Octapeptide with immunity improving effect and preparation and application thereof

Abstract

The invention discloses an octapeptide with an immunity improving effect as well as preparation and application thereof, and belongs to the technical field of biological medicines. The amino acid sequence of the octapeptide is Gly-Phe-Asn-Asp-Leu-Gly-Lys-Arg, and the amino acid sequence of the octapeptide is shown in the description. The peptide fragment can be obtained through artificial synthesis or directional enzymolysis of wheat protein, and functional verification shows that the octapeptide has the function of promoting immune cell proliferation, and the function is mainly characterized in that the number of macrophages and neutrophils is remarkably increased, and the level of immune factor interferon-gamma is increased. The invention provides a novel active substance for improving the immune function, which can be applied to preparation of food or medicines for preventing and improving the poor phenomenon of immunosuppression, and has good market prospect and application potential.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an octapeptide having an immunity-enhancing effect and a preparation and application thereof. Background Art

[0002] The immune system is the body's key mechanism for defending against foreign pathogens and abnormal cells. It is responsible for identifying and eliminating potential threats and maintaining a healthy state. It protects the body through three lines of defense: tissue barriers, innate immunity, and adaptive immunity, to prevent the occurrence of infectious diseases and cancer.

[0003] Low immunity is a common sub-health condition that can easily lead to symptoms such as colds and fevers, and may even cause cancer in severe cases. In particular, due to stress, irregular work and rest, and unhealthy diet, people face the problem of impaired immune system. Therefore, improving immune function has become a focus of public attention.

[0004] Bioactive peptides refer to a class of small molecule peptides with biological functions, usually composed of less than 20 amino acid residues. They are widely used in the fields of food, medicine and cosmetics, and have diverse functions, including antibacterial, anti-tumor, antihypertensive, anti-lipid and immunomodulatory. For example, the thick-shelled mussel active peptides prepared by enzymatic hydrolysis can achieve immunomodulatory effects through the NF-κB / MAPK signaling pathway (He Kang. Preparation of thick-shelled mussel active peptides and research on their immunomodulatory effects [D]. Zhejiang Ocean University, 2021.); calf thymosin can increase the level of immune-related cytokines in immunosuppressed mice and increase the activity of mouse killer immune cells (Li Lanzhou. Research on the anti-colorectal cancer activity and hematopoietic function of calf thymosin based on immunomodulation [D]. Jilin University, 2021.).

[0005] Wheat peptides are made from wheat protein (gluten powder) through bio-enzymatic hydrolysis, refining, spray drying and other processes. Studies have shown 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 properties 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 to enhance immunity. Therefore, by analyzing the peptide spectrum of wheat peptides and screening out high-efficiency peptides related to immune enhancement, it will provide a basis for the development of new immunomodulators. Summary of the invention

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

[0007] To achieve the above object, the present invention adopts the following technical solution: The present invention uses LC-MS / MS peptide mapping technology to analyze the polypeptide sequence in wheat peptides, and then uses molecular docking technology to explore the interaction between peptides and acetylcholine receptors, and screens out a candidate peptide. After mass spectrometry identification, its amino acid sequence is Gly-Phe-Asn-Asp-Leu-Gly-Lys-Arg (GFNDLGKR), and its molecular weight is 906.456 Da, which is named GR8. Further, through artificial synthesis of octapeptide GR8, functional verification found that the octapeptide showed a significant immune-enhancing effect in an immunocompromised animal model.

[0008] Therefore, the present invention provides an octapeptide GR8 having an immunity-enhancing effect, wherein the amino acid sequence of the octapeptide GR8 is Gly-Phe-Asn-Asp-Leu-Gly-Lys-Arg.

[0009] The present invention also provides a method for preparing the octapeptide GR8. The octapeptide GR8 can be prepared by solid phase synthesis. The specific method includes: adopting Fmoc solid phase synthesis strategy, using Fmoc protected amino acids as raw materials, selecting Wang resin as a solid phase carrier, sequentially introducing arginine, lysine, glycine, leucine, aspartic acid, asparagine, phenylalanine, and glycine residues, extending the peptide chain from the C-terminus to the N-terminus, and solid phase synthesizing the octapeptide GR8.

