An octapeptide with immune enhancing effect, its preparation and application

Abstract

The present invention discloses an octapeptide with immune-enhancing effect, its preparation and application, belonging to the technical field of biomedicine. The amino acid sequence of the octapeptide is Gly-Phe-Asn-Asp-Leu-Gly-Lys-Arg. This peptide segment can be obtained by artificial synthesis or directional enzymatic hydrolysis of wheat protein. Function verification shows that this octapeptide has the effect of promoting the proliferation of immune cells, mainly manifested in significantly increasing the number of macrophages and neutrophils, and increasing the level of immune factor interferon-γ. The present invention provides a new active substance for enhancing immune function, which can be applied to the preparation of health foods or drugs for preventing and improving immune hypofunction, and has good market prospects and application potential.

Description

Technical Field

[0001] The present invention relates to the field of biological medicine technology, and particularly relates to an octapeptide with immune-enhancing effect, its preparation and application. Background Art

[0002] The immune system is a key mechanism for the body to resist foreign pathogens and abnormal cells, responsible for recognizing and removing potential threats and maintaining a healthy state. It protects the body through three lines of defense, namely tissue barriers, innate immunity and adaptive immunity, to prevent the occurrence of infectious diseases and cancers.

[0003] Immune hypofunction is a common sub-healthy state, which is prone to symptoms such as colds and fevers, and may even cause cancer in severe cases. Especially due to reasons such as stress, irregular work and rest, and unhealthy diet, the problem of damaged immune system is faced. Therefore, enhancing immune function has become the focus of public attention.

[0004] Bioactive peptides are a class of small peptides with biological functions, usually composed of less than 20 amino acid residues. They have a wide range of applications in the fields of food, medicine and cosmetics, with diverse functions, including antibacterial, anti-tumor, blood pressure lowering, blood lipid lowering and immune regulation. For example, the active peptides of Mytilus coruscus prepared by enzymatic hydrolysis can achieve immune regulation through the NF-κB / MAPK signaling pathway (He Kang. Study on the preparation and immune regulation of active peptides of Mytilus coruscus [D]. Zhejiang Ocean University, 2021.); calf thymosin can increase the levels of immune-related cytokines in immune hypofunction mice and improve the activity of cytotoxic immune cells in mice (Li Lanzhou. Study on the anti-colorectal cancer activity and hematopoiesis-promoting function of calf thymosin based on immune regulation [D]. Jilin University, 2021.).

[0005] Wheat peptides are made from wheat protein (gluten) through processes such as biological enzymatic hydrolysis, refining, and spray drying. Research shows that wheat peptides prepared by hydrolyzing wheat protein with alkaline protease can enhance the phagocytic ability of macrophages and promote the secretion of immune factors such as interferon-γ (INF-γ) (Dai Hui et al. Study on the immune activity and antioxidant effect of wheat peptides [J]. Natural Product Research and Development, 2009, 21(03): 473-476.). The functional characteristics of bioactive peptides are closely related to their amino acid composition and arrangement order, which makes the rational design and synthesis of bioactive peptides an effective strategy for enhancing immunity. Therefore, by analyzing the peptide spectrum of wheat peptides and screening out high-efficiency peptide segments related to immune enhancement, it will provide a basis for the development of new immune regulators. Summary of the Invention

[0006] The object of the present invention is to provide a natural small molecule bioactive peptide with immune enhancing effect and apply it to the development of safe and efficient immunomodulatory products.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] The present invention uses LC-MS / MS peptide mapping analysis technology to analyze the polypeptide sequence in wheat peptides, and then uses molecular docking technology to explore the interaction between peptide segments and acetylcholine receptors. A candidate peptide segment is screened out. After mass spectrometry identification, its amino acid sequence is Gly-Phe-Asn-Asp-Leu-Gly-Lys-Arg (GFNDLGKR), and the molecular weight is 906.456 Da. It is named GR8. Further, the octapeptide GR8 is synthesized artificially, and functional verification shows that the octapeptide exhibits a significant immune enhancing effect in an immune-deficient animal model.

[0009] Therefore, the present invention provides an octapeptide GR8 with immune enhancing effect, and the amino acid sequence of the octapeptide GR8 is Gly-Phe-Asn-Asp-Leu-Gly-Lys-Arg.

