Giant salamander immunoregulation meat peptide and application thereof

Abstract

The invention discloses a giant salamander immune regulation meat peptide and application thereof. The meat peptide comprises one or more than two polypeptides selected from SEQ ID NOs: 1-5. The polypeptide can enhance cellular immune response, such as enhancing macrophage proliferation activity, improving macrophage nitric oxide secretion level, enhancing macrophage phagocytic ability, enhancing macrophage antigen presentation ability, enhancing immune organ functions, regulating T cell subpopulation, improving cellular immune factor level and enhancing immune globulin level.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of polypeptide preparation, and particularly relates to a giant salamander immune-regulating meat peptide and application thereof. BACKGROUND

[0002] Andrias davidianus, which is the largest amphibian in the world, has extremely high edible and medicinal values and great development and application potential in the field of life and health. Wild giant salamanders are a national second-class protected aquatic animal, but in the past two decades, the artificial breeding technology of giant salamanders has gradually matured, and the giant salamander industry has rapidly developed. A large-scale breeding giant salamander industry and market has been formed in many provinces and regions such as Shaanxi, Hunan, Henan and Sichuan, which provides sustainable biological resources for extracting life and health products with medical and health care values from giant salamander meat. SUMMARY

[0003] The present application provides a giant salamander immune-regulating meat peptide and application thereof. The giant salamander meat peptide extract or polypeptide can enhance cell immune response, such as enhancing macrophage proliferation activity, improving macrophage nitric oxide secretion level, enhancing macrophage phagocytosis, enhancing macrophage antigen presentation ability, enhancing immune organ function, regulating T cell subgroups, improving cell immune factor level and enhancing immunoglobulin level.

[0004] The present application relates to the following contents:

[0005] 1. A giant salamander meat peptide extract, wherein the giant salamander bone peptide extract contains 30-80% of polypeptides with a molecular weight less than 1000 Da, 15-40% of polypeptides with a molecular weight of 1000-2000 Da, 3-20% of polypeptides with a molecular weight of 2000-3000 Da, 1-15% of polypeptides with a molecular weight of 3000-5000 Da and 0.3-10% of polypeptides with a molecular weight greater than or equal to 5000 Da, in terms of mass percentage in the giant salamander meat peptide extract.

[0006] 2. The giant salamander meat peptide extract according to item 1, wherein the giant salamander bone peptide extract contains 35-70% of polypeptides with a molecular weight less than 1000 Da, 15-35% of polypeptides with a molecular weight of 1000-2000 Da, 4-13% of polypeptides with a molecular weight of 2000-3000 Da, 2-12% of polypeptides with a molecular weight of 3000-5000 Da and 0.4-7% of polypeptides with a molecular weight greater than or equal to 5000 Da, in terms of mass percentage in the giant salamander meat peptide extract.

[0007] 3. The Andrias meat peptide extract of any one of claims 1 or 2, comprising a polypeptide comprising any one or more of SEQ ID NOs: 1-5 or a polypeptide comprising any one or more of SEQ ID NOs: 1-5.

[0008] 4. An Andrias meat peptide extract comprising a polypeptide comprising any one or more of SEQ ID NOs: 1-5 or a polypeptide comprising any one or more of SEQ ID NOs: 1-5.

[0009] 5. A polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 1-5 or an amino acid of any one of SEQ ID NOs: 1-5.

[0010] 6. A nucleic acid comprising a nucleic acid for encoding the polypeptide of claim 5.

[0011] 7. A composition comprising the Andrias meat peptide extract of any one of claims 1-4 or the polypeptide of claim 5.

[0012] 8. The composition of claim 7, further comprising a pharmaceutically, a food- scientifically, or a nutraceutically acceptable carrier, optionally an excipient, and optionally an active ingredient.

[0013] 9. Use of the Andrias meat peptide extract of any one of claims 1-4 or the polypeptide of claim 5 or the composition of claim 7 in the manufacture of a medicament for enhancing immune response.

[0014] 10. The use of claim 9, wherein the enhancing immune response comprises enhancing macrophage proliferative activity or elevating macrophage nitric oxide secretion level or enhancing macrophage phagocytic ability or enhancing macrophage antigen presentation ability.

[0015] 11. The use of claim 9, wherein the enhancing immune response comprises enhancing immune organ function or modulating T cell subpopulation or improving cellular immune factor level or enhancing immunoglobulin level.

[0016] 12. Use of the Andrias meat peptide extract of any one of claims 1-4 or the polypeptide of claim 5 or the composition of claim 7 in enhancing immune response.

[0017] 13. The use of claim 12, wherein the enhancing immune response comprises enhancing macrophage proliferative activity or elevating macrophage nitric oxide secretion level or enhancing macrophage phagocytic ability or enhancing macrophage antigen presentation ability.

[0018] 14. The use according to item 12, wherein the enhanced immune response comprises enhancing immune organ function or modulating T cell subpopulation or improving cytokine level or enhancing immunoglobulin level.

[0019] 15. A medicament for enhancing immune response, comprising the Andrias davidianus meat peptide extract of any one of items 1-4 or the polypeptide of item 5 or the composition of item 7.

[0020] 16. The medicament according to item 15, wherein the enhanced immune response comprises enhancing macrophage proliferation activity or increasing macrophage nitric oxide secretion level or enhancing macrophage phagocytosis ability or enhancing macrophage antigen presentation ability.

[0021] 17. The medicament according to item 15, wherein the enhanced immune response comprises enhancing immune organ function or modulating T cell subpopulation or improving cytokine level or enhancing immunoglobulin level.

[0022] Effects of the Invention

[0023] The Andrias davidianus meat peptide extract or the polypeptide described in the present application can enhance cellular immune response, such as: enhancing macrophage proliferation activity, increasing macrophage nitric oxide secretion level, enhancing macrophage phagocytosis ability, enhancing macrophage antigen presentation ability, enhancing immune organ function, modulating T cell subpopulation, improving cytokine level, and enhancing immunoglobulin level. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 is a schematic diagram of the appearance characteristics of Andrias davidianus cooked meat powder and Andrias davidianus meat peptide powder after enzymatic hydrolysis.

[0025] Figure 2 is a schematic diagram of the enzymatic hydrolysis rate of Andrias davidianus meat peptide (n = 3).

[0026] Figure 3 is a schematic diagram of the degree of hydrolysis of Andrias davidianus meat peptide (n = 3).

[0027] Figures 4a-4b is a schematic diagram of the molecular weight of Andrias davidianus meat peptide extract, wherein Figure 4a is a schematic diagram of the GPC profile of Andrias davidianus meat peptide extract; Figure 4b is a schematic diagram of the molecular weight distribution accumulation of Andrias davidianus meat peptide extract (n = 3).

[0028] Figure 5 is a schematic diagram of the effect of Andrias davidianus meat peptide extract (ADMP) on the proliferation rate of RAW264.7 macrophages (n = 3).

[0029] Figure 6 is a schematic diagram of the standard curve of nitrite concentration.

[0030] Figure 7 is a schematic diagram of the effect of Andrias davidianus meat peptide extract (ADMP) on NO secretion of RAW264.7 macrophages (n = 3, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001).

[0031] Figure 8 is a schematic diagram of the effect of Andrias davidianus meat peptide extract (ADMP) on phagocytic ability of RAW264.7 macrophages (n = 3, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001).

[0032] Figure 9 is a schematic diagram of the effect of Andrias davidianus meat peptide extract (ADMP) on MHC-II expression of RAW264.7 macrophages (n = 3, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001).

[0033] Figure 10 is a schematic diagram of the binding strength of predicted polypeptides to MHC-II molecules by NetMHCIIpan 4.0.

[0034] Figures 11a-11b is a schematic diagram of the MHC-II affinity prediction results of the Andrias davidianus meat whole-transcriptome peptide library, wherein Figure 11a is a schematic diagram of the number of strong bindings of predicted polypeptides to different MHC-II phenotypes; Figure 11b is a heatmap of the binding of the top 10 predicted polypeptides to common MHC-II phenotypes in the general population (%Rank values ≤ 2 indicate strong binding (SB) to MHC-II molecules, while values > 2 and ≤ 10 indicate weak binding (WB), and values > 10 indicate no binding).

[0035] Figures 12a-12b is a schematic diagram of the MHC-II affinity prediction results of the Andrias davidianus meat enzymatic component peptide library, wherein Figure 12a is a schematic diagram of the number of strong bindings of predicted polypeptides to different MHC-II phenotypes; Figure 12b is a heatmap of the binding of the top 10 predicted polypeptides to common MHC-II phenotypes in the general population (%Rank values ≤ 2 indicate strong binding (SB) to MHC-II molecules, while values > 2 and ≤ 10 indicate weak binding (WB), and values > 10 indicate no binding).

[0036] Figure 13 is a schematic diagram of the effect of Andrias davidianus meat synthetic peptides on the proliferation rate of RAW264.7 macrophages (n = 3).

[0037] Figure 14This is a schematic diagram showing the effect of giant salamander meat peptide synthesis on NO secretion in RAW264.7 macrophages (n=3, * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001).

[0038] Figure 15 This is a schematic diagram showing the effect of giant salamander meat peptide synthesis on the phagocytic capacity of RAW264.7 macrophages (n=3, * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001).

