Nonapeptide with immunomodulatory effect as well as preparation and application of nonapeptide

Abstract

The invention discloses a nonapeptide with an immunomodulatory effect as well as preparation and application thereof, and belongs to the technical field of biological medicines. The amino acid sequence of the nonapeptide QT9 is Gln-Pro-Gln-Pro-Pro-Phe-Ser-His-Thr, and the amino acid sequence of the nonapeptide QT9 is shown in the specification. The peptide fragment can be obtained through artificial synthesis or directional enzymolysis of wheat protein. Functional verification shows that the nonapeptide has the effects of promoting immune cell proliferation, improving the immune factor level, improving the immune organ index and improving the immune function of intestinal mucosa. Moreover, the nonapeptide QPQPPFSHT is a natural peptide fragment and is high in biological safety, so that the nonapeptide QPQPPFSHT can be applied to preparation of food or medicines for preventing and improving the undesirable phenomenon of immunosuppression. A new solution is provided for improving the immune function, the requirements of the public for immune health care are met, and good market prospects and application potential are achieved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a nonapeptide with immunomodulatory effect and a preparation and application thereof. Background Art

[0002] The immune system is an important system for the body to perform immune responses and immune functions. It is composed of immune organs, immune cells and immune molecules. The immune system has the function of identifying and eliminating antigenic foreign bodies, coordinating with other systems of the body, and jointly maintaining the stability of the body's internal environment and physiological balance.

[0003] Immunosuppression is a common sub-health condition. People with low immune function have difficulty resisting the invasion of bacteria, viruses, fungi, etc., which can easily lead to various serious infections, such as upper respiratory tract infection, urinary tract infection, sepsis, meningitis, chickenpox, measles, tuberculosis, etc., and may even cause cancer in severe cases. Compared with people with normal immune function, people with low immune function are more likely to get sick or their condition is more likely to worsen in the same environment.

[0004] According to the different causes of the disease, low immune function can be divided into primary and secondary types. Primary is caused by congenital maldevelopment, most of which are related to genetics and occur mostly in children; secondary is caused by infections such as viruses, bacteria, fungi, or drugs, tumors, fatigue, insomnia, malnutrition, excessive stress, etc., and can be seen in people of all ages. Therefore, improving immune function has become the focus of public attention.

[0005] 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, antilipidemic and immune regulation. 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. For example: Thymopentin is a fragment of amino acid residues 32 to 36 of thymopoietin II, which has the function of inducing T lymphocyte differentiation, maturation and activation, enhancing the activity of natural killer cells (NK) and the phagocytic function of macrophages, and is mainly used as an immunomodulator in clinical practice.

[0006] Other studies have shown that some small molecule peptides produced by protein hydrolysis have immunological activity. They can not only enhance the body's immunity, but also stimulate the proliferation of lymphocytes and enhance the phagocytic ability of macrophages. For example, Wu et al. extracted a novel peptide from wheat germ globulin and purified it to obtain a peptide with the sequence of Glu-Cys-Phe-Ser-Thr-Ala (ECFSTA). After immunological evaluation, they found that it can activate macrophages and can be used as an immunomodulator (Characterization and Immunomodulatory Activity of a Novel Peptide, ECFSTA, from Wheat GermGlobulin. Journal of Agricultural and Food Chemistry , 2017,65:5561-5569.).

[0007] Wheat peptides are made from wheat protein (gluten powder) through bio-enzymatic hydrolysis, refining, spray drying and other processes, such as patent document CN 112226478 A. Studies have shown that wheat peptides have the effect of enhancing immunity. Therefore, by analyzing the peptide spectrum of wheat peptides and screening out high-efficiency peptide segments related to immunity enhancement, it will provide a basis for the development of new immunomodulators. Summary of the invention

[0008] The purpose of the present invention is to provide a natural small molecule bioactive peptide with immunomodulatory effect, which can be used to develop safe and efficient immunomodulatory products.

[0009] 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 peptide sequence in wheat peptides, and then uses molecular docking technology to explore the interaction between the peptide and M1-muscarinic acetylcholine receptor (CHRM1), and screens out a candidate peptide. After mass spectrometry identification, its amino acid sequence is Gln-Pro-Gln-Pro-Pro-Phe-Ser-His-Thr (QPQPPFSHT), and its molecular weight is 1037.49 Da, which is named QT9. Further, through artificial synthesis of nonapeptide QT9, functional verification found that nonapeptide QT9 showed a significant effect of enhancing immune function in immunosuppressed animal models.

