A nonapeptide with immunomodulatory effects, its preparation and application

Abstract

The present invention discloses a nonapeptide with immunomodulatory effects, its preparation and application, belonging to the technical field of biomedicine. The amino acid sequence of the nonapeptide QT9 is Gln-Pro-Gln-Pro-Pro-Phe-Ser-His-Thr. This peptide segment can be obtained by artificial synthesis or directional enzymatic hydrolysis of wheat protein. Functional verification shows that this nonapeptide has the effects of promoting the proliferation of immune cells, increasing the levels of immune factors, increasing the immune organ index, and improving the intestinal mucosal immune function. Moreover, the nonapeptide QPQPPFSHT 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 the adverse phenomenon of immune deficiency. The present invention provides a new solution for enhancing immune function, meets the public's demand for immune health care, and has good market prospects and application potential.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and particularly relates to a nonapeptide with immunomodulatory effects and its preparation and application. Background Art

[0002] The immune system is an important system for the body to execute immune responses and immune functions. It consists of immune organs, immune cells, and immune molecules. The immune system has the functions of recognizing and eliminating antigenic foreign substances, and coordinating with other systems of the body to jointly maintain the stability of the internal environment and physiological balance of the body.

[0003] Immune hypofunction is a common sub-healthy state. Those with immune hypofunction are difficult to resist the invasion of bacteria, viruses, fungi, etc., and are prone to various serious infections, such as upper respiratory tract infections, urinary tract infections, septicemia, meningitis, chickenpox, measles, tuberculosis, etc. In severe cases, it may even cause cancer. Compared with those with normal immune function, those with immune hypofunction are more likely to get sick or their condition is more likely to worsen in the same environment.

[0004] According to different causes of onset, immune hypofunction can be divided into two categories: primary and secondary. Primary is caused by congenital hypoplasia, and most of it is related to genetics, mostly occurring in children; secondary is caused by infections such as viruses, bacteria, fungi, or reasons such as drugs, tumors, fatigue, insomnia, malnutrition, excessive stress, etc., and can be seen in people of all ages. Therefore, enhancing immune function has become the focus of public attention.

[0005] Bioactive peptides refer to 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 immunomodulation. The functional characteristics of bioactive peptides are closely related to their amino acid composition and sequence, which makes the rational design and synthesis of bioactive peptides an effective strategy for enhancing immunity. For example: thymopentin is the fragment of amino acid residues 32 - 36 of thymopoietin II, which has the function of inducing the differentiation, maturation, and activation of T lymphocytes, enhancing the activity of natural killer cells (NK) and the phagocytic function of macrophages, and is mainly used as an immunomodulator clinically.

[0006] Other studies have shown that some small molecular peptides produced by proteolysis have immunomodulatory effects. They can not only enhance the body's immunity, but also stimulate the proliferation of lymphocytes in the body and enhance the phagocytic ability of macrophages. For example, Wu et al. extracted a novel peptide from wheat germ globulin, purified it to obtain the sequence Glu-Cys-Phe-Ser-Thr-Ala (ECFSTA), and evaluated its immunity. It was found that it could activate macrophages and could be used as an immunomodulator (Characterization and Immunomodulatory Activity of a Novel Peptide, ECFSTA, from Wheat Germ Globulin. Journal of Agricultural and Food Chemistry , 2017, 65:5561 - 5569.).

[0007] Wheat peptides are made from wheat protein (gluten) through processes such as biological enzymatic hydrolysis, refining, and spray drying, such as patent document CN 112226478 A. Research has shown that wheat peptides have an immunopotentiating effect. Therefore, by analyzing the peptide profile of wheat peptides and screening out highly efficient peptide segments related to immune enhancement, it will provide a basis for the development of novel immunomodulators. Summary of the Invention

[0008] The purpose of the present invention is to provide a natural small molecule bioactive peptide with immunomodulatory effects and apply it to the development of safe and highly efficient immunomodulatory products.

[0009] To achieve the above purpose, the present invention adopts the following technical solutions:

[0010] The present invention uses LC-MS / MS peptide profile analysis technology to analyze the peptide sequences in wheat peptides, and then uses molecular docking technology to explore the interaction between the peptide segments and the M1-muscarinic acetylcholine receptor (CHRM1). A candidate peptide segment is screened out. 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. It is named QT9. Further, the nonapeptide QT9 is synthesized artificially, and functional verification shows that the nonapeptide QT9 exhibits a significant effect of enhancing immunity in an immunosuppressed animal model.