[0039] Figure 16 This is a schematic diagram showing the effect of giant salamander meat peptide synthesis on MHC-II expression in RAW264.7 macrophages (n=3, * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001).

[0040] Figure 17 This is a schematic diagram showing the effect of giant salamander meat peptide synthesis on the proliferation rate of DC2.4 cells (n=3).

[0041] Figure 18 This is a schematic diagram showing the effect of giant salamander meat peptide synthesis on MHC-II expression in DC2.4 cells (n=3, * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001).

[0042] Figure 19 This is a schematic diagram of the dosing regimen for a cyclophosphamide (CTX)-induced immunosuppressed mouse model.

[0043] Figure 20 This is a schematic diagram showing the effect of giant salamander meat peptides on the body weight of cyclophosphamide (CTX)-induced immunosuppressed mice (n=6).

[0044] Figures 21a-21b This is a schematic diagram illustrating the effect of giant salamander meat peptides on the immune organ indices of cyclophosphamide (CTX)-induced immunosuppressed mice. Figure 21a A schematic diagram showing the spleen index in mice. Figure 21b A schematic diagram representing the thymus index in mice (n=6, *p<0.05, **p<0.01, ***p<0.001 vs. model group); # p<0.05, ## p<0.01, ### p<0.001 (vs. control group). The model group represents the CTX treatment group; QL represents the low-dose QR14 treatment group; QH represents the high-dose QR14 treatment group; AL represents the low-dose giant salamander peptide extract treatment group; and AH represents the high-dose giant salamander peptide extract treatment group.

[0045] Figures 22a-22dis a schematic diagram of the effect of andouder meat peptide on the subtypes of T cells in the spleen of cyclophosphamide (CTX)-induced immunosuppressed mice, wherein Figure 22a represents a representative flow cytometry diagram of spleen T lymphocyte typing, Figure 22b represents a schematic diagram of the proportion of CD4+ T cells, Figure 22c represents a schematic diagram of the proportion of CD8+ T cells, Figure 22d represents a schematic diagram of the proportion of CD4+ / CD8+ T cells (n=3, *p<0.05, **p<0.01, ***p<0.001 vs. model group; # p<0.05, ## p<0.01, ### p<0.001 vs. control group). The model group represents the CTX treatment group; QL represents the QR14 low-dose treatment group; QH represents the QR14 high-dose treatment group; AL represents the andouder meat peptide extract low-dose treatment group; and AH represents the andouder meat peptide extract high-dose treatment group.

[0046] Figures 23a-23d is a schematic diagram of the effect of andouder meat peptide on serum cytokines in cyclophosphamide (CTX)-induced immunosuppressed mice, wherein Figure 23a represents a schematic diagram of the effect of andouder meat peptide on serum cytokine IL-6 in cyclophosphamide (CTX)-induced immunosuppressed mice, Figure 23b represents a schematic diagram of the effect of andouder meat peptide on serum cytokine IL-1β in cyclophosphamide (CTX)-induced immunosuppressed mice, Figure 23c represents a schematic diagram of the effect of andouder meat peptide on serum cytokine IFN-γ in cyclophosphamide (CTX)-induced immunosuppressed mice, Figure 23d represents a schematic diagram of the effect of andouder meat peptide on serum cytokine TNF-α in cyclophosphamide (CTX)-induced immunosuppressed mice (n=6, *p<0.05, **p<0.01, ***p<0.001 vs. model group; # p<0.05, ## p<0.01, ### p<0.001 vs. control group). The model group represents the CTX treatment group; QL represents the QR14 low-dose treatment group; QH represents the QR14 high-dose treatment group; AL represents the andouder meat peptide extract low-dose treatment group; and AH represents the andouder meat peptide extract high-dose treatment group.

[0047] Figures 24a-24c is a schematic diagram of the effect of andouder meat peptide on serum immunoglobulin in cyclophosphamide (CTX)-induced immunosuppressed mice, wherein Figure 24a represents a schematic diagram of the effect of andouder meat peptide on serum immunoglobulin IgG in CTX-induced immunosuppressed mice, Figure 24bFigure 25 is a diagram showing the effect of andouder meat peptide on serum immunoglobulin IgM in CTX-induced immunosuppressed mice, Figure 24c Figure 26 is a diagram showing the effect of andouder meat peptide on serum immunoglobulin IgA in CTX-induced immunosuppressed mice (n = 6, *p < 0.05, **p < 0.01, ***p < 0.001 vs. model group; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. control group). The model group represents the CTX-treated group; QL represents the QR14 low-dose treatment group; QH represents the QR14 high-dose treatment group; AL represents the andouder meat peptide extract low-dose treatment group; and AH represents the andouder meat peptide extract high-dose treatment group.

[0048] Figures 25a-25f Figure 27 is a diagram showing the effect of andouder meat peptide on blood routine in CTX-induced immunosuppressed mice, wherein Figure 25a Figure 28 is a diagram showing the effect of andouder meat peptide on the number of white blood cells in blood routine in CTX-induced immunosuppressed mice, Figure 25b Figure 29 is a diagram showing the effect of andouder meat peptide on the number of lymphocytes in blood routine in CTX-induced immunosuppressed mice, Figure 25c Figure 30 is a diagram showing the effect of andouder meat peptide on the number of neutrophils in blood routine in CTX-induced immunosuppressed mice, Figure 25d Figure 31 is a diagram showing the effect of andouder meat peptide on the number of red blood cells in blood routine in CTX-induced immunosuppressed mice, Figure 25e Figure 32 is a diagram showing the effect of andouder meat peptide on hemoglobin in blood routine in CTX-induced immunosuppressed mice, and Figure 33 is a diagram showing the effect of andouder meat peptide on the number of platelets in blood routine in CTX-induced immunosuppressed mice (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001 vs. model group; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. control group). The model group represents the CTX-treated group; QL represents the QR14 low-dose treatment group; QH represents the QR14 high-dose treatment group; AL represents the andouder meat peptide extract low-dose treatment group; and AH represents the andouder meat peptide extract high-dose treatment group.

[0049] Figure 26 Figure 34 is a diagram showing the effect of andouder meat peptide on the morphology of immune organs in CTX-induced immunosuppressed mice. QL represents the QR14 low-dose treatment group; QH represents the QR14 high-dose treatment group; AL represents the andouder meat peptide extract low-dose treatment group; and AH represents the andouder meat peptide extract high-dose treatment group.

[0050] Figure 27is a schematic diagram of H&E staining results of mouse spleen and thymus. QL represents QR14 low-dose treatment group; QH represents QR14 high-dose treatment group; AL represents Megalobatrachus amurensis meat peptide extract low-dose treatment group; AH represents Megalobatrachus amurensis meat peptide extract high-dose treatment group.

[0051] Figure 28 is a schematic diagram of H&E staining results of mouse ileum tissue. QL represents QR14 low-dose treatment group; QH represents QR14 high-dose treatment group; AL represents Megalobatrachus amurensis meat peptide extract low-dose treatment group; AH represents Megalobatrachus amurensis meat peptide extract high-dose treatment group.

[0052] Figure 29 is a schematic diagram of immunohistochemical staining results of mouse ileum tissue. QL represents QR14 low-dose treatment group; QH represents QR14 high-dose treatment group; AL represents Megalobatrachus amurensis meat peptide extract low-dose treatment group; AH represents Megalobatrachus amurensis meat peptide extract high-dose treatment group. DETAILED DESCRIPTION

[0053] The embodiments described below are merely exemplary in nature and are not intended to limit the scope of the application, as described. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. The description below is presented to enable any person skilled in the art to make and use the application.

[0054] As used throughout this description and in the claims, the terms "comprise" and "comprising" are to be interpreted inclusively rather than exclusively. The description that follows is intended to be a thorough description of the preferred embodiments of the application, and as such, is not intended to limit the scope of the application. The only true limit of the scope of the application is to be found in the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0055] The present application provides a giant salamander meat peptide extract, wherein the giant salamander bone peptide extract comprises, in terms of the mass percentage in the giant salamander meat peptide extract, 30-80% of polypeptides with a molecular weight less than 1000 Da, 15-40% of polypeptides with a molecular weight of 1000-2000 Da, 3-20% of polypeptides with a molecular weight of 2000-3000 Da, 1-15% of polypeptides with a molecular weight of 3000-5000 Da, and 0.3-10% of polypeptides with a molecular weight greater than or equal to 5000 Da. In some embodiments, the giant salamander bone peptide extract comprises, in terms of the mass percentage in the giant salamander meat peptide extract, 35-70% of polypeptides with a molecular weight less than 1000 Da, 15-35% of polypeptides with a molecular weight of 1000-2000 Da, 4-13% of polypeptides with a molecular weight of 2000-3000 Da, 2-12% of polypeptides with a molecular weight of 3000-5000 Da, and 0.4-7% of polypeptides with a molecular weight greater than or equal to 5000 Da.