[0010] The present invention also provides a method for preparing the octapeptide GR8. The octapeptide GR8 can be prepared by solid-phase synthesis method. The specific method includes: adopting Fmoc solid-phase synthesis strategy, using Fmoc-protected amino acids as raw materials, selecting Wang resin as the solid-phase carrier, and introducing arginine, lysine, glycine, leucine, aspartic acid, asparagine, phenylalanine, and glycine residues in sequence to extend the peptide chain from the C-terminus to the N-terminus, and solid-phase synthesizing the octapeptide GR8.

[0011] The octapeptide GR8 can also be obtained by enzymatic hydrolysis of wheat gluten. Specifically, the reaction kettle is charged according to the mass ratio of water to wheat protein (gluten powder) of 10:1. After the feeding is completed, the pH of the feed liquid is adjusted to 8.0 ± 0.2, and then alkaline protease (accounting for 1.0% of the total weight of gluten powder) is added for enzymatic hydrolysis for 30 min. During the enzymatic hydrolysis process, the pH of the feed liquid is continuously maintained at ≥ 7.5. After completion, neutral protease (accounting for 1.5% of the total weight of gluten powder) is added for enzymatic hydrolysis for 60 min. After the enzymatic hydrolysis is completed, flavor protease (accounting for 0.5% of the total weight of gluten powder) is used for 30 min, and then the enzyme is inactivated at 100 °C for 30 min. Subsequently, it is concentrated and dried to obtain wheat peptide powder, which contains the octapeptide GR8.

[0012] The present invention provides the application of the octapeptide GR8 in the preparation of immunomodulatory products, and the immunomodulation includes at least one of enhancing the proliferation of immune cells and increasing the level of immune factors. The research of the present invention shows that in an immunocompromised animal model, treatment with octapeptide GR8 can significantly increase the number of immune cells such as macrophages and neutrophils; significantly increase the content of immune factors such as INF-γ, and it can be applied to the development of related products for regulating immune function.

[0013] Furthermore, the immune cells include macrophages and neutrophils; the immune factors include interferon-γ.

[0014] Furthermore, the product is a drug for treating immunodeficiency or a health food for enhancing immunity. The drug can be used to treat immunodeficiency diseases, and the diseases can refer to those accompanied by a state of immunodeficiency, for which an immune enhancer needs to be given or it is more beneficial to relieve symptoms by giving an immune enhancer. The health food can be used to improve the sub-healthy state of immunodeficiency.

[0015] Furthermore, the manifestations of immunodeficiency include: a decrease in the number of immune cells in the body and a decrease in the level of interferon-γ.

[0016] Furthermore, the product is a drug for relieving immunodeficiency in mammals or a health food for enhancing the immunity of mammals. The mammals can be, but are not limited to, humans.

[0017] The present invention also provides a pharmaceutical composition for treating immunodeficiency diseases, and the active ingredient of the pharmaceutical composition includes the octapeptide GR8 with the amino acid sequence of Gly-Phe-Asn-Asp-Leu-Gly-Lys-Arg.

[0018] The present invention is prepared with the octapeptide GR8 as the main active ingredient and added with pharmaceutically acceptable carriers, and can be made into a preparation according to the preparation methods of preparations recorded in pharmacy. The octapeptide GR8 in the pharmaceutical composition provided by the present invention can be used as the only active ingredient for enhancing immunity, or can be compounded with other active ingredients having immunomodulatory effects such as sea cucumber peptides and soybean peptides.

[0019] Furthermore, the pharmaceutical composition also includes pharmaceutically acceptable carriers. The pharmaceutically acceptable carriers include one or more of fillers, wetting agents, disintegrants, binders or lubricants.

[0020] Furthermore, the preparation form of the pharmaceutical composition can be, but is not limited to, oral liquid, capsule, microcapsule powder, tablet, granule or emulsion.

[0021] The specific dosage of the pharmaceutical composition is adjusted according to the type of disease, the degree of disease, age and the purpose of administration. In the immunosuppressed zebrafish model, the effective concentration of octapeptide GR8 is 1-10 μg / mL, and no toxic or side effects are shown under this condition.

[0022] The present invention also provides a health food for enhancing immunity, which includes octapeptide GR8 as an active ingredient and food-grade acceptable excipients. The amino acid sequence of the octapeptide GR8 is Gly-Phe-Asn-Asp-Leu-Gly-Lys-Arg.