[0010] Therefore, the present invention provides a nonapeptide QT9 with immunomodulatory effect, wherein the amino acid sequence of the nonapeptide QT9 is Gln-Pro-Gln-Pro-Pro-Phe-Ser-His-Thr.

[0011] The present invention also provides a method for preparing the nonapeptide QT9. The nonapeptide QT9 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, and sequentially introducing threonine, histidine, serine, phenylalanine, proline, proline, glutamine, proline, and glutamine residues to extend the peptide chain from the C-terminus to the N-terminus, and solid phase synthesizing the nonapeptide QT9.

[0012] The nonapeptide QT9 can also be obtained by enzymolysis of wheat gluten. Specifically, water and wheat protein (gluten powder) are mixed in a mass ratio of 10:1, and the pH of the liquid is adjusted to 8.0 ± 0.2, and then 1.0% of the total weight of gluten powder is added for enzymolysis for 30 minutes. The pH of the liquid is maintained at ≥ 7.5 during the enzymolysis process. After the enzymolysis is completed, 1.5% of the total weight of gluten powder is added for enzymolysis for 60 minutes. After the enzymolysis is completed, 0.5% of the total weight of gluten powder is used for flavor protease for 30 minutes, and then the enzyme is inactivated at 100 ° C for 30 minutes. Subsequently, the wheat peptide powder is concentrated and dried to obtain the wheat peptide powder, which contains the nonapeptide QT9.

[0013] The present invention provides the use of the nonapeptide QT9 in the preparation of an immunomodulatory product, wherein the immunomodulation includes at least one of increasing the proliferation of immune cells, increasing the level of immune factors, increasing the immune organ index, and increasing the content of intestinal short-chain fatty acids. The present invention's research shows that the nonapeptide QT9 treatment in an immunosuppressed animal model can significantly increase the number of immune cells such as macrophages and neutrophils; significantly increase the content of immune factors such as immunoglobulin IgA and interferon-γ; significantly increase the immune organ index such as thymus index and spleen index; and significantly increase the content of intestinal short-chain fatty acids such as acetate, propionate, and butyrate, indicating that the nonapeptide QT9 has immunomodulatory activity and promotes the recovery of immune cells, so it can be used to develop related products for regulating immune function.

[0014] Furthermore, the immune cells include at least one of macrophages and neutrophils; the immune factors include at least one of immunoglobulin IgA and interferon-γ; the immune organ index includes a thymus index and a spleen index; and the intestinal short-chain fatty acids include at least one of acetate, propionate, and butyrate.

[0015] Furthermore, the product is a drug for preventing or treating immunosuppression. Specifically, the drug can be used to treat immunosuppression diseases, which may refer to diseases accompanied by a state of immunosuppression that require the administration of immunosuppressants or that are more conducive to relieving symptoms, such as immunodeficiency diseases, recurrent or refractory infectious diseases, which are caused by the body's low immune function, resulting in repeated attacks and a long treatment medication cycle. The drug can also be used to treat immunosuppression caused by the use of immunosuppressants such as glucocorticoids and cyclophosphamide.

[0016] Furthermore, the manifestations of immunosuppression include: a decrease in at least one of the following indicators: the number of immune cells, the level of immune factors, and the content of intestinal short-chain fatty acids.

[0017] As a specific embodiment of the present invention, the immunosuppression is immunosuppression caused by the use of immunosuppressants. Specifically, the immunosuppressant may be, but is not limited to, cyclophosphamide.

[0018] As a specific embodiment of the present invention, the immunosuppression is immunosuppression caused by the use of chloramphenicol. The toxic and side effects of chloramphenicol include a decrease in the number of immune cells leading to immunosuppression.

[0019] Furthermore, the product is a food for enhancing immunity. Specifically, the food is used to improve the sub-healthy state of low immune function.

[0020] 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.

[0021] The present invention provides a pharmaceutical composition for preventing or treating immunosuppression, comprising an effective dose of nonapeptide QT9 and a pharmaceutically acceptable carrier, wherein the amino acid sequence of the nonapeptide QT9 is Gln-Pro-Gln-Pro-Pro-Phe-Ser-His-Thr.