[0056] For example, the giant salamander meat peptide extract comprises, in terms of the mass percentage in the giant salamander meat peptide extract, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, or 80% of polypeptides with a molecular weight less than 1000 Da;

[0057] 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, or 40% of polypeptides with a molecular weight of 1000-2000 Da;

[0058] 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% of polypeptides with a molecular weight of 2000-3000 Da;

[0059] 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, or 12%, 13%, 14%, or 15% of polypeptides with a molecular weight of 3000-5000 Da; and

[0060] 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, or 7%, 8%, 9%, or 10% of polypeptides with a molecular weight greater than or equal to 5000 Da.

[0061] In the present application, for polypeptides with a molecular weight of 1000-2000 Da, it refers to polypeptides with a molecular weight greater than or equal to 1000 Da and a molecular weight less than 2000 Da;

[0062] For polypeptides with a molecular weight of 2000-3000 Da, it refers to polypeptides with a molecular weight greater than or equal to 2000 Da and a molecular weight less than 3000 Da;

[0063] For polypeptides with a molecular weight of 3000-5000 Da, it refers to polypeptides with a molecular weight greater than or equal to 3000 Da and a molecular weight less than 5000 Da.

[0064] In the present application, for the determination method of the content of polypeptides with different molecular weights in the Andrias meat peptide extract, the present application does not make any limitation, which can be determined according to the conventional method in the art, for example, the high performance liquid chromatography is used to analyze the molecular weight distribution of the Andrias meat peptide extract. In some embodiments, the content of different peptides in the Andrias meat peptide extract is determined by using standard samples to prepare a standard curve. In some embodiments, the standard samples are cytochrome C (12,384 Da), aprotinin (6511 Da), an artificially synthesized polypeptide VPSGPLGPEGPR (1161.6 Da), glutathione (307 Da), and glutamate (147 Da). In some embodiments, the chromatographic column of the high performance liquid chromatography is TSK-Gel G2000SWXL (300 mm x 7.8 mm). In some embodiments, the mobile phase is 30% acetonitrile solution (containing 0.1% trifluoroacetic acid). In some embodiments, the column temperature is 30°C, the flow rate is 0.5 mL / min, and the detection wavelength is 220 nm.

[0065] In some embodiments, the Andrias meat peptide extract is obtained by a preparation method comprising the following steps:

[0066] (1) preparing Andrias cooked meat powder;

[0067] (2) subjecting the Andrias cooked meat powder to protease enzymolysis and centrifugation to obtain supernatant;

[0068] (3) subjecting the supernatant to concentration treatment and freeze-drying to obtain the Andrias meat peptide extract after enzymolysis.

[0069] In the present application, the preparation of Andrias mature meat powder is not limited, and methods known to those skilled in the art can be selected. For example, the muscle part of the fresh Andrias raw material after natural thawing can be obtained after blanching, freezing, drying, and crushing.

[0070] In the present application, the selection of protease is not limited, and proteases known to those skilled in the art can be selected and subjected to enzymolysis at the optimum pH and enzymolysis temperature. For example, flavor protease, papain, neutral protease, trypsin, and alkaline protease can be used for enzymolysis. For example, for flavor protease, enzymolysis can be carried out at pH 5.0-pH 7.0, 40°C-60°C; for papain, enzymolysis can be carried out at pH 6.0-pH 8.0, 40°C-60°C; for neutral protease, enzymolysis can be carried out at pH 6.0-pH 8.0, 40°C-60°C; for trypsin, enzymolysis can be carried out at pH 6.0-pH 7.0, 30°C-50°C; for alkaline protease, enzymolysis can be carried out at pH 7.0-pH 9.0, 45°C-65°C.

[0071] In the present application, the enzymolysis step of Andrias mature meat powder is not limited, and methods known to those skilled in the art can be selected. For example, the constant temperature water bath can be preheated to the optimum temperature of the enzyme, then the freeze-dried Andrias meat powder is weighed, the protease and ultrapure water are added and stirred, the enzymolysis reaction is carried out under the conditions of maintaining the optimum temperature and pH of the enzyme, after the enzymolysis is completed, the enzyme is inactivated, centrifuged, the supernatant is collected, and finally the supernatant is concentrated, cooled, and freeze-dried to obtain Andrias meat peptide powder. The instruments used in the process, including constant temperature water bath, magnetic stirrer, evaporator, and reagents used for pH adjustment, such as HCl or NaOH, are all conventional instruments or reagents known to those skilled in the art.

[0072] The present application provides an Andrias meat peptide extract, which comprises a polypeptide comprising any one or two or more of SEQ ID NOs: 1-5 or any one or two or more of the polypeptides of SEQ ID NOs: 1-5.

[0073] wherein,

[0074] The amino acid sequence of SEQ ID NO: 1 is as follows: QQQPIVSFQTAPAR (abbreviated as QR14)

[0075] The amino acid sequence of SEQ ID NO: 2 is as follows: IDLFIVNETHPNDPR (abbreviated as IR15)

[0076] The amino acid sequence of SEQ ID NO: 3 is as follows: IDGGQYILQVSNVAGTK (abbreviated as IK17)

[0077] The amino acid sequence of SEQ ID NO: 4 is as follows: MVNHFVAEFKRKHK (abbreviated as MK14)

[0078] The amino acid sequence of SEQ ID NO: 5 is as follows: NEWRMAANIPAK (abbreviated as NK12)

[0079] In some embodiments, based on the prediction and mining of the immune-regulating peptides from the meat of Andrias davidianus by bioinformatics tools in the early stage, combined with the affinity evaluation between the polypeptide and different allelic genotypes of MHC-II molecules, molecular docking, molecular dynamics simulation and multi-parameter screening results, the top three candidate peptides with the highest binding capacity in the whole transcriptome-derived peptide library are screened as SEQ ID NOs: 1-3; the top two peptides with the highest binding capacity in the enzyme digestion group-derived peptide library are screened as SEQ ID NOs: 4-5, a total of 5 peptides are synthesized by solid phase synthesis, and are used for subsequent verification of the immune-regulating function in the RAW264.7 macrophage model.

[0080] In some embodiments, the bioinformatics tool is NetMHCIIpan 4.0 server, which uses artificial neural network (ANN) for prediction, so as to analyze the affinity between the peptide segment and MHC-II molecules. This tool can not only predict the binding affinity between different MHC-II alleles and polypeptides, but also be used for analysis of peptide sequences of different lengths, thereby providing more comprehensive epitope prediction ability. The prediction result is output in the form of prediction score, indicating whether the analyzed peptide is more likely to be naturally presented by the selected MHC-II receptor.

[0081] In some embodiments, the NetMHCIIpan 4.0 model is used to screen potential immune-regulating peptides from the peptide library obtained from the whole transcriptome of Andrias davidianus meat and the enzyme digestion group of Andrias davidianus meat by affinity with different MHC-II phenotypes.

[0082] In some embodiments, 677 MHC-II phenotype sites are selected, of which 672 are from humans (i.e. 607 HLA-DRB1, 32 HLA-DRB3, 6 HLA-DRB4, 15 HLA-DRB5, 6 HLA-DP and 6 HLA-DQ epitopes) and 5 are from mice (i.e. epitopes H-2-IAb, H-2-IAd, H-2-IEd, H2-IAu, H-2-IEk from mice are selected for prediction.

[0083] In the present application, the method for synthesizing the peptide segment in solid phase is not limited in the present application, and can be synthesized according to the method known in the art.

[0084] The present application provides a polypeptide comprising an amino acid sequence as shown in any one of SEQ ID NOs: 1-5 or an amino acid as shown in any one of SEQ ID NOs: 1-5.

[0085] The present application provides a nucleic acid comprising a nucleic acid for encoding the above-mentioned polypeptide.

[0086] The present application provides a composition comprising the above-mentioned Andrias meat peptide extract or polypeptide. In some embodiments, it further comprises a pharmaceutically, food or health product acceptable carrier, optional adjuvant and optional active ingredient.

[0087] In the present application, the pharmaceutically, food or health product acceptable carrier, optional adjuvant and optional active ingredient are not limited in the present application, and can be the carrier, adjuvant or active ingredient commonly used in the art.

[0088] The present application provides the use of the above-mentioned Andrias meat peptide extract or polypeptide or composition in the preparation of a medicament for enhancing immune response.

[0089] In some embodiments, the enhancing immune response comprises enhancing macrophage proliferation activity or increasing macrophage nitric oxide secretion level or enhancing macrophage phagocytosis ability or enhancing macrophage antigen presentation ability.

[0090] In some embodiments, the enhancing immune response comprises enhancing immune organ function or regulating T cell subpopulation or improving cytokine level or enhancing immunoglobulin level.

[0091] The present application provides the use of the above-mentioned Andrias meat peptide extract or polypeptide or composition in enhancing immune response.

[0092] In some embodiments, the enhancing immune response comprises enhancing macrophage proliferation activity or increasing macrophage nitric oxide secretion level or enhancing macrophage phagocytosis ability or enhancing macrophage antigen presentation ability.

[0093] In some embodiments, the enhancing immune response comprises enhancing immune organ function or regulating T cell subpopulation or improving cytokine level or enhancing immunoglobulin level.

[0094] The present application provides a medicament for enhancing immune response, comprising the above-mentioned Andrias meat peptide extract or polypeptide or composition.

