Immunoregulatory active peptide RWEF derived from carya illinoensis as well as screening method and application of immunoregulatory active peptide RWEF

Abstract

The invention discloses an immunomodulatory active peptide derived from carya illinoensis as well as a screening method and application of the immunomodulatory active peptide. According to the immunoregulation active peptide RWEF derived from the carya illinoensis, the active peptide is extracted from protein of the carya illinoensis through a specific enzymolysis process, and the obtained peptide molecule has good biological activity and stability. Experimental verification shows that the immunoregulation active peptide RWEF can effectively improve the functions of immune organs (such as spleen and thymus), enhance the activity of immune cells, promote secretion of immune factors and regulate inflammatory response. The immunomodulatory active peptide RWEF provided by the invention has a wide application prospect in health foods, pharmaceutical preparations and nutritional supplements.

Description

Technical Field

[0001] The invention belongs to the field of plant biotechnology and relates to an immunomodulatory active peptide RWEF derived from Carya sylvestris and a screening method and application thereof. Background Art

[0002] Bioactive peptides are small molecule polymers composed of different amino acids, usually with a molecular weight between 50 and 10,000 Da, between amino acids and proteins. As a functional biomacromolecule, bioactive peptides have a wide range of application potentials in the fields of medicine, nutrition and food science. Bioactive peptides can perform a variety of physiological functions in the body, including anti-oxidation, anti-hypertension, hypolipidemic, immune regulation, etc. Therefore, they have shown important value in the prevention and treatment of some modern epidemics (such as diabetes and cardiovascular diseases).

[0003] Immunomodulatory peptides are bioactive peptides with immunomodulatory functions, usually with a small molecular weight, generally below 3000Da. Since immunomodulatory peptides have strong stability, low side effects, and good bioavailability, they have become one of the current focuses of bioactive peptide research. Immunomodulatory peptides can regulate the innate and adaptive immune responses of the immune system, enhance the body's immune function, promote the proliferation and activation of immune cells, and help the body resist pathological conditions such as infection and tumors.

[0004] Many studies have shown that food-derived protein hydrolysates play an important role in the immune system, especially some plant-derived peptides that have shown significant immunomodulatory activity. Plant-derived immunomodulatory peptides generally regulate the body's immune response by directly acting on immune cells or by changing the secretion of immune factors. For example, soy hydrolysates have been found to regulate the rate of leukocyte production and immunoglobulin levels in mice, and affect the levels of proinflammatory cytokines (such as IL-1β and TNF-α). Walnut protein hydrolysates can exhibit their immunomodulatory effects through multiple mechanisms such as increasing NK cell activity, activating macrophages, promoting antibody production, and enhancing intestinal mucosal immunity.

[0005] The activity of immunomodulatory peptides is affected by their physicochemical properties, including the composition, sequence, length, hydrophobicity, degree of hydrolysis, and three-dimensional structure of the peptide molecule. Studies have shown that peptides containing hydrophobic amino acids (such as glycine, valine, leucine, proline, and phenylalanine) usually have strong immunomodulatory effects, which may be due to the fact that these amino acids can enhance the interaction between peptides and cell membranes, thereby affecting downstream immune signaling pathways. In addition, arginine-rich peptides can also activate T cells to produce lymphokines after binding to T cell membrane receptors, further enhancing immune function.

[0006] Carya illinoinensis (Wangenh.) K.Koch, native to the United States and northern Mexico, is a deciduous tree of the genus Carya in the Juglandaceae family. Carya illinoinensis is grown in Yunnan, Fujian, Jiangsu, Anhui, Zhejiang and other places in China, and is widely used in food, health food and related fields. Carya illinoinensis nuts are rich in protein and a variety of amino acids that are beneficial to the human body, and are an ideal natural health raw material.