[0023] Furthermore, the health food can be a beverage, oral liquid, capsule, microcapsule powder, tablet, granule or emulsion.

[0024] The beneficial effects of the present invention are as follows:

[0025] The present invention provides an octapeptide GFNDLGKR with an immunomodulatory effect. This peptide can be obtained by artificial synthesis or directional enzymatic hydrolysis of wheat protein. Functional verification in animal models shows that this polypeptide has the effect of promoting the proliferation of immune cells, mainly manifested in significantly increasing the number of macrophages and neutrophils, and increasing the content of the immune factor interferon-γ. Moreover, octapeptide GFNDLGKR is a natural peptide segment with high biological safety. Therefore, it can be applied to the preparation of health foods or drugs for preventing and improving adverse phenomena of immunosuppression. The present invention provides a new active substance for enhancing immune function, meeting the public's demand for immune health care, and having good market prospects and application potential. Description of the Drawings

[0026] Figure 1 It is the secondary mass spectrometry diagram of octapeptide GR8.

[0027] Figure 2 It is the schematic diagram of the binding effect between octapeptide GR8 and CHRM1.

[0028] Figure 3 It is the photo of the head macrophages observed under a stereomicroscope after the zebrafish model is treated with octapeptide GR8.

[0029] Figure 4 It is Figure 3 The statistical chart of the number of head macrophages in

[0030] Figure 5 It is the photo of the fluorescence of neutrophils from the cloaca to the end of the tail observed under a stereomicroscope after the zebrafish model is treated with octapeptide GR8.

[0031] Figure 6 It is Figure 5 The statistical chart of the fluorescence intensity of neutrophils in

[0032] Figure 7 Effect of octapeptide GR8 on zebrafish INF-γ Specific implementation mode

[0033] The present invention will be further described below in conjunction with specific embodiments. The following embodiments are only used to illustrate the present invention and are not used to limit the scope of application of the present invention. Without departing from the spirit and essence of the present invention, any modification or replacement of the methods, steps or conditions of the present invention shall fall within the scope of the present invention.

[0034] Unless otherwise specified, the test methods used in the following examples are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, are reagents and materials that can be obtained from commercial channels.

[0035] Wheat protein (gluten) was purchased from Binzhou Zhongyu Food Co., Ltd.; alkaline protease (derived from Bacillus licheniformis) was purchased from Angel Enzyme Preparation (Yichang) Co., Ltd.; neutral protease (derived from Bacillus subtilis) was purchased from Nanning Pangbo Bioengineering Co., Ltd.; flavor protease was purchased from Angel Enzyme Preparation (Yichang) Co., Ltd.

[0036] Example 1: Screening of active peptide segments

[0037] 1. Preparation of wheat peptides

[0038] According to the mass ratio of water to wheat protein (gluten) of 10:1, add them into the reaction kettle. After the feeding is completed, adjust the pH of the feed liquid to 8.0 ± 0.2, then add alkaline protease (accounting for 1.0% of the total weight of gluten) and enzymolyze for 30 min. During the enzymolysis process, continuously maintain the pH of the feed liquid ≥ 7.5. After the end, add neutral protease (accounting for 1.5% of the total weight of gluten) and enzymolyze for 60 min. After the enzymolysis is completed, use flavor protease (accounting for 0.5% of the total weight of gluten) to act for 30 min, and then place it at 100 °C to inactivate the enzyme for 30 min. Subsequently, concentrate the sugar degree of the feed liquid to 25 °Bx and then perform spray drying to obtain wheat peptide powder.

[0039] 2. Screening of active peptide segments

[0040] Perform LC-MS / MS peptide spectrum analysis on wheat peptides, organize the obtained peptide segments of wheat peptides according to the screening conditions, and then further use molecular docking to determine the final theoretical effective peptide segments according to the scores. The specific analysis process is as follows:

[0041] (a) Identification of wheat peptide sequence

[0042] Dissolve the wheat peptide sample in NH 4 HCO 3In the solution, add dithiothreitol solution and place it in a water bath at 56 °C for 1 h for reduction. Subsequently, add iodoacetamide solution and react in the dark for 40 min. After desalting, evaporate the solvent to dryness, and then dissolve the sample with 10 μL of mobile phase A (0.1% formic acid) into a liquid chromatography injection vial. Subsequently, LC-MS / MS analysis is carried out.