[0095] In some embodiments, the enhancing immune response comprises enhancing macrophage proliferative activity or increasing macrophage nitric oxide secretion level or enhancing macrophage phagocytosis or enhancing macrophage antigen presentation.

[0096] In some embodiments, the enhancing immune response comprises enhancing immune organ function or modulating T cell subpopulation or improving cytokine level or enhancing immunoglobulin level.

[0097] Examples

[0098] The materials used in the experiments and the experimental methods are generally and / or specifically described in this application. In the following examples, % means wt%, i.e. weight percentage, if no other specific description is given. The reagents or instruments used are not specified by the manufacturer, and are all conventional reagent products that can be obtained commercially.

[0099] Example 1 Preparation of Andrias davidianus meat peptide extract

[0100] 1.1 Preparation of Andrias davidianus cooked meat powder

[0101] (1) Take the fresh Andrias davidianus raw material after natural thawing, remove its internal organs, epidermis and adipose tissue, and only keep the muscle part. After being washed clean with flowing water, cut it into small pieces with a size of about 1 cm x 1 cm x 1 cm;

[0102] (2) Put the above fish pieces into boiling water for blanching treatment, and heat at 100°C for 20 min until the fish is completely cooked;

[0103] (3) Take out the cooked fish, cool it to room temperature, drain the surface water, and lay it flat in a clean container (the thickness is controlled at about 0.5 cm). After sealing the film, freeze it in a -80°C refrigerator for 24 h;

[0104] (4) After freezing, dehydrate it by vacuum freeze-drying method. After drying, crush the freeze-dried sample using a crushing device, and sieve it through a 40-mesh sieve to obtain Andrias davidianus cooked meat powder;

[0105] (5) Determine the water content and dry weight ratio of the obtained meat powder. Pack the sample in airtight containers and store it at -20°C for standby use.

[0106] 1.2. Enzymatic preparation of Andrias davidianus meat peptide extract

[0107] (1) Preheat the constant temperature water bath to the optimum temperature corresponding to the enzyme, and maintain it stable for 5 min;

[0108] (2) Take 5 g of freeze-dried Andrias davidianus meat powder, add the corresponding protease according to the enzyme addition amount of 2500 U / g, and add ultrapure water according to the solid-liquid ratio of 1:10 (g / mL);

[0109] The proteases are respectively selected as flavor protease (optimum pH 6.0, 50℃), papain (optimum pH 7.0, 50℃), neutral protease (optimum pH 7.0, 50℃), trypsin (optimum pH 7.0, 40℃) and alkaline protease (optimum pH 8.0, 55℃) to perform the enzymatic reaction.

[0110] (3) Start the magnetic stirrer, set the stirring speed to 500 r / min, and use 1.0 mol / L HCl or NaOH solution to adjust the solution to the optimum pH value of each enzyme;

[0111] (4) Perform 4h enzymatic reaction under constant temperature conditions, and continuously add 1.0 mol / L NaOH solution during the reaction to maintain pH stability;

[0112] (5) After the enzymolysis is completed, transfer to a 100℃ water bath for 15min, and then centrifuge at 5000xg at 25℃ for 15min, and collect the supernatant;

[0113] (6) Concentrate the obtained supernatant in a rotary evaporator (speed: 60 rpm, water bath temperature: 50℃, pressure: 15 hPa), and collect the concentrated polypeptide solution;

[0114] (7) Cool the concentrated solution to room temperature, freeze it in a-80℃ refrigerator for 24h, and then perform vacuum freeze-drying treatment;

[0115] (8) Record the dry weight of the product after freeze-drying, and reserve it.

[0116] The appearance characteristics of the Andrias japonicus cooked meat powder and the Andrias japonicus meat peptide extract obtained by enzymolysis are shown in Table 1. Figure 1 The Andrias japonicus cooked meat powder is light yellow, with a water content of 2.91%, good dispersibility, delicate texture and dryness, and a slight fishy smell. The coarse enzymatic hydrolysate shows obvious stratification after standing, with brownish yellow suspension on the upper layer and milky yellow mud-like precipitate on the lower layer, accompanied by a strong fishy smell. The enzymatic supernatant is light yellow and transparent, with no stratification phenomenon and a significantly reduced fishy smell. The Andrias japonicus meat peptide obtained by freeze-drying is white and granular, with uneven particle size. After grinding, a fine powder can be obtained, with a lighter fishy smell.

[0117] The degree of hydrolysis (DH) of the Andrias japonicus meat peptide extract was determined by the pH-stat method, and the results are shown in Table 2. Figure 2 The calculation formula of the degree of hydrolysis is as follows:

[0118] DH(%) = [B x Nb / (Mp x a x h tot )] x 100%

[0119] Where B is the volume of alkali consumed during pH maintenance (mL), Nb is the alkali concentration (mol / L), Mp is the substrate protein content (g), htot is the total number of peptide bonds in the protein (7.72 mmol / g in this experiment), and a is the dissociation degree of a-amino groups during enzymatic hydrolysis, which is calculated according to the following formula:

[0120] a = 10 pH-pK / 1 + 10 pH-pK

[0121] From Figure 2 it can be seen that among the five enzyme preparations, the flavor protease group showed the highest degree of hydrolysis, reaching 21.95 ± 4.40%, indicating that its enzymatic efficiency is superior and the proportion of small molecule peptides in the product is higher; the papain group had the lowest degree of hydrolysis, which was 9.37 ± 0.67%.

[0122] The yield of giant salamander meat peptide enzymatic hydrolysate was calculated using the following formula, and the results are shown in Figure 3 .

[0123] Giant salamander meat peptide enzymatic hydrolysate yield = M1 / M0 x 100%

[0124] Where M1 is the dry weight of the freeze-dried enzymatic supernatant product (g), and M0 is the dry weight of the substrate giant salamander meat powder (g). All samples were tested in triplicate, and the standard deviation was calculated to assess the stability of the data.

[0125] From Figure 3 it can be seen that the yield of the alkaline protease treatment group was the highest, reaching 72.5 ± 2.7%, indicating that it has high output efficiency and practical application potential in large-scale enzymatic hydrolysis applications. In comparison, the yield of papain was the lowest, only 49.5 ± 1.2%.

[0126] The following method was used to determine the molecular weight distribution of giant salamander meat peptides:

[0127] An Agilent 1260 high-performance liquid chromatograph was used, equipped with a TSK-GEL G2000 SWXL gel exclusion chromatography column (7.8 mm x 300 mm), a mobile phase of 30% acetonitrile solution (containing 0.1% trifluoroacetic acid), a column temperature of 30°C, a flow rate of 0.5 mL / min, and a detection wavelength of 220 nm.

[0128] To establish a standard curve, the following standard samples were used for calibration: cytochrome C (12,384 Da), aprotinin (6,511 Da), synthetic polypeptide VPSGPLGPEGPR (1,161.6 Da), glutathione (307 Da), and glutamic acid (147 Da).

[0129] The linear regression was performed with the standard retention time as the independent variable and the natural logarithm of the molecular weight as the dependent variable to establish the molecular weight-retention time relationship curve. The retention time points of 5 kDa, 3 kDa, 2 kDa and 1 kDa were used to quantitatively analyze the components in the sample, and the proportion of the peak area in each molecular weight interval was calculated to determine the polypeptide distribution in different molecular weight intervals (<1 kDa, 1-2 kDa, 2-3 kDa, 3-5 kDa, >5 kDa), and the average molecular weight of the sample was calculated accordingly, and the results are shown in Table 1 and FIG. 2. Figures 4a-4b and Table 1

[0130] From Figure 4a It can be seen that the overall chromatographic signal intensity of the Andrias davidianus meat powder enzymolysis group is low, and the main retention time is concentrated in the 18-20 min region, which is speculated to be high molecular weight and not subjected to fine separation treatment. From Figure 4b and Table 1, the molecular weight distribution of the Andrias davidianus meat enzymolysis group can be seen, and the results show that the proportion of low molecular weight peptides (<1000 Da) in the products obtained by flavor enzymes and alkaline enzymes is significantly higher, which is suitable as a raw material for bioactive peptide research.

[0131] Table 1 Molecular weight distribution of Andrias davidianus meat enzymolysis group

[0132]

[0133] Example 2 Immunomodulatory effect of immunized Andrias davidianus meat peptides on RAW264.7 cells

[0134] 2.1 Effect of Andrias davidianus meat peptide extract on the proliferation activity of RAW264.7 macrophages

[0135] To evaluate the effect of Andrias davidianus meat peptide extract (ADMP) on the proliferation activity of macrophages, an in vitro functional experiment was carried out using a RAW264.7 cell model. In the experiment, an appropriate amount of ADMP freeze-dried powder was weighed and dissolved using high-sugar DMEM complete medium (purchased from Thermo Fisher Scientific / Gibco (USA)), and five concentrations of 125, 250, 500, 1000 and 2000 μg / mL of ADMP stock solution were prepared, which were filtered through a 0.22 μm microporous filter to remove bacteria and used as a reserve.

[0136] RAW264.7 cells (mouse-derived macrophages RAW264.7 were donated by Professor Chang Zhijie of Tsinghua University School of Medicine) in the logarithmic growth phase were taken, and 1.0×10 5The cells / mL density preparation cell suspension, and 100 μL evenly inoculated in 96-well culture plates. To prevent evaporation effects, 100 μL PBS was added to the edge of the well. The cells were incubated at 37°C, 5% CO2 incubator for 24 h, so that they fully adhere to the wall.

