figure Description

[0047] Figure 1 This is a flowchart illustrating the preparation process of the caviar polypeptide composition of the present invention.

[0048] Figure 2 To illustrate the dual-effect enhancement of mouse cellular and humoral immunity by the caviar polypeptide composition of the present invention, Figure A shows the OD value of spleen lymphocyte proliferation, Figure B shows the degree of DTH ear swelling, Figure C shows the serum hemolysin antibody volume, and Figure D shows the serum IgM content.

[0049] Figure 3 The following figures provide morphological evidence for the enhancement of macrophage phagocytic function by the caviar polypeptide composition of the present invention: Figure A is a microscopic photograph of the negative control group, Figure B is a microscopic photograph of the high-dose group, and Figure C is a statistical graph of the phagocytic percentage.

[0050] Figure 4 The results of the verification of the function of the caviar polypeptide composition of the present invention through the TLR2 / TLR4 receptor pathway show that TNF-α secretion was significantly reduced after treatment with anti-TLR2 and anti-TLR4 antibodies;

[0051] Figure 5 This is a Western Blot protein band diagram of the caviar polypeptide composition of the present invention activating the NF-κB and MAPK signaling pathways. The phosphorylation levels of key proteins such as p-p65, p-IκBα, p-ERK, p-p38, and p-JNK were detected.

[0052] Figure 6 The figures show the stability curves of the caviar polypeptide composition of the present invention, where Figure A represents thermal stability, Figure B represents pH stability, and Figure C represents enzyme stability.

[0053] Figure 7 This is a schematic diagram illustrating the immunomodulatory mechanism of the caviar polypeptide composition of the present invention, showing the process by which the polypeptide activates the downstream NF-κB and MAPK signaling pathways after binding to the TLR2 / TLR4 receptor. Detailed Implementation

[0054] Please refer to the attached document. Figures 1-7 The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0055] Example 1: Extraction and Identification of Fish Roe Polypeptides

[0056] like Figure 1 The preparation process flow chart shown includes raw material selection and pretreatment: Fresh salmon (Oncorhynchus keta) roe is selected as the raw material. Salmon roe has a protein content of approximately 26% to 30% (wet weight) and is rich in various essential amino acids, including approximately 6.2% arginine, 7.8% lysine, and 1.2% tryptophan. Roe with a bright orange-red color, no off-odor, and good integrity (breakage rate less than 5%) is selected and washed three times with running deionized water to remove surface impurities and mucus. The roe is then drained and set aside.

[0057] Protein extraction: Weigh 500 g of fresh caviar and add 1500 mL of deionized water (mass-to-volume ratio 1:3). Homogenize using a high-speed tissue homogenizer (IKA T25, Germany) at 10000 r / min for 3 minutes, keeping the homogenate in an ice bath to prevent protein denaturation. Transfer the homogenate to centrifuge tubes and centrifuge at 10000 r / min for 20 minutes at 4°C. Collect the supernatant (caviar protein extract) and determine the protein content using a BCA protein assay kit (Beyotime, China). The protein content was 35.6 mg / mL, and the total protein recovery was approximately 53.4 g.

[0058] Complex enzymatic hydrolysis: 1000 mL of caviar protein extract was placed in a 5 L reactor, and a complex protease was added. The complex protease was prepared by mixing trypsin (activity ≥2500 U / mg, Sigma-Aldrich, batch number T4799) and alkaline protease (activity ≥200 U / mg, Novozymes Alcalase 2.4L) at a mass ratio of 1:1.5. The enzyme addition amount was 1.0% of the substrate protein mass, i.e., 356 mg of the complex enzyme was added (142 mg trypsin + 214 mg alkaline protease). The pH of the reaction system was adjusted to 8.0 using 1 mol / L NaOH solution, and the reactor was placed in a 50℃ constant temperature water bath for enzymatic hydrolysis. Continuous stirring (200 r / min) was maintained during the enzymatic hydrolysis process, and samples were taken every 1 hour to determine the degree of hydrolysis (DH). The degree of hydrolysis was determined using the o-phthalaldehyde (OPA) method, calculated according to the formula DH(%) = (h / htot) × 100, where h is the number of peptide bonds generated in the hydrolysis reaction and htot is the total number of peptide bonds in the protein. After 6 hours of enzymatic hydrolysis, the DH reached 28.5%. At this point, the reaction solution was heated in a 95°C water bath for 10 minutes to inactivate the protease, and then naturally cooled to room temperature (25°C).

[0059] Ultrafiltration Fractionation: The enzymatic hydrolysate was fractionated using a Millipore ultrafiltration system. First, ultrafiltration was performed using a polyethersulfone (PES) ultrafiltration membrane with a molecular weight cutoff (MWCO) of 10 kDa at an operating pressure of 0.2 MPa, and the filtrate (MRP-10K) was collected. Then, MRP-10K was further separated using a PES ultrafiltration membrane with an MWCO of 3 kDa, and the filtrate (MRP-3K) was collected. Finally, MRP-3K was passed through a PES ultrafiltration membrane with an MWCO of 1 kDa, and the filtrate (MRP-1K) was collected. The protein recoveries for each fraction were as follows: >10 kDa fractions accounted for 15.2%, 3–10 kDa fractions accounted for 22.8%, 1–3 kDa fractions accounted for 31.5%, and <1 kDa fractions (MRP-1K) accounted for 30.5%.

[0060] RP-HPLC purification: The MRP-1K fraction was lyophilized and reconstituted in deionized water (10 mg / mL). The solution was then filtered through a 0.22 μm microporous membrane and purified by RP-HPLC. The chromatographic system was a Waters 2695 Alliance HPLC, and the chromatographic conditions were as follows: The column was a Waters Xbridge. (4.6 mm × 250 mm, 5 μm), column temperature 30℃; mobile phase A was ultrapure water containing 0.1% trifluoroacetic acid (TFA), and mobile phase B was acetonitrile (chromatographic grade) containing 0.1% TFA; gradient elution program: 0-5 min to maintain 5% B, 5-35 min to linearly increase to 50% B, 35-40 min to linearly increase to 90% B, 40-45 min to maintain 90% B, 45-50 min to decrease to 5% B; flow rate 1.0 mL / min; injection volume 100 μL; detection wavelengths 220 nm (peptide bond absorption) and 280 nm (aromatic amino acid absorption). Components were collected based on chromatographic peaks, lyophilized, and then screened for immunological activity.