[0011] Therefore, the present invention provides a nonapeptide QT9 with immunomodulatory effects, and the amino acid sequence of the nonapeptide QT9 is Gln-Pro-Gln-Pro-Pro-Phe-Ser-His-Thr.

[0012] 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 the Fmoc solid-phase synthesis strategy, using Fmoc-protected amino acids as raw materials, selecting Wang resin as the 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, thereby solid-phase synthesizing the nonapeptide QT9.

[0013] The nonapeptide QT9 can also be obtained by enzymatic hydrolysis of wheat gluten. Specifically, it is mixed according to the mass ratio of water to wheat protein (gluten powder) of 10:1, 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 pH≥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 it is placed at 100 °C for 30 min to inactivate the enzyme. Subsequently, it is concentrated and dried to obtain wheat peptide powder, which contains the nonapeptide QT9.

[0014] The present invention provides the application of the nonapeptide QT9 in the preparation of immunomodulatory products. The immunomodulation includes at least one of enhancing the proliferation of immune cells, increasing the levels of immune factors, increasing the immune organ index, and increasing the content of intestinal short-chain fatty acids. The research of the present invention shows that in an immune-suppressed animal model, treatment with the nonapeptide QT9 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; significantly increase the content of intestinal short-chain fatty acids such as acetic acid, propionic acid, and butyric acid, indicating that the nonapeptide QT9 has immunomodulatory activity and promotes the recovery of immune cells. Therefore, it can be applied to the development of related products for regulating immune function.

[0015] 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 thymus index and spleen index; the intestinal short-chain fatty acids include at least one of acetic acid, propionic acid, and butyric acid.

[0016] Further, the product is a drug for preventing or treating immunodeficiency. Specifically, the drug can be used to treat immunodeficiency diseases, which refer to diseases accompanied by a state of low immune function and require the administration of immunopotentiators or for which the administration of immunopotentiators is more beneficial for symptom relief, such as immunodeficiency diseases, recurrent or refractory infectious diseases. These diseases are characterized by repeated attacks and a long treatment cycle due to the low immune function of the body. The drug can also be used to treat immunodeficiency caused by the use of immunosuppressants such as glucocorticoids and cyclophosphamide.

[0017] Further, the manifestations of immunodeficiency include a decrease in at least one of the indicators of the number of immune cells, the level of immune factors, and the content of intestinal short-chain fatty acids.

[0018] As a specific embodiment of the present invention, the immunodeficiency is immunodeficiency caused by the use of immunosuppressants. Specifically, the immunosuppressant can be, but is not limited to, cyclophosphamide.

[0019] As a specific embodiment of the present invention, the immunodeficiency is immunodeficiency caused by the use of chloramphenicol. The toxic and side effects of chloramphenicol include a decrease in the number of immune cells, resulting in immunodeficiency.

[0020] Further, the product is a health food for enhancing immunity. Specifically, the health food is used to improve the sub-healthy state of immunodeficiency.

[0021] Further, 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.

[0022] The present invention provides a pharmaceutical composition for preventing or treating immunodeficiency, which comprises an effective dose of nonapeptide QT9 and a pharmaceutically acceptable carrier. The amino acid sequence of the nonapeptide QT9 is Gln-Pro-Gln-Pro-Pro-Phe-Ser-His-Thr.

[0023] The present invention is prepared with nonapeptide QT9 as the main active ingredient and added with a pharmaceutically acceptable carrier, and can be prepared into a preparation according to the preparation methods of preparations recorded in pharmacy. In the pharmaceutical composition provided by the present invention, nonapeptide QT9 can be used as the only active ingredient that exerts immunomodulatory effects, or can be compounded with other active ingredients with immunomodulatory effects such as sea cucumber peptide and soybean peptide.

[0024] Furthermore, 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 capable of delivering the active substance in an effective dose of the present invention, without interfering with the biological activity of the active substance and having no toxic or side effects on the host or subject.

[0025] Furthermore, the dosage form of the pharmaceutical composition may be, but is not limited to, oral liquid, capsule, microcapsule powder, tablet, granule, or emulsion.

[0026] 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 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 - 0.5 g / kg of mouse body weight. No toxic or side effects were observed under the above action conditions.