[0043] Chromatographic conditions: Analytical column (Acclaim PepMap RPLC C18, 150×150 mm, 3 μm); mobile phase A (0.1% formic acid); mobile phase B (0.1% formic acid and 80% acetonitrile); flow rate (600 nL / min). Gradient elution program: 0 - 2 min, 4% B - 8% B; 2 - 45 min, 8% B - 40% B; 45 - 55 min, 40% B - 60% B; 55 - 56 min, 60% B - 95% B; 56 - 66 min, 95% B.

[0044] Mass spectrometry conditions: For full-scan MS, Orbitrap is used for the first-level scan, the scan range is (100~1500 m / z), the resolution is (70000), the maximum ion injection time is (100 ms), and the automatic gain control is (3×10 6 ); High-energy collision dissociation is used to fragment the top 20 precursor ions that meet the tandem (MS / MS) fragmentation conditions and scan with Orbitrap, the resolution is (17500), the maximum ion injection time is (50 ms), and the automatic gain control is (1×10 5 ). The raw data obtained by mass spectrometry is analyzed using the De novo software of PEAKS Studio for polypeptide sequence parsing.

[0045] (b) Screening of peptides with potential immunomodulatory function

[0046] Acetylcholine receptors play an important role in the immune system. Research shows that acetylcholine can inhibit the inflammatory response and enhance the function of immune cells by binding to nicotinic receptors on immune cells. In this example, the peptide segments of wheat peptides are molecularly docked with acetylcholine receptors to screen out peptide segments with potential immunomodulatory functions.

[0047] Peptide segments that meet the conditions are screened according to the conditions that the average local confidence (ALC) is greater than 95%, the peak area is greater than 2×10 6 , and the PeptideRanker score is greater than 0.8. Subsequently, the peptide segments are molecularly docked with the M1-muscarinic acetylcholine receptor (CHRM1).

[0048] First, download the crystal structure (5CXV) of CHRM1 from the Protein Data Bank (PDB). After removing water molecules and adding hydrogen atoms to the receptor target through Discovery Studio software, define its active center. The structure of the selected wheat peptide segments was constructed by Discovery Studio, and its energy was minimized by the CHARMm force field. These peptides were defined as ligands. The constructed peptides were docked with CHRM1 using CDOCKER to simulate the binding mode, site, and amino acid residues involved with the lowest binding energy and highest binding degree, and screened according to the binding energy and the number of hydrogen bonds. Finally, an octapeptide (GFNDLGKR) was determined, as shown in Table 1.

[0049] Table 1. Peptide segments with potential immunomodulatory activity in wheat peptides

[0050] Peptide sequence ALC score (%) Length Mass-to-charge ratio (m / z) Charge number (z) Relative abundance Molecular weight (Da) PeptideRanker score Docking energy (kcal / mol) GFNDLGKR 96.8 8 454.234 2 4.90E+06 906.456 0.697 -161.15

[0051] The secondary mass spectrum of octapeptide GR8 is as shown in Figure 1 . The cleavage fragment ions of peptides include: N-terminal fragment ions (types a, b, c) and C-terminal fragment ions (types x, y, z). The side chain cleavage of a, y, and z type ions forms d, v, w type ions respectively. In addition, there are internal ions formed by the cleavage at both ends, etc. The b and y series ions are the most common. The primary structure of the peptide can be deduced and analyzed based on the b or y series fragment ions of the peptide. 454.234 m / z is the [M+H]+ ion signal of octapeptide GR8, with a charge number (z) of 2 and a molecular weight of 906.456 Da, which is basically consistent with octapeptide GFNDLGKR. Further, the octapeptide was analyzed by in-source collision-induced dissociation technology for secondary mass spectrometry (see Figure 1 ), and the primary structure of the octapeptide was determined to be Gly-Phe-Asn-Asp-Leu-Gly-Lys-Arg.

[0052] The 2D and 3D maps of the molecular docking of octapeptide GR8 with CHRM1 are as shown in Figure 2 . Analysis of the chemical bonds found that it binds to CHRM1 mainly through van der Waals forces, hydrogen bonds (including conventional hydrogen bonds and carbon-hydrogen bonds), hydrophobic interactions (alkyl groups), and electrostatic interactions (including salt bridges and attractive charges), with a docking energy of -161.15 kcal / mol.