[0022] The present invention uses nonapeptide QT9 as the main active ingredient, adds a pharmaceutically acceptable carrier, and can be prepared according to the preparation method recorded in pharmacy. In the pharmaceutical composition provided by the present invention, nonapeptide QT9 can be used as the only active ingredient that exerts an immunomodulatory effect, or it can be compounded with other active ingredients that have an immunomodulatory effect, such as sea cucumber peptides and soybean peptides.

[0023] Further, the pharmaceutically acceptable carrier includes: one or more of a filler, a wetting agent, a disintegrant, a binder or a lubricant. The pharmaceutically acceptable carrier is any preparation or carrier medium that can deliver an effective dose of the active substance of the present invention, does not interfere with the biological activity of the active substance, and has no toxic side effects on the host or the subject.

[0024] Furthermore, the pharmaceutical composition may be in the form of, but not limited to, oral liquid, capsule, microcapsule powder, tablet, granule or emulsion.

[0025] 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 nonapeptide QT9 is 5 μg / mL; in the immunocompromised mouse model, the effective concentration of the wheat peptide containing the nonapeptide QT9 is 0.25~0.5g / kg mouse body weight. No toxic side effects were observed under the above conditions.

[0026] The present invention also provides a health food for enhancing immunity, comprising nonapeptide QT9 as an active ingredient and a food-based acceptable auxiliary material, wherein the amino acid sequence of the nonapeptide QT9 is Gln-Pro-Gln-Pro-Pro-Phe-Ser-His-Thr. The food-based acceptable auxiliary material is any preparation or carrier medium that can deliver an effective dose of the active substance of the present invention, does not interfere with the biological activity of the active substance, and has no toxic side effects on the host or the subject.

[0027] The present invention has the following beneficial effects: The present invention provides a nonapeptide QPQPPFSHT with immunomodulatory effects, and the peptide can be obtained by artificial synthesis or directed enzymatic hydrolysis of wheat protein. Animal model function verification shows that the peptide has the effect of promoting the proliferation of immune cells, which is mainly reflected in promoting the proliferation of macrophages and neutrophils, increasing the levels of immune factors IgA and IFN-γ, increasing the immune organ index, and improving the immune function of intestinal mucosa. Moreover, the nonapeptide QPQPPFSHT prepared by enzymatic hydrolysis of wheat protein is a natural peptide with high biosafety. Therefore, it can be used to prepare food or medicine for preventing and improving the adverse phenomena of immunosuppression. The present invention provides a new solution for improving immune function, meets the public's demand for immune health care, and has good market prospects and application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1This is the secondary mass spectrum of the nonapeptide QT9, in which #1:1 represents the number, Q / P / Q / P / P / F / S / H / T represents the nonapeptide sequence; the ordinate Intensity (%) represents the relative abundance; y1 represents the first fragment ion produced by the cleavage of the C-terminus of the peptide; b2 / b4[2+] represents the second fragment ion produced by the cleavage of the N-terminus of the peptide and the fourth fragment ion produced by the cleavage of the N-terminus of the peptide have two charges, b3 represents the third fragment ion produced by the cleavage of the N-terminus of the peptide, b4 represents the fourth fragment ion produced by the cleavage of the N-terminus of the peptide, b5 represents the fifth fragment ion produced by the cleavage of the N-terminus of the peptide, y5 represents the fifth fragment ion produced by the cleavage of the C-terminus of the peptide; y6 represents the sixth fragment ion produced by the cleavage of the C-terminus of the peptide, b6 represents the sixth fragment ion produced by the cleavage of the N-terminus of the peptide, y7 represents the seventh fragment ion produced by the cleavage of the C-terminus of the peptide, and y8-H2O represents the eighth dehydrated fragment ion produced by the cleavage of the C-terminus of the peptide.

[0029] Figure 2 Schematic diagram of the binding interaction between nonapeptide QT9 and CHRM1.

[0030] Figure 3 These are photos of head macrophages observed under a stereomicroscope after treatment with the nonapeptide QT9 in the zebrafish model.

[0031] Figure 4 for Figure 3 Statistical chart of the number of macrophages in the head, where * indicates a significant difference compared with the normal group, and * indicates p <0.05, **** means p <0.0001; # indicates significant difference compared with the modeling group, # indicates p <0.05.