[0010] The octapeptide GR8 can also be obtained by enzymolysis of wheat gluten protein. Specifically, water and wheat protein (gluten powder) are added into a reactor at a mass ratio of 10:1. After the feeding is completed, the pH of the feed solution 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 enzymolysis for 30 minutes. The pH of the feed solution is maintained at ≥7.5 during the enzymolysis process. After the enzymolysis is completed, neutral protease (accounting for 1.5% of the total weight of gluten powder) is added for enzymolysis for 60 minutes. After the enzymolysis is completed, flavor protease (accounting for 0.5% of the total weight of gluten powder) is used for 30 minutes, and then the enzyme is inactivated at 100°C for 30 minutes. Subsequently, the wheat peptide powder containing the octapeptide GR8 is obtained by concentration and drying.

[0011] The present invention provides the use of the octapeptide GR8 in the preparation of an immunomodulatory product, wherein the immunomodulation includes at least one of increasing the proliferation of immune cells and increasing the level of immune factors. The present invention's research shows that the treatment of the octapeptide GR8 in an immunosuppressed animal model can significantly increase the number of immune cells such as macrophages and neutrophils; and significantly increase the content of immune factors such as INF-γ, and it can be used to develop related products for regulating immune function.

[0012] Furthermore, the immune cells include macrophages and neutrophils; and the immune factors include interferon-γ.

[0013] Furthermore, the product is a drug for treating immunosuppression or a food for enhancing immunity. The drug can be used to treat immunosuppression diseases, and the disease can refer to a disease accompanied by a state of immunosuppression, which requires administration of an immunopotentiator or is more beneficial for alleviating symptoms by administration of an immunopotentiator. The food can be used to improve the sub-healthy state of immunosuppression.

[0014] Furthermore, the manifestations of immunosuppression include: a decrease in the number of immune cells in the body and a decrease in the level of interferon-γ.

[0015] Furthermore, the product is a medicine for alleviating immunosuppression in mammals or a food for enhancing immunity in mammals. The mammals may be, but are not limited to, humans.

[0016] The present invention also provides a pharmaceutical composition for treating immunodeficiency diseases, wherein the active ingredient of the pharmaceutical composition comprises octapeptide GR8 having an amino acid sequence of Gly-Phe-Asn-Asp-Leu-Gly-Lys-Arg.

[0017] The present invention uses the octapeptide GR8 as the main active ingredient, adds a pharmaceutically acceptable carrier, and can be prepared according to the preparation method recorded in pharmacy. The octapeptide GR8 in the pharmaceutical composition provided by the present invention can be used as the only active ingredient that plays an immune-enhancing role, or it can be compounded with other active ingredients that have immune-regulating effects, such as sea cucumber peptides and soybean peptides.

[0018] Furthermore, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, which comprises one or more of a filler, a wetting agent, a disintegrant, a binder or a lubricant.

[0019] Furthermore, the pharmaceutical composition may be in the form of, but not limited to, oral liquid, capsule, microcapsule powder, tablet, granule or emulsion.

[0020] The specific dosage of the pharmaceutical composition is adjusted according to the type of disease, the severity of the disease, the age and the purpose of administration. In the immunocompromised zebrafish model, the effective concentration of the octapeptide GR8 is 1-10 μg / mL, and no toxic side effects are shown under this condition.

[0021] The present invention also provides a health food for enhancing immunity, comprising 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.

[0022] Furthermore, the health food can be a beverage, oral liquid, capsule, microcapsule powder, tablet, granule or emulsion.

[0023] The present invention has the following beneficial effects: The present invention provides an octapeptide GFNDLGKR with an immune-enhancing effect. The peptide can be obtained by artificial synthesis or directional enzymatic hydrolysis of wheat protein. Functional verification of animal models shows that the polypeptide has the effect of promoting the proliferation of immune cells, which is mainly manifested in significantly increasing the number of macrophages and neutrophils, and increasing the content of immune factor interferon-γ. Moreover, the octapeptide GFNDLGKR is a natural peptide with high biological safety. Therefore, it can be used to prepare food or medicine for preventing and improving the adverse phenomenon of immunosuppression. The present invention provides a new active substance for enhancing immune function, which meets the public's demand for immune health care and has good market prospects and application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the secondary mass spectrum of the octapeptide GR8.