[0053] The octapeptide GR8 forms 13 van der Waals forces with amino acid residues GLN1068, ILE1008, GLY1011, THR1141, THR1020, MET1105, TYR1023, GLY1106, THR1025, ASP1019, ARG1147, ASN1143 and PRO1142, 9 hydrogen bonds with PHE1103, GLY1029, ASP1009, ARG1144, GLU1010, LYS1034 and GLN1104, 1 hydrophobic interaction with LEU1031, and 4 electrostatic interactions with GLU1021, ASP1069, LYS1034 and ARG1144.

[0054] From the molecular docking results, it can be seen that the octapeptide GR8 can bind to CHRM1, thereby activating CHRM1 and then exerting an immunomodulatory effect.

[0055] The peptide segment GFNDLGKR with a purity of ≥98% was synthesized by Shenzhen Borun Sida Biotechnology Co., Ltd. for subsequent functional verification.

[0056] Example 2: Proliferation effect of octapeptide (GFNDLGKR) on macrophages

[0057] In this invention, a zebrafish model was used to characterize the immune function of the peptide segment. The zebrafish has a genomic similarity of about 87% with humans and a high homology. Therefore, the immune system of zebrafish is similar to that of humans, and its immune response can reliably simulate and predict human physiological characteristics. The immune system of zebrafish is complete, with an innate immune system and an adaptive immune system. The main cells in the innate immune system of zebrafish are macrophages and neutrophils, which are similar to those in mammals and develop early. Macrophages and neutrophils appear in the embryo 30 h after fertilization. T lymphocytes and B lymphocytes start to develop after 4 dpf, and the adaptive immune system is not fully mature until four to six weeks after fertilization. That is to say, the immune function of zebrafish in the embryonic stage is mainly based on the innate immune system. Zebrafish have immune molecules participating in the immune response in the embryonic stage, such as TLRs, TNF and ILs. TLRs with MyD88 as the receptor protein can bind to the pattern recognition receptors of pathogens, thereby activating signal pathways such as MAPK and NF-κB. Therefore, some substances with immunomodulatory activity can affect the innate immune system of zebrafish embryos through these immune molecules, and indicators such as the number of macrophages, the number of neutrophils and the content of certain cytokines such as IFN-γ can measure the immunomodulatory activity of these substances on zebrafish.

[0058] Macrophages are an important part of the innate immune system, originating from progenitor cells in the bone marrow and tissue-resident macrophages. Macrophages can kill bacteria and eliminate healthy and abnormal cells through phagocytosis, and secrete cytokines with different functions. When pathogens invade or cells are damaged, macrophages polarize into different phenotypes according to the physiological conditions of the body: pro-inflammatory macrophages (M1 type) and anti-inflammatory macrophages (M2 type). M1 type can secrete pro-inflammatory factors such as IL-6, IL-1β and TNF-α, as well as substances such as NO and reactive oxygen species that enhance the bactericidal ability; M2 type can secrete anti-inflammatory cytokines and participate in processes such as angiogenesis and fibrosis and repair tissues. Therefore, the characterization of the number of macrophages is an important indicator of immune enhancement.

[0059] In this example, wild AB zebrafish were modeled with chloramphenicol to reduce the number of macrophages and create an immune deficiency model, and the octapeptide GFNDLGKR was used for intervention to characterize the effect of the octapeptide on the number of macrophages, which are important immune cells.

[0060] At 24 hpf, 10 μL / mL of phenylthiourea mother liquor (PTU) was added to wild AB zebrafish embryos. Demembranation was carried out at 48 hpf. The demembranated embryos were placed in a six-well plate, with 2 parallel wells in each group and 10 embryos in each well. The groups were as follows:

[0061] (1) Blank control group (NC): 0.5% DMSO + system water + PTU;

[0062] (2) Model group (MC): 125 μg / mL chloramphenicol (dissolved in DMSO) + system water + PTU, with the volume fraction of DMSO being 0.5%;

[0063] (3) Wheat peptide control group (WP-100): WP 100 μg / mL + 125 μg / mL chloramphenicol (dissolved in DMSO) + system water + PTU, with the volume fraction of DMSO being 0.5%;

[0064] (4) Wheat peptide control group (WP-10): WP 10 μg / mL + 125 μg / mL chloramphenicol (dissolved in DMSO) + system water + PTU, with the volume fraction of DMSO being 0.5%;

[0065] (5) Octapeptide intervention groups (GR8-50, GR8-10, GR8-5, GR8-1, GR8-0.1): The GFNDLGKR octapeptide (GR8) was added at 50 / 10 / 5 / 1 / 0.1 μg / mL respectively + 125 μg / mL chloramphenicol (dissolved in DMSO) + system water + PTU, with the volume fraction of DMSO being 0.5%.