[0027] The present invention also provides a health food for enhancing immunity, comprising the nonapeptide QT9 as an active ingredient and a food - acceptable excipient. The amino acid sequence of the nonapeptide QT9 is Gln - Pro - Gln - Pro - Pro - Phe - Ser - His - Thr. The food - acceptable excipient is any preparation or carrier medium capable of delivering the active substance in an effective dose of the present invention, without interfering with the biological activity of the active substance and having no toxic or side effects on the host or subject.

[0028] The beneficial effects of the present invention are as follows:

[0029] The present invention provides a nonapeptide QPQPPFSHT with immunomodulatory effects. This peptide segment can be obtained by artificial synthesis or directional enzymatic hydrolysis of wheat protein. Functional verification in animal models shows that this peptide segment has the effect of promoting the proliferation of immune cells, 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 intestinal mucosal immune function. Moreover, the nonapeptide QPQPPFSHT prepared by enzymatic hydrolysis of wheat 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 the adverse phenomena of immunodeficiency. The present invention provides a new solution for enhancing immune function, meets the public's demand for immune health care, and has good market prospects and application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] 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 peptide C-terminus; b2 / b4[2+] represents the second fragment ion produced by the cleavage of the peptide N-terminus and the fourth fragment ion produced by the cleavage of the peptide N-terminus with two charges, b3 represents the third fragment ion produced by the cleavage of the peptide N-terminus, b4 represents the fourth fragment ion produced by the cleavage of the peptide N-terminus, b5 represents the fifth fragment ion produced by the cleavage of the peptide N-terminus, y5 represents the fifth fragment ion produced by the cleavage of the peptide C-terminus; y6 represents the sixth fragment ion produced by the cleavage of the peptide C-terminus, b7 represents the sixth fragment ion produced by the cleavage of the peptide N-terminus, y7 represents the seventh fragment ion produced by the cleavage of the peptide C-terminus, y8-H represents the sixth fragment ion produced by the cleavage of the peptide 2 O represents the eighth dehydrated fragment ion generated by the C-terminal cleavage of the peptide.

[0031] Figure 2 Schematic diagram of the binding interaction between nonapeptide QT9 and CHRM1.

[0032] Figure 3 These are photos of head macrophages observed under a stereomicroscope after treatment with the nonapeptide QT9 in the zebrafish model.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] 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; The # sign indicates a significant difference compared with the model group, and ## indicates p < 0.01.

[0037] Figure 8 Effect on food intake after treatment with wheat peptides containing QT9 in a mouse model.

[0038] Figure 9 Effect on body weight after treatment with wheat peptides containing QT9 in a mouse model, where the * sign indicates a significant difference compared with the normal group, and * indicates p < 0.05, *** indicates p < 0.001.

[0039] Figure 10 Effect on spleen index after treatment with wheat peptides containing QT9 in a mouse model, where the * sign indicates a significant difference compared with the normal group, and **** indicates p < 0.0001; The # sign indicates a significant difference compared with the model group, and # indicates p < 0.05.

[0040] Figure 11 Effect on thymus index after treatment with wheat peptides containing QT9 in a mouse model, where the * sign indicates a significant difference compared with the normal group, and ** indicates p < 0.01, **** indicates p < 0.0001; The # sign indicates a significant difference compared with the model group, and ## indicates p < 0.01.

[0041] Figure 12 Effect on serum immunoglobulin IgA after treatment with wheat peptides containing QT9 in a mouse model, where the * sign indicates a significant difference compared with the normal group, and **** indicates p < 0.0001; The # sign indicates a significant difference compared with the model group, and ## indicates p < 0.01, # indicates p < 0.0001.

[0042] Figure 13 Effect on serum cytokine IFN-γ after treatment with wheat peptides containing QT9 in a mouse model, where the * sign indicates a significant difference compared with the normal group, and ** indicates p < 0.01, **** indicates p < 0.0001; The # sign indicates a significant difference compared with the model group, and # indicates p < 0.0001.

[0043] Figure 14Effect of wheat peptides containing QT9 on acetic acid content in feces after treatment in a mouse model, where the * symbol indicates a significant difference compared with the normal group, * indicates p <0.05, ** indicates p <0.01; the # symbol indicates a significant difference compared with the model group, # indicates p <0.0001.

[0044] Figure 15 Effect of wheat peptides containing QT9 on propionic acid content in feces after treatment in a mouse model, where the * symbol indicates a significant difference compared with the normal group, * indicates p <0.05, ** indicates p <0.01; the # symbol indicates a significant difference compared with the model group, # indicates p <0.0001.

