A cervus nippon meat-derived active peptide and application thereof

Abstract

The application discloses a sika deer meat-derived active peptide and application, and belongs to the technical field of bioactive peptides. The amino acid sequence of the active peptide is AAVI; the application further provides a prodrug type coupling compound alpha-D-Man-PEG8-cisAconitic-GFLG-AAVI based on AAVI. The coupling compound contains an alpha-D-mannosyl targeting unit, can specifically recognize and combine with a CD206 receptor on a macrophage surface, realizes active targeted delivery, a cis-aconitic acid sensitive linker is broken in an acid environment to realize pH response release, and a GFLG enzyme sensitive linker is specifically cut by an intracellular cathepsin B to realize enzymatic drug release. The AAVI active peptide and the coupling compound can effectively enhance the immune function of an immunocompromised mouse, significantly increase a thymus index and a spleen index, up-regulate serum IgG, IgA, IgM and cytokine TNF-alpha, IL-1beta and IL-2 levels, and regulate a TLR-4 / MyD88 / NF-kappa B signal path, and has important application value in the preparation of an immunoregulation health care food or medicine.

Description

Technical Field

[0001] This invention belongs to the field of bioactive peptide technology, specifically relating to an active peptide derived from sika deer meat and its applications. Background Technology

[0002] Sika deer (Cervus nippon) is an important medicinal animal resource in my country. Deer meat, a high-protein, low-fat, and low-cholesterol meat, is rich in essential amino acids and various trace elements, possessing extremely high nutritional and medicinal value. The protein content of sika deer meat is approximately 20-22%, with essential amino acids accounting for over 40% of its amino acid composition, making it exceptionally nutritious. Currently, research on sika deer meat mainly focuses on traditional medicinal parts such as antlers and blood, while research on the in-depth development and comprehensive utilization of sika deer meat protein resources is relatively limited. Studies have shown that enzymatic hydrolysis of sika deer meat protein can release bioactive polypeptide fragments, which exhibit potential application value in immunomodulation, antioxidation, and blood pressure reduction. However, systematic research on immunomodulatory peptides derived from sika deer meat is still lacking, particularly in the isolation and identification of single active peptides, their immunomodulatory mechanisms, and pre-drug chemical modification research.

[0003] Immunomodulatory peptides (IMPs) are a class of bioactive peptides that regulate the body's immune function. They activate or inhibit immune signaling pathways by binding to specific receptors on the surface of immune cells, thereby regulating the proliferation, differentiation, and function of immune cells. Studies have shown that the mechanisms of action of IMPs mainly include: activating Toll-like receptor (TLR) signaling pathways and promoting cytokine secretion; regulating B cell proliferation and antibody production; enhancing natural killer (NK) cell activity; and promoting macrophage phagocytosis and antigen presentation. At the molecular level, IMPs primarily exert their effects by regulating the TLR-4 / MyD88 / NF-κB signaling pathway, a core signal transduction pathway of the innate immune response closely related to inflammatory responses and immune cell activation. In recent years, the application of targeted delivery systems in IMPs has become a research hotspot. By conjugating IMPs with targeted ligands, nanocarriers, or acid-sensitive / enzyme-sensitive linkers, precise delivery and controlled release of IMPs can be achieved, improving their bioavailability and therapeutic efficacy.

[0004] Although bioactive peptides have shown promising application prospects in the field of immunomodulation, existing technologies still have the following shortcomings: (1) There is a lack of systematic research on single bioactive peptides derived from sika deer meat. Existing studies are mostly focused on the immunomodulatory effects of mixtures of deer meat peptides, and the isolation and identification of specific bioactive peptide sequences are not in-depth enough; (2) The direct application of natural bioactive peptides has problems such as low bioavailability, short half-life, and easy degradation by proteases, which limit their clinical application; (3) There is a lack of active targeted delivery strategies for immune cells, resulting in disordered drug distribution and poor targeting; (4) There is a lack of prodrug design with both pH response and enzyme sensitivity, making it difficult to achieve precise release of immunomodulatory peptides at the lesion site. Therefore, developing immunomodulatory bioactive peptides derived from sika deer meat with clear amino acid sequences, and combining them with prodrug modification and active targeting technology to construct an intelligent responsive delivery system, is of great significance for improving the development and utilization value of sika deer meat resources and expanding the application fields of immunomodulatory peptides. Summary of the Invention

[0005] Technical problems to be solved: To address the above-mentioned problems, the purpose of this invention is to provide an active peptide derived from sika deer meat and its application, solving the problems of unclear sequences and unclear immune regulation mechanisms of active peptides derived from sika deer meat in the prior art; at the same time, it provides a prodrug conjugate based on AAVI and its preparation method, by introducing macrophage targeting units, acid-sensitive linkers and enzyme-sensitive linkers, to construct an intelligent delivery system with active targeting and dual stimulation response functions, solving the technical problems of low bioavailability, poor targeting and lack of intelligent drug release function of immunomodulatory peptides.