[0025] Figure 2 Schematic diagram of the binding interaction between octapeptide GR8 and CHRM1.

[0026] Figure 3 This is a photograph of head macrophages observed under a stereomicroscope after the zebrafish model was treated with the octapeptide GR8.

[0027] Figure 4 for Figure 3 Statistical chart of the number of macrophages in the head.

[0028] Figure 5 This is a photograph of neutrophil fluorescence from the cloaca to the end of the tail observed under a stereomicroscope after the zebrafish model was treated with the octapeptide GR8.

[0029] Figure 6 for Figure 5 Statistical graph of neutrophil fluorescence intensity in .

[0030] Figure 7 This is the effect of octapeptide GR8 on zebrafish INF-γ. DETAILED DESCRIPTION

[0031] 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 intended to limit the scope of application of the present invention. Without departing from the spirit and essence of the present invention, modifications or replacements made to the inventive method, steps or conditions all belong to the scope of the present invention.

[0032] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0033] Wheat protein (gluten powder) was purchased from Binzhou Zhongyu Food Co., Ltd.; alkaline protease (derived from Bacillus licheniformis) was purchased from Angel Enzyme (Yichang) Co., Ltd.; neutral protease (derived from Bacillus subtilis) was purchased from Nanning Pangbo Bioengineering Co., Ltd.; flavor protease was purchased from Angel Enzyme (Yichang) Co., Ltd.

[0034] Example 1: Screening of active peptides 1. Preparation of wheat peptide Water and wheat protein (gluten) were added into the reactor at a mass ratio of 10:1. After the feeding was completed, the pH of the feed solution was adjusted to 8.0±0.2, and alkaline protease (accounting for 1.0% of the total weight of gluten) was added for enzymolysis for 30 minutes. The pH of the feed solution was maintained at ≥7.5 during the enzymolysis process. After the enzymolysis was completed, neutral protease (accounting for 1.5% of the total weight of gluten) was added for enzymolysis for 60 minutes. After the enzymolysis was completed, flavor protease (accounting for 0.5% of the total weight of gluten) was used for 30 minutes, and then the enzyme was inactivated at 100°C for 30 minutes. Subsequently, the sugar content of the feed solution was concentrated to 25°Bx and spray-dried to obtain wheat peptide powder.

[0035] 2. Screening of active peptides The wheat peptides were analyzed by LC-MS / MS peptide spectrum, and the peptide segments of the wheat peptides were sorted according to the screening conditions, and then molecular docking was further used to determine the final theoretical effective peptide segments according to the scores. The specific analysis process is as follows: (a) Wheat peptide sequence identification The wheat peptide samples were dissolved in NH 4 HCO 3 Add dithiothreitol solution to the solution and place it in a 56 °C water bath for 1 h. Then add iodoacetamide solution and react for 40 min in the dark. After desalting, evaporate the solvent and then dissolve the sample in 10 μL mobile phase A (0.1% formic acid) into a liquid injection bottle. Then perform LC-MS / MS analysis.

[0036] 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.

[0037] Mass spectrometry conditions: Full scan MS using Orbitrap for primary scanning, scan range (100-1500 m / z), resolution (70,000), maximum ion introduction time (100 ms), automatic gain control (3×10 6 ); The top 20 parent ions that met the MS / MS fragmentation conditions were fragmented using high-energy collision dissociation and scanned using Orbitrap, with a resolution of 17500, a maximum ion introduction time of 50 ms, and an automatic gain control of 1×10 5 The raw data obtained by mass spectrometry were analyzed for peptide sequence using the De novo (de novo sequencing) software of PEAKS Studio.

[0038] (b) Screening of peptides with potential to regulate immune function Acetylcholine receptors play an important role in the immune system. Studies have shown that acetylcholine can inhibit inflammatory responses and enhance the function of immune cells by binding to nicotinic receptors on immune cells. In this example, peptide segments of wheat peptides were molecularly docked with acetylcholine receptors to screen out peptide segments with potential immune regulation function.