[0137] After adhering, the culture medium was discarded, and different concentrations of ADMP treatment solution were added for intervention treatment; the control group and the blank group only added the same volume of culture medium without ADMP. After 24 h of continuous incubation, the supernatant was discarded, 100 μL of fresh complete culture medium (10% fetal bovine serum (FBS) (purchased from Thermo Fisher Scientific / Gibco (USA)) and 1% penicillin-streptomycin (purchased from Thermo Fisher Scientific / Gibco (USA)) were added to the DMEM high-sugar culture medium, and the complete culture medium was obtained by thoroughly mixing and uniformly storing in a 4°C refrigerator for standby) containing 10 μL CCK-8 reagent (purchased from American Lun Biotech Co., Ltd. (Dalian, China)) was added to each well, and incubated at 37°C for 2 h. Subsequently, the absorbance (A 450 ) was measured at 450 nm wavelength, and the proliferation rate was calculated according to the following formula:

[0138]

[0139] A0 is the absorbance of the blank well, A is the absorbance of the control group, and A1 is the absorbance of the sample group.

[0140] The experimental results are shown in Figure 5 .

[0141] As can be seen from Figure 5 , the ADMP components obtained by five different protease enzymolysis do not exhibit cytotoxicity in the above concentration range, and can promote the proliferation of RAW264.7 cells, showing good biocompatibility. Especially in the range of 500-1000 μg / mL, the cell activity is significantly enhanced, indicating that ADMP has a certain cell activation potential, and is suitable for the development of immune function enhancing active peptides.

[0142] 2.2, Effect of Andrias davidianus Peptide on Nitric Oxide (NO) Secretion Level of RAW264.7 Macrophage Cells

[0143] To further evaluate the regulatory effect of ADMP on the immune function of macrophages, a nitric oxide (NO) detection kit (purchased from Biyun Tian Biotechnology Co., Ltd. (Shanghai, China)) based on the Griess reaction method was used to indirectly detect the concentration of nitrite (NO2 - ) in the cell culture supernatant to calculate the NO secretion level.

[0144] The specific procedure is as follows: 250, 500, and 1000 μg / mL of ADMP were added to RAW264.7 cells along with 1 μg / mL of lipopolysaccharide (LPS), respectively. After treatment for 24 h, 50 μL of supernatant was collected. 50 μL of I Lysse Reagent I (from the Nitric Oxide Kit, purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China)) was added sequentially, and the cells were incubated at room temperature for 10 min. Then, 50 μL of II Lysse Reagent II (from the Nitric Oxide Kit, purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China)) was added. Finally, the absorbance value was read at a wavelength of 540 nm.

[0145] A standard curve for NaNO2 was established, and the fitted equation was: y = 0.0063x + 0.0526 (R² = 0.999). Figure 6 As shown, the correlation is good.

[0146] Where x is the NaNO2 concentration (μmol / L) and y is the absorbance value.

[0147] Calculate NO2 in cell supernatant based on standard curve. - The concentration, thus reflecting the NO secretion level of RAW264.7 cells, is shown in the following results. Figure 7 As shown.

[0148] Depend on Figure 7 It can be seen that treatment with 250, 500, and 1000 μg / mL ADMP significantly increased NO release from RAW264.7 cells (p<0.001), which was statistically significant. The flavor protease, trypsin, and neutral protease components induced the most significant increase in NO levels, suggesting that ADMP has the ability to activate macrophages and enhance the immune response.

[0149] 2.3 Effect of giant salamander meat peptides on the phagocytic capacity of RAW264.7 macrophages

[0150] To assess the effect of ADMP on macrophage phagocytic activity, the phagocytic capacity of RAW264.7 cells was detected using the neutral red uptake assay. This method indirectly evaluates phagocytic function by measuring the amount of neutral red dye absorbed by the cells.

[0151] In the experiment, RAW264.7 cells were seeded in 96-well plates and incubated at 37°C and 5% CO2 for 24 h, after which the culture medium was discarded. ADMP samples at concentrations of 250, 500, and 1000 μg / mL and lipopolysaccharide (LPS) at concentrations of 1 μg / mL were added, respectively, and treated for 24 h. Subsequently, 20 μL of medium containing neutral red was added to each well, and incubation continued for 2 h. After treatment, 200 μL of cell lysis buffer was added to each well to release the dye.

[0152] Read the absorbance A1 at 540 nm wavelength, the absorbance of the blank control hole is A0, and the phagocytosis index is calculated as follows:

[0153]

[0154] The experimental results are shown in Figure 8

[0155] As can be seen from Figure 8 After treatment with different concentrations (250, 500, 1000 pg / mL) of ADMP, the phagocytosis index was significantly higher than that of the control group, with statistical significance (p < 0.001), and the trypsin enzymatic component had the most significant effect. The phagocytosis index of the trypsin enzymatic component at concentrations of 250, 500, and 1000 pg / mL was 1.46 ± 0.03, 2.07 ± 0.10, and 2.76 ± 0.13, respectively, indicating that it can effectively enhance the phagocytic ability of macrophages and exhibit good immune enhancement potential.

[0156] 2.4, Effect of Andrias davidianus Meat Peptide Extract on MHC-II molecule expression of RAW264.7 macrophages

[0157] To explore the regulatory effect of ADMP on MHC-II molecule expression on the membrane surface of macrophages, flow cytometry was used to quantitatively detect the expression of MHC-II protein on the surface of RAW264.7 cells.

[0158] MHC-II molecules are key markers in the process of antigen presentation and immune activation of macrophages, and their expression level directly reflects the antigen presentation ability and immune response state of cells. In this experiment, the expression level of MHC-II protein on the surface of RAW264.7 cells after ADMP treatment was detected by flow cytometry.

[0159] The specific steps are as follows: RAW264.7 cells were treated with 1000 pg / mL of ADMP for 24 h, and then washed twice with pre-cooled staining buffer. PE fluorescently labeled anti-mouse MHC-II antibody (MHC-Class-II-I-A-I-E-clone-M5-114-15-2 antibody purchased from Thermo Fisher Scientific (USA)) was added, and the cells were stained at 4°C for 30 min in the dark. After staining, the cells were washed again with phosphate buffered saline (PBS) (purchased from Amercan Biotech (Dalian, China)) and resuspended in 400 pL of PBS.

[0160] CytoFLEX flow cytometer (Beckman Coulter) was used to collect data, and FlowJov 7.6 software was used for data analysis. The change in fluorescence intensity of MHC-II was used to evaluate the regulatory effect of macrophage immune activity.​

[0161] The experimental results are shown in Figure 9

[0162] As can be seen from Figure 9 It can be seen that different ADMP components can significantly improve the expression of MHC-II molecules (p<0.001), indicating that ADMP has the mechanism of enhancing the antigen presentation ability of macrophages, which supports its immune regulation potential.

[0163] Example 3 Synthesis of Andrias davidianus meat peptide

[0164] 3.1, MHC-II affinity screening and NetMHCIIpan 4.0 prediction process

[0165] MHC-II molecules have an open binding groove that can interact with peptides of varying lengths, most commonly 11-17 amino acids. The binding groove of MHC-II molecules mainly interacts with a fragment of 9 amino acids in length in polypeptides, which is called the binding core. The NetMHCIIpan 4.0 server uses artificial neural networks (ANN) for prediction, thereby analyzing the affinity of peptide segments to MHC-II molecules. This tool not only predicts the binding affinity of different MHC-II alleles to polypeptides, but also can be used for analysis of peptide sequences of different lengths, thereby providing more comprehensive epitope prediction capabilities. The prediction results are output in the form of prediction scores, indicating whether the analyzed peptide is more likely to be naturally presented by the selected MHC-II receptor. This method can effectively improve the screening efficiency of immunogenic peptides and is widely used in the fields of immune regulatory peptide discovery, vaccine design, and autoimmune disease research. The process of NetMHCIIpan 4.0 predicting the binding strength of polypeptides to MHC-II molecules is shown in Figure 10

[0166] Computer simulation prediction of immune regulatory peptide MHC-II binding The peptide library obtained from the Andrias davidianus meat full transcriptome and Andrias davidianus meat enzymatic components was screened using NetMHCIIpan 4.0 to obtain peptide sequences. Specifically, for polypeptide sequences obtained from the Andrias davidianus full transcriptome, due to the input limit requirement of NetMHCIIpan 4.0 that the polypeptide length should be greater than 8 amino acids, on the basis of virtual enzyme digestion, polypeptide sequences with a sequence length greater than 8 amino acids and a frequency of more than 5 times were selected for binding prediction; for polypeptide sequences obtained from the Andrias davidianus meat enzymatic components, the polypeptides were directly sorted and predicted.