[0006] Technical solution: A bioactive peptide derived from sika deer meat, characterized in that the amino acid sequence of the bioactive peptide derived from sika deer meat is AAVI (Ala-Ala-Val-Ile).

[0007] This invention also provides the application of the above-mentioned active peptides derived from sika deer meat in the preparation of immunomodulatory products.

[0008] Furthermore, the immune-modulating product is a health food that helps enhance immunity or a medicine with immune-modulating effects.

[0009] This invention also provides a sika deer meat-derived active peptide conjugate compound with the general structural formula: XT-L3-L2-P, wherein: P represents the activity of the above amino acid sequence AAVI, with its N-terminus being a free amino group; L2 is an enzyme-cleavable linker that can be specifically cleaved by intracellular proteases; L3 is an acid-sensitive linker that breaks down under acidic pH conditions via an intramolecular cyclization mechanism; T stands for macrophage targeting unit, which contains ligands that target receptors on the surface of macrophages; X is an optional vector or reporter molecule.

[0010] Furthermore, the enzyme can cleave the linker as a substrate peptide sequence of cathepsin B.

[0011] Furthermore, the substrate peptide sequence of the cathepsin B is Gly-Phe-Leu-Gly (GFLG).

[0012] Furthermore, the acid-sensitive linker is a cis-aconitine diamine linker, which breaks the amide bond through intramolecular cyclization of the free carboxyl group under acidic conditions of pH ≤ 5.5.

[0013] Furthermore, the macrophage targeting unit is α-D-mannose, which is connected to L3 via a PEG spacer arm and targets the CD206 mannose receptor on the surface of macrophages.

[0014] Furthermore, X is a long chain of polyethylene glycol, liposomes, nanoparticles, or a fluorescent reporter group; or X is absent.

[0015] Furthermore, the structure of the active peptide conjugate derived from sika deer meat is: α-D-Man-PEG8-cisAconitic-GFLG-AAVI, namely: α-D-mannopyranosyl-octaethylene glycol spacer arm-cis-aconitine linker-GFLG-AAVI.

[0016] This invention also provides a method for preparing the above-mentioned active peptide-coupled compound derived from sika deer meat. The method involves preparing a peptide containing L2-P using a solid-phase peptide synthesis method or a liquid-phase peptide synthesis method, then linking L3 via a cis-aconitine ring-opening reaction, and finally functionalizing the spacer arm containing the macrophage targeting unit T with an amino group via a disulfide bond exchange reaction. The resulting compound is then coupled to the L3 terminal carboxyl group via an amide condensation reaction and purified to obtain the target compound.

[0017] This invention also provides the application of the above-mentioned active peptides derived from sika deer meat in the preparation of immunomodulatory products, wherein the regulation of the body's immune function includes enhancing the immune function of individuals with low immunity, or regulating the TLR-4 / MyD88 / NF-κB signaling pathway.

[0018] A pharmaceutical composition comprising a therapeutically effective amount of the above-mentioned sika deer meat-derived active peptide or the above-mentioned sika deer meat-derived active peptide conjugate, and a pharmaceutically acceptable carrier or excipient. Beneficial effects

[0019] 1. This invention is the first to identify and isolate a tetrapeptide AAVI (Ala-Ala-Val-Ile) with a defined amino acid sequence from the enzymatic hydrolysate of sika deer meat. This peptide has significant immunomodulatory activity and can effectively enhance the immune function of immunocompromised mice, providing a new direction for the in-depth development and utilization of sika deer meat resources.

[0020] 2. The α-D-Man-PEG8-cisAconitic-GFLG-AAVI conjugate constructed in this invention achieves active targeted delivery to macrophages. α-D-mannose, as a targeting ligand, can specifically recognize and bind to the CD206 receptor, thereby increasing the accumulation of the drug in macrophages and reducing systemic toxicity.

[0021] 3. The cis-aconitine diamine-sensitive linker in the coupling compound of the present invention can achieve pH-responsive cleavage in response to the slightly acidic environment of tumor / inflammatory environment (pH 5.5≤), and the GFLG enzyme-sensitive linker can be specifically cleaved by cathepsin B in macrophage lysosomes to achieve enzymatic drug release, thus forming a dual stimulus-response drug release system and improving the precise release of drugs at the target site.

[0022] 4. The AAVI active peptide and conjugated compounds of the present invention can exert immunomodulatory effects by regulating the TLR-4 / MyD88 / NF-κB signaling pathway, significantly increasing the thymus index and spleen index of immunocompromised mice, and upregulating the serum levels of IgG, IgA, IgM and cytokines TNF-α, IL-1β and IL-2, with the effects being dose-dependent.