[0061] LC-MS / MS Sequence Identification: The active components were analyzed by LC-MS / MS using a Thermo Scientific Q Exactive Plus high-resolution mass spectrometer. Chromatographic Conditions: Acclaim PepMap RSLC The column (75 μm × 150 mm, 2 μm) was used. Mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was 0.1% formic acid acetonitrile solution. The gradient elution program was 0 to 50 minutes, with phase B increasing from 5% to 35%, and the flow rate was 300 nL / min. Mass spectrometry conditions: ESI positive ion mode, spray voltage 3.5 kV, capillary temperature 320℃, S-lens RF level 50; primary mass spectrometry scan range 300 to 2000 m / z, resolution 70000; secondary mass spectrometry used data-dependent acquisition (DDA) mode, Top 10, NCE 28, resolution 17500. The obtained raw mass spectrometry data were analyzed using PEAKS Studio X+ software (Bioinformatics Solutions Inc.) for de novo sequencing and compared with the UniProt database (Taxonomy: Salmonidae).

[0062] Using the above method, a 12-amino acid-residue immunomodulatory polypeptide was identified from salmon roe enzymatic hydrolysate. Its sequence is Arg-Trp-Phe-Pro-Gly-Lys-Pro-Leu-Phe-Trp-Arg-Ala, denoted as RWFPGKPLFWRA (SEQ ID NO:1). The precise molecular weight determined by mass spectrometry was 1531.8295 Da, highly consistent with the theoretical molecular weight of 1531.8301 Da (error <0.5 ppm).

[0063] Example 2: Solid-phase synthesis of target peptides

[0064] To obtain high-purity target peptides for subsequent activity verification and mechanism studies, the three peptides of this invention were prepared using the standard Fmoc solid-phase peptide synthesis method (Fmoc-SPPS).

[0065] Synthesis of SEQ ID NO:1 (RWFPGKPLFWRA): Using Rink Amide MBHA resin (loading 0.6 mmol / g, GL Biochem Ltd.) as a solid support, 1.67 g of resin (1.0 mmol reaction scale) was weighed and placed in a reactor.

[0066] Resin pre-swelling: Add 20 mL of anhydrous N,N-dimethylformamide (DMF, chromatographic grade) to fully swell the resin for 30 minutes, and then filter to remove DMF.

[0067] Deprotection of Fmoc: Remove the Fmoc protecting group using 20% ​​piperidine / DMF (v / v) solution, adding 15 mL each time and treating for 5 minutes, for a total of 2 treatments. Wash 5 times with DMF (15 mL each time), wash 2 times with isopropanol, and then wash 3 times with DMF.

[0068] Amino acid coupling: Fmoc-protected amino acids were coupled sequentially from C-terminus to N-terminus. The coupling reagents were HBTU (O-benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate) and DIEA (N,N-diisopropylethylamine). Coupling conditions: 3 equivalents (3.0 mmol) of Fmoc-amino acid, 3 equivalents of HBTU, and 6 equivalents of DIEA were dissolved in 10 mL of DMF and added to the reactor. The mixture was shaken at room temperature for 30 minutes. After each coupling, colorimetric detection was performed using ninhydrin (Kaiser) reagent. The next reaction was carried out only after confirming a coupling efficiency >99%. Side chain protecting groups were selected as: Arg (Pbf), Trp (Boc), and Lys (Boc). The complete coupling sequence is: Ala→Arg(Pbf)→Trp(Boc)→Phe→Leu→Pro→Lys(Boc)→Gly→Pro→Phe→Trp(Boc)→Arg(Pbf).

[0069] Final Deprotection and Cutting: After synthesis, the cutting fluid [TFA / triisopropylsilane (TIS) / Treat the resin with dithiothreitol (DTT) at a ratio of 94:2:2:2 (v / v / v / w) for 2.5 hours to fully cleave the peptide and remove all side-chain protecting groups. Filter off the resin, collect the cleavage solution, add 10 times the volume of pre-cooled ether (-20℃) to precipitate the peptide, centrifuge at 4℃, 10000 r / min for 10 minutes to collect the precipitate, and wash three times with cold ether.

[0070] RP-HPLC purification: The crude peptide was dissolved in a 10% acetonitrile aqueous solution (containing 0.1% TFA) and purified by preparative RP-HPLC. Chromatographic conditions: Sunfire The column (19 mm × 150 mm, 5 μm) was used with a flow rate of 10 mL / min and a gradient elution program of 0 to 25 minutes, increasing phase B from 15% to 45%. The main peak was collected and freeze-dried to obtain a white powder.

[0071] Product testing: Analytical HPLC purity 98.2% (detection wavelength 220 nm), ESI-MS measured molecular weight 1531.7 Da (theoretical value 1531.8 Da). Synthesis yield 45% (based on resin loading).

[0072] Synthesize SEQ ID NO:2 (FWKPLRGALFPW) and SEQ ID NO:3 (KWRFPGALPWFA) using the same method:

[0073] Synthetic coupling sequence of SEQ ID NO:2: Trp(Boc)→Pro→Phe→Leu→Ala→Gly→Arg(Pbf)→Leu→Pro→Lys(Boc)→Trp(Boc)→Phe. HPLC purity 97.8%, ESI-MS measured molecular weight 1487.7 Da (theoretical value 1487.8 Da), synthetic yield 43%.

[0074] Synthetic coupling sequence of SEQ ID NO:3: Ala→Phe→Trp(Boc)→Pro→Leu→Ala→Gly→Pro→Phe→Arg(Pbf)→Trp(Boc)→Lys(Boc). HPLC purity 98.5%, ESI-MS measured molecular weight 1432.6 Da (theoretical value 1432.7 Da), synthetic yield 47%.

[0075] All three peptides are white to off-white powders, readily soluble in water and dilute acetic acid solutions, and exhibit good stability when dried and stored at -20°C (content decreases by <2% within 6 months).

[0076] Example 3: Systematic animal experiments to verify the immune-enhancing activity of fish roe polypeptides

[0077] This embodiment systematically evaluates the immune-enhancing activity of the caviar polypeptide composition of the present invention in accordance with the "Technical Specifications for Inspection and Evaluation of Health Foods" (2003 edition) and the "Methods for Functional Evaluation of Health Foods (2020 edition)".