[0032] Figure 5 These are photos of neutrophil fluorescence from the cloaca to the tail end observed under a stereomicroscope after treatment with the nonapeptide QT9 in the zebrafish model.

[0033] Figure 6 for Figure 5 Statistical graph of neutrophil fluorescence intensity in the neutrophil group, where * indicates significant difference compared with the normal group, **** indicates p <0.0001; # indicates significant difference compared with the modeling group, ### indicates p <0.001.

[0034] Figure 7 The effect of nonapeptide QT9 on zebrafish model INF-γ, where * indicates significant difference compared with the normal group, *** indicates p<0.001; # indicates significant difference compared with the modeling group, ## indicates p <0.01.

[0035] Figure 8 The effect of wheat peptide containing QT9 on food intake in a mouse model.

[0036] Fig. 9 The effect of wheat peptide containing QT9 on body weight in mouse model, where * indicates significant difference compared with the normal group, and * indicates p <0.05, *** indicates p <0.001.

[0037] Fig.10 The effect of wheat peptide containing QT9 on spleen index in mouse model, where * indicates significant difference compared with the normal group, **** indicates p <0.0001; # indicates significant difference compared with the modeling group, # indicates p <0.05.

[0038] Fig.11 The effect of wheat peptide containing QT9 on thymus index in mouse model, where * indicates significant difference compared with normal group, ** indicates p <0.01,**** indicates p <0.0001; # indicates significant difference compared with the modeling group, ## indicates p <0.01.

[0039] Fig.12 The effect of wheat peptide containing QT9 on serum immunoglobulin IgA in mouse model, where * indicates significant difference compared with the normal group, **** indicates p <0.0001; # indicates significant difference compared with the modeling group, ## indicates p <0.01, #### indicates p <0.0001.

[0040] Fig.13 The effect of wheat peptide containing QT9 on serum cytokine IFN-γ in mouse model, where * indicates significant difference compared with the normal group, ** indicates p <0.01,**** indicates p <0.0001; # indicates significant difference compared with the modeling group, #### indicates p <0.0001.

[0041] Fig.14The effect of wheat peptide containing QT9 on the acetic acid content in feces in the mouse model, where * indicates a significant difference compared with the normal group, and * indicates p <0.05,** indicates p <0.01; # indicates significant difference compared with the modeling group, #### indicates p <0.0001.

[0042] Fig.15 The effect of wheat peptide containing QT9 on the propionic acid content in feces in the mouse model, where * indicates a significant difference compared with the normal group, and * indicates p <0.05,** indicates p <0.01; # indicates significant difference compared with the modeling group, #### indicates p <0.0001.

[0043] Fig.16 The effect of wheat peptide containing QT9 on butyric acid content in feces in mice model, where * indicates significant difference compared with the normal group, and * indicates p <0.05,** indicates p <0.01, *** indicates p <0.001; # indicates significant difference compared with the modeling group, #### indicates p <0.0001. DETAILED DESCRIPTION

[0044] 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.

[0045] 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.

[0046] 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.

[0047] Example 1: Screening of active peptides 1. Preparation of wheat peptide (glutamine 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 enzymatic hydrolysis for 30 minutes. The pH of the feed solution was maintained at ≥ 7.5 during the enzymatic hydrolysis process. After the enzymatic hydrolysis was completed, neutral protease (accounting for 1.5% of the total weight of gluten) was added for enzymatic hydrolysis for 60 minutes. After the enzymatic hydrolysis 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 (glutamine peptide) powder.

[0048] 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 sample was dissolved in NH4HCO3 solution, dithiothreitol solution was added, and the sample was reduced in a 56 ℃ water bath for 1 h. Then iodoacetamide solution was added and reacted for 40 min in the dark. After desalting, the solvent was evaporated, and then 10 μL of mobile phase A (0.1% formic acid) was used to dissolve the sample into a liquid injection bottle. LC-MS / MS analysis was then performed.

[0049] 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.

[0050] 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 5The raw data obtained by mass spectrometry were analyzed for peptide sequence using the De novo (de novo sequencing) software of PEAKS Studio.

[0051] (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.

[0052] According to the average local confidence (ALC) greater than 95%, the peak area is 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).

[0053] 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 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, a nonapeptide (QPQPPFSHT) was determined, as shown in Table 1.