[0007] Although the nutritional components of walnuts have been widely studied, the research on the immunomodulatory activity of their protein hydrolysates is still relatively scarce. In view of the complexity of the immunomodulatory mechanism, it is rare to be able to isolate active peptides with immunomodulatory effects from walnuts. Therefore, the present invention studies the immunomodulatory active peptides from walnuts, and finds that the peptides (such as Arg-Trp-Glu-Phe, RWEF) from them can significantly enhance the immune function of immunosuppressed mice, which is specifically manifested in protecting spleen and thymus tissue, enhancing immune indicators, and showing its effective immunomodulatory potential. Summary of the invention

[0008] In order to solve the above problems, the present invention provides an immunomodulatory active peptide RWEF derived from walnuts and a screening method and application thereof.

[0009] The invention provides an immunomodulatory active peptide RWEF derived from Carya sylvestris. The amino acid sequence of the immunomodulatory active peptide RWEF is SEQ ID No.1.

[0010] In one embodiment, the immunomodulatory active peptide RWEF is derived from pecan protein.

[0011] The present invention also provides the use of the immunoregulatory active peptide RWEF in preparing food for improving the immunoregulatory ability of human body.

[0012] The present invention also provides the use of the immunomodulatory active peptide RWEF in the preparation of immunomodulatory drugs.

[0013] The present invention also provides a method for screening the immunomodulatory active peptide RWEF derived from Carya syringae, the method comprising the following steps:

[0014] (1) Enzymatic hydrolysis: using alkaline protease and neutral protease to hydrolyze the walnut protein to obtain protein hydrolysate;

[0015] (2) Ultrafiltration: Use a 3 kDa ultrafiltration membrane to filter the protein hydrolyzate to obtain a thin-shell pecan small molecule peptide with a molecular weight of less than 3 kDa;

[0016] (3) Liquid chromatography-mass spectrometry (LC-MS / MS) analysis: The pecan small molecule peptides were subjected to reduction alkylation and desalting, and then the treated samples were analyzed by LC-MS / MS to obtain the raw file of the original mass spectrometry results, and the sequence analysis results of the pecan small molecule peptides were obtained by PEAKS Studio 10.6 De novo analysis;

[0017] (4) Screening: Virtual screening of the obtained peptide sequences is performed, and peptides with no toxicity, high biological activity, and immunomodulatory function are screened according to bioinformatics prediction scores;

[0018] (5) In vivo assay: The immunomodulatory function of candidate peptides was determined in cyclophosphamide-induced immunosuppressive model mice, and the immunomodulatory active peptide RWEF was screened.

[0019] In one embodiment, the amino acid sequence of the above-mentioned walnut small molecule peptide contains the sequence of SEQ ID No.1.

[0020] In one embodiment, virtual screening performs biological activity prediction based on the PeptideRanker platform, and the biological activity prediction value of the immunomodulatory active peptide RWEF is 0.899911.

[0021] In one embodiment, virtual screening is performed based on the IFNepitope platform to predict immunomodulatory activity, and the immunomodulatory active peptide RWEF has immunomodulatory activity.

[0022] The present invention also provides an application of an immunomodulatory active peptide RWEF, which plays a role in improving the function of the immune system, repairing the pathological structure of the immune organs of immunosuppressed mice, restoring the level of immunoglobulins, improving the phagocytic ability of immune cells, and promoting immune response, wherein the amino acid sequence of the immunomodulatory active peptide is SEQID No.1.

[0023] In one embodiment, the immunomodulatory active peptide RWEF can improve the body weight, spleen index and thymus index of immunosuppressed mice, repair the pathological tissue structure of the spleen and thymus, and restore the level of immunoglobulins (IgM, IgG).

[0024] The immunomodulatory active peptide RWEF derived from the thin-shelled pecan of the present invention is extracted from the protein of the thin-shelled pecan through a specific enzymatic hydrolysis process, and the obtained peptide molecule has good biological activity and stability. Experimental verification shows that the immunomodulatory active peptide RWEF can effectively improve the function of immune organs (such as spleen and thymus), and can enhance the activity of immune cells, promote the secretion of immune factors and regulate inflammatory reactions. The immunomodulatory active peptide RWEF provided by the present invention has a wide range of application prospects in health foods, pharmaceutical preparations and nutritional supplements. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The drawings described herein are used to provide a further understanding of the present application and constitute a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute an improper limitation of the present application. In the drawings: NC is the normal group, MC is the model control group, PC is the positive drug control group, and RWEF is the RWEF peptide treatment group. The different letters above each bar graph are significant differences between different treatments (P<0.05).