[0045] Figure 16 Effect of wheat peptides containing QT9 on butyric acid content in feces after treatment in a mouse model, where the * symbol indicates a significant difference compared with the normal group, * indicates p <0.05, ** indicates p <0.01, *** indicates p <0.001; the # symbol indicates a significant difference compared with the model group, # indicates p <0.0001. Specific implementation mode

[0046] 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 method, steps or conditions of the present invention belongs to the scope of the present invention.

[0047] 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.

[0048] 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.

[0049] Example 1: Screening of active peptide segments

[0050] 1. Preparation of wheat peptides (glutamine peptides)

[0051] Put into the reaction kettle according to the mass ratio of water to wheat protein (gluten) of 10:1. 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 content of the feed liquid to 25°Bx and then carry out spray drying to obtain wheat peptide (glutamine peptide) powder.

[0052] 2. Screen active peptide segments

[0053] Perform LC-MS / MS peptide spectrum analysis on wheat peptides, organize the 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:

[0054] (a) Identification of wheat peptide sequence

[0055] Dissolve the wheat peptide sample in NH 4 HCO 3 solution, add dithiothreitol solution, and place it in a 56 °C water bath for reduction for 1 h. 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 phase injection vial. Subsequently, perform LC-MS / MS analysis.

[0056] 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 - 66min, 95%B.

[0057] Mass spectrometry conditions: Full scan MS uses Orbitrap for primary scan, scan range (100~1500 m / z), resolution (70000), maximum ion introduction time (100 ms), automatic gain control (3×10 6); The top 20 precursor ions that meet the tandem (MS / MS) fragmentation conditions were fragmented using high-energy collision dissociation and scanned with an Orbitrap at a resolution of 17,500, a maximum ion injection time of 50 ms, and an automatic gain control of 1×10 5 ). The raw data obtained from the mass spectrometry was analyzed for peptide sequence using the De novo software of PEAKS Studio.

[0058] (b) Screening of peptides with potential immunomodulatory activity

[0059] 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, the peptide segments of wheat peptides were molecularly docked with acetylcholine receptors to screen for peptide segments with potential immunomodulatory functions.

[0060] Peptide segments that met the criteria were screened according to the conditions of an average local confidence (ALC) greater than 95%, a peak area greater than 2×10 6 , and a PeptideRanker score greater than 0.8. Subsequently, the peptide segments were molecularly docked with the M1-muscarinic acetylcholine receptor (CHRM1).

[0061] First, the crystal structure (5CXV) of CHRM1 was downloaded from the PDB protein database. After removing water molecules and adding hydrogen atoms to the receptor target through Discovery Studio software, its active center was defined. The structures of the peptide segments of the selected wheat peptides were constructed by Discovery Studio, and their energies were minimized using 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 the highest binding degree, and were screened according to the binding energy and the number of hydrogen bonds. Finally, one nonapeptide (QPQPPFSHT) was identified, as shown in Table 1.

[0062] Table 1. Peptide segments in wheat peptides with potential immunomodulatory activity

[0063] 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

[0064] Among them, the secondary mass spectrometry of the nonapeptide QT9 Figure 1As shown, 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. According to the b or y series fragment ions of the peptide, the primary structure of the peptide can be deduced. 519.752 m / z is the [M+H]+ ion signal of the nonapeptide, the charge number (z) is 2, and the molecular weight is 1037.49 Da. Further, the nonapeptide was analyzed by in-source collision-induced dissociation technology for the second mass spectrometry (as Figure 1 shown), and the primary structure of the nonapeptide was determined to be Gln-Pro-Gln-Pro-Pro-Phe-Ser-His-Thr.

[0065] The 2D and 3D maps of the molecular docking of the nonapeptide QT9 and CHRM1 are as Figure 2 shown. By analyzing the chemical bonds, it was found that the nonapeptide QT9 binds to CHRM1 through van der Waals forces, hydrogen bonds (including conventional hydrogen bonds and carbon-hydrogen bonds), and hydrophobic interactions (alkyl groups), and the docking energy is -88.16 kcal / mol. The nonapeptide QT9 forms 12 van der Waals forces with amino acid residues ALA1073, VAL1102, GLU1021, THR1020, ARG1136, ARG1007, GLU1010, TYR1023, GLY1029, ARG1144, HIS1030, and ASP1069, and forms 16 hydrogen bonds with GLY1106, MET1105, THR1141, TRP1137, GLN1140, ILE1008, ASP1009, GLY1011, GLN1104, PHE1103, ASP1019, TYR1017, and THR1025, and forms 1 hydrophobic interaction with LEU1031.