[0054] 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) QPQPPFSHT 95.8 9 519.752 2 3.92E+06 1037.49 0.71 -88.16 The secondary mass spectrometry of the nonapeptide QT9 Figure 1 As shown in the figure, 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. 519.752 m / z is the [M+H]+ ion signal of the nonapeptide, with a charge number (z) of 2 and a molecular weight of 1037.49 Da. The nonapeptide was further analyzed by secondary mass spectrometry using the in-source collision-induced dissociation technique (such as Figure 1 As shown in the figure, the primary structure of the nonapeptide was determined to be Gln-Pro-Gln-Pro-Pro-Phe-Ser-His-Thr.

[0055] Molecular docking 2D and 3D images of nonapeptide QT9 and CHRM1 are shown in the figure. Figure 2 Analysis of chemical bonds revealed that the nonapeptide QT9 was bound to CHRM1 through van der Waals forces, hydrogen bonds (including conventional hydrogen bonds and carbon-hydrogen bonds) and hydrophobic interactions (alkyl groups), with a docking energy of -88.16 kcal / moL. The nonapeptide QT9 formed 12 van der Waals forces with amino acid residues ALA1073, VAL1102, GLU1021, THR1020, ARG1136, ARG1007, GLU1010, TYR1023, GLY1029, ARG1144, HIS1030, and ASP1069, 16 hydrogen bonds with GLY1106, MET1105, THR1141, TRP1137, GLN1140, ILE1008, ASP1009, GLY1011, GLN1104, PHE1103, ASP1019, TYR1017, and THR1025, and 1 hydrophobic interaction with LEU1031.

[0056] The molecular docking results show that the nonapeptide QT9 can bind to CHRM1 and activate CHRM1, thereby exerting an immunomodulatory effect.

[0057] Shenzhen Borunsida Biotechnology Co., Ltd. was commissioned to synthesize the peptide QPQPPFSHT with a purity of ≥98% for subsequent functional verification.

[0058] Example 2: Effect of Nonapeptide (QPQPPFSHT) 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.

[0059] 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.

[0060] In this example, wild AB zebrafish were modeled with chloramphenicol to reduce the number of macrophages, thereby creating an immunodeficiency model. The QPQPPFSHT nonapeptide was used for intervention to characterize the effect of the nonapeptide on the number of important immune cells, macrophages.

[0061] 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) Normal control group: 0.5% DMSO + system water + PTU; (2) Modeling group: 125 μg / mL chloramphenicol (dissolved in DMSO) + system water + PTU, DMSO volume fraction is 0.5%; (3) Nonapeptide QT9 intervention group: 5 μg / mL QPQPPFSHT nonapeptide (QT9) + 125 μg / mL chloramphenicol (dissolved in DMSO) + system water + PTU, with a DMSO volume fraction of 0.5%.

[0062] After 24 hours of drug addition, 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 modeling 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.

[0063] 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 5 μg / mL QPQPPFSHT nonapeptide was used for intervention, it could significantly promote the proliferation of macrophages ( p< 0.05), indicating that QPQPPFSHT nonapeptide has a promoting effect on important immune cells macrophages.

[0064] Example 3: Effect of Nonapeptide (QPQPPFSHT) on the Proliferation of Neutrophils Among the white blood cells circulating in the blood, neutrophils derived from bone marrow stem cells account for the highest proportion. When pathogens invade the body, neutrophils are the first to arrive at the site of infection, killing and engulfing pathogens by secreting a variety of cytotoxic granules and proteases. In addition, they also play a role in recruiting monocytes and repairing damaged tissues. 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 various parts of the body. Therefore, neutrophils are also an important type of immune cell. In immunological experiments, neutrophil fluorescent transgenic zebrafish larvae are usually used as a model to directly observe the fluorescence intensity or number of neutrophils from the excretion hole to the end of the tail under a fluorescence microscope.

[0065] 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 QPQPPFSHT nonapeptide was used for intervention to characterize the effect of the nonapeptide on the number of neutrophils, an important immune cell.