[0026] Figure 1 This is the mass spectrometry analysis of the immunomodulatory active peptide RWEF.

[0027] Figure 2 The effect of immunomodulatory active peptide RWEF on the body weight of immunosuppressed mice.

[0028] Figure 3 The effect of immunomodulatory active peptide RWEF on immune organ indexes of immunosuppressed mice.

[0029] Figure 4 This is the effect of the immunomodulatory active peptide RWEF on the changes in spleen tissue in immunosuppressed mice.

[0030] Figure 5 The effect of the immunomodulatory active peptide RWEF on the changes in thymus tissue of immunosuppressed mice.

[0031] Figure 6 The effect of the immunomodulatory active peptide RWEF on immunoglobulins (IgM, IgG) in the serum of immunosuppressed mice.

[0032] Figure 7 The effect of the immunomodulatory active peptide RWEF on the serum cytokine IFN-γ concentration in immunosuppressed mice.

[0033] Figure 8 The effect of the immunomodulatory active peptide RWEF on the concentrations of ACPase and LDH in the spleen homogenate of immunosuppressed mice.

[0034] Fig. 9The effect of the immunomodulatory active peptide RWEF on the concentrations of ACPase, LDH, CAT and GSH in the liver homogenate of immunosuppressed mice.

[0035] Fig.10 The effect of immunomodulatory active peptide RWEF on delayed-type hypersensitivity in immunosuppressed mice.

[0036] Fig.11 The effect of the immunomodulatory active peptide RWEF on the phagocytic ability of macrophages in immunosuppressed mice. DETAILED DESCRIPTION

[0037] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0038] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0039] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0040] Example 1

[0041] To illustrate the extraction of pecan protein, the steps are as follows:

[0042] 1. Raw material preparation: Select defatted walnut meal from thin shells, add it into distilled water at a ratio of 1:50 (w / v), and disperse it fully.

[0043] 2. Dissolution and pH adjustment: After heating the mixture to 55°C, adjust the pH to 11.0 using 1 mol / L sodium hydroxide solution and continue stirring for 90 minutes to ensure that the protein is completely dissolved.

[0044] 3. Centrifugation: Centrifuge at 8000 rpm for 10 minutes, collect the supernatant and remove impurities.

[0045] 4. Precipitate protein: Use 1 mol / L hydrochloric acid to adjust the pH of the supernatant to 4.0, let it stand and precipitate, and finally take the precipitate, which is the thin-shell pecan protein.

[0046] Example 2

[0047] The steps for describing the preparation of the walnut immunomodulatory active peptide RWEF are as follows:

[0048] 1. Protein dissolution and heating: The thin-shell pecan protein obtained in Example 1 was dissolved in distilled water at a ratio of 1:50 (w / v), and heated to 90° C. for 15 minutes to remove possible contaminants.

[0049] 2. Enzyme hydrolysis: First, hydrolyze with 9000U / g neutral protease at 53°C, control the pH value at 7.2, and enzymolysis for 120 minutes. Then, inactivate the enzyme, raise the solution temperature to 90°C, and keep it for 15 minutes.

[0050] 3. Second enzymatic hydrolysis: adjust the pH value of the protein solution to 8.4, add 7000U / g alkaline protease, enzymatic hydrolysis for 180 minutes, keep the enzymatic hydrolysis temperature at 56℃, and adjust the pH to be stable during the process. After the end, put it in a 90℃ water bath for 15 minutes to kill the enzyme, adjust the pH value to 7.0, centrifuge at 8000r / min, 4℃ for 10 minutes, and the supernatant is the thin-shell pecan peptide.