[0066] After the addition of the drug for 24 h, 2.5 μg / mL neutral red dye and PTU were added to each well, and the cells were stained for 6 h in the dark. After washing with system water in the dark, the embryos were fixed with 6% methyl cellulose after anesthesia, and the number of head macrophages was photographed and counted under a stereomicroscope. Using the model group as a control, data analysis was performed using SPSS 22.0 and GraphPad Prism 8.0 software, and Tukey's test in One-way ANOVA was used to compare the differences between multiple groups.

[0067] The results were as Figure 3 and Figure 4 shown. After modeling with chloramphenicol (125 μg / mL), an immunosuppressive model was successfully established, and the number of macrophages was significantly lower than that of the normal control group ( p <0.0001). When the intervention concentration of wheat peptides was 100 μg / mL, it could significantly promote the proliferation of macrophages ( p <0.05), while when the intervention concentration of wheat peptides was 10 μg / mL, there was no promoting effect on macrophage proliferation. However, when the octapeptide GFNDLGKR (GR8) was used to intervene in immunosuppressed zebrafish, when the intervention concentration was 1 - 10 μg / mL, it could significantly promote the proliferation of macrophages ( p <0.05), indicating that from the perspective of the important immune cell macrophages, the immune-enhancing effect of the octapeptide GFNDLGKR was about 100 times that of wheat peptides.

[0068] Example 3: Proliferation effect of octapeptide (GFNDLGKR) on neutrophils

[0069] Among the white blood cells in blood circulation, neutrophils derived from bone marrow stem cells account for the highest proportion. When pathogens invade the body, neutrophils arrive at the infection site earliest and kill and phagocytose pathogens by secreting a variety of cytotoxic granules and proteases. In addition, they also play roles such as recruiting monocytes and repairing damaged tissues. Therefore, neutrophils are also an important type of immune cell.

[0070] Neutrophils in zebrafish embryos originate from myeloid progenitor cells in the embryonic liver or yolk sac. These cells differentiate into neutrophils at the front end of the hematopoietic region, enter the blood circulation, and then reach all parts of the body. In immunological experiments, fluorescent transgenic zebrafish larvae with neutrophils are usually used as a model, and the fluorescence intensity or number of neutrophils from the excretory pore to the end of the tail is directly observed under a fluorescence microscope.

[0071] In this example, Tg(Lyz:DsRed) neutrophil fluorescent transgenic zebrafish larvae were used as a model. Chloramphenicol was used to establish a model to reduce the number of neutrophils and create an immune deficiency model. The octapeptide GFNDLGKR was used for intervention to characterize the effect of the octapeptide on the number of neutrophils, which are important immune cells.

[0072] At 24 hpf of Tg(Lyz:DsRed) zebrafish embryos, 10 μL / mL of phenylthiourea mother liquor (PTU) was added. Demembranation was performed at 48 hpf. Embryos expressing the fluorescent gene were screened under a stereomicroscope with fluorescence. The demembranated embryos with the fluorescent gene were placed in a six-well plate, with 2 parallel wells in each group and 10 embryos in each well. The grouping was as follows:

[0073] (1) Blank control group (NC): 0.5% DMSO + system water + PTU;

[0074] (2) Model group (MC): 125 μg / mL chloramphenicol (dissolved in DMSO) + system water + PTU, with the volume fraction of DMSO being 0.5%;

[0075] (3) Wheat peptide control group (WP-100): 100 μg / mL WP + 125 μg / mL chloramphenicol (dissolved in DMSO) + system water + PTU, with the volume fraction of DMSO being 0.5%;

[0076] (4) Wheat peptide control group (WP-10): 10 μg / mL WP + 125 μg / mL chloramphenicol (dissolved in DMSO) + system water + PTU, with the volume fraction of DMSO being 0.5%;

[0077] (5) Octapeptide intervention groups (GR8-50, GR8-10, GR8-5, GR8-1, GR8-0.1): GFNDLGKR octapeptide (GR8) was added at 50 / 10 / 5 / 1 / 0.1 μg / mL respectively + 125 μg / mL chloramphenicol (dissolved in DMSO) + system water + PTU, with the volume fraction of DMSO being 0.5%.