[0023] 5. The AAVI active peptide of the present invention has a small molecular weight (approximately 372 Da), a simple structure, and is easy to synthesize and modify. Furthermore, the introduction of the PEG spacer arm can improve the water solubility and stability of the compound and prolong its circulation time in vivo, thus showing good prospects for drug development. Attached Figure Description

[0024] Figure 1 The total ion chromatogram of sika deer meat polypeptides; Figure 2 The effect of AAVI on the body weight of immunocompromised mice. Detailed Implementation

[0025] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. In the present invention, unless otherwise specified, the equipment and raw materials used can be purchased from the market or are commonly used in the art. Unless otherwise specified, the methods in the following embodiments are conventional methods in the art.

[0026] The alkaline protease activity is 400,000 U / g.

[0027] Example 1 A method for preparing sika deer meat polypeptide includes the following steps: (1) Take sika deer meat, thaw and thoroughly clean it, remove excess fat, cut the meat into evenly sized pieces, and use a high-speed hand-held homogenizer to grind it into a homogenate; (2) Adjust the pH, add 5% alkaline protease, put it into a microwave-ultrasound combined synthesis / extraction instrument, adjust the microwave power to 200W, the ultrasonic power to 650W, the reaction temperature to 50℃, and react for 30min to obtain the enzymatic hydrolysate; (3) Boil for 5 minutes to inactivate the enzyme, cool naturally to room temperature, centrifuge at 10000 r / min for 15 minutes, then collect the supernatant and obtain a clear enzyme hydrolysate by filtration; (4) The clarified enzymatic hydrolysate was subjected to vacuum freeze-drying to obtain sika deer meat polypeptide powder.

[0028] The yield of polypeptides from sika deer meat was measured to be 3.8448 mg / mL.

[0029] Example 2: Structural Identification of Sika Deer Meat Polypeptides 1. The peptide sequences of sika deer meat peptides were identified after separation and desalting by ultrafiltration and gel chromatography. Figure 1 The total ion chromatogram of sika deer meat polypeptides is shown.

[0030] 2. Molecular docking: Toll-like receptor 4 (TLR-4) is a key pattern recognition receptor in the innate immune system, capable of recognizing pathogen-associated molecular patterns and initiating downstream signaling cascades. Upon activation, TLR-4 recruits the adaptor protein MyD88 via a MyD88-dependent pathway, thereby activating the IRAK4 / IRAK1 / TRAF6 signaling complex, ultimately activating the NF-κB transcription factor and promoting the expression of various immune-related cytokines such as TNF-α, IL-1β, and IL-6, playing a central role in the body's immune defense and regulation. Therefore, this invention uses TLR-4 as a molecular docking target for screening immunomodulatory activity.

[0031] Based on the peptides in the peptide profiling data of Example 2, according to their relative abundance (≥10), 7 Peptides were screened based on criteria such as peptide score (PeptideRanker, ≥0.7), peptide amino acid sequence length ≤10, and good water solubility. These peptides were then used for modeling and hydrogenation treatment using AutoDockVina. Molecular docking was then performed with the TLR-4 receptor protein (PDB ID: 3FXI). Peptides with a docking score below -7.5 kcal / mol with TLR-4 were considered as potential immunomodulatory peptides (Table 1) and used for experiments.

[0032] Table 1. Peptides with potential immunomodulatory effects after docking with TLR-4 AAVI 0.892 -8.73 373.47 1.0 <![CDATA[2.75×10 6 ]]> Ala-Ala-Val-Ile AGLM 0.756 -7.86 391.51 1.0 <![CDATA[1.42×10 6 ]]> Ala-Gly-Leu-Met 3. We commissioned Polypeptide Bioengineering (Shanghai) Co., Ltd. to obtain Ala-Ala-Val-Ile and Ala-Gly-Leu-Met using solid-phase synthesis for evaluation of their immunomodulatory effects.

[0033] Example 3: Immunomodulatory Effects of Peptide AAVI 1. Laboratory animals and grouping Forty male ICR mice, 2-3 weeks old and weighing (20.0±1.0) g, were selected. After 3 days of acclimatization, the mice were allowed free access to food and water. The ambient temperature was (22±2) ℃, the relative humidity was 50-60%, and the light and dark were alternated for 12 hours. The mice were randomly divided into 5 groups of 8 mice each: (1) Control group: saline was injected into the tail vein; (2) Model group: saline was injected into the tail vein and cyclophosphamide was used to establish the model; (3) Low-dose AAVI group (AAVI-L): AAVI 10 mg / kg was injected into the tail vein and cyclophosphamide was used to establish the model; (4) Medium-dose AAVI group (AAVI-M): AAVI 20 mg / kg was injected into the tail vein and cyclophosphamide was used to establish the model; (5) High-dose AAVI group (AAVI-H): AAVI 40 mg / kg was injected into the tail vein and cyclophosphamide was used to establish the model.