[0039] According to the average local confidence (ALC) greater than 95%, the peak area greater than 2×10 6 , the peptides with a PeptideRanker score greater than 0.8 were screened to obtain qualified peptides. The peptides were then docked with the M1-muscarinic acetylcholine receptor (CHRM1).

[0040] First, the crystal structure of CHRM1 (5CXV) was downloaded from the PDB protein database. The active center was defined by removing water molecules and adding hydrogen atoms from the receptor target using the Discovery Studio software. The structure of the peptide segment of the selected wheat peptide was constructed by Discovery Studio, and its energy was minimized by the CHARMm force field, and these peptides were defined as ligands. The constructed peptide was docked with CHRM1 by CDOCKER to simulate the binding mode, site and amino acid residues with the lowest binding energy and the highest binding degree, and screened according to the binding energy and the number of hydrogen bonds. Finally, the octapeptide (GFNDLGKR) was determined, as shown in Table 1.

[0041] Table 1. Peptides with potential immune-regulating activity in wheat peptides 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 The secondary mass spectrum of the octapeptide GR8 is as follows Figure 1 As shown, the fragment ions of peptides include: N-terminal fragment ions (a, b, c types) and C-terminal fragment ions (x, y, z types). The side chains of a, y, and z-type ions break to form d, v, and w-type ions, respectively. In addition, there are internal ions formed by the breakage of both ends. The b and y series ions are the most common. The primary structure of the peptide can be analyzed and inferred based on the b or y series fragment ions of the peptide. 454.234 m / z is the [M+H]+ ion signal of the octapeptide GR8, with a charge number (z) of 2 and a molecular weight of 906.456 Da, which is basically consistent with the octapeptide GFNDLGKR. The octapeptide was further analyzed by secondary mass spectrometry using the in-source collision-induced dissociation technique (see Figure 1 ), the primary structure of the octapeptide was determined to be Gly-Phe-Asn-Asp-Leu-Gly-Lys-Arg.

[0042] The 2D and 3D images of the molecular docking of GR8 octapeptide and CHRM1 are shown in Figure 2 The chemical bond analysis revealed that it is mainly combined with CHRM1 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.

[0043] The octapeptide GR8 formed 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.

[0044] The molecular docking results show that the octapeptide GR8 can bind to CHRM1 and activate CHRM1, thereby exerting an immunomodulatory effect.

[0045] Shenzhen Borunsida Biotechnology Co., Ltd. was commissioned to synthesize the peptide GFNDLGKR with a purity of ≥98% for subsequent functional verification.

[0046] Example 2: Effect of Octapeptide (GFNDLGKR) on the Proliferation of Macrophages The present invention uses a zebrafish model to characterize the immune function of peptides. The zebrafish genome is about 87% similar to that of humans, and has high homology, so the zebrafish immune system is similar to that of humans, and its immune response can reliably simulate and predict the physiological characteristics of humans. The zebrafish immune system is complete, with an innate immune system and an adaptive immune system. The main cells in the zebrafish innate immune system are macrophages and neutrophils, which are similar to mammals and develop early. Macrophages and neutrophils appear in the embryo 30 hours after fertilization. T lymphocytes and B lymphocytes begin to develop after 4 dpf, and the adaptive immune system is fully mature four to six weeks after fertilization. In other words, the immune function of zebrafish in the embryonic stage is mainly based on the innate immune system. Zebrafish have immune molecules involved in immune responses in the embryonic stage, such as TLRs, TNF and ILs. TLRs with MyD88 as receptor protein can bind to the pattern recognition receptors of pathogens, thereby activating signaling 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. 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.

[0047] 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 pathogens and remove healthy abnormal cells through phagocytosis, and secrete cytokines with different functions. When pathogens invade or cells are damaged, macrophages will 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 NO, reactive oxygen species and other substances that have improved 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.

[0048] In this example, chloramphenicol was used to model wild AB zebrafish to reduce the number of macrophages, thereby creating an immunodeficiency model. The GFNDLGKR octapeptide was used for intervention to characterize the effect of the octapeptide on the number of important immune cells, macrophages.