[0066] From the molecular docking results, it can be seen that the nonapeptide QT9 can bind to CHRM1, thereby activating CHRM1 and then exerting an immunomodulatory effect.

[0067] The peptide segment QPQPPFSHT was synthesized by Shenzhen Borun Sida Biotechnology Co., Ltd. with a purity ≥ 98% for subsequent functional verification.

[0068] Example 2: Proliferation effect of the nonapeptide (QPQPPFSHT) on macrophages

[0069] The present invention uses a zebrafish model to characterize the immune function of peptide segments. The similarity between the zebrafish genome and the human genome is approximately 87%, with 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 begin 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. Immune molecules such as TLRs, TNF, and ILs are involved in the immune response in the zebrafish embryo. TLRs with MyD88 as the 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, 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.

[0070] 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 remove 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 can improve the bactericidal ability; M2 type can secrete anti-inflammatory cytokines and participate in processes such as angiogenesis, fibroplasia, and tissue repair. Therefore, the characterization of the number of macrophages is an important indicator of immune enhancement.

[0071] In this example, a wild AB strain of zebrafish was used to establish a model with chloramphenicol to reduce the number of macrophages and create an immune deficiency model. The nonapeptide QPQPPFSHT was used for intervention to characterize the effect of the nonapeptide on the number of macrophages, an important immune cell.

[0072] At 24 hpf, 10 μL / mL of phenylthiourea mother liquor (PTU) was added to the embryos of wild AB strain zebrafish. Demembranation was performed 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 grouping was as follows:

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

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

[0075] (3) Intervention group with nonapeptide QT9: 5 μg / mL nonapeptide QPQPPFSHT (QT9) + 125 μg / mL chloramphenicol (dissolved in DMSO) + system water + PTU, with the volume fraction of DMSO being 0.5%.

[0076] After 24 h of drug addition, 2.5 μg / mL neutral red dye and PTU were added to each well, and stained for 6 h in the dark. It was eluted with system water in the dark. After anesthesia, the embryos were fixed with 6% methylcellulose, and the number of head macrophages was photographed and counted under a stereomicroscope. Taking the model group as the control, data analysis was performed using SPSS 22.0 and GraphPad Prism 8.0 software, and Tukey test in One-way ANOVA was used to compare the differences among multiple groups.

[0077] The results are 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 intervened with 5 μg / mL nonapeptide QPQPPFSHT, it could significantly promote the proliferation of macrophages ( p < 0.05), indicating that nonapeptide QPQPPFSHT has a promoting effect on macrophages, an important immune cell.

[0078] Example 3: Proliferation effect of nonapeptide (QPQPPFSHT) on neutrophils

[0079] 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 reach the infection site earliest and kill and phagocytize the pathogens by secreting a variety of cytotoxic granules and proteases. In addition, they also play roles such as 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 region, enter the blood circulation, and then reach all parts of the body. Therefore, neutrophils are also an important type of immune cell. In immunological experiments, usually using fluorescent transgenic zebrafish larvae of neutrophils as a model, the fluorescence intensity or number of neutrophils from the excretory pore to the tail end is directly observed under a fluorescence microscope.

[0080] In this example, Tg(Lyz:DsRed) neutrophil fluorescent transgenic zebrafish larvae were used as a model. Chloramphenicol was used to establish the model to reduce the number of neutrophils and create an immune deficiency model. The nonapeptide QPQPPFSHT was used for intervention to characterize the effect of the nonapeptide on the number of neutrophils, which are important immune cells.

[0081] At 24 hpf of Tg(Lyz:DsRed) zebrafish embryos, 10 μL / mL of phenylthiourea mother liquor (PTU) was added. Demembranation was carried out 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:

[0082] (1) Normal control group: 0.5% DMSO + system water + PTU;

[0083] (2) Model group: 125 μg / mL chloramphenicol (dissolved in DMSO) + system water + PTU, with the volume fraction of DMSO being 0.5%;

[0084] (3) Nonapeptide QT9 intervention group: 5 μg / mL QPQPPFSHT nonapeptide (QT9) + 125 μg / mL chloramphenicol (dissolved in DMSO) + system water + PTU, with the volume fraction of DMSO being 0.5%.

[0085] 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 between the model group and other groups.