[0066] 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) Normal control group: 0.5% DMSO + system water + PTU; (2) Modeling group: 125 μg / mL chloramphenicol (dissolved in DMSO) + system water + PTU, DMSO volume fraction is 0.5%; (3) Nonapeptide QT9 intervention group: 5 μg / mL QPQPPFSHT nonapeptide (QT9) + 125 μg / mL chloramphenicol (dissolved in DMSO) + system water + PTU, with a DMSO volume fraction of 0.5%.

[0067] 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 SPSS 22.0 software was used to perform a significance test between the modeling group and other groups.

[0068] 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 5 μg / mL QPQPPFSHT nonapeptide was used for intervention, it could significantly promote the proliferation of neutrophils ( p< 0.001), and there was no significant difference with the normal control group ( p> 0.05), indicating that from the perspective of important immune cells, neutrophils, QPQPPFSHT nonapeptide also has an immune-enhancing effect.

[0069] Example 4: Upregulation of the immune factor IFN-γ by nonapeptide (QPQPPFSHT) 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 (IFN-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, IFN-γ is an immunomodulatory interferon, and its immunomodulatory effect is dozens of times stronger than that of antiviral interferon. Therefore, IFN-γ is an important immune factor that characterizes immune effects.

[0070] In this example, chloramphenicol was used to model wild AB zebrafish to reduce the level of IFN-γ, thereby creating an immunodeficiency model. The QPQPPFSHT nonapeptide was used for intervention to characterize the effect of the nonapeptide on the level of IFN-γ, an important immune factor.

[0071] 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) Normal control group: 0.5% DMSO + system water + PTU; (2) Modeling group: 150 μg / mL chloramphenicol (dissolved in DMSO) + system water + PTU, DMSO volume fraction is 0.5%; (3) Nonapeptide QT9 intervention group: 5 μg / mL QPQPPFSHT nonapeptide (QT9) + 150 μg / mL chloramphenicol (dissolved in DMSO) + system water + PTU, with a DMSO volume fraction of 0.5%.

[0072] 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, 5000 r / min, 5 min). The supernatant was taken and the IFN-γ content in the supernatant was determined using an IFN-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.

[0073] The results are as follows Figure 7As shown in the figure, the immunosuppression model was successfully established after the use of chloramphenicol (150 μg / mL), and the IFN-γ level was significantly lower than that of the normal control group ( p <0.001). When 5 μg / mL QPQPPFSHT nonapeptide was used for intervention, the IFN-γ level was significantly increased ( p <0.01), indicating that from the perspective of the level of important immune factor IFN-γ, QPQPPFSHT nonapeptide also has an immune-enhancing effect.

[0074] Example 5: Immunoenhancing effect of wheat peptides in immunocompromised mouse model This example uses an immunocompromised mouse model modeled with cyclophosphamide to explore the immune-enhancing effect of wheat peptides (prepared in Example 1, containing the peptide segment QPQPPFSHT). Cyclophosphamide, as a commonly used anticancer chemotherapy drug, can cause dysfunction of the immune system in important immune organs throughout the body (such as the spleen, thymus, and intestinal mucosa). This immunosuppressive state not only affects the proliferation and activity of immune cells in mice, but also makes them susceptible to various infections and diseases. Therefore, the establishment of this model provides an important basis for evaluating the effect of wheat peptides in immunoregulation. During the study, immunocompromised mice will be treated with different doses of wheat peptides to observe their effects on immune cell proliferation. By detecting the levels of immune factors in mice, changes in immune organs, and the functions of related immune cells, it is intended to determine whether wheat peptides can effectively reverse the immunosuppression caused by cyclophosphamide and promote the recovery of immune cells.

[0075] 1. Experimental methods (1) Mice and husbandry 45 SPF male ICR mice (age: 6-11 weeks, weight: 35±2 g) were purchased from Zhejiang Chinese Medical University from Shanghai Slake Company, animal experiment ethics approval number: IACUC-20230206-07. The mouse breeding environment is as follows: 22±2 ℃, 50±10% relative humidity, 12 h light / dark cycle.

[0076] (2) Grouping and dosing method The experiment set up a normal control group, three wheat peptide treatment groups (the dosages were high dose 0.5 g / kg mouse body weight, recorded as high dose of wheat peptide; medium dose 0.25 g / kg, recorded as medium dose of wheat peptide; low dose 0.1 g / kg, recorded as low dose of wheat peptide) and one cyclophosphamide modeling control group (modeling group), a total of 5 groups. The high, medium and low wheat peptide treatment groups were gavaged with corresponding doses of wheat peptide, while the normal group and modeling group were gavaged with the same amount of normal saline. Gavage once a day for 30 consecutive days. However, starting from the 28th day, the modeling group and the treatment group were treated with 50 mg / kg cyclophosphamide for modeling (days 28-30), and the normal control group was treated with the same amount of normal saline. The body weight and food intake before and after cyclophosphamide modeling were recorded.