[0051] 4. Separation and purification: The thin-shell pecan peptide liquid was ultrafiltered using a 3kDa ultrafiltration membrane and a peristaltic pump at 120rpm. The ultrafiltration fractions with molecular weight <3kDa in the filtrate were collected and freeze-dried in a vacuum to obtain thin-shell pecan dry powder containing the immunomodulatory active peptide RWEF.

[0052] Example 3

[0053] To illustrate the structural identification of pecan protein peptide, the steps are as follows:

[0054] (1) Sample pretreatment: Weigh 1 mg of pecan peptide powder, add an appropriate amount of 50 mmol / L ammonium bicarbonate solution to dissolve the sample, add dithiothreitol solution to make the final concentration 10 mmol / L, and reduce it in a 56°C water bath for 1 h. Add iodoacetamide solution to make the final concentration 55 mmol / L, react in the dark for 40 min, desalt using a self-filled desalting column, and evaporate the solvent in a 45°C vacuum centrifugal concentrator.

[0055] (2) LC-MS / MS detection: Liquid chromatography conditions: pre-column: 150 μm idx 5 cm, packing: Reprosil-Pur 120C18-AQ 3 μm; analytical column: 100 μm idx 180 mm, packing: Reprosil-Pur 120C18-AQ 3 μm; mobile phase A: 0.1% formic acid aqueous solution; mobile phase B: 80% ACN / 0.1% formic acid aqueous solution; flow rate: 600 nL / min; analysis time for each component: 60 min.

[0056] Mass spectrometry conditions: primary mass spectrometry parameters: resolution: 70,000; AGC target: 3e6; Maximum IT: 60 ms; Scan range: 100 to 1500 m / z. Secondary mass spectrometry parameters: resolution: 17,500; AGC target: 5e4; Maximum IT: 80 ms; NCE / stepped NCE: 27.

[0057] (3) Database search: The mass spectrometry raw files were analyzed for peptide sequences using the PEAKS Studio 10.6 De novo method. The search parameters were as follows: fixed modifications; variable modifications; enzyme; maximum missed cleavages; primary mass spectrometry error; secondary mass spectrometry error. The amino acid sequence of the pecan peptide was identified by LC-MS / MS as Arg-Trp-Glu-Phe (RWEF). The RWEF mass spectrometry results are attached. Figure 1 .

[0058] Example 4

[0059] The steps for bioinformatics test of RWEF, an immunomodulatory peptide from pecan, are as follows:

[0060] (1) Prediction of the potential biological activity of the immunomodulatory active peptide RWEF: The potential biological activity of the obtained peptide sequences was analyzed using the PeptideRanker online platform (http: / / distilldeep.ucd). The peptides were ranked according to their predicted probability of biological activity. The preset threshold of the PeptideRanker prediction model was 0.5, and a threshold greater than 0.5 was considered to have biological activity. The predicted value of the new peptide RWEF screened in this application was 0.899911. The results of bioinformatics prediction analysis showed that the immunomodulatory active peptide RWEF had high potential biological activity.

[0061] (2) Prediction of potential toxicity of immunomodulatory active peptide RWEF: using Toixinpred

[0062] (https: / / webs.iiitd.edu.in / raghava / toxinpred / index.h-tml) platform, based on the SVM (Swiss-Port) algorithm to predict the potential toxicity of new peptides. After potential toxicity analysis, the immunomodulatory active peptide RWEF was non-toxic (Non-Toxin).

[0063] (3) Prediction of the potential immunomodulatory activity of immunomodulatory active peptide RWEF: using IFNepitope

[0064] (https: / / webs.iiitd.edu.in / raghava / ifnepitope / index.php) platform, based on the SVM algorithm to predict the potential of peptides to induce IFN-γ. After potential immunomodulatory activity analysis, the immunomodulatory active peptide RWEF was positive (POSITIVE) and had potential immunomodulatory activity.