[0086] The results are as Figure 5 and Figure 6 shown. After establishing the 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 intervened with 5 μg / mL QPQPPFSHT nonapeptide, it could significantly promote the proliferation of neutrophils ( p < 0.001), and there was no significant difference compared with the normal control group ( p > 0.05), indicating that from the perspective of neutrophils, which are important immune cells, the QPQPPFSHT nonapeptide also has the effect of enhancing immunity.

[0087] Example 4: Upregulation effect of the nonapeptide (QPQPPFSHT) on the immune factor IFN-γ

[0088] Interferon (INF) is a class of glycoproteins with a variety of 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 (IFN-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, 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 characterizing immune function.

[0089] In this example, a wild AB strain zebrafish was used to establish a model with chloramphenicol to reduce the level of IFN-γ, creating an immune deficiency model, and the nonapeptide QPQPPFSHT was used for intervention to characterize the effect of the nonapeptide on the level of the important immune factor IFN-γ.

[0090] At 24 hpf, 10 μL / mL of phenylthiourea mother liquor (PTU) was added to the embryos of wild AB strain zebrafish. 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 grouping was as follows:

[0091] (1) Normal control group: 0.5 % DMSO + system water + PTU;

[0092] (2) Model group: 150 μg / mL chloramphenicol (dissolved in DMSO) + system water + PTU, and the volume fraction of DMSO was 0.5%;

[0093] (3) Nonapeptide QT9 intervention group: 5 μg / mL QPQPPFSHT nonapeptide (QT9) + 150 μg / mL chloramphenicol (dissolved in DMSO) + system water + PTU, and the volume fraction of DMSO was 0.5%.

[0094] After culturing in an incubator at 28.5 °C for 24 h, 120 embryos in each group were placed in 1.5 mL centrifuge tubes. The embryos were washed with PBS buffer at a volume 1 time that of the total volume of the liquid in each well. 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. The samples were homogenized by a high-speed tissue grinder for 90 s and then centrifuged (4 °C, 5000 r / min, 5 min). The supernatant was taken, and the content of IFN-γ in the supernatant was measured using an IFN-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 Tukey's test in One-way ANOVA was used to compare the differences between multiple groups.

[0095] The results are as Figure 7As shown, an immunosuppressed model was successfully established after modeling with chloramphenicol (150 μg / mL), and the IFN-γ level was significantly lower than that of the normal control group ( p < 0.001). After intervention with 5 μg / mL QPQPPFSHT nonapeptide, the IFN-γ level was significantly up-regulated ( p < 0.01), indicating that from the perspective of the important immune factor IFN-γ level, QPQPPFSHT nonapeptide also has an immune enhancing effect.

[0096] Example 5: Immune enhancing effect of wheat peptide in an immunosuppressed mouse model

[0097] In this example, an immunosuppressed mouse model established with cyclophosphamide was used to explore the immune enhancing effect of wheat peptide (prepared in Example 1, which contains the peptide segment QPQPPFSHT). As a commonly used anti-cancer chemotherapy drug, cyclophosphamide can cause disorders in the immune system functions of important immune organs throughout the body (such as the spleen, thymus, and intestinal mucosa). This immunosuppressed state not only affects the proliferation and activity of immune cells in mice but also makes them susceptible to various infections and diseases. Therefore, establishing this model provides an important basis for evaluating the effect of wheat peptide in immune regulation. During the research process, immunosuppressed mice will receive different doses of wheat peptide treatment to observe its effect on immune cell proliferation. By detecting the immune factor levels in mice, the changes in immune organs, and the functions of related immune cells, the aim is to determine whether wheat peptide can effectively reverse the immunosuppression caused by cyclophosphamide and promote the recovery of immune cells.

[0098] 1. Experimental method

[0099] (1) Mice and feeding

[0100] Forty-five SPF-grade male ICR mice (age: 6 - 11 weeks old, weight: 35 ± 2 g) were purchased from Zhejiang Chinese Medical University, sourced from Shanghai Slack Co., Ltd., and the animal experiment ethics batch number: IACUC-20230206-07. The mouse breeding environment was as follows: 22 ± 2 °C, 50 ± 10% relative humidity, and a 12-hour light / dark cycle.