[0077] (3) Determination of immune organ index The collected thymus and spleen were washed in pre-cooled phosphate buffer solution (PBS, 0.01 mol / L, pH=7.4) and then dried with filter paper. The spleen and thymus indexes were calculated according to the following formula: Spleen index (%) = spleen weight / body weight × 100%; Thymus index (%) = thymus weight / body weight × 100%.

[0078] (4) Elisa assay for determination of serum immune-related cytokines and immunoglobulin A (IgA) The levels of serum cytokine interferon-γ (IFN-γ) and immunoglobulin A (IgA) were measured using a commercial Elisa analysis kit from Genentech, and the measurement method was carried out according to the instructions of the kit.

[0079] (5) Determination of fecal short-chain fatty acid content Sample preparation: Accurately weigh about 50 mg of mouse feces, add 250 μL of sterile ultrapure water, vortex for 5 minutes, then add 10 μL of 5 mol / L hydrochloric acid solution to adjust the pH of the suspension to 2-3, vortex for 1 minute, let it stand at room temperature for 5 minutes, then centrifuge at 4 °C, 12000 rpm for 30 minutes, take 200 μL of supernatant and place it in 0.5 μL of 20-fold diluted 2-ethylbutyric acid (internal standard), vortex for 1 minute, and centrifuge at 4 °C, 12000 rpm for 5 minutes, take 150 μL of supernatant for gas chromatography analysis.

[0080] GC conditions: Agilent DB-FFAP125-3237 (30 m×0.52 mm×0.50 mm) gas column; the temperature program of the gas column was as follows: the initial temperature was 100 °C and maintained for 0.5 min, then the temperature was programmed to 180 °C at 8 °C / min and maintained for 1 min, and finally the temperature was programmed to 240 °C at 20 °C / min and maintained for 15 min; the injection port temperature and hydrogen ion flame detector temperature were 200 °C and 240 °C, respectively; the flow rates of hydrogen, air, and nitrogen were set to 30 mL / min, 300 mL / min, and 20 mL / min, respectively; the injection volume was 1 μL.

[0081] 2. Results Analysis 2.1 Growth performance Depend on Figure 8 It can be seen that the administration of cyclophosphamide had no significant effect on the food intake of mice.

[0082] Depend on Fig. 9 It can be seen that after the administration of cyclophosphamide, the body weight of the model group was significantly reduced compared with the normal control group ( p< 0.001), indicating that the cyclophosphamide immunosuppression treatment model has been preliminarily established. However, there was no significant difference between the high-dose and medium-dose wheat peptide groups and the normal control group ( p> 0.05), indicating that wheat peptide treatment containing peptide QPQPPFSHT can improve the weight loss of mice caused by cyclophosphamide.

[0083] 2.2 Immune organ index Cyclophosphamide modeling significantly reduces the production of immune cells in the body, which is manifested in the atrophy of primary and secondary immune organs that produce immune cells. Fig.10 and Fig.11 As shown, cyclophosphamide modeling significantly reduced the thymus and spleen indexes of mice ( p< 0.0001), indicating that the ability of immune organs to produce immune cells decreased, while high-dose wheat peptides significantly increased the spleen index and thymus index ( p< 0.05, p< 0.01), medium dose wheat peptide significantly increased the thymus index ( p< 0.01), indicating that wheat peptides can significantly restore the proliferation capacity of immune cells in immunosuppressed mice.

[0084] 2.3 Immune factors Immunoglobulins and cytokines in serum often play a role in activating immune cells and clearing antigens in body fluid circulation. For example, IFN-γ can directly clear antigens in the blood, and IgA can resist infection and promote the body's mucosal immunity. Fig.12 and Fig.13 As shown in Figure 2, the immunosuppression induced by cyclophosphamide significantly downregulated the serum immunoglobulin IgA and cytokine IFN-γ in the modeling group compared with the normal control group ( p< 0.0001), indicating that the immune capacity of mice was suppressed. Supplementation of wheat peptides can significantly reverse the decrease of these immune-related proteins in serum: compared with the modeling group, IgA of high, medium and low doses of wheat peptides were significantly upregulated ( p< 0.0001, p< 0.01, p< 0.01); IFN-γ in high and medium dose wheat peptides was significantly upregulated ( p< 0.0001). This indicates that wheat peptides can significantly enhance immunity.