[0065] Example 5

[0066] The steps used to illustrate the chemical synthesis of the walnut immunomodulatory active peptide RWEF and its ability to improve the immune ability of cyclophosphamide-induced immunosuppression model mice are as follows:

[0067] (1) Peptide synthesis: The small peptide (Arg-Trp-Glu-Phe) RWEF was chemically synthesized using amino acid solid phase synthesis technology. It was synthesized by chemical solid phase synthesis by Jiangsu Jitai Peptide Technology Co., Ltd. The purity of the peptide was >95%.

[0068] (2) Verification of the immunomodulatory function of RWEF peptide: Specific pathogen free (SPF) grade, male BALB / c mice, weight (22±2) g, were provided by SPF (Suzhou) Biotechnology Co., Ltd. The experimental animal production license number is SCXK (Suzhou) 2022-0006. The animal experiments were approved by the Animal Ethics Committee of Zhejiang A&F University (approval number: ZAFUAC202430), ensuring that the animal experiments were in compliance with relevant regulations and were approved by the ethics committee.

[0069] All mice were placed in the experimental feeding room under the following conditions: temperature 25±1℃, humidity 50±5%, light cycle 12h (9:00-21:00), and mice had free access to water and food. The mice were divided into four groups (n=10 / group): mice were cultured for 7 days, namely normal group (Normal Control, NC, NC), model control group (Model Control, MC), positive drug control group (Positive control, PC) and RWEF peptide treatment group (RWEF) treatment group. On the first 1-3 days of the experiment, except for the NC group, the other 5 groups of mice were intraperitoneally injected with 80mg / kg.bw cyclophosphamide, and the NC group was injected with an equal amount of normal saline for 3 consecutive days. On the 4th to 24th day, the NC group was gavaged with an equal amount of normal saline, the PC group was gavaged with 20mg / kg.bw levamisole hydrochloride; the RWEF group was gavaged with 100mg / kg.bw RWEF peptide for 21 consecutive days. Cyclophosphamide, levamisole hydrochloride and RWEF peptide were all prepared with normal saline.

[0070] ①Effects of RWEF peptide on body weight and immune organ indexes in immunosuppressed mice

[0071] After CO2 inhalation anesthesia, mice were killed by cervical dislocation, and spleen, thymus, liver and other organs were taken, their wet weights were weighed, and spleen index and thymus index were calculated according to the formula. Reference formula: organ index = organ weight / body weight.

[0072] In animal model experiments, cyclophosphamide has a certain immunosuppressive effect on mice, causing a decrease in body weight, immune organ index and other indicators of mice. Figure 2 It can be seen that the initial weight of mice in each group was not significantly different, but the modeling weight and final weight were significantly different. After cyclophosphamide modeling, the modeling weight of mice in the MC group, PC group and RWEF peptide group was significantly lower than that in the NC group (P<0.05). Compared with the model group, the final weight of mice in the RWEF peptide group increased significantly (P<0.05), an increase of 22.7%. The results of this study showed that the RWEF peptide group can improve the weight of mice in the cyclophosphamide-induced immunosuppression model.

[0073] The spleen and thymus are important components of the immune system, and immune organ indicators can reflect the body's immune regulation level to a certain extent. Figure 3It can be seen that compared with the NC group, the spleen and thymus indexes of the MC group were significantly reduced (P<0.05), by 23.5% and 72.8%, respectively. The spleen and thymus indexes of different thin-shelled pecan immunomodulatory active peptides RWEF were significantly improved compared with the model group (P<0.05). Compared with the spleen and thymus indexes of the MC group, the RWEF group increased by 90.6% and 315.1%, respectively. The results of this study show that the RWEF peptide group can improve the immune organs of cyclophosphamide-induced immunosuppression model mice.

[0074] ②Effects of RWEF peptide on pathological sections of immune organs in immunosuppressed mice

[0075] Using the hematoxylin-eosin staining method, mouse spleen and thymus tissue blocks (about 0.5 cm × 0.5 cm × 0.3 cm) fixed with 10% formaldehyde solution were taken. After dehydration and paraffin embedding, the spleen tissue was cut into 5 μm thick blocks using a freezing microtome, and then H&E staining was performed, and pathological morphology was observed under an optical microscope.