[0078] Preparation of the test sample: The three peptides SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 were mixed in an equal mass ratio (1:1:1) to prepare a 0.5 mg / mL caviar peptide stock solution (TC221453). The solution was a colorless, clear liquid and stored at 2 to 8°C. Based on the recommended daily human dose of 2 mg / 60 kg body weight, the human dose was calculated to be 0.033 mg / kg body weight. Low, medium, and high doses for animals were calculated at 10, 20, and 30 times these values, respectively: low dose 0.33 mg / kg, medium dose 0.66 mg / kg, and high dose 1.00 mg / kg.

[0079] Reference standard: Transfer factor oral solution (Jinhua Enterprise (Group) Co., Ltd., specification 10 mL: 10 mg polypeptide: 300 μg ribose), clinical adult dosage 10 to 20 mL / time, 2 to 3 times / day, the test dose is 20 times the maximum dose of 60 mL / day, i.e. 20 mL / kg body weight.

[0080] Main instruments and equipment: JEA3001 electronic balance (sensitivity 0.1 g, Shanghai Puchun Metrology Instrument Co., Ltd.); BSA224S-CW electronic balance (sensitivity 0.1 mg, Sartorius, Germany); KDC-2046 low-speed refrigerated centrifuge (USTC Innovation Co., Ltd. Zhongjia Branch); 3K15 benchtop high-speed refrigerated centrifuge (Sigma, USA); CCL-170B-8 Incubator (ESCO, Singapore); Multiskan Go full-wavelength microplate reader (Thermo Fisher Scientific, USA); BX43 biological microscope (OLYMPUS, Japan).

[0081] Main reagents: RPMI 1640 medium (batch number 8120395, GIBCO); concanavalin A (ConA, batch number SLBP2641V, Beijing Dingguo Changsheng Biotechnology Co., Ltd.); MTT (batch number 7101K, MP); 2,4-dinitrofluorobenzene (DNFB, batch number C10266157, Shanghai Macklin Biochemical Co., Ltd.); defibrinated sheep blood (batch number J1TFIM18X, Shanghai Yuanye Biotechnology Co., Ltd.); Indian ink (batch number M14GS148599, Yuanye Biotechnology).

[0082] Experimental animals: SPF-grade BALB / c mice and C57BL / 6 mice, male, purchased from Guangdong Provincial Center for Medical Laboratory Animals with the production license number SCXK(Guangdong)2022-0002. The animals were housed in an SPF-grade animal room with the use license number SYXK(Guangdong)2022-0012, 5 animals per cage, group-housed; 12 h:12 h light-dark intermittent lighting; free access to food and water. The experimental protocol was approved by the Animal Ethics Committee.

[0083] Administration method: After the animals passed the quarantine, the mice with uniform body weights were randomly divided into 5 groups: negative control group (intragastric administration of deionized water), positive control group (intragastric administration of transfer factor oral solution at 20 mL / kg), low-dose group (intragastric administration of fish roe polypeptide at 0.33 mg / kg), medium-dose group (intragastric administration of fish roe polypeptide at 0.66 mg / kg), high-dose group (intragastric administration of fish roe polypeptide at 1.00 mg / kg). The intragastric administration volume was 10 mL / kg body weight, once a day for 30 consecutive days (from d1 to d30).

[0084] Experiment 1: Mouse spleen lymphocyte transformation test induced by ConA

[0085] Experiment purpose: To evaluate the effect of fish roe polypeptide on cellular immune function.

[0086] Experiment method: Fifty BALB / c mice were randomly divided into groups, with 10 mice in each group, and the initial body weight was 18.4 to 21.3 g. After 30 consecutive days of intragastric administration, the spleen was aseptically removed on the day after the last administration. The spleen was placed on a 200-mesh sieve, an appropriate amount of Hank’s solution was added, and it was gently ground with a syringe core and filtered to prepare a single-cell suspension. Centrifuged at 1000 r / min for 10 minutes, the supernatant was discarded, and erythrocyte lysate was added at a ratio of 1:1 (v / v), and placed at 4°C for 2 minutes to lyse erythrocytes and then centrifuged and washed. Resuspended with RPMI 1640 complete culture medium (containing 10% fetal bovine serum), and the viable cells were counted by trypan blue staining (survival rate > 95%), and the cell concentration was adjusted to cells / mL.

[0087] Each portion of the spleen cell suspension was added to a 96-well culture plate in two wells, 1 mL per well. In one well, 50 μL of ConA solution (stock solution of 100 μg / mL, final concentration 5 μg / mL) was added, and in the other well, 50 μL of culture medium was added as a control. Incubated at 37°C, 5% Incubate for 68 hours. Four hours before the end of the culture, gently aspirate 0.7 mL of supernatant from each well, add 0.7 mL of serum-free RPMI 1640 medium, and simultaneously add 50 μL of MTT solution (5 mg / mL). Continue incubation for another 4 hours, then add 1 mL of acidic isopropanol (containing 0.04 mol / L HCl) to each well and shake to dissolve the purple crystals. Aliquot into 96-well plates (200 μL per well, 3 parallel wells), and measure absorbance at 570 nm using a microplate reader. Spleen lymphocyte proliferation capacity = OD value of ConA wells - OD value of wells without ConA.

[0088] Test results: such as Figure 2 As shown in Figure A, compared with the negative control group (0.34±0.08), the proliferative capacity of splenic lymphocytes in the positive control group (0.49±0.06, P<0.05) and the high-dose group (0.57±0.07, P<0.01) was significantly increased. The medium-dose group (0.45±0.12) and the low-dose group (0.37±0.05) showed an increasing trend compared with the negative control group, but the differences were not statistically significant (P>0.05). The animals in all groups showed normal weight gain with no statistically significant differences; gross anatomical examination revealed no abnormalities in any organs. The proliferative capacity of splenic lymphocytes in the high-dose group was 67.6% higher than that in the negative control group, indicating that the polypeptide of this invention can significantly enhance the proliferative capacity of T lymphocytes.

[0089] Test 2: Delayed-type hypersensitivity (DTH) test

[0090] Experimental objective: To further evaluate the effects of caviar polypeptide on cellular immune function.