[0049] At 24 hpf, 10 μL / mL phenylthiourea stock solution (PTU) was added to wild AB zebrafish embryos. The embryos were demembraned at 48 hpf and placed in a six-well plate. Each group had 2 parallel wells, 10 strips per well, and the groups were as follows: (1) Blank control group (NC): 0.5% DMSO + system water + PTU; (2) Model group (MC): 125 μg / mL chloramphenicol (dissolved in DMSO) + system water + PTU, DMSO volume fraction was 0.5%; (3) Wheat peptide control group (WP-100): WP 100 μg / mL + 125 μg / mL chloramphenicol (dissolved in DMSO) + system water + PTU, DMSO volume fraction is 0.5%; (4) Wheat peptide control group (WP-10): WP 10 μg / mL + 125 μg / mL chloramphenicol (dissolved in DMSO) + system water + PTU, DMSO volume fraction is 0.5%; (5) Octapeptide intervention group (GR8-50, GR8-10, GR8-5, GR8-1, GR8-0.1): GFNDLGKR octapeptide (GR8) was added with 50 / 10 / 5 / 1 / 0.1 μg / mL + 125 μg / mL chloramphenicol (dissolved in DMSO) + system water + PTU, respectively, and the volume fraction of DMSO was 0.5%.

[0050] After 24 hours of drug treatment, 2.5 μg / mL neutral red dye and PTU were added to each well and stained in the dark for 6 hours. The cells were washed with system water in the dark, and the embryos were fixed with 6% methylcellulose after anesthesia. The embryos were photographed under a stereomicroscope and the number of macrophages in the head was counted. The model group was used as the control, and SPSS 22.0 and GraphPad Prism 8.0 software were used for data analysis. Tukey test in One-way ANOVA was used to compare the differences among multiple groups.

[0051] The results are as follows Figure 3 and Figure 4 As shown in the figure, the immunosuppression model was successfully established after the use of chloramphenicol (125 μg / mL), and the number of macrophages was significantly lower than that of the normal control group ( p <0.0001). When the wheat peptide intervention concentration was 100 μg / mL, it could significantly promote the proliferation of macrophages ( p <0.05), when the wheat peptide intervention concentration was 10 μg / mL, it did not promote macrophage proliferation. However, when GFNDLGKR octapeptide (GR8) intervened in immunocompromised zebrafish, it could significantly promote macrophage proliferation when the intervention concentration was 1~10 μg / mL ( p <0.05), indicating that from the perspective of important immune cells macrophages, the immune-enhancing effect of GFNDLGKR octapeptide is about 100 times that of wheat peptide.

[0052] Example 3: Effect of octapeptide (GFNDLGKR) on the proliferation of neutrophils Among the white blood cells circulating in the blood, neutrophils derived from bone marrow stem cells account for the largest proportion. When pathogens invade the body, neutrophils are the first to arrive at the site of infection, killing and engulfing the pathogens by secreting a variety of cytotoxic granules and proteases. In addition, they also play a role in recruiting monocytes and repairing damaged tissues. Therefore, neutrophils are also an important type of immune cell.

[0053] 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 zone, enter the blood circulation, and then reach all parts of the body. In immunological experiments, neutrophil fluorescent transgenic zebrafish larvae are usually used as models to directly observe the fluorescence intensity or number of neutrophils from the excretory pore to the end of the tail under a fluorescence microscope.

[0054] In this example, Tg (Lyz: DsRed) neutrophil fluorescence transgenic zebrafish larvae were used as a model, chloramphenicol was used to create a model to reduce the number of neutrophils, creating an immunodeficiency model, and GFNDLGKR octapeptide was used for intervention to characterize the effect of the octapeptide on the number of neutrophils, an important immune cell.