[0076] The spleen is the largest peripheral lymphoid organ in the body and the habitat of mature T / B lymphocytes. Its structure includes white pulp, red pulp and marginal zone. The white pulp contains a large number of lymphocytes and is the main organ for the body to produce specific immunity. The marginal zone is located at the junction of the red pulp and white pulp. There are many macrophages in this area, which is an important part of the spleen for capturing and recognizing antigens. Effect of RWEF peptide on spleen histopathology in immunosuppressed mice Figure 4 As shown, the number of lymphocytes and pathological status of spleen tissue were identified by hematoxylin-eosin staining, and the state of spleen sections of mice in each group was observed under an optical microscope. The red and white pulp margins of the spleen of mice in the NC group were clearly defined, the white pulp area was large and uniform, the splenic cord network structure was good, and there were no obvious pathological changes; the number of spleen cells and the area of ​​white pulp in the MC group were significantly reduced, the cells were of different sizes, sparsely and disorderly arranged, and the boundary between the red pulp and white pulp was blurred. Compared with the MC group, the number of spleen cells in the RWEF peptide group increased to a certain extent, the cells were neatly arranged, the nucleoli were clear, and there were few gaps. The margin between the red pulp and white pulp of the mouse spleen was relatively clear, and it was close to that of the NC group. RWEF peptide has a good improvement on the pathological state of the spleen in immunosuppressed mice. RWEF peptide can improve the immune system organ index of cyclophosphamide-induced immunosuppressive model mice and repair the pathological tissue structure of the spleen.

[0077] The thymus is an important immune organ of the body. It is the site of differentiation and maturation of T lymphocytes and plays an important role in the body's immune regulation. Figure 5It can be seen that the hematoxylin-eosin staining method identified the changes in the thymic structure of each group. The thymic cortical medulla of the NC group had clear boundaries and thicker cortex, while the thymic cortical medulla of the MC group mice had unclear boundaries, disordered cortical medulla structure, a large reduction in thymocytes, and lighter coloring. Compared with the MC group, the cortical medulla boundary of the RWEF peptide group was restored to a clear state, and the cortical medulla structure was stable, but compared with the NC group, the thymic medulla area was reduced and the cortex was thickened. These results indicate that RWEF peptide can restore the disorder of the thymic structure of mice with cyclophosphamide-induced immunosuppression.

[0078] ③Effects of RWEF peptide on blood cells in immunosuppressed mice

[0079] 24h after the last treatment, 50μL of blood was collected from the eye sockets of mice with sodium ethylenediaminetetraacetic acid as anticoagulant on the day of sacrifice. The red blood cell (RBC), white blood cell (WBC), platelet (PLT), hemoglobin (Hb) and other indicators were measured by automatic blood cell analyzer. The immune cells and immune active substances in the blood together constitute the blood immune barrier of the body, which plays an important role in the body's defense against the invasion of external pathogens and in the immune defense. As shown in Table 1, the RBCs, WBCs and PLTs in the blood of mice in the model group were significantly decreased (P<0.05). RWEF peptide can significantly increase the levels of RBCs, WBCs, PLTs and Hb in the blood of immunosuppressed mice (P<0.05). Compared with the MC group, RBCs, WBCs, PLTs and Hb in the RWEF peptide group increased by 89%, 546%, 49.5% and 104.3%. The above results show that RWEF peptide can restore the blood cell function of cyclophosphamide-induced immunosuppressive model mice.

[0080] Table 1. Effects of RWEF peptides on blood cells in immunosuppressed mice

[0081]

[0082] ④Effects of RWEF peptide on serum immunoglobulins (IgM, IgG) in immunosuppressed mice

[0083] Serum proteins such as immunoglobulin M (IgM) and immunoglobulin G (IgG) mediate a variety of protective functions through interactions with specific receptors and immune mediators. Figure 6It can be seen that compared with the normal group, the IgM and IgG levels in the model group decreased significantly (P<0.05), indicating that cyclophosphamide can significantly inhibit the secretion and expression of IgM and IgG antibodies in mice. Compared with the IgM and IgG in the MC group, the RWEF peptide group increased by 457.5% and 161.2%, respectively. The RWEF peptide group showed a significant ability to resist the decrease in IgM and IgG concentrations in immunosuppressed mice induced by cyclophosphamide (P<0.05).