[0034] 2. Animal handling methods Establishment of an immunosuppressed mouse model: Except for the control group, all other groups of mice underwent immunosuppression by intraperitoneal injection of cyclophosphamide (100 mg / kg) for 7 consecutive days. The control group received an equal volume of physiological saline. After modeling, each AAVI dose group was given a tail vein injection of the corresponding concentration of AAVI solution (dissolved in physiological saline), while the control and model groups received an equal volume of physiological saline, administered every other day for 24 consecutive days. Changes in mouse weight and food intake were recorded every 3 days during this period. After the experiment, mice were fasted for 12 hours, euthanized by cervical dislocation after blood collection from the orbital fossa, and the thymus and spleen were quickly removed. The surface bloodstains were rinsed with pre-cooled physiological saline, blotted dry with filter paper, and weighed. Blood samples were allowed to stand at 4°C for 2 hours, then centrifuged at 3000 rpm for 15 minutes. The supernatant serum was collected and stored at -80°C for later use. A portion of liver tissue was fixed in 4% paraformaldehyde for histopathological analysis, while the remainder was flash-frozen in liquid nitrogen and stored at -80°C for Western blot and RT-qPCR detection.

[0035] 3. Changes in mouse body weight The results are as follows Figure 2As shown, in the early stages of the experiment, the body weight of mice in the control group showed a continuous increasing trend with prolonged feeding time. The body weight of mice in the model group, however, showed a trend of first increasing and then decreasing. In the low, medium, and high dose groups, the body weight of mice all showed a trend of first increasing, then decreasing, and then gradually recovering, with the recovery effect becoming more pronounced with increasing dose. Behavioral and physiological observations showed that mice in the control group had smooth, soft fur, moved quickly, had strong limb movements, normal feeding behavior, and good mental condition; while mice in the model group had dull fur, were lethargic, had reduced spontaneous activity, and significantly decreased appetite. In contrast, the overall condition of mice in all three dose groups was better than that of the model group, with increased activity and a slight recovery in food intake, indicating that AAVI intervention can improve cyclophosphamide-induced immunosuppression and promote the recovery of physiological functions in mice.

[0036] 4. Immune organ index measurement The formulas for calculating the thymus index and spleen index are: thymus index (mg / g) = thymus weight (mg) / body weight (g); spleen index (mg / g) = spleen weight (mg) / body weight (g).

[0037] Table 2 Blank group 1.303±0.048 2.931±0.093 Model group 0.580±0.040 2.051±0.117 AAVI-L Group 0.706±0.061 2.296±0.073 AAVI-M Group 0.813±0.046 2.601±0.081 AAVI-H group 1.135±0.057 2.996±0.047 As shown in Table 2 above, compared with the control group, the thymus index and spleen index of mice in the model group were significantly decreased (P<0.01), indicating that the cyclophosphamide-induced immunosuppression model was successfully established. Compared with the model group, the thymus index and spleen index of mice in each AAVI dose group were significantly increased (P<0.05 or P<0.01), and this increase was dose-dependent. The results indicate that AAVI can effectively improve the atrophy of immune organs in immunosuppressed mice and has a significant immunoprotective effect.

[0038] 5. Blood biochemical index determination Serum levels of immunoglobulins IgG, IgA, and IgM, and cytokines TNF-α, IL-1β, and IL-2 were determined using enzyme-linked immunosorbent assay (ELISA). The ELISA kits were purchased from Wuhan Boster Biological Engineering Co., Ltd., and the procedures were strictly followed according to the kit instructions. OD values ​​at 450 nm were read using a BioTek Synergy H1 microplate reader, and the concentrations of each indicator were calculated based on the standard curve.

[0039] Table 3 Blank group 10.23±0.68 3.52±0.24 2.56±0.18 156.8±10.2 98.6±7.5 58.3±4.5 Model group 5.23±0.42 1.87±0.15 1.42±0.11 86.5±7.2 52.3±4.8 28.7±2.9 AAVI-L Group 6.58±0.48 2.31±0.18 1.68±0.13 105.2±8.6 64.8±5.2 35.2±3.1 AAVI-M Group 7.42±0.52 2.62±0.19 1.92±0.14 124.6±9.1 76.5±5.8 43.8±3.6 AAVI-H group 8.76±0.58 2.94±0.21 2.18±0.16 142.3±9.8 89.6±6.7 51.4±4.2 As shown in Table 3, compared with the model group, the serum levels of IgG, IgA, and IgM in each AAVI dose group were significantly increased (P<0.05 or P<0.01), with the high-dose group showing the most significant effect. IgG increased from 5.23±0.42 mg / mL in the model group to 8.76±0.58 mg / mL, IgA increased from 1.87±0.15 mg / mL to 2.94±0.21 mg / mL, and IgM increased from 1.42±0.11 mg / mL to 2.18±0.16 mg / mL. Simultaneously, serum TNF-α, IL-1β, and IL-2 levels in each AAVI dose group increased in a dose-dependent manner. Specifically, TNF-α increased from 86.5±7.2 pg / mL in the model group to 142.3±9.8 pg / mL in the high-dose group, IL-1β increased from 52.3±4.8 pg / mL to 89.6±6.7 pg / mL, and IL-2 increased from 28.7±2.9 pg / mL to 51.4±4.2 pg / mL. These results indicate that AAVI can promote humoral immune responses and cytokine secretion in immunocompromised mice, effectively enhancing the body's immune function.