[0078] After co-action for 24 h, the embryos were washed, anesthetized, and photographed under a stereomicroscope with fluorescence. The fluorescence intensity from the cloaca to the end of the tail was statistically analyzed. The SPSS 22.0 software was used to perform a significance test on the model group and other groups.

[0079] The results are as Figure 5 and Figure 6 shown. After establishing a model with chloramphenicol (125 μg / mL), an immune deficiency model was successfully created, and the number of neutrophils was significantly lower than that of the normal control group ( p<0.0001). When the intervention concentration of wheat peptides was 100 μg / mL, although there was no significant difference in the fluorescence intensity of neutrophils compared with the model group ( p >0.05), there was no significant difference between the group intervened with 100 μg / mL wheat peptides and the normal group ( p >0.05); when the concentration of wheat peptides was 10 μg / mL, there was still a significant difference compared with the normal group ( p <0.05). However, when the octapeptide GFNDLGKR (GR8) intervened in immunocompromised zebrafish, it could significantly promote the proliferation of neutrophils when the intervention concentration was 1-10 μg / mL ( p <0.05), indicating that from the perspective of neutrophils, an important immune cell, the immune-enhancing effect of the octapeptide GFNDLGKR is at least 100 times that of wheat peptides.

[0080] Example 4: Up-regulation effect of octapeptide (GFNDLGKR) on immune factor INF-γ

[0081] Interferon (INF) is a class of glycoproteins with various biological activities. Interferons are divided into type I and type II. Type I mainly includes interferon-β and interferon-α, and type II only includes one kind, interferon-γ (INF-γ). IFN-γ can be secreted by T cells, NK cells, macrophages and neutrophils, and is the main macrophage-activating factor in mammals. Different from interferon-β and interferon-α, INF-γ is an immune regulatory interferon, and its immune regulatory effect is dozens of times stronger than that of antiviral interferon. Therefore, INF-γ is an important immune factor characterizing immune function.

[0082] In this example, wild AB-strain zebrafish were used to establish a model with chloramphenicol to reduce the level of INF-γ and create an immunocompromised model, and the octapeptide GFNDLGKR was used for intervention to characterize the effect of the octapeptide on the level of the important immune factor INF-γ.

[0083] When wild AB-strain zebrafish embryos reached 24 hpf, 10 μL / mL of phenylthiourea mother liquor (PTU) was added, and the embryos were dechorionated at 48 hpf. The dechorionated embryos were placed in a six-well plate, with 2 parallel wells in each group and 10 embryos in each well. The grouping was as follows:

[0084] (1) Blank control group (NC): 0.5 % DMSO + system water + PTU;

[0085] (2) Model group (MC): 150 μg / mL chloramphenicol (dissolved in DMSO) + system water + PTU, with the volume fraction of DMSO being 0.5%;

[0086] (3) Wheat peptide control group (WP-100): WP 100 μg / mL + 150 μg / mL chloramphenicol (dissolved in DMSO) + system water + PTU, with the volume fraction of DMSO being 0.5%;

[0087] (4) Wheat peptide control group (WP-10): WP 10 μg / mL + 150 μg / mL chloramphenicol (dissolved in DMSO) + system water + PTU, with the volume fraction of DMSO being 0.5%;

[0088] (4) Octapeptide intervention groups (GR8-50, GR8-10, GR8-5, GR8-1, GR8-0.1): The octapeptide GFNDLGKR (GR8) was added at 50 / 10 / 5 / 1 / 0.1 μg / mL respectively + 150 μg / mL chloramphenicol (dissolved in DMSO) + system water + PTU, with the volume fraction of DMSO being 0.5%.