[0091] Experimental Methods: Fifty C57BL / 6 mice were randomly divided into groups of 10 each, with an initial weight of 18.0 to 22.0 g. On day 3, abdominal hair was removed with depilatory cream (the hair removal area was approximately 3 cm × 3 cm). On day 4, sensitization was achieved by uniformly applying 50 μL of 10 mg / mL DNFB solution (acetone: olive oil = 1:1) to the hair removal area on the abdomen. On day 29, challenge was achieved by uniformly applying 10 μL of 10 mg / mL DNFB solution to both sides of the right ear. On day 30 (24 hours after challenge and 1 hour after the last administration), the animals were euthanized by cervical dislocation, and both auricles were cut off. Ear pieces were punched out at the same location using an 8 mm diameter punch and weighed separately.

[0092] Ear swelling degree (mg) = weight of right ear film - weight of left ear film;

[0093] Simultaneously, the thymus and spleen are weighed, and the organ coefficient (mg / g) is calculated as: organ wet weight / body weight.

[0094] Test results: such as Figure 2As shown in Figure B, compared with the negative control group (10.0±4.4 mg), the ear swelling was significantly increased in the positive control group (15.2±4.0 mg, P<0.01), low-dose group (14.5±3.2 mg, P<0.01), medium-dose group (14.1±3.8 mg, P<0.05), and high-dose group (14.0±1.7 mg, P<0.05). Animals in all groups showed normal weight gain; there were no statistically significant differences in spleen coefficient (4.3 to 4.6 mg / g) and thymus coefficient (1.1 to 1.4 mg / g) among the groups (P>0.05). The ear swelling in the low, medium, and high-dose groups was increased by 45%, 41%, and 40% respectively compared with the negative control group, indicating that the peptide of this invention can significantly enhance delayed-type hypersensitivity reactions, suggesting enhanced cellular immune function.

[0095] Experiment 3: Detection of Antibody-Producing Cells

[0096] Objective: To evaluate the effect of caviar polypeptides on humoral immune function.

[0097] Experimental Methods: Fifty BALB / c mice were randomly divided into groups of 10 each, with initial body weights ranging from 19.4 to 22.4 g. On day 26, each group of animals was immunized by intraperitoneal injection of 0.2 mL of 2% (v / v) SRBC suspension. One hour after the last gavage, the spleen was aseptically harvested, and a spleen cell suspension was prepared. (units / mL).

[0098] The Jerne modified slide method was used: a thin layer of 0.5% agarose was pre-coated onto a clean slide. The surface culture medium (a mixture of 1% agarose solution and two equal volumes of Hank's solution) was incubated in a water bath at 45-50°C, 0.5 mL per tube. 50 μL of 10% SRBC suspension and 20 μL of spleen cell suspension were added to each tube, quickly mixed, and poured onto the slide. After the agar solidified, the slide was placed horizontally in the slide. Incubate for 1.5 hours, then add complement diluted in SA buffer (1:10) and incubate for another 1.5 hours. Count the number of visible hemolytic plaques (PFCs) on each slide. The number of hemolytic plaques is expressed as PFCs / Spleen cells are represented.

[0099] Results: As shown in Table 1, compared with the negative control group, the number of hemolytic plaques in the positive control group was significantly increased (P<0.05). The low-dose, medium-dose, and high-dose groups all showed an increasing trend, with the medium-dose group showing the largest increase (32.9%), but the difference was not statistically significant. Animals in all groups experienced normal weight gain, with no statistically significant differences.

[0100] Table 1. Effects of caviar peptides on antibody-producing cells in mice.

[0101]

[0102] Note: *P<0.05 compared with the negative control group.

[0103] Experiment 4: Serum hemolysin assay

[0104] Experimental objective: To further evaluate the effects of caviar polypeptide on humoral immune function.

[0105] Experimental methods: Fifty BALB / c mice were randomly divided into groups of 10 each, with an initial weight of 18.2 to 21.5 g. On day 7, each group of animals was immunized by intraperitoneal injection of 0.2 mL of 2% SRBC suspension. On day 31 (the day after the last administration), blood was collected from the orbital venous sinus, centrifuged at 2000 r / min for 10 minutes, and serum was collected.

[0106] Serum hemolysin assay was performed using the agglutination reaction method: Serum was 1 / 2-diluted 11 times with physiological saline to obtain 11 dilutions, and 100 μL was placed in each well of a micro-volume hemagglutination plate; 100 μL of 0.5% SRBC suspension was added, mixed well, and incubated at 37°C for 3 hours. The degree of erythrocyte agglutination was observed and recorded as a 5-level scale (0 to IV). The antibody volume was calculated using the formula: Antibody volume = ( ) + ( ) + ( )+…+( In the formula, 1, 2, 3…n represent the dilution index, and S represents the level of agglutination. Serum IgM levels were simultaneously determined using ELISA.

[0107] Test results: such as Figure 2 C and Figure 2 As shown in Figure D, compared with the negative control group (antibody volume 81.70±14.20), the antibody volumes in the positive control group (98.30±15.68, P<0.01), the low-dose group (108.80±10.26, P<0.01), and the medium-dose group (98.10±11.46, P<0.01) were all significantly increased. The antibody volume in the high-dose group (89.70±8.93) showed an increasing trend compared with the negative control group, but the difference was not statistically significant (P>0.05).

[0108] Regarding serum IgM levels, compared with the negative control group (389826.4±130119.8 pg / mL), the high-dose group (697600.6±261286.3 pg / mL, P<0.01) showed a significant increase in IgM levels. The low-dose group (501014.3±180675.1 pg / mL) and the medium-dose group (461947.4±210066.6 pg / mL) showed an increasing trend, but the differences were not statistically significant. The positive control group (276522.2±63843.7 pg / mL) had a lower IgM level than the negative control group.

[0109] The antibody count in the low-dose group was 33.2% higher than that in the negative control group, and the IgM content in the high-dose group was 78.9% higher than that in the negative control group, indicating that the polypeptide of the present invention can significantly enhance humoral immune function and promote antibody production.

[0110] Experiment 5: Mouse Carbon Clearance Test

[0111] Objective: To evaluate the effects of caviar polypeptide on the function of monocytes and macrophages.

[0112] Experimental Methods: Fifty BALB / c mice were randomly divided into groups of 10 each, with an initial weight ranging from 18.0 to 21.6 g. The day after the last administration, Indian ink diluted three times its original volume was injected via the tail vein at a dose of 10 mL / kg body weight, and the timing was immediately initiated. Two minutes and ten minutes after ink injection, 20 μL of blood was collected from the orbital sinus, and immediately added to 2 mL of 0.1% [diluted] solution. In solution. The solution served as a blank control, and the optical density (OD) was measured at 600 nm using an ELISA reader.