[0101] (2) Grouping and drug administration method

[0102] An experimental setup included a normal control group, three groups treated with wheat peptides (high dose of 0.5 g / kg of mouse body weight, denoted as high-dose wheat peptide; medium dose of 0.25 g / kg, denoted as medium-dose wheat peptide; low dose of 0.1 g / kg, denoted as low-dose wheat peptide), and one cyclophosphamide-induced modeling control group (modeling group), for a total of five groups. The high, medium, and low wheat peptide treatment groups were gavaged with the corresponding doses of wheat peptides, while the normal group and the modeling group were gavaged with an equal volume of normal saline. Gavage was performed once a day for 30 consecutive days. However, starting from the 28th day, the modeling group and the treatment groups were treated with 50 mg / kg of cyclophosphamide for modeling (from the 28th to the 30th day), and the normal control group was treated with an equal volume of normal saline. Body weight and food intake were recorded before and after cyclophosphamide-induced modeling.

[0103] (3)Determination of immune organ indices

[0104] The collected thymus and spleen were washed in pre-cooled phosphate buffer solution (PBS, 0.01 mol / L, pH = 7.4), and then blotted dry with filter paper. The spleen and thymus indices were calculated according to the following formulas:

[0105] Spleen index (%) = spleen weight / body weight × 100%;

[0106] Thymus index (%) = thymus weight / body weight × 100%.

[0107] (4)ELISA determination of serum immune-related cytokines and immunoglobulin A (IgA)

[0108] The contents of serum cytokines interferon-γ (IFN-γ) and immunoglobulin A (IgA) were determined using a GeneMe commercial ELISA analysis kit, and the determination method was carried out according to the kit instructions.

[0109] (5)Determination of fecal short-chain fatty acid content

[0110] Sample preparation: Approximately 50 mg of mouse feces was accurately weighed, 250 μL of sterile ultrapure water was added, vortexed for 5 min, then 10 μL of 5 mol / L hydrochloric acid solution was added to adjust the pH of the suspension to 2 - 3, vortexed for 1 min, and then allowed to stand at room temperature for 5 min. Then, it was centrifuged at 4 °C and 12,000 rpm for 30 min. 200 μL of the supernatant was taken and placed in a container containing 0.5 μL of 2-ethylbutyric acid (internal standard) diluted 20 times, vortexed for 1 min, and then centrifuged at 4 °C and 12,000 rpm for 5 min. 150 μL of the supernatant was taken for gas chromatography analysis.

[0111] Gas chromatography conditions: Agilent DB-FFAP 125-3237 (30 m × 0.52 mm × 0.50 mm) gas column; The gas column temperature programming conditions were an initial temperature of 100 °C for 0.5 min, then programmed to 180 °C at a rate of 8 °C / min and held for 1 min, and finally programmed to 240 °C at a rate of 20 °C / min and held for 15 min; The inlet temperature and the hydrogen flame detector temperature were 200 °C and 240 °C respectively; The flow rates of hydrogen, air and nitrogen were set at 30 mL / min, 300 mL / min and 20 mL / min respectively; The injection volume was 1 μL.

[0112] 2. Results analysis

[0113] 2.1 Growth performance

[0114] From Figure 8 it can be seen that the administration of cyclophosphamide had no significant difference in the food intake of mice.

[0115] From Figure 9 it can be known that after the administration of cyclophosphamide, compared with the normal control group, the body weight of the model group was significantly decreased ( p <0.001), indicating that the cyclophosphamide immunosuppressive treatment model was initially established. While there was no significant difference between the high-dose and medium-dose wheat peptide groups and the normal control group (>0.05), indicating that the wheat peptide containing the peptide segment QPQPPFSHT could improve the body weight loss of mice caused by cyclophosphamide. p <0.001), indicating that the cyclophosphamide immunosuppressive treatment model was initially established. While there was no significant difference between the high-dose and medium-dose wheat peptide groups and the normal control group (>0.05), indicating that the wheat peptide containing the peptide segment QPQPPFSHT could improve the body weight loss of mice caused by cyclophosphamide. p >0.05), indicating that the wheat peptide containing the peptide segment QPQPPFSHT could improve the body weight loss of mice caused by cyclophosphamide.