[0084] ⑤Effect of RWEF peptide on serum cytokine IFN-γ concentration in immunosuppressed mice

[0085] Each immune response requires the cooperation of cells. The communication between cells depends on diffusion media, the most important of which is cytokines. The concentration of cytokine IFN-γ is detected by ELISA. Figure 7 It can be seen that the RWEF peptide group showed a significant ability to resist the cyclophosphamide-induced decrease in the serum cytokine IFN-γ concentration in immunosuppressive mice (P<0.05).

[0086] ⑥Effects of RWEF peptide on enzyme and glutathione concentrations in liver and spleen homogenates of immunosuppressed mice

[0087] Under sterile conditions, the spleen and liver of mice were removed, placed in cold phosphate buffer, chopped and passed through 0.074 mm medical gauze, and the corresponding cell suspension was collected and homogenized with a polytetrafluoroethylene glass homogenizer to prepare spleen homogenate and liver homogenate. Centrifuged at 2000 rpm and 4 ° C for 10 min, the supernatant was taken, and the concentrations of acid phosphatase (ACPase, ACP), lactate dehydrogenase (LDH), catalase (CAT) and reduced glutathione (GSH) were detected.

[0088] Cyclophosphamide can promote macrophage apoptosis, reduce macrophage activity, inhibit macrophage defense function, and lead to a decrease in immune active substances LDH and ACPase. Figure 8 As shown in the figure, after the immunosuppressive effect of CTX, the ACPase and LDH concentrations in the spleen of the MC group were significantly reduced compared with those of the NC group (P<0.05). The RWEF peptide group was able to significantly increase the ACPase and LDH concentrations in the spleen compared with the MC group (P<0.05). Compared with the ACPase and LDH concentrations in the MC group, the RWEF group increased by 30.5% and 37.8%, respectively. Fig. 9As shown in the results, the RWEF group was able to significantly increase the concentrations of liver ACPase, LDH, GSH and CAT compared with the MC group (P<0.05), and the RWEF group increased liver ACPase, LDH, GSH and CAT by 36.6%, 20.8%, 87.0% and 34.3% respectively compared with the MC group. The above results indicate that RWEF peptide can restore the liver and spleen enzyme function activity of cyclophosphamide-induced immunosuppression model mice.

[0089] ⑦RWEF peptide immunosuppressed delayed hypersensitivity in mice

[0090] On day 14, the mice were shaved and their abdomens were coated with 50 μL of 10 g.L -1 2,4-Dinitrofluorobenzene was dissolved in acetone-olive oil (acetone:olive oil = 1:1 (v:v)) for initial immune stimulation. 24 h after administration on day 21, 20 μL of 10 g.L was applied to the right ear of the mouse. -1 2,4-dinitrofluorobenzene was used as an antigen to attack the mice. One hour after application, the mice were killed by cervical dislocation. The left and right ears were cut off, and the ear pieces with a diameter of 8 mm were taken out with a puncher. The ear swelling degree was weighed and calculated. The ear swelling degree = the mass of the right ear piece - the mass of the left ear piece. Fig.10 As shown, the hypersensitivity index of the RWEF peptide group increased by 218.6% compared with the MC group, which can significantly improve the immune response ability of mice (P<0.05).