[0055] At 24 hpf, 10 μL / mL phenylthiourea mother solution (PTU) was added to Tg (Lyz:DsRed) zebrafish embryos. The embryos were decapped at 48 hpf. The embryos expressing the fluorescent gene were screened under a stereofluorescence microscope. The decapped embryos with the fluorescent gene were placed in a six-well plate, with 2 parallel wells per group and 10 strips per well. The groups were as follows: (1) Blank control group (NC): 0.5% DMSO + system water + PTU; (2) Model group (MC): 125 μg / mL chloramphenicol (dissolved in DMSO) + system water + PTU, DMSO volume fraction was 0.5%; (3) Wheat peptide control group (WP-100): WP 100 μg / mL + 125 μg / mL chloramphenicol (dissolved in DMSO) + system water + PTU, DMSO volume fraction is 0.5%; (4) Wheat peptide control group (WP-10): WP 10 μg / mL + 125 μg / mL chloramphenicol (dissolved in DMSO) + system water + PTU, DMSO volume fraction is 0.5%; (5) Octapeptide intervention group (GR8-50, GR8-10, GR8-5, GR8-1, GR8-0.1): GFNDLGKR octapeptide (GR8) was added with 50 / 10 / 5 / 1 / 0.1 μg / mL + 125 μg / mL chloramphenicol (dissolved in DMSO) + system water + PTU, respectively, and the volume fraction of DMSO was 0.5%.

[0056] After 24 h of co-action, the embryos were washed, anesthetized, and photographed under a stereofluorescence microscope. The fluorescence intensity from the cloaca to the end of the tail was counted, and a significance test was performed between the model group and other groups using SPSS 22.0 software.

[0057] The results are as follows Figure 5 and Figure 6 As shown in the figure, the immunosuppression model was successfully established after the use of chloramphenicol (125 μg / mL), and the number of neutrophils was significantly lower than that of the normal control group ( p <0.0001). When the wheat peptide intervention concentration was 100 μg / mL, although there was no significant difference in the fluorescence intensity of neutrophils compared with the model group ( p >0.05), but there was no significant difference between the intervention group and the normal group after 100 μg / mL wheat peptide ( p >0.05); when the concentration of wheat peptide was 10 μg / mL, there was still a significant difference compared with the normal group ( p<0.05). However, when GFNDLGKR octapeptide (GR8) intervened in immunocompromised zebrafish, the intervention concentration was 1~10 μg / mL and could significantly promote the proliferation of neutrophils ( p <0.05), indicating that from the perspective of important immune cells, neutrophils, the immune-enhancing effect of GFNDLGKR octapeptide is at least 100 times that of wheat peptide.

[0058] Example 4: Upregulation of the immune factor INF-γ by the octapeptide (GFNDLGKR) Interferon (INF) is a class of glycoproteins with multiple biological activities. Interferons are divided into type I and type II. Type I is mainly interferon-β and interferon-α, and type II only includes interferon-γ (INF-γ). IFN-γ can be secreted by T cells, NK cells, macrophages, and neutrophils, and is the main macrophage activation factor in mammals. Unlike interferon-β and interferon-α, INF-γ is an immunomodulatory interferon, and its immunomodulatory effect is dozens of times stronger than that of antiviral interferon. Therefore, INF-γ is an important immune factor that characterizes immune effects.

[0059] In this example, wild AB zebrafish were modeled with chloramphenicol to reduce the level of INF-γ, thereby creating an immunodeficiency model. The GFNDLGKR octapeptide was used for intervention to characterize the effect of the octapeptide on the level of the important immune factor INF-γ.

[0060] At 24 hpf, 10 μL / mL phenylthiourea stock solution (PTU) was added to wild AB zebrafish embryos. The embryos were demembraned at 48 hpf and placed in a six-well plate. Each group had 2 parallel wells, 10 strips per well, and the groups were as follows: (1) Blank control group (NC): 0.5% DMSO + system water + PTU; (2) Model group (MC): 150 μg / mL chloramphenicol (dissolved in DMSO) + system water + PTU, DMSO volume fraction was 0.5%; (3) Wheat peptide control group (WP-100): WP 100 μg / mL + 150 μg / mL chloramphenicol (dissolved in DMSO) + system water + PTU, DMSO volume fraction is 0.5%; (4) Wheat peptide control group (WP-10): WP 10 μg / mL + 150 μg / mL chloramphenicol (dissolved in DMSO) + system water + PTU, DMSO volume fraction is 0.5%; (4) Octapeptide intervention group (GR8-50, GR8-10, GR8-5, GR8-1, GR8-0.1): GFNDLGKR octapeptide (GR8) was added with 50 / 10 / 5 / 1 / 0.1 μg / mL + 150 μg / mL chloramphenicol (dissolved in DMSO) + system water + PTU, respectively, and the DMSO volume fraction was 0.5%.