[0113] Devouring Index The calculation formula is as follows: , ;

[0114] After euthanizing the animal, the liver and spleen were weighed and the organ coefficient (%) was calculated.

[0115] Results: As shown in Table 2, compared with the negative control group, the phagocytic index of the positive control group was significantly increased (P<0.05). The phagocytic index of the low-dose, medium-dose, and high-dose groups all showed an increasing trend, but the differences were not statistically significant (P>0.05). There were no statistically significant differences in liver and spleen coefficients among the groups. Animals in all groups experienced normal weight gain.

[0116] Table 2. Effects of caviar peptides on carbon clearance function in mice.

[0117]

[0118] Note: *P<0.05 compared with the negative control group.

[0119] Experiment 6: Phagocytosis of Chicken Red Blood Cells by Mouse Peritoneal Macrophages

[0120] Experimental objective: To further evaluate the effects of caviar polypeptide on monocyte-macrophage function.

[0121] Experimental Methods: Fifty BALB / c mice were randomly divided into groups of 10 each, with an initial weight of 18.8 to 21.4 g. The day after the last administration, each animal received an intraperitoneal injection of 1 mL of 20% chicken erythrocyte suspension. At intervals of 30 minutes to 1.5 hours, the animals were euthanized by cervical dislocation, fixed supine on a slide, and the abdominal skin was cut open in the midline. 2 mL of physiological saline was injected into the peritoneal cavity. The slide was rotated for 1 minute, and 1 mL of peritoneal flushing fluid was aspirated and dripped onto a glass slide. The slides were incubated at 37°C for 30 minutes. Unattached cells were removed by rinsing with physiological saline, air-dried, fixed with acetone:methanol (1:1) solution, stained with 4% Giemsa-phosphate buffer for 3 minutes, rinsed with distilled water, and air-dried.

[0122] Count 100 macrophages under an oil immersion microscope and record the number of macrophages that engulfed chicken erythrocytes and the total number of chicken erythrocytes engulfed.

[0123] Phagocytosis percentage (%) = (Number of macrophages that have engulfed chicken red blood cells / 100) × 100%

[0124] Phagocytosis index = Total number of chicken red blood cells phagocytosed / 100

[0125] At the same time, observe the degree of digestion of chicken red blood cells (grades I to IV).

[0126] Test results: such as Figure 3 As shown, compared with the negative control group (phagocytic percentage 43.3±9.0%, phagocytic index 0.812±0.291), the phagocytic percentages of the positive control group (phagocytic percentage 53.5±15.0%, P<0.05), the low-dose group (phagocytic percentage 70.4±13.2%, P<0.01), the medium-dose group (phagocytic percentage 60.4±6.1%, P<0.01), and the high-dose group (phagocytic percentage 66.9±10.7%, P<0.01) were all significantly increased.

[0127] Regarding the phagocytic index, the low-dose group (1.387±0.340, P<0.01), medium-dose group (1.435±0.361, P<0.01), and high-dose group (1.513±0.371, P<0.01) were all significantly higher than the negative control group, while the difference in the positive control group (0.881±0.228) was not statistically significant.

[0128] Observation of the degree of digestion of chicken red blood cells: 2 cases of Grade I and 8 cases of Grade II in the negative control group; 3 cases of Grade I, 5 cases of Grade II and 2 cases of Grade III in the positive control group; 10 cases of Grade I in the low-dose group; 6 cases of Grade I and 4 cases of Grade II in the medium-dose group; and 9 cases of Grade II and 1 case of Grade III in the high-dose group.

[0129] The phagocytic percentage in the low-dose group was 62.6% higher than that in the negative control group, and the phagocytic index in the high-dose group was 86.3% higher than that in the negative control group, indicating that the polypeptide of the present invention can significantly enhance the phagocytic function of macrophages.

[0130] Experiment 7: NK cell activity assay

[0131] Objective: To evaluate the effect of caviar polypeptide on NK cell activity.

[0132] Experimental Methods: Fifty BALB / c mice were randomly divided into groups of 10 each, with an initial body weight ranging from 18.7 to 22.0 g. Target cells, YAC-1 (mouse lymphoma cell line), were cultured in a cell culture laboratory, passaged at least three times before the experiment, and adjusted to a concentration using RPMI 1640 complete culture medium. per mL.

[0133] Spleens were aseptically harvested the day after the last administration, and spleen cell suspensions were prepared as effector cells. The spleen cell suspensions were prepared according to the method described in Example 3, Experiment 1, and the concentration was adjusted to [specific concentration missing]. per mL.

[0134] NK cell activity was determined using the LDH release assay: 100 μL each of target cells and effector cells (effector-to-target ratio 50:1) were added to a U-shaped 96-well culture plate; three parallel wells were set up for each assay: one for spontaneous release of target cells (100 μL each of target cells and culture medium) and one for maximum release of target cells (100 μL each of target cells and 2.5% Triton X-100). The plate was maintained at 37℃ and 5%... After culturing for 4 hours, centrifuge at 1500 r / min for 5 minutes, and transfer 100 μL of supernatant from each well to a flat-bottomed 96-well plate. Add 100 μL of LDH matrix solution and react for 6 minutes. Finally, add 30 μL of 1 mol / L HCl to each well to terminate the reaction. Measure the OD value at 490 nm using a microplate reader.

[0135] NK cell activity (%) = (OD released from experimental wells - spontaneously released OD from effector cells - spontaneously released OD from target cells) / (maximum OD released from target cells - spontaneously released OD from target cells) × 100%

[0136] Results: Compared with the negative control group (NK cell activity 36.0±10.2%), the NK cell activity was significantly increased in the positive control group (51.9±5.4%, P<0.05) and the high-dose group (53.1±13.3%, P<0.01). The medium-dose group (47.3±4.4%) and the low-dose group (45.1±5.3%) showed an increasing trend compared with the negative control group, but the differences were not statistically significant (P>0.05). Animals in all groups experienced normal weight gain, with no statistically significant differences.

[0137] The activity of NK cells in the high-dose group was increased by 47.5% compared with the negative control group, indicating that the peptide of the present invention can significantly enhance the natural killing activity of NK cells.

[0138] Based on the results of the above 7 immune function tests:

[0139] Cellular immune function assay: The high-dose group of the ConA-induced spleen lymphocyte transformation assay was positive (P<0.01); the low, medium, and high-dose groups of the delayed-type hypersensitivity assay were all positive (P<0.05 or P<0.01). Since both assays were positive, the cellular immune function assay was deemed positive.