[0091] ⑧Effect of RWEF peptide on phagocytic ability of macrophages in immunosuppressive mice

[0092] The phagocytic ability of mouse macrophages was evaluated by carbon clearance test. After the last administration, the mice were weighed and injected with 0.1mL / 10g.bw of India ink (5-fold dilution, India ink: saline = 1:4, w / v) through the tail vein. 20μL of retro-orbital venous blood was collected at 2min (T) and 10min (Tz) after injection and mixed with 2mL of 0.1% Na2CO3 solution. 0.1% Na2CO3 solution was used as a blank control, and the absorbance of each sample at 600nm was measured. Afterwards, the mice were killed by cervical dissection, and the liver and spleen of the mice were dissected and weighed. The carbon clearance index (K) and phagocytic index (a) were calculated using the following formula:

[0093] Carbon Clearance Index

[0094] Phagocytic index

[0095] Where: OD1 represents the absorbance of the sample at 600nm at T1 (2min); OD2 represents the absorbance of the sample at 600nm at T2 (10min); M体重 Indicates the mass of mice; M 脾脏 Represents the spleen mass of mice; M 肝脏 Mouse liver macrophages are phagocytic cells derived from monocytes, which participate in nonspecific immunity. Exogenous stimuli can activate macrophages, thereby causing the host's immune response. Fig.10 As shown, it is also involved in specific immunity. Compared with the MC group, the phagocytic ability of macrophages in the RWEF peptide group increased by 29.5%. RWEF peptide can restore the phagocytic ability of macrophages in cyclophosphamide-induced immunosuppressive model mice.

[0096] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An immunomodulatory active peptide RWEF derived from Carya sylvestris, characterized in that: The amino acid sequence of the immunomodulatory active peptide RWEF is SEQ ID No.

1.

2. The immunomodulatory active peptide RWEF according to claim 1, characterized in that: The immunomodulatory active peptide RWEF is derived from walnut protein.

3. Use of the immunomodulatory active peptide RWEF according to claim 1 in the preparation of food for improving human immune regulation ability.

4. Use of the immunomodulatory active peptide RWEF according to claim 1 in the preparation of immunomodulatory drugs.

5. A method for screening the immunomodulatory active peptide RWEF derived from Carya sylvestris, characterized in that: The method comprises the following steps: (1) Enzymatic hydrolysis: using alkaline protease and neutral protease to hydrolyze the walnut protein to obtain protein hydrolysate; (2) ultrafiltration: filtering the protein hydrolyzate using a 3 kDa ultrafiltration membrane to obtain a thin-shell pecan small molecule peptide with a molecular weight of less than 3 kDa; (3) Liquid chromatography-mass spectrometry (LC-MS / MS) analysis: The pecan small molecule peptide is subjected to reduction alkylation and desalting, and then the treated sample is analyzed by LC-MS / MS to obtain a raw file of the original mass spectrometry result, and the pecan small molecule peptide sequence analysis result is obtained by PEAKS Studio 10.6 De novo analysis; (4) Screening: Virtual screening of the obtained small molecule peptide sequences is performed, and peptides with no toxicity, high biological activity, and immunomodulatory function are screened according to bioinformatics prediction and scoring; (5) In vivo assay: The immunomodulatory function of the candidate peptides was assayed in cyclophosphamide-induced immunosuppressive model mice, and the immunomodulatory active peptide RWEF was screened.

6. The method according to claim 5, characterized in that The amino acid sequence of the walnut nut small molecule peptide contains the sequence of SEQ ID No.

1.

7. The method according to claim 5, characterized in that The virtual screening performed biological activity prediction based on the PeptideRanker platform, and the biological activity prediction value of the immunomodulatory active peptide RWEF was 0.899911.

8. The method according to claim 5, characterized in that The virtual screening predicts immunomodulatory activity based on the IFNepitope platform, and the immunomodulatory active peptide RWEF has immunomodulatory activity.

9. An application of an immunomodulatory active peptide RWEF, characterized in that: The immunomodulatory active peptide RWEF plays a role in improving the function of the immune system, repairing the pathological structure of the immune organs of immunosuppressed mice, restoring the level of immunoglobulins, improving the phagocytic ability of immune cells and promoting immune response, wherein the amino acid sequence of the immunomodulatory active peptide RWEF is SEQ ID No.

1.

10. The use according to claim 9, characterized in that: The immunomodulatory active peptide RWEF can improve the body weight, spleen index and thymus index of immunosuppressed mice, repair the pathological tissue structure of the spleen and thymus, and restore the level of immunoglobulins (IgM, IgG).

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