[0089] After culturing in an incubator at 28.5 °C for 24 h, 120 embryos of each group were placed into 1.5 mL centrifuge tubes. The embryos were washed with PBS buffer at a volume 1-fold of the total volume of each well, and after sucking out the residual PBS buffer, PBS buffer was added according to the ratio of 50 μL of liquid per 10 mg of sample. After homogenizing by high-speed tissue grinder for 90 s and centrifuging (4 °C, 5000 r / min, 5 min), the supernatant was taken. The content of INF-γ in the supernatant was measured using an INF-γ ELISA kit (Wuhan Gene Beauty Biotechnology Co., Ltd.) according to the kit instructions. Data analysis was performed using SPSS 22.0 and GraphPad Prism 8.0 software, and the Tukey test in One-way ANOVA was used to compare the differences among multiple groups.

[0090] The results were as Figure 7 shown. After modeling with chloramphenicol (150 μg / mL), an immune deficiency model was successfully established, and the INF-γ level was significantly lower than that of the normal control group ( p < 0.001). When the intervention concentration of wheat peptide was 100 μg / mL, it could significantly up-regulate the INF-γ level ( p < 0.001). When the intervention concentration of wheat peptide was 10 μg / mL, it had no effect on regulating the INF-γ level. However, when the octapeptide GFNDLGKR (GR8) intervened in immune-deficient zebrafish, when the intervention concentration was between 5 - 10 μg / mL, it could significantly up-regulate the INF-γ level ( p < 0.0001). When the intervention concentration was 1 μg / mL, although there was no significant difference from the model group ( p > 0.05), there was also no significant difference from the normal group ( p> 0.05), indicating that the immune enhancement effect of the octapeptide GFNDLGKR is about 20 times that of wheat peptide in terms of the level of the important immune factor INF-γ.

[0091] In summary, through mass spectrometry identification and molecular docking of peptide segments, the present invention screened the octapeptide GFNDLGKR from wheat peptides. This peptide segment showed highly efficient immunity-enhancing activity in an immunocompromised zebrafish animal model, mainly reflected in promoting the proliferation of macrophages and neutrophils and increasing the level of the immune factor INF-γ. The present invention provides a theoretical basis for the development of new active substances with immune-enhancing effects and is used to prepare health foods or drugs for preventing and improving adverse phenomena of immunodeficiency. Specifically, the octapeptide GFNDLGKR can be used alone to prepare health products or drugs for enhancing immunity, or can be used in combination with other active ingredients with immune-enhancing effects. The active components with immune-enhancing effects may include plant extracts and other protein peptides (such as sea cucumber peptides, soybean peptides, etc.). The preparation form of the health food or drug can be made into microcapsules to improve the gastrointestinal digestion stability, bioavailability and extend the shelf life of the polypeptide, so that it can be better applied to the food industry and the health care field.

Claims

1. An octapeptide GR8 having an immune-enhancing effect, characterized in that: The amino acid sequence of the octapeptide GR8 is Gly-Phe-Asn-Asp-Leu-Gly-Lys-Arg.

2. The method for preparing the octapeptide GR8 according to claim 1, characterized in that: The octapeptide GR8 is prepared by solid phase synthesis; or obtained by sequentially enzymatically hydrolyzing wheat gluten protein with alkaline protease, neutral protease and flavor protease.

3. The use of the octapeptide GR8 according to claim 1 in the preparation of an immunomodulatory product, characterized in that: The product is a drug for treating immunodeficiency or a health food for enhancing immunity; the immune regulation is at least one of increasing the proliferation of immune cells and increasing the level of immune factors, the immune cells are macrophages and neutrophils; the immune factor is interferon-γ.

4. The use according to claim 3, characterized in that The manifestations of the immunodeficiency are: a decrease in the number of immune cells in the body and a decrease in the level of interferon-γ.

5. A pharmaceutical composition for treating immunodeficiency diseases, characterized in that: The active ingredient of the pharmaceutical composition is octapeptide GR8 with an amino acid sequence of Gly-Phe-Asn-Asp-Leu-Gly-Lys-Arg.

6. The pharmaceutical composition according to claim 5, characterized in that Also included are pharmaceutically acceptable carriers.

7. The pharmaceutical composition according to claim 6, characterized in that The pharmaceutically acceptable carrier includes one or more of a filler, a wetting agent, a disintegrant, a binder or a lubricant.

8. A health food for enhancing immunity, characterized in that: The invention comprises octapeptide GR8 as an active ingredient and excipients acceptable in food science, wherein the amino acid sequence of the octapeptide GR8 is Gly-Phe-Asn-Asp-Leu-Gly-Lys-Arg.

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