[0040] 6. RT-qPCR detection Total RNA was extracted from liver tissue using the TRIzol method and analyzed using a Thermo NanoDrop spectrophotometer. One method was used to determine RNA concentration and purity (A260 / 280 values ​​between 1.8 and 2.0 are considered acceptable). 1 μg of RNA was used to synthesize cDNA via reverse transcription according to the instructions of the reverse transcription kit (Takara, RR047A). Using the cDNA as a template, qPCR amplification was performed using a real-time PCR instrument (Bio-Rad CFX96). Primer sequences were synthesized by Sangon Biotech (Shanghai) Co., Ltd., and the specific sequences are as follows: TLR-2 upstream: 5'-GCCTCTGCCTCCTCTGAC-3', downstream: 5'-GGCTGCTTGATGATGTTGTTG-3'; TLR-4 upstream: 5'-GGCAGAAATTGCCTTTACC-3', downstream: 5'-GATACATTATCTTGCGCTTG-3'; IL-2 upstream: 5'-CCTGAGCAGGATGGAGAATTACA-3', downstream: 5'-CCAGAACATGCCGCAGAG-3'; IL-3 upstream: 5'-TTCTCCAGCGTCTTCCACTC-3', downstream: 5'-GGTCACCAGCTGCTTGAG-3'; IL-6 upstream: 5'-ACCACTTCACAAGTCGGAGGC-3'. Downstream: 5'-CTGCAAGTGCATCATCGTTGTTC-3'; GAPDH was used as an internal reference gene, upstream: 5'-AGGTCGGTGTGAACGGATTTG-3', downstream: 5'-TGTAGACCATGTAGTTGAGGTCA-3'. The relative expression levels of each gene were calculated using the 2⁻ΔΔCt method.

[0041] Table 4 Blank group 1.00±0.00 1.00±0.00 1.00±0.00 1.00±0.00 1.00±0.00 Model group 0.42±0.04 0.54±0.06 0.48±0.05 0.51±0.05 1.89±0.15 AAVI-L Group 0.62±0.05 0.76±0.07 0.68±0.06 0.72±0.06 1.42±0.12 AAVI-M Group 0.85±0.07 1.02±0.08 0.92±0.07 0.96±0.08 1.08±0.09 AAVI-H group 1.18±0.09 1.32±0.11 1.18±0.09 1.24±0.10 0.72±0.08 As shown in Table 4, compared with the model group, the expression levels of TLR-2, TLR-4, IL-2, and IL-3 mRNA were significantly increased in each AAVI dose group (P<0.05 or P<0.01), while the expression level of IL-6 mRNA was significantly decreased (P<0.05 or P<0.01), showing a dose-dependent effect. In the high-dose group, the relative expression level of TLR-4 mRNA increased from 0.54±0.06 in the model group to 1.32±0.11, IL-2 mRNA increased from 0.48±0.05 to 1.18±0.09, and IL-6 mRNA decreased from 1.89±0.15 to 0.72±0.08. This indicates that AAVI can exert an immunomodulatory effect by activating the TLR-2 / TLR-4 signaling pathway and regulating the expression of downstream inflammatory factors. It is noteworthy that IL-6, as a pro-inflammatory factor, showed a compensatory increase in expression level in the cyclophosphamide-induced immunosuppression model, which was significantly reduced after AAVI intervention, indicating that AAVI can exert an immunomodulatory effect by inhibiting excessive inflammatory response and restoring immune balance.

[0042] Comparative Example 1: Immunomodulatory Effect of Peptide AGLM To compare the differences in immunomodulatory activity of different bioactive peptides derived from sika deer meat, the same experimental method as in Example 3 was used to evaluate the immunomodulatory effect of the peptide AGLM (Ala-Gly-Leu-Met). The experimental groups were: blank group, model group, low-dose AGLM group (10 mg / kg), medium-dose AGLM group (20 mg / kg), and high-dose AGLM group (40 mg / kg).