[0167] ​​The predicted values of the peptide sequences are expressed in %Rank values, which rank the predicted affinities of the reference peptides. The prediction conditions are as follows: 677 MHC-II molecule sites, of which 672 are from humans (i.e. 607 HLA-DRB1, 32 HLA-DRB3, 6 HLA-DRB4, 15 HLA-DRB5, 6 HLA-DP and 6 HLA-DQ epitopes) and 5 are from mice (i.e. epitopes H-2-IAb, H-2-IAd, H-2-IEd, H2-IAu, H-2-IEk from mice are selected for prediction). A %Rank value < 2 indicates strong binding (SB) to the MHC-II molecule, while a value > 2 and < 10 indicates weak binding (WB); a value > 10 indicates no binding and is filtered out. Peptides showing SB with multiple (≥ 3) MHC-II molecules are then selected for further study.

[0168] On this basis, the andoua meat peptide and MHC-II binding assay selects a group of high frequency MHC-II molecules (HLA-DRB1*0101, *0301, *0401, *0701, *0801, *0901, *1101, *1301, *1402 and *1501) for affinity prediction result display. These HLA phenotypes cover about 70% of individuals in the general population who have at least one allele. In NetMHCIIpan 4.0, these phenotypes are selected, and for the top 10 polypeptide inputs, the affinity results of each polypeptide for each phenotype are again predicted and presented in the form of a heat map.

[0169] 3.2, andoua meat peptide and MHC-II affinity screening

[0170] Potential immunomodulatory peptides are screened from the peptide library obtained from the andoua meat whole transcriptome and andoua meat enzymatic group using the NetMHCIIpan 4.0 model by affinity to different MHC-II phenotypes. First, for the results of the whole transcriptome, the NetMHCIIpan 4.0 model is used to select 677 MHC-II phenotypes, and the number of strong binding (SB) for each polypeptide is arranged. The MHC-II affinity prediction results of the andoua meat whole transcriptome peptide library are shown in Figures 11a to 11b , wherein the statistics of the number of strong bindings are shown in Figure 11a , and the results of the top 10 polypeptides with the most bindings are shown in Table 2, of which the optimal QR14 has a strong binding number of 430. On this basis, the common MHC-II phenotypes covering 70% of the general population are selected, and the docking results are presented in the form of a heat map, as shown in Figure 11b , it can be seen that the top 3 peptides all have excellent binding performance.

[0171] Table 2 andoua meat whole transcriptome peptide library predicted MHC-II affinity immunopeptides

[0172]

[0173]

[0174] For the results of enzymatic components, the same affinity simulation was carried out as before, and the results are shown in Figure 12. Similarly, the top 10 polypeptides with strong binding were sorted out, as shown in Table 3. From the results of strong binding, the best two polypeptides, MK14, reached 152 in number; NK12 reached 100 in number. For 70% of the common phenotypes in the human population, MK14 and NK12 both have more strong binding results, indicating the potential immunomodulatory activity of the two polypeptide sequences derived from the meat enzymatic components of Andrias davidianus.

[0175] Table 3 MHC-II affinity immunopeptides predicted by Andrias davidianus meat enzymatic component peptide library

[0176]

[0177]

[0178] In summary, based on the prediction and mining of immunomodulatory peptides derived from Andrias davidianus meat by bioinformatics tools, combined with the affinity evaluation between polypeptides and different allelic genotypes of MHC-II, molecular docking, molecular dynamics simulation and multi-parameter screening results, the top 3 candidate peptides with strong binding ability were screened from the whole transcriptome-derived peptide library, and SEQ ID NOs: 1-3 were obtained. The remaining 7 peptides with low binding ability are SEQ ID NOs: 6-12.

[0179] Among them:

[0180] The amino acid sequence of SEQ ID NO: 1 is as follows: QQQPIVSFQTAPAR (abbreviated as QR14)

[0181] The amino acid sequence of SEQ ID NO: 2 is as follows: IDLFIVNETHPNDPR (abbreviated as IR15)

[0182] The amino acid sequence of SEQ ID NO: 3 is as follows: IDGGQYILQVSNVAGTK (abbreviated as IK17)

[0183] The amino acid sequence of SEQ ID NO: 6 is as follows: VIQYFASIAASGDK (abbreviated as VK14-1)

[0184] The amino acid sequence of SEQ ID NO: 7 is as follows: HATEIAILNANYMAR (abbreviated as HR15)

[0185] The amino acid sequence of SEQ ID NO: 8 is as follows: DTGEYLLTVANIAGSK (DK16 for short)

[0186] The amino acid sequence of SEQ ID NO: 9 is as follows: GYAFVHFETQDAADR (GR15 for short)

[0187] The amino acid sequence of SEQ ID NO: 10 is as follows: VIQYFASIAATSDK (VK14-2 for short)

[0188] The amino acid sequence of SEQ ID NO: 11 is as follows: VIQYFASIAASSDK (VK14-3 for short)

[0189] The amino acid sequence of SEQ ID NO: 12 is as follows: NFDDAFVPILKPHLER (NR16 for short)

[0190] In addition, two peptides with the highest binding ability were screened from the enzymatic group-derived peptide library, and SEQ ID NOs: 4-5 were obtained. The remaining eight peptides with low binding ability are SEQ ID NOs: 13-20.

[0191] The amino acid sequence of SEQ ID NO: 4 is as follows: MVNHFVAEFKRKHK (MK14 for short)

[0192] The amino acid sequence of SEQ ID NO: 5 is as follows: NEWRMAANIPAK (NK12 for short)

[0193] The amino acid sequence of SEQ ID NO: 13 is as follows: DSYVGDEAQSKR (DR12 for short)

[0194] The amino acid sequence of SEQ ID NO: 14 is as follows: FTILKENVPLTSWK (FK14 for short)

[0195] The amino acid sequence of SEQ ID NO: 15 is as follows: LVLKEMVNEVGVVK (LK14 for short)

[0196] The amino acid sequence of SEQ ID NO: 16 is as follows: SNKIYVMSAVYR (SR12 for short)

[0197] The amino acid sequence of SEQ ID NO: 17 is as follows: REAAMSWLEYNTVSR (RR15 for short)

[0198] The amino acid sequence of SEQ ID NO: 18 is as follows: RGMFKSNCILFWPAQKER (abbreviated as RR18)

[0199] The amino acid sequence of SEQ ID NO: 19 is as follows: DDNHFRGLGFGFGPR (abbreviated as DR15)

[0200] The amino acid sequence of SEQ ID NO: 20 is as follows: LSLPVEDAPASHHSAGR (abbreviated as LR17)

[0201] The 3 candidate peptides with the highest binding capacity in the whole transcriptome-derived peptide library and the 2 peptides with the highest binding capacity in the enzymatic group-derived peptide library, i.e., SEQ ID NOs: 1-5, a total of 5 peptides, were synthesized by solid phase synthesis and used for subsequent verification of the immunomodulatory function of RAW264.7 macrophage cell model.

[0202] Example 4 Immunomodulatory effect of andoua meat peptide synthetic peptides on RAW264.7 cells

[0203] 4.1 Effect of andoua meat peptide synthetic peptides on the proliferation activity of RAW264.7 macrophages

[0204] The same experimental method as in Example 2.1 was used, and the CCK-8 method was used to detect the effect of synthetic peptides on the proliferation activity of RAW264.7 cells. In the experiment, the synthetic peptide treatment concentrations were set to 50, 100, 200, 400, and 800 μg / mL, respectively.

[0205] By measuring the absorbance changes of cells at different concentrations, the cell proliferation rate was calculated to evaluate the proliferation-promoting or toxic effects and provide basic data support for subsequent polypeptide function screening.

[0206] The experimental results are shown in Figure 13 .

[0207] As Figure 13 can be seen, the 5 synthetic peptides did not show cytotoxicity when treating RAW264.7 cells for 24 h at a concentration range of 50-400 μg / mL, and all showed a tendency to promote cell proliferation, indicating that they had the potential to activate macrophages. At a concentration of 800 μg / mL, the QR14 and IR15 treatment groups showed a decrease in cell viability, suggesting that there may be a cell inhibition effect at high doses. This result shows that most synthetic peptides have good biocompatibility within an appropriate dose range and can effectively enhance the activity of macrophages.

[0208] 4.2 Effect of andoua meat peptide synthetic peptides on the NO secretion level of RAW264.7 macrophages

[0209] This experiment employed the same experimental method as in Example 2.2, utilizing the Griess reaction system to evaluate the regulatory effect of the synthetic peptide on NO secretion levels in macrophages. Three concentrations of 100, 200, and 400 μg / mL were selected for intervention.

[0210] By detecting nitrite (NO2) in cell culture supernatant - The content of ) indirectly reflects the effect of synthetic peptide treatment on NO release levels, in order to determine its immune activation potential. The results are as follows: Figure 14 As shown.

[0211] Depend on Figure 14 It can be seen that treatment of RAW264.7 cells with different concentrations (100, 200, and 400 μg / mL) of giant salamander meat peptide significantly increased NO secretion levels compared to the control group (p<0.01), and this increase was clearly dose-dependent. These results indicate that the synthetic peptide can effectively promote macrophage activation and enhance NO secretion, further demonstrating its potential ability to regulate immune factors.

[0212] 4.3 Effects of giant salamander meat peptide synthesis on the phagocytic capacity of RAW264.7 macrophages

[0213] This experiment used the same experimental method as in Example 2.3, employing the neutral red uptake method to evaluate the phagocytic capacity of RAW264.7 cells treated with the synthetic peptide. The treatment concentrations were set at 100, 200, and 400 μg / mL.