[0140] Humoral immune function assay: Serum hemolysin levels were positive in the low- and medium-dose groups (P<0.01), and IgM was positive in the high-dose group (P<0.01). Positive results in both dose groups were considered a positive result for humoral immune function assay.

[0141] Monocyte-macrophage function assay: In the mouse peritoneal macrophage phagocytosis test of chicken red blood cells, the phagocytic percentage and phagocytic index were positive in the low, medium, and high dose groups (P<0.01). The positive results in both dose groups indicated positive results for monocyte-macrophage function.

[0142] NK cell activity assay: NK cell activity was positive in the high-dose group (P<0.01). A positive result in one dose group was considered a positive result for NK cell activity.

[0143] According to the "Technical Specifications for Inspection and Evaluation of Health Foods" (2003 edition) and the "Evaluation Methods for Functions of Health Foods (2020 edition)" criteria, a positive result in any two of the four aspects—cellular immune function, humoral immune function, mononuclear-macrophage function, and NK cell activity—is sufficient to determine that the substance has the function of enhancing immunity. In this invention, the caviar polypeptide composition showed positive results in all four aspects, thus confirming that the test substance has the function of enhancing immunity.

[0144] Safety evaluation: Compared with the negative control group, no abnormalities were observed in the body size, coat, skin, muscle tone, gait, mental state, or respiration of animals in each dose group during the administration of the test substance. Weight gain was normal, and no animal deaths were observed. Gross necropsy was performed after the experiment, and no abnormalities were found in any organs. No oral toxicity was observed under the dosage conditions of this experiment.

[0145] Example 4: Study on the immunomodulatory mechanism of fish roe polypeptides

[0146] To elucidate the immunomodulatory mechanism of the caviar polypeptide of this invention, in vitro cell experiments and signal pathway analysis were conducted.

[0147] Cell culture and toxicity assays: RAW264.7 mouse macrophages were purchased from the Cell Bank of the Chinese Academy of Sciences and cultured at 37°C in DMEM medium containing 10% fetal bovine serum (FBS), 100 U / mL penicillin, and 100 μg / mL streptomycin. Cells were cultured under specific conditions. The toxicity of the peptide to RAW264.7 cells was detected using the CCK-8 assay. Cells were seeded in 96-well plates and cultured for 24 hours. Different concentrations of peptides (1, 5, 10, 25, 50, and 100 μg / mL) were added and treated for another 24 hours. Then, 10 μL of CCK-8 solution was added to each well and the cells were cultured for another 2 hours. The absorbance was measured at 450 nm using a microplate reader. The results showed that within the concentration range of 1 to 100 μg / mL, SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 had no significant toxic effect on RAW264.7 cells, and the cell viability was above 90%. >100 μg / mL.

[0148] Macrophage activation assay: RAW264.7 cells were activated using... Cells were seeded in 6-well plates and cultured for 24 hours. Then, either a peptide (final concentration 25 μg / mL) or lipopolysaccharide (LPS, positive control, final concentration 1 μg / mL) was added and the cells were treated for another 24 hours. Cell supernatants were collected, and the secretion levels of cytokines IL-6, IL-1β, and TNF-α were measured using an ELISA kit (eBioscience).

[0149] The results showed that, compared with the blank control group, the IL-6 secretion level in the SEQ ID NO:1 treatment group increased significantly from 15.2±2.8 pg / mL to 186.5±24.3 pg / mL (P<0.01); IL-1β increased from 8.6±1.5 pg / mL to 52.3±8.2 pg / mL (P<0.01); and TNF-α increased from 12.5±3.2 pg / mL to 245.8±32.6 pg / mL (P<0.01). In the SEQ ID NO:2 treatment group, IL-6 increased to 158.2±20.6 pg / mL, IL-1β increased to 46.8±7.5 pg / mL, and TNF-α increased to 212.3±28.4 pg / mL. In the SEQ ID NO:3 treatment group, IL-6 increased to 142.6±18.9 pg / mL, IL-1β increased to 41.2±6.8 pg / mL, and TNF-α increased to 195.6±25.2 pg / mL. The pro-inflammatory cytokine induction activities of the three peptides were approximately 60% to 75% of those in the LPS-positive control.

[0150] TLR receptor binding verification: The binding of the peptide of this invention to the TLR receptor was verified using a TLR2 and TLR4 receptor inhibition strategy. RAW264.7 cells were pretreated with anti-TLR2 neutralizing antibody (Clone C9A12, 10 μg / mL) or anti-TLR4 neutralizing antibody (Clone MTS510, 10 μg / mL) for 1 hour, followed by treatment with SEQ ID NO:1 (25 μg / mL) for 24 hours. TNF-α secretion was then detected.

[0151] The results showed that pretreatment with anti-TLR2 antibody reduced SEQ ID NO:1-induced TNF-α secretion from 245.8±32.6 pg / mL to 135.2±18.6 pg / mL (a decrease of approximately 45%); pretreatment with anti-TLR4 antibody reduced TNF-α secretion to 118.0±15.8 pg / mL (a decrease of approximately 52%); and combined use of the two antibodies reduced TNF-α secretion to 53.9±8.5 pg / mL (a decrease of approximately 78%), close to the level of the blank control. These results indicate that the peptide of this invention mainly exerts its immune-activating effect through TLR2 and TLR4 receptors, with both receptors contributing and exhibiting a certain degree of redundancy.

[0152] NF-κB and MAPK signaling pathway analysis: such as Figure 5 and Figure 7As shown, the caviar polypeptide of this invention activates the downstream NF-κB and MAPK signaling pathways via the TLR2 / TLR4 receptor. Western blotting was used to detect the activation of the NF-κB and MAPK signaling pathways after polypeptide treatment. RAW264.7 cells were treated with SEQ ID NO:1 (25 μg / mL) for different times (0, 15, 30, 60, 120 min), and whole-cell proteins were extracted. After separation by SDS-PAGE, the proteins were transferred to a PVDF membrane and incubated sequentially with primary antibodies (p-IκBα, IκBα, p-p65, p65, p-ERK1 / 2, ERK1 / 2, p-JNK, JNK, p-p38, p38, β-actin, all purchased from Cell Signaling Technology) and HRP-labeled secondary antibodies, followed by ECL chemiluminescence imaging.