[0043] Some of the results are shown in Table 5 below: Table 5 Blank group 1.303±0.048 2.931±0.093 10.23±0.68 156.8±10.2 58.3±4.5 Model group 0.580±0.040 2.051±0.117 5.23±0.42 86.5±7.2 28.7±2.9 AGLM-L group 0.631±0.048 2.152±0.082 6.12±0.45 96.8±7.8 32.5±2.8 AGLM-M group 0.712±0.052 2.368±0.089 6.98±0.48 108.5±8.4 38.2±3.2 AGLM-H group 0.958±0.068 2.756±0.086 7.89±0.52 125.6±9.2 43.7±3.8 The results of immune organ index analysis showed that the thymus index and spleen index also increased in all AGLM dose groups, but the increase was less than that in the AAVI group.

[0044] Blood biochemical results showed that serum IgG, IgA, IgM, TNF-α, IL-1β, and IL-2 levels were all increased in each AGLM dose group, but the increase was less than that in the AAVI group.

[0045] RT-qPCR results showed that the expression levels of TLR-2, TLR-4, IL-2, and IL-3 mRNA increased in each AGLM dose group, while the expression level of IL-6 mRNA decreased, but the magnitude of the changes was lower than that in the AAVI group.

[0046] The above results indicate that AGLM possesses certain immunomodulatory activity, but its effect is weaker than that of AAVI. AAVI, as the tetrapeptide with the strongest immunomodulatory activity among sika deer meat-derived bioactive peptides, shows better application prospects.

[0047] Example 4 The preparation method of α-D-Man-PEG8-cisAconitic-GFLG-AAVI is as follows: 1. The GFLG-AAVI peptide was obtained by solid-phase synthesis from Polypeptide Bioengineering (Shanghai) Co., Ltd.

[0048] 2. Ring-opening reaction of cis-aconitine anhydride to connect acid-sensitive linkers 100 mg of cis-aconitic anhydride was dissolved in anhydrous tetrahydrofuran (THF) and slowly added dropwise to a solution of GFLG-AAVI peptide (200 mg dissolved in anhydrous DMF) under ice bath conditions, while triethylamine (TEA) was added dropwise as an acid-binding agent. The reaction was carried out at room temperature for 4 hours. After the reaction was completed, the organic solvent was removed by vacuum distillation. The residue was extracted with ethyl acetate, the organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain cisAconitic-GFLG-AAVI.

[0049] 3. α-D-Mannose-PEG8 spacer coupling 50 mg of α-D-aminomannose was dissolved in anhydrous DMSO, and succinimide-3-pyridyl dithiopropionate (SPDP) was added at a molar ratio of 1:1.2. The reaction was carried out at room temperature for 2 h to generate an α-D-mannosyl-pyridyl dithiopropionamide derivative (α-D-Man-PDP). After the reaction was completed, the solution was concentrated under reduced pressure and purified by silica gel column chromatography to obtain α-D-Man-PDP. Simultaneously, 30 mg of amino-octaethylene glycol-amino (H2N-PEG8-NH2, purity ≥95%) was dissolved in borate buffer at pH 8.0, and Traut's reagent (2-iminothione, molar ratio 1:5) was added. The reaction was carried out at room temperature for 1 h, and purified by desalting column chromatography to obtain HS-PEG8-NH2. α-D-Man-PDP and HS-PEG8-NH2 were reacted in phosphate buffer at pH 7.4 at room temperature for 4 h. The mixture was then purified by Sephadex G-25 desalting column and lyophilized to obtain α-D-Man-SS-PEG8-NH2. α-D-Man-SS-PEG8-NH2 was dissolved in anhydrous DMF, and cisAconitic-GFLG-AAVI (molar ratio 1:1) was added. Using EDC / HOBt (molar ratio 1:1.5:1.5) as a coupling agent, the mixture was reacted in MES buffer at pH 6.0 at room temperature for 6 h. The free amino group of α-D-Man-SS-PEG8-NH2 formed an amide bond with the terminal carboxyl group of cisAconitic-GFLG-AAVI, generating α-D-Man-SS-PEG8-cisAconitic-GFLG-AAVI.

[0050] 4. Purification and Characterization The crude product was purified using semi-preparative high-performance liquid chromatography (HPLC, Agilent 1260 Infinity II) to obtain the target compound α-D-Man-PEG8-cisAconitic-GFLG-AAVI. Molecular weight characterization was performed using electrospray ionization mass spectrometry (ESI-MS, Thermo Q Exactive), with a measured value of [M+H]. + m / z 1689.82 (calculated value 1689.78), confirming it as the target compound. Structural confirmation was achieved using 1H NMR (DMSO-d6), with chemical shifts and peak shapes consistent with the target structure. HPLC analysis showed a purity ≥95%, meeting pharmaceutical standards.