[0214] The phagocytic index was calculated by measuring the absorbance of the phagocytic dye to quantitatively analyze the effect of synthetic peptides on the non-specific immune function of macrophages. The results are as follows: Figure 15 As shown.

[0215] Depend on Figure 15 It can be seen that all synthetic peptides can significantly increase the phagocytic index of RAW264.7 macrophages under treatment conditions of 100–400 μg / mL, which is statistically significant (p<0.05), and the phagocytic capacity increases with increasing concentration in a dose-dependent manner.

[0216] Among them, the phagocytic index of the QR14 treatment group with a concentration of 400 μg / mL reached 1.56±0.17, showing the most significant effect, indicating that the peptide has strong activity in immune activation.

[0217] 4.4 Effects of giant salamander meat peptide synthesis on MHC-II protein expression in RAW264.7 macrophages

[0218] The same flow cytometry method as in Example 2.4 was used to detect the regulatory effect of the synthetic peptide on the expression level of MHC-II molecules on the surface of macrophages. 400 pg / mL was selected as the uniform treatment concentration in the experiment.

[0219] After the cells were treated with the synthetic peptide, they were stained with fluorescently labeled MHC-II antibodies, and the change in fluorescence intensity was detected by flow cytometry to evaluate the regulatory effect of the synthetic peptide on antigen presentation function.

[0220] The experimental results are shown in Figure 16 .

[0221] As Figure 16 can be seen, the five synthetic peptides at a concentration of 400 pg / mL can all increase the expression level of MHC-II molecules, and the expression ratio of the QR14 treatment group is as high as 20.8 ± 0.2%, which is significantly higher than that of the control group (p < 0.05). This result shows that the synthetic peptide can significantly enhance the antigen presentation function of macrophages and activate their immune response potential, thereby verifying the scientificity and effectiveness of computer-aided screening of immunologically active peptides.

[0222] Example 5 Immune regulatory effect of andrias davidianus meat peptide synthetic peptides on DC2.4 cells

[0223] To further verify the immune activation potential of the synthetic immune peptide in the dendritic cell (DC) model, DC2.4 cells (mouse-derived DC2.4 dendritic cells purchased from Fuhui Biotechnology Co., Ltd. (Hunan, China)) were used as the research object, and the effects of the synthetic peptide on the proliferation activity, immune factor secretion, and MHC-II molecule expression of the cells were systematically evaluated to expand their functional adaptability in innate immune regulation.

[0224] 5.1, Effect of andrias davidianus meat peptide synthetic peptides on the proliferation activity of DC2.4 cells

[0225] The same CCK-8 detection method as in Example 2.1 was used in this experiment, and the experimental object was replaced with DC2.4 mouse dendritic cells, and the culture medium was selected as RPMI-1640. The cell density was adjusted to 1.0 x 10 5 cells / mL, and the cells were uniformly inoculated in a 96-well plate, with 100 pL of cell suspension added to each well.

[0226] The synthetic peptide treatment concentrations were set as 50, 100, 200, 400, and 800 pg / mL, respectively. After 24 h of treatment, CCK-8 reagent was added for incubation for 2 h, and then the absorbance value (A 450 ) was measured at a wavelength of 450 nm, the cell proliferation rate was calculated, and the effect of the synthetic peptide on the activity of DC2.4 cells and its biocompatibility were evaluated.

[0227] The experimental results are shown in Figure 17As shown.

[0228] Depend on Figure 17 It can be seen that the synthetic peptides from the five giant salamanders, when treated for 24 hours at concentrations ranging from 50 to 400 μg / mL, did not exhibit toxicity. On the contrary, they significantly enhanced the activity of DC2.4 cells, showing a proliferation-promoting effect. Among them, the treatment group with a concentration of 400 μg / mL QR14 showed the most significant effect, with a cell proliferation rate of 125.62 ± 1.87%. These results indicate that the synthetic peptides have good potential for activating immune cells and the ability to promote dendritic cell activation.

[0229] 5.2 Effect of giant salamander meat peptide synthesis on the expression of MHC-II protein on the surface of DC2.4 cell membrane

[0230] To detect the regulatory effect of synthetic peptides on the antigen presentation ability of DC2.4 cells, the same flow cytometry method as in Example 2.4 was used to quantitatively analyze the expression level of MHC-II molecules on the cell membrane surface.

[0231] DC2.4 cells were used in the experiment. The culture medium was RPMI-1640 (purchased from Thermo Fisher Scientific / Gibco, USA), and the synthetic peptide concentration was uniformly 400 μg / mL. After 24 h of treatment, the cells were washed twice with pre-cooled PBS and incubated for 30 min with PE-labeled anti-mouse MHC-II monoclonal antibody. Fluorescence signals were acquired using a flow cytometer (CytoFLEX, Beckman Coulter), and data analysis was performed using FlowJo 7.6 software.

[0232] Experimental results are as follows Figure 18 As shown.

[0233] Depend on Figure 18 It can be seen that after treatment with synthetic peptides at a concentration of 400 μg / mL, the expression level of MHC-II in DC2.4 cells was significantly increased in all treatment groups (p<0.001). Among them, the MHC-II expression level in the QR14 treatment group reached 30.7±1.0%, which was significantly higher than that in the control group.

[0234] These results indicate that the synthetic peptides from giant salamander meat can not only activate DC2.4 cells to secrete immune factors, but also promote the expression of their antigen-presenting molecules, suggesting that they have significant potential in inducing DC cell maturation and enhancing immune responses. This further verifies the functional effectiveness of the immunomodulatory peptides obtained through computer-aided screening.

[0235] Example 6: Therapeutic effect of giant salamander peptides on cyclophosphamide (CTX)-induced immunosuppressed mice

[0236] 6.1 Laboratory Animals and Ethical Statement

[0237] Sixty 7-week-old, 18-22 g healthy male BALB / c SPF mice were selected and provided by Vantianlihua Experimental Animal Technology Co., Ltd. (Beijing, China). All experimental operations strictly followed the relevant regulations of animal experiment ethics and were approved by the Life Ethics Committee of Tsinghua University Shenzhen Graduate School (Ethics No. F133, 2024). During the experiment, all feeding supplies, including bedding, water sources, water bottles, feed, etc., were treated with high-temperature sterilization. The mice were raised in a SPF-grade animal facility with environmental control conditions of temperature 20-26°C, relative humidity 40%-70%, and light cycle of 12h∶12h alternating illumination.

[0238] 6.2, Immunosuppression model construction and grouping

[0239] The mice were adaptively fed for 7 days before the experiment, with free access to food and water. The mouse immunosuppression model was established by intraperitoneal injection of cyclophosphamide (CTX), and QR14 synthetic peptide and giant salamander meat peptide extract (ADMP) were given by gavage to evaluate their immune enhancement potential.

[0240] The experiment was divided into 6 groups, 10 mice in each group, and the treatment schemes were as follows:

[0241] Control group (Control): intraperitoneal injection of 200 μL of normal saline on days 1-3; gavage with 200 μL of normal saline on days 4-17.

[0242] Model group (Model): intraperitoneal injection of 200 μL of 80 mg / kg CTX on days 1-3; gavage with 200 μL of normal saline on days 4-17.

[0243] QR14 low-dose group (QL): intraperitoneal injection of 200 μL of 80 mg / kg CTX on days 1-3; gavage with 200 μL of 25 mg / kg QR14 synthetic peptide on days 4-17.

[0244] QR14 high-dose group (QH): intraperitoneal injection of 200 μL of 80 mg / kg CTX on days 1-3; gavage with 200 μL of 100 mg / kg QR14 synthetic peptide on days 4-17.

[0245] ADMP low-dose group (AL): intraperitoneal injection of 200 μL of 80 mg / kg CTX on days 1-3; gavage with 200 μL of 400 mg / kg ADMP on days 4-17.

[0246] ADMP high-dose group (AH): intraperitoneal injection of 200 μL of 80 mg / kg CTX on days 1-3; gavage with 200 μL of 800 mg / kg ADMP on days 4-17.

[0247] Figure 19 Dosing regimen for CTX-induced immunosuppressed mouse model.

[0248] The dose of each group was set according to previous studies and literature reports. During the experiment, the individuals were distinguished by ear tags, and no abnormal behavior or stress response was observed during the observation process.

[0249] 6.3, Mouse weight determination

[0250] Starting from the first day of CTX injection (Day 1), the mouse weight was measured and recorded every 2 days using an electronic balance as a basic physiological indicator for evaluating the general condition.

[0251] After the experimental mice were adaptively fed and treated with CTX by intraperitoneal injection for 3 consecutive days, they showed significant weight loss, reduced activity, and poor mental state, indicating impaired physiological function and successful construction of the immunosuppressed model. Subsequently, QR14 synthetic peptides and Andrias davidianus meat peptide extract (ADMP) were administered according to the set gavage intervention scheme, and the body weight changes were recorded every 2 days.

[0252] The experimental results are shown in Figure 20 .