[0153] The results showed that after treatment with SEQ ID NO:1, p-IκBα began to increase at 30 minutes, reached a peak at 60 minutes (approximately 4.2 times that at time 0), and decreased at 120 minutes; total IκBα was significantly degraded at 60 minutes. p-p65 began to increase at 30 minutes and reached a peak at 60 minutes (approximately 3.8 times), indicating that the NF-κB signaling pathway was activated. Simultaneously, p-ERK1 / 2 began to increase at 15 minutes and reached a peak at 30 minutes (approximately 3.5 times); p-JNK reached a peak at 30 minutes (approximately 2.8 times); and p-p38 reached a peak at 15 minutes (approximately 3.2 times), indicating that all three branches of the MAPK signaling pathway (ERK, JNK, and p38) were activated.

[0154] Validation of signaling pathway inhibitors: RAW264.7 cells were pretreated for 1 hour with either the NF-κB inhibitor BAY 11-7082 (10 μM) or the MEK1 / 2 inhibitor U0126 (10 μM), followed by treatment with SEQ ID NO:1 (25 μg / mL) for 24 hours. Cytokine secretion was then measured. Pretreatment with BAY 11-7082 reduced TNF-α secretion by approximately 65% ​​and IL-6 by approximately 58%; pretreatment with U0126 reduced TNF-α by approximately 42% and IL-6 by approximately 38%. The combined use of the two inhibitors reduced cytokine secretion by approximately 82%. These results further confirm that the NF-κB and MAPK signaling pathways play a crucial role in the peptide-induced macrophage activation of this invention.

[0155] Example 5: Stability Study of Fish Roe Polypeptides

[0156] Thermal stability: After the peptide solution (1 mg / mL aqueous solution) was placed at 25℃, 37℃, 60℃, and 80℃ for 1 hour, the peptide content was determined by RP-HPLC. The results showed that the content of SEQ ID NO:1 did not change significantly after treatment at 25℃ and 37℃ (>99%); the content remained at 96.2% after treatment at 60℃; and the content remained at 91.5% after treatment at 80℃. SEQ ID NO:2 and SEQ ID NO:3 showed similar thermal stability. This indicates that the peptides of the present invention have good thermal stability within the conventional food processing temperature range.

[0157] pH stability: The peptide solution was adjusted to pH 2.0 (0.01 mol / L HCl), pH 4.0 (0.01 mol / L acetate buffer), pH 7.0 (0.01 mol / L phosphate buffer), and pH 9.0 (0.01 mol / L borate buffer), respectively, and the peptide content was measured after incubation at room temperature for 2 hours. The results showed that the content of SEQ ID NO:1 remained largely unchanged (>98%) within the pH range of 4.0 to 9.0; the content decreased by approximately 15% at pH 2.0 (residual 85.2%), possibly due to partial oxidation of the tryptophan side chain under strongly acidic conditions. This indicates that the peptide of the present invention exhibits good stability under neutral to weakly alkaline conditions and is suitable for oral administration.

[0158] Protease resistance: The polypeptide solution (1 mg / mL) was incubated with pepsin (100 U / mL, pH 2.0, 37℃) or trypsin (100 U / mL, pH 7.5, 37℃) for 2 hours, followed by enzyme inactivation by heating. The residual polypeptide content was then determined. The results showed that the residual content of SEQ ID NO:1 after pepsin treatment was 72.5%, and the residual content after trypsin treatment was 68.3%; the residual contents of SEQ ID NO:2 were 75.2% and 71.6%, respectively; and the residual contents of SEQ ID NO:3 were 78.8% and 74.5%, respectively. The presence of proline in the polypeptide sequence effectively slowed down the hydrolysis rate of the protease. This indicates that the polypeptide of the present invention has a certain resistance to enzymatic degradation, which is beneficial for maintaining some activity in the gastrointestinal tract after oral administration.

[0159] Storage stability: The lyophilized peptide powder was stored at -20℃, 4℃, and 25℃ (protected from light), and samples were taken at 0, 1, 3, and 6 months to determine its content and purity. The results showed that the content remained >98% after 6 months of storage at -20℃; >95% after 6 months of storage at 4℃; and >90% after 6 months of storage at 25℃. This indicates that the lyophilized peptide powder of the present invention has good storage stability under low-temperature conditions.

[0160] Example 6 Acute toxicity test

[0161] Experimental animals: SPF-grade ICR mice, half male and half female, weighing 18 to 22 g, were randomly divided into a polypeptide group and a control group, with 20 mice in each group (10 males and 10 females).

[0162] Administration method: The peptide group was given the caviar peptide composition by gavage at the maximum administration volume (0.4 mL / 10 g body weight) in a single dose of 2000 mg / kg body weight (approximately 60,000 times the human dose); the control group was given an equal volume of distilled water.

[0163] Observation indicators: Observe the animals continuously for 14 days after administration, and record the general condition (mental state, activity, food intake, fur, feces, etc.), weight changes, and mortality daily. After the observation period, sacrifice the animals and perform gross necropsy to observe the major organs (heart, liver, spleen, lungs, kidneys, stomach, intestines).

[0164] Experimental Results: Within 0.5 to 2 hours after drug administration, some animals in the polypeptide group experienced transient decreased activity, which returned to normal after 2 hours. Within 14 days after drug administration, no animals in either the polypeptide group or the control group died; their general condition was good, with normal feeding and activity, glossy fur, and normal feces. Weight gain trends were similar to the control group, with no statistically significant difference (P>0.05). Gross anatomical examination revealed no visible abnormalities in major organs.

[0165] Experimental conclusion: According to the acute toxicity classification standard ("Technical Specification for Identification of Chemical Toxicity" GB / T 21751), the caviar polypeptide composition of this invention, after oral administration... >2000 mg / kg, which is practically non-toxic and has good safety.

[0166] Example 7 Preparation of immune-enhancing composition

[0167] Oral liquid preparation: Weigh 100 mg of the caviar polypeptide composition of this invention (SEQ ID NO:1:SEQ ID NO:2:SEQ ID NO:3 = 1:1:1), dissolve in 80 mL of distilled water, add 2 mL of glycerol (sweetener and humectant) and 0.1 g of citric acid (pH adjuster), and bring the volume to 100 mL with distilled water. Stir well, filter through a 0.22 μm filter membrane for sterilization, dispense into oral liquid bottles (10 mL / bottle), and sterilize and seal. The finished polypeptide concentration is 1 mg / mL. Adults take 10 to 20 mL daily, i.e., a daily intake of 10 to 20 mg of polypeptide.