[0061] After culturing in a 28.5°C incubator for 24 h, 120 embryos in each group were placed in 1.5 mL centrifuge tubes and washed with PBS buffer (1 times the total volume of the liquid in each well). After the residual PBS buffer was aspirated, PBS buffer was added at a ratio of 50 μL of liquid for every 10 mg of sample. The homogenate was ground with a high-speed tissue grinder for 90 s and then centrifuged (4°C, 5 000 r / min, 5 min). The supernatant was taken and the INF-γ content in the supernatant was determined using an INF-γ ELISA kit (Wuhan Gene Beauty Biotechnology Co., Ltd.) according to the kit instructions. SPSS 22.0 and GraphPad Prism 8.0 software were used for data analysis, and the Tukey test in One-way ANOVA was used to compare the differences among multiple groups.

[0062] The results are as follows Figure 7 As shown in Figure 2, the immunodeficiency model was successfully established using chloramphenicol (150 μg / mL), and the INF-γ level was significantly lower than that in the normal control group ( p <0.001). When the wheat peptide intervention concentration was 100 μg / mL, it could significantly increase the level of INF-γ ( p <0.001), when the wheat peptide intervention concentration was 10 μg / mL, there was no effect on regulating INF-γ levels. However, when GFNDLGKR octapeptide (GR8) intervened in immunocompromised zebrafish, the INF-γ level was significantly increased when the intervention concentration was 5-10 μg / mL ( p <0.0001), when the intervention concentration was 1 μg / mL, there was no significant difference with the modeling group ( p >0.05), but there was no significant difference with the normal group ( p >0.05), indicating that from the perspective of the important immune factor INF-γ level, the immune-enhancing effect of GFNDLGKR octapeptide is about 20 times that of wheat peptide.

[0063] In summary, the present invention screened out the octapeptide GFNDLGKR from wheat peptides through mass spectrometry identification and molecular docking of peptide segments. The peptide segment showed high-efficiency activity in improving immunity in the zebrafish animal model with low immunity, which was mainly reflected in promoting the proliferation of macrophages and neutrophils and increasing the level of immune factor INF-γ. The present invention provides a theoretical basis for the development of new active substances that enhance immunity, and is used to prepare foods or drugs that prevent and improve the adverse phenomena of low immunity. Specifically, the octapeptide GFNDLGKR can be used alone to prepare health products or drugs that enhance immunity, or it can be used in combination with other active ingredients that enhance immunity. The active components that enhance immunity can include plant extracts and other protein peptides (sea cucumber peptides, soybean peptides, etc.). The preparation form of the health product or drug can be microcapsule to improve the gastrointestinal digestion stability, bioavailability and shelf life of the polypeptide, so that it can be better used in the food industry and health care fields.

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 enzymatic hydrolysis of wheat gluten protein.

3. The use of the octapeptide GR8 according to claim 1 in the preparation of an immunomodulatory product, characterized in that: The immune regulation includes at least one of increasing the proliferation of immune cells and increasing the level of immune factors.

4. The use according to claim 3, characterized in that The immune cells include macrophages and neutrophils; the immune factors include interferon-γ.

5. The use according to claim 3, characterized in that The product is a drug for treating immunodeficiency or a food for enhancing immunity.

6. The use according to claim 5, characterized in that The manifestations of immunosuppression include: a decrease in the number of immune cells in the body and a decrease in the level of interferon-γ.

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

8. The pharmaceutical composition according to claim 7, characterized in that Also included are pharmaceutically acceptable carriers.

9. The pharmaceutical composition according to claim 8, characterized in that The pharmaceutically acceptable carrier includes one or more of a filler, a wetting agent, a disintegrant, a binder or a lubricant.

10. 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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