[0051] Example 5: Immunomodulatory Effect of α-D-Man-PEG8-cisAconitic-GFLG-AAVI 1. Experimental Grouping and Methods To evaluate the immunomodulatory activity of the α-D-Man-PEG8-cisAconitic-GFLG-AAVI conjugate, in vivo pharmacodynamic evaluation was performed using a method similar to that in Example 3. The experiment consisted of six groups: (1) Control group; (2) Model group; (3) AAVI naked peptide group (AAVI-H, 40 mg / kg); (4) AGLM naked peptide group (AGLM-H, 40 mg / kg); (5) Low-dose conjugate group (Conj-L, 50 mg / kg); and (6) High-dose conjugate group (Conj-H, 100 mg / kg). Administration was via tail vein injection, every other day for 24 consecutive days.

[0052] 2. Results of immune organ index Table 6 Blank group 1.303±0.048 2.931±0.093 Model group 0.580±0.040 2.051±0.117 AAVI-H group 1.135±0.057 2.996±0.047 AGLM-H group 0.958±0.068 2.756±0.086 Conj-L group 1.168±0.058 2.856±0.072 Conj-H group 1.287±0.061 3.124±0.068 As shown in Table 6, the thymus index of mice in the high-dose conjugate group was 1.287±0.061 mg / g, and the spleen index was 3.124±0.068 mg / g, which were significantly higher than those in the high-dose AAVI naked peptide group (thymus index 1.135±0.057 mg / g, spleen index 2.996±0.047 mg / g) (P<0.01). The conjugate compound enhanced the distribution and accumulation of AAVI in immune organs through macrophage targeting mediated by α-D-mannosyl, thereby improving the immunomodulatory effect.

[0053] 1. Blood biochemical indicators results Table 7 Blank group 10.23±0.68 3.52±0.24 2.56±0.18 156.8±10.2 98.6±7.5 58.3±4.5 Model group 5.23±0.42 1.87±0.15 1.42±0.11 86.5±7.2 52.3±4.8 28.7±2.9 AAVI-H group 8.76±0.58 2.94±0.21 2.18±0.16 142.3±9.8 89.6±6.7 51.4±4.2 AGLM-H group 7.89±0.52 2.68±0.19 1.98±0.15 125.6±9.2 78.5±6.1 43.7±3.8 Conj-L group 9.12±0.62 3.15±0.22 2.32±0.17 152.6±10.5 95.2±7.2 55.8±4.6 Conj-H group 10.23±0.72 3.42±0.25 2.58±0.19 168.5±11.2 106.8±8.3 62.7±5.1 Table 7 shows that the serum IgG levels in the high-dose conjugate group were 10.23±0.72 mg / mL, IgA was 3.42±0.25 mg / mL, and IgM was 2.58±0.19 mg / mL, significantly higher than those in the AAVI naked peptide group (P<0.01). Simultaneously, the TNF-α levels in the high-dose conjugate group were 168.5±11.2 pg / mL, IL-1β was 106.8±8.3 pg / mL, and IL-2 was 62.7±5.1 pg / mL, also significantly higher than those in the AAVI naked peptide group. These results indicate that the conjugate significantly enhanced the immunomodulatory activity of AAVI through targeted delivery and controlled release.

[0054] 2. Western blot detection of the MyD88 / TLR-4 / NF-κB signaling pathway The expression levels of proteins related to the TLR-4 / MyD88 / NF-κB signaling pathway in liver tissue were detected by Western blot. Primary antibodies were used for TLR-4 (1:1000, Abcam, ab13556), MyD88 (1:1000, Abcam, ab219413), and NF-κB p65 (1:1000, Abcam, ab16502), and secondary antibody was HRP-labeled anti-rabbit IgG (1:5000).

[0055] Table 8 Blank group 1.00±0.08 1.00±0.07 1.00±0.08 Model group 0.52±0.05 0.48±0.04 0.58±0.05 AAVI-H group 1.18±0.09 0.92±0.07 1.05±0.08 AGLM-H group 0.98±0.08 0.78±0.06 0.88±0.07 Conj-L group 1.52±0.11 1.22±0.09 1.38±0.10 Conj-H group 1.87±0.12 1.54±0.11 1.72±0.13 As shown in Table 8, compared with the AAVI naked peptide group, the expression levels of TLR-4, MyD88, and NF-κB p65 proteins were significantly increased in the high-dose conjugate group (P<0.01). The results indicate that the conjugate exerts its immunomodulatory effect by activating the TLR-4 / MyD88 / NF-κB signaling pathway, and its effect is superior to that of the AAVI naked peptide.