[0085] 2.4 Short-chain fatty acids Short-chain fatty acids regulate the absorption of various nutrients and hormone production in the intestine, and play a role in the body's energy metabolism and intestinal barrier repair and synthesis. Their content and composition changes are closely related to intestinal mucosal immunity. Figure 14-16 As shown. The fecal acetic acid, propionic acid and butyric acid in the cyclophosphamide modeling group were significantly lower than those in the normal group ( p< 0.05, p< 0.05, p< 0.001). Compared with the normal group and the modeling group, high and medium doses of wheat peptide significantly increased the content of acetic acid, propionic acid and butyric acid in the feces of mice ( p< 0.0001), indicating that the immune-enhancing effect of wheat peptides also has a beneficial effect on the intestinal mucosa.

[0086] In summary, the present invention screened out the nonapeptide QPQPPFSHT from wheat peptides through mass spectrometry identification and molecular docking of peptide segments. The nonapeptide molecule showed efficient immunomodulatory effects in both the zebrafish model and the mouse model of immunosuppression, which was mainly reflected in promoting the proliferation of immune cells and increasing the content of immune factors. It also had a beneficial effect on the intestinal mucosa while improving immunity. The present invention provides a new solution for improving immune function. The nonapeptide QPQPPFSHT can be applied to the preparation of food or medicine for preventing and improving the adverse phenomena of immunosuppression. Specifically, the nonapeptide QPQPPFSHT can be used alone in health products or medicines for improving immunity, or it can be compounded with other active ingredients with immune-enhancing effects. The active components with immune-enhancing effects can include plant extracts and other protein peptides (sea cucumber peptides, soybean peptides, etc.). The preparation form of the health product or medicine can be microcapsule to improve the gastrointestinal digestion stability, bioavailability and shelf life of the polypeptide, so that it can be better applied to the food industry and health care fields.

Claims

1. A nonapeptide QT9 with immunomodulatory effect, characterized in that: The amino acid sequence of the nonapeptide QT9 is Gln-Pro-Gln-Pro-Pro-Phe-Ser-His-Thr.

2. The method for preparing the nonapeptide QT9 according to claim 1, characterized in that: The nonapeptide QT9 is prepared by solid phase synthesis; or obtained by enzymatic hydrolysis of wheat gluten protein.

3. The use of the nonapeptide QT9 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, increasing the level of immune factors, increasing the immune organ index, and increasing the content of intestinal short-chain fatty acids.

4. The use according to claim 3, characterized in that The immune cells include at least one of macrophages and neutrophils; the immune factors include at least one of immunoglobulin IgA and interferon-γ; the immune organ index includes a thymus index and a spleen index; and the intestinal short-chain fatty acids include at least one of acetic acid, propionic acid, and butyric acid.

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

6. The use according to claim 5, characterized in that The manifestations of immunodeficiency include: a decrease in at least one of the following indicators: the number of immune cells, the level of immune factors, and the content of intestinal short-chain fatty acids.

7. The use according to claim 5, characterized in that The immunosuppression is a weakened immune function caused by the use of immunosuppressants.

8. The use according to claim 5, characterized in that The immunosuppression is immunosuppression caused by the use of chloramphenicol or cyclophosphamide.

9. A pharmaceutical composition for preventing or treating immunosuppression, characterized in that: The pharmaceutical composition comprises an effective dose of nonapeptide QT9 and a pharmaceutically acceptable carrier, wherein the amino acid sequence of the nonapeptide QT9 is Gln-Pro-Gln-Pro-Pro-Phe-Ser-His-Thr.

10. A health food for enhancing immunity, characterized in that: The invention comprises nonapeptide QT9 as an active ingredient and excipients acceptable in food science, wherein the amino acid sequence of the nonapeptide QT9 is Gln-Pro-Gln-Pro-Pro-Phe-Ser-His-Thr.

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