[0116] 2.2 Immune organ index

[0117] Cyclophosphamide modeling will significantly reduce the production of immune cells in the body, which is manifested in the atrophy of the primary and secondary immune organs that produce immune cells. As Figure 10 and Figure 11 shown, cyclophosphamide modeling significantly reduced the thymus and spleen indices of mice ( p <0.0001), indicating a decrease in the ability of immune organs to produce immune cells, while high-dose wheat peptide significantly increased the spleen index and thymus index ( p <0.05, p <0.01), and medium-dose wheat peptide significantly increased the thymus index ( p <0.01), indicating that wheat peptide could significantly restore the immune cell proliferation ability of immunosuppressed mice. p <0.0001), indicating a decrease in the ability of immune organs to produce immune cells, while high-dose wheat peptide significantly increased the spleen index and thymus index ( p <0.05, p <0.01), and medium-dose wheat peptide significantly increased the thymus index ( p <0.01), indicating that wheat peptide could significantly restore the immune cell proliferation ability of immunosuppressed mice. p <0.05, p <0.01), and medium-dose wheat peptide significantly increased the thymus index ( p <0.01), indicating that wheat peptide could significantly restore the immune cell proliferation ability of immunosuppressed mice. p <0.01), indicating that wheat peptide could significantly restore the immune cell proliferation ability of immunosuppressed mice.

[0118] 2.3 Immune factors

[0119] Immunoglobulins and cytokines in serum often play roles in activating immune cells and clearing antigens in the humoral circulation. For example, IFN-γ can directly clear antigens in the blood, and IgA can resist infections and promote mucosal immunity of the body. The effects of wheat peptides on serum immunoglobulin IgA and cytokine IFN-γ are as Figure 12 and Figure 13 shown. Due to the immunosuppression induced by cyclophosphamide, compared with the normal control group, the serum immunoglobulin IgA and cytokine IFN-γ in the model group were significantly down-regulated ( p < 0.0001), indicating that the immune ability of the mice was inhibited. Supplementing wheat peptides could significantly reverse the decrease of these immune-related proteins in serum: compared with the model group, IgA in the high, medium, and low-dose wheat peptide groups was significantly up-regulated ( p < 0.0001, p < 0.01, p < 0.01); IFN-γ in the high and medium-dose wheat peptide groups was significantly up-regulated ( p < 0.0001). This shows that wheat peptides can significantly enhance the immune function.

[0120] 2.4 Short-chain fatty acids

[0121] Short-chain fatty acids regulate the absorption of various nutrients in the intestine and hormone production, and play roles in participating in the body's energy metabolism and intestinal barrier repair and synthesis. The changes in their content and composition are closely related to intestinal mucosal immunity. The effects of wheat peptides on the content of short-chain fatty acids in mouse feces are as Figure 14 - Figure 16 shown. Under the cyclophosphamide-induced modeling treatment, the acetic acid, propionic acid, and butyric acid in feces were significantly decreased compared with the normal group ( p < 0.05, p < 0.05, p < 0.001). Compared with the normal group and the model group, the high and medium-dose wheat peptides significantly increased the content of acetic acid, propionic acid, and butyric acid in mouse feces ( p < 0.0001), indicating that the immune-enhancing effect of wheat peptides also has beneficial effects on the intestinal mucosa.

[0122] In summary, through mass spectrometry identification of peptide segments and molecular docking, the nonapeptide QPQPPFSHT was screened from wheat peptides. This nonapeptide molecule exhibits efficient immunomodulatory effects in both immunocompromised zebrafish models and mouse models, mainly manifested in promoting the proliferation of immune cells, increasing the content of immune factors, and also having beneficial effects on the intestinal mucosa while enhancing immunity. The present invention provides a new solution for enhancing immune function. The nonapeptide QPQPPFSHT can be applied to the preparation of health foods or drugs for preventing and improving adverse phenomena of immunodeficiency. Specifically, the nonapeptide QPQPPFSHT can be used alone in health products or drugs for enhancing immunity, or can be compounded 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. 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, wherein the enzymatic hydrolysis conditions are: water and wheat gluten are mixed in a mass ratio of 10:1, the pH of the feed solution is adjusted to 8.0±0.2, and then 1.0% of alkaline protease accounting for the total weight of the gluten is added for enzymatic hydrolysis for 30 minutes, and the pH of the feed solution is continuously maintained at ≥7.5 during the enzymatic hydrolysis process. After the end, 1.5% of neutral protease accounting for the total weight of the gluten is added for enzymatic hydrolysis for 60 minutes, and after the end of the enzymatic hydrolysis, 0.5% of flavor protease accounting for the total weight of the gluten is used for 30 minutes.

3. The use of the nonapeptide QT9 according to claim 1 in the preparation of an immunomodulatory product, characterized in that: The product is a health food for enhancing immunity.

4. 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.

5. 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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