[0056] 3. Macrophage targeting validation The macrophage targeting ability of the conjugated compound was verified using immunofluorescence colocalization. Frozen sections of liver tissue from mice in the high-dose conjugated compound group were subjected to CD206 (macroplast mannose receptor) immunofluorescence staining. Results showed that significant green fluorescence (FITC-labeled AAVI) was observed in the liver macrophages of mice in the conjugated compound group, and it exhibited significant colocalization with the red fluorescence of CD206 (yellow fluorescence), with a colocalization coefficient of 0.87±0.05. In contrast, the green fluorescence of the naked AAVI peptide group was more diffuse, with a colocalization coefficient of only 0.32±0.04 with CD206. These results indicate that the α-D-Man-PEG8 spacer arm endows the conjugated compound with good macrophage targeting ability, achieving precise enrichment of AAVI in macrophages.

[0057] 4. Validation of pH response and enzyme-sensitive drug release characteristics To verify the pH-responsive and enzyme-sensitive drug release characteristics of the conjugate, in vitro release experiments were conducted. The conjugate was placed in phosphate-buffered saline (pH 7.4 or pH 5.0) with or without cathepsin B and incubated at 37°C with shaking. AAVI release was measured periodically. Results showed that at pH 7.4 (simulating normal tissue), the AAVI release rate was only 8.5 ± 1.2% within 24 hours; at pH 5.0 (simulating an acidic microenvironment), the release rate was 23.7 ± 2.1% within 24 hours; and at pH 5.0 with cathepsin B (simulating a lysosomal environment), the release rate reached 78.3 ± 4.6% within 24 hours and 92.1 ± 3.8% within 48 hours. These results indicate that the conjugate exhibits both pH-responsive and enzyme-sensitive dual-stimulus release characteristics, enabling intelligent release of AAVI at the target site.

[0058] In summary, the α-D-Man-PEG8-cisAconitic-GFLG-AAVI conjugate provided by this invention achieves precise delivery and intelligent controlled release of AAVI immunomodulatory peptides through α-D-mannosyl-mediated macrophage active targeting, pH-responsive release mediated by a cis-aconitine-sensitive linker, and enzymatic release mediated by a GFLG enzyme-sensitive linker, significantly enhancing its immunomodulatory effects. Compared with naked AAVI peptides, the conjugate exhibits significant advantages in immune organ indices, serum immunoglobulin and cytokine levels, and activation of the TLR-4 / MyD88 / NF-κB signaling pathway, demonstrating promising clinical application prospects.

[0059] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention, or modify them into equivalent embodiments, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention, without departing from the content of the technical solutions of the present invention, shall still fall within the scope of protection of the present invention.

Claims

1. An active peptide derived from sika deer meat, characterized in that, The amino acid sequence of the active peptide derived from sika deer meat is AAVI.

2. The application of the sika deer meat-derived active peptide according to claim 1 in the preparation of immunomodulatory products.

3. The application according to claim 2, characterized in that, The immune-modulating products mentioned are health foods that help enhance immunity or medicines with immune-modulating effects.

4. A sika deer meat-derived active peptide conjugate compound, characterized in that, Its general structural formula is: XT-L3-L2-P, where: P represents the activity of the amino acid sequence AAVI as described in claim 1, wherein the N-terminus is a free amino group; L2 is an enzyme-cleavable linker that can be specifically cleaved by intracellular proteases; L3 is an acid-sensitive linker that breaks down under acidic pH conditions via an intramolecular cyclization mechanism; T stands for macrophage targeting unit, which contains ligands that target receptors on the surface of macrophages; X is an optional vector or reporter molecule.

5. The active peptide conjugate derived from sika deer meat according to claim 4, characterized in that, The enzyme can cleave the linker as a substrate peptide sequence of cathepsin B.

6. The active peptide conjugate derived from sika deer meat according to claim 4, characterized in that, The acid-sensitive linker is a cis-aconitine linker.

7. The active peptide conjugate derived from sika deer meat according to claim 4, characterized in that, The macrophage targeting unit is α-D-mannose.

8. The active peptide conjugate derived from sika deer meat according to claim 4, characterized in that, X is a long chain of polyethylene glycol, liposome, nanoparticle, or fluorescent reporter group; or X is absent.

9. A method for preparing the active peptide-coupled compound derived from sika deer meat according to any one of claims 4-8, characterized in that, The peptide containing L2-P was prepared by solid-phase peptide synthesis or liquid-phase peptide synthesis, and then L3 was linked by a cis-aconitine ring-opening reaction. Finally, the spacer arm containing the macrophage targeting unit T was functionalized with amino groups by disulfide bond exchange reaction, and then coupled with the L3 terminal carboxyl group by amide condensation reaction. The target compound was obtained after purification.

10. The application of the sika deer meat-derived bioactive peptide according to any one of claims 4-8 in the preparation of immunomodulatory products, characterized in that, The regulation of the body's immune function includes enhancing the immune function of immunocompromised individuals or regulating the TLR-4 / MyD88 / NF-κB signaling pathway.

Images