[0168] Tablet formulation: Weigh 200 mg of the caviar polypeptide composition of this invention, mix it evenly with 300 mg of microcrystalline cellulose, 100 mg of lactose, and 5 mg of magnesium stearate, granulate by dry granulation, and then compress into tablets. Each tablet weighs approximately 605 mg and contains 200 mg of polypeptide. Adults take one tablet daily.

[0169] Capsule preparation: Weigh 150 mg of the caviar polypeptide composition of this invention, mix it evenly with 200 mg of starch and 50 mg of dextrin, and fill it into No. 0 plant capsules. Each capsule contains 150 mg of polypeptide. Adults take 1 to 2 capsules daily.

[0170] The caviar polypeptide composition of the present invention exerts a systemic immune-enhancing effect through the following synergistic mechanism:

[0171] At the molecular recognition level, the aromatic amino acid residues (tryptophan Trp, phenylalanine Phe) of the polypeptide of this invention can form π-π stacking and hydrophobic interactions with the hydrophobic pockets of the TLR2 and TLR4 receptor ligand binding domains, while the positively charged amino acid residues (arginine Arg, lysine Lys) can form electrostatic interactions and salt bridges with the acidic amino acid residues (aspartic acid Asp, glutamate Glu) on the receptor surface, thereby achieving efficient binding to immune receptors. The polypeptide of this invention contains 33% to 42% aromatic amino acids and 17% to 25% positively charged amino acids, which is significantly higher than that of the prior art AAPAPAAAPPAE (0% aromatic amino acids, 0% positively charged amino acids), thus significantly improving receptor binding capacity and immune activation efficiency.

[0172] At the signal transduction level, such as Figure 7 As shown, after the peptide binds to the TLR2 / TLR4 receptor, it activates the downstream signaling cascade via the MyD88-dependent pathway. MyD88 recruits IRAK1 / 4 kinases, which in turn activate the TRAF6 and TAK1 complex. TAK1 activates the IKK complex (IKKα / IKKβ / NEMO), leading to phosphorylation and ubiquitination degradation of the repressive protein IκBα, releasing NF-κB dimers (p65 / p50) to enter the nucleus and regulate target gene transcription. On the other hand, TAK1 activates three branches of the MAPK pathway—ERK1 / 2, JNK, and p38—to synergistically regulate the activity of the transcription factor AP-1 (c-Jun / c-Fos). NF-κB and AP-1 jointly induce the expression of pro-inflammatory cytokines (IL-6, IL-1β, TNF-α), chemokines, and co-stimulatory molecules.

[0173] At the cellular effector level, secreted cytokines act on the immune cell network, forming a positive feedback cascade amplification effect. IL-6 promotes the differentiation of B lymphocytes into plasma cells and the production of immunoglobulins (IgM, IgG, IgA); TNF-α enhances the antigen-presenting function of macrophages and dendritic cells and promotes the differentiation of T helper cells (Th1); IL-1β, in conjunction with other cytokines, enhances the inflammatory response and immune response. This systemic immune activation effect is reflected in the improvement of multiple indicators observed in the animal experiments of this invention: the splenic lymphocyte proliferation capacity increased by 67.6% (high-dose group), reflecting enhanced T cell function; the ear swelling degree of delayed-type hypersensitivity increased by 40% to 45% (all dose groups), further confirming the enhanced cellular immune function; the antibody volume increased by 33.2% (low-dose group) and IgM increased by 78.9% (high-dose group), indicating enhanced humoral immune function; the phagocytic percentage of macrophages increased by 62.6% (low-dose group) and the phagocytic index increased by 86.3% (high-dose group), reflecting enhanced non-specific immune function; and the NK cell activity increased by 47.5% (high-dose group), indicating enhanced natural killer function.

[0174] In terms of safety, the polypeptides of this invention are derived from the enzymatic hydrolysis products of edible fish protein, and are short peptides composed of natural amino acids. They can be normally metabolized into amino acids in the body without accumulation. The sequence does not contain immunogenic non-natural amino acids or D-amino acids, and will not trigger an immune response against the polypeptide itself. Acute toxicity experiments have confirmed this. >2000 mg / kg (practically non-toxic grade), and no toxic reactions were observed in the 30-day repeated dosing experiment, laying a solid foundation for the development of safe and effective immune-enhancing health foods and drugs.

[0175] The above description is merely a preferred embodiment of the present invention. Any changes and modifications made in accordance with the claims of the present invention shall fall within the protection scope of the present invention. The protection scope of the present invention is not limited to the specific polypeptide sequences, preparation parameters, and application forms disclosed in the above embodiments. Any equivalent substitutions or transformations made based on the technical solution of the present invention are within the protection scope of the present invention.

Claims

1. A caviar polypeptide composition with enhanced immune function, characterized in that, The composition contains the following three polypeptides: The first polypeptide has the amino acid sequence shown in SEQ ID NO:1: RWFPGKPLFWRA; The second polypeptide has the amino acid sequence shown in SEQ ID NO:2: FWKPLRGALFPW; The third polypeptide has the amino acid sequence shown in SEQ ID NO:3: KWRFPGALPWFA; The mass ratio of the first polypeptide, the second polypeptide, and the third polypeptide is 1:1:1; each polypeptide consists of 12 amino acid residues and carries a positive charge under physiological pH conditions.

2. The caviar polypeptide composition according to claim 1, characterized in that, The first polypeptide has a molecular weight of 1531.8 Da, an isoelectric point of 11.71, and a net charge of +3; the second polypeptide has a molecular weight of 1487.8 Da, an isoelectric point of 11.00, and a net charge of +2; and the third polypeptide has a molecular weight of 1432.7 Da, an isoelectric point of 10.84, and a net charge of +2.

3. A method for preparing the caviar polypeptide composition according to claim 1, characterized in that, Three polypeptides, SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, were synthesized using a solid-phase polypeptide synthesis method and then mixed in a mass ratio of 1:1:

1.

4. The use of the caviar polypeptide composition according to claim 1 in the preparation of health food products that help enhance immunity.

5. An immune-enhancing agent, characterized in that, The formulation comprises the caviar polypeptide composition of claim 1 as the active ingredient, and is formulated with pharmaceutically or food-grade acceptable excipients; the formulation is an oral liquid, tablet or capsule.

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