[0253] As can be seen from Figure 20 , CTX treatment significantly inhibited the weight gain of mice, while after QR14 and ADMP intervention, the body weight of mice gradually recovered, and the recovery effect of the high-dose QR14 group was better than that of other groups.

[0254] 6.4, Immune organ index

[0255] Twenty-four hours after the last gavage, the mice were weighed and euthanized. The spleen and thymus were separated in a clean bench, the fascia was stripped, and the surface moisture was rinsed with PBS and dried. The organ index was calculated as follows:

[0256]

[0257] The experimental results are shown in Figure 21.

[0258] As can be seen from Figure 21, CTX significantly reduced the spleen and thymus indices of mice (p<0.01), indicating that it inhibited the immune system. The QR14 and ADMP intervention groups significantly improved this index (p<0.001), with the high-dose group showing particularly significant improvement. The spleen index of the QH group was 3.68±0.10, and the thymus index was 1.48±0.12; the AH group was 3.34±0.27 and 1.30±0.12, respectively, indicating that Andrias davidianus meat peptide had a good effect on restoring immune organ function. (QH represents the high-dose QR14 treatment group, and AH represents the high-dose Andrias davidianus meat peptide extract treatment group).

[0259] 6.5, Spleen T lymphocyte subpopulation detection

[0260] The spleen was taken under sterile conditions, and the fat and connective tissue was removed. After rinsing with PBS, the tissue was cut into small pieces and ground into a single cell suspension using a 70 μm filter screen. After hemolysis and PBS washing, the cell concentration was adjusted to 1.0 × 10 6 / mL. Anti-CD4 and CD8 fluorescently labeled antibodies (FITC anti-mouse CD3, PE / Cyanine7 anti-mouse CD4 Antibody, BrilliantViolet 421 TM anti-mouse CD8a, APC / Cy7 Rat anti-Mouse CD45, RBC Lysis Buffer (10X) were purchased from BioLegend (USA). ) were added, and incubated for 20 minutes in the dark. After PBS washing, the proportion of CD4 + and CD8 + T cells was detected by flow cytometry. The proportion of T cell subpopulations reflects immune system activity. CD4 + is a helper T cell, and CD8 + is an inhibitory T cell.

[0261] The experimental results are shown in FIG. 22.

[0262] As can be seen from FIG. 22, in the CTX induction group, the proportion of CD4 + T cells decreased, and the proportion of CD8 + T cells increased. The CD4 + / CD8 + ratio decreased from 4.27 ± 0.22 to 2.89 ± 0.23 (p < 0.001). After QR14 and ADMP treatment, the proportion of CD4 + T cells increased significantly, the proportion of CD8 + T cells decreased, and the CD4 + / CD8 + ratio increased (QH was 3.73 ± 0.13, and AH was 3.55 ± 0.11), indicating that Andrias davidianus meat peptide has a significant T cell subpopulation regulation ability.

[0263] 6.6, Serum cytokine determination

[0264] Mouse orbital blood was collected, and the serum was separated by centrifugation at 880 × g for 10 min at 4°C and stored at -80°C. The levels of IL-6, IL-1β, IFN-γ, and TNF-α were detected by ELISA to analyze the immune factor regulation effect of Andrias davidianus peptide.

[0265] The experimental results are shown in FIG. 23.

[0266] CTX treatment significantly inhibited serum IL-6, IL-1β, IFN-γ and TNF-α levels (p<0.001). As can be seen from Figure 23, after QR14 and ADMP treatment, the levels of various cytokines were significantly restored (p<0.01), and in a dose-dependent manner, indicating that andouves meat peptides have a positive effect on the regulation of cytokines.

[0267] 6.7, Determination of serum immunoglobulin levels

[0268] According to the method of Example 6.6 above, mouse serum was collected, and the contents of IgG, IgM and IgA were detected using a commercial kit (purchased from Boyun Biotechnology Co., Ltd. (Nanjing, China)) according to the instructions to evaluate the immune function of the body.

[0269] The experimental results are shown in Figure 24.

[0270] As can be seen from Figure 24, CTX treatment caused a significant decrease in the levels of IgG, IgM and IgA (p<0.001), while QR14 and ADMP treatment significantly increased the content of immunoglobulin in serum (p<0.05) in a dose-dependent manner, reflecting its immune-enhancing function.

[0271] 6.8, Detection of routine blood parameters

[0272] 150 μL of mouse anticoagulant whole blood was collected, and an automatic blood analyzer was used to detect blood cell count, red blood cell parameters, platelet-related indicators, etc. The detection items are shown in Table 4.

[0273] Table 4. Detection items of mouse blood routine test

[0274]

[0275] The experimental results are shown in Figure 25.

[0276] As can be seen from Figure 25, the WBC, Lymph and Gran of the CTX group decreased significantly, and the HGB also changed to varying degrees (p<0.01). After QR14 and ADMP treatment, the WBC was restored significantly (p<0.001), and the Lymph was also increased, indicating that andouves meat peptides can effectively improve hematopoiesis and immune status.

[0277] 6.9, Hematoxylin-eosin staining (H&E) tissue staining

[0278] The mouse spleen, thymus and ileum tissues were fixed with 4% paraformaldehyde, paraffin-embedded, sectioned, and stained with H&E. The tissue structure and pathological changes were observed under a microscope to evaluate the repair of immune organs by andouves peptides.

[0279] The H&E results of the spleen and thymus are shown in Figures 26 and 27, respectively. Figure 26 andFigure 27 As shown, the ileum staining results are as follows: Figure 28 As shown.

[0280] Depend on Figure 26 and 27 It can be seen that CTX treatment caused damage to the spleen and thymus tissue structure, with blurred red and white pulp boundaries and sparse cells. After intervention with QR14 and ADMP, the tissue structure gradually recovered and the cells were densely arranged. The high-dose group showed particularly significant improvement, verifying the immune organ repair potential of giant salamander peptides.

[0281] Depend on Figure 28 It can be seen that CTX treatment led to atrophy and structural disorder of the ileal villi in mice. After treatment with QR14 and ADMP, the intestinal tissue structure was significantly restored, especially in the QH and AH groups, indicating that it has the ability to promote intestinal barrier repair and immune function recovery.

[0282] 6.10. Ileal immunohistochemical analysis

[0283] After dewaxing, hydration, and hydrogen peroxide blocking, non-specific sites were blocked with BSA, and primary antibody (CD4, catalog number: ab183685, dilution ratio: 1 / 1000) was added and incubated overnight at 4°C. The next day, after washing with PBS, secondary antibody (i.e., HRP-labeled universal rabbit secondary antibody, catalog number: K5007, retrieval: EDTA (pH 9.0)) was added and incubated for 50 min. Diaminobenzidine (DAB) (purchased from Dako) was used for staining, and hematoxylin was used for counterstaining. Brownish-yellow positive expression was observed under a microscope to reveal the immune regulatory mechanism.

[0284] Experimental results are as follows Figure 29 As shown.

[0285] Depend on Figure 29 It can be seen that CD4 + T cells were actively expressed in the control group and the giant salamander peptide treatment group, while their expression was extremely low in the model group. These results suggest that giant salamander peptide may enhance intestinal immunity by regulating the local intestinal immune microenvironment and promoting T cell differentiation and antigen presentation.

[0286] In summary, both QR14 synthetic peptide and giant salamander meat peptide extract exhibit good in vivo immunomodulatory activity, especially in enhancing the function of immune organs, regulating T cell subsets, and improving cytokine and immunoglobulin levels, demonstrating their potential as immune function improvers.

[0287] The description in this disclosure is provided for illustrative and descriptive purposes only and is not intended to be exhaustive or to limit the disclosure to its forms. Many modifications and variations will be apparent to those skilled in the art. The embodiments were chosen and described in order to better illustrate the principles and practical application of this disclosure and to enable those skilled in the art to understand this disclosure and to design various embodiments with various modifications suitable for a particular purpose.

Claims

1. A giant salamander meat peptide extract, wherein, Based on the mass percentage of the giant salamander meat peptide extract, the giant salamander meat peptide extract comprises 30-80% peptides with a molecular weight less than 1000 Da, 15-40% peptides with a molecular weight of 1000-2000 Da, 3-20% peptides with a molecular weight of 2000-3000 Da, 1-15% peptides with a molecular weight of 3000-5000 Da, and 0.3-10% peptides with a molecular weight greater than or equal to 5000 Da. Preferably, based on the mass percentage of the giant salamander peptide extract, the giant salamander peptide extract comprises 35-70% peptides with a molecular weight less than 1000 Da, 15-35% peptides with a molecular weight of 1000-2000 Da, 4-13% peptides with a molecular weight of 2000-3000 Da, 2-12% peptides with a molecular weight of 3000-5000 Da, and 0.4-7% peptides with a molecular weight greater than or equal to 5000 Da. Optionally, it comprises one or more polypeptides as shown in SEQ ID NOs:1-5.

2. A giant salamander meat peptide extract, comprising one or more polypeptides as shown in SEQ ID NOs:1-5 or one or more polypeptides as shown in SEQ ID NOs:1-5.

3. A polypeptide comprising an amino acid sequence as shown in any one of SEQ ID NOs:1-5 or an amino acid as shown in any one of SEQ ID NOs:1-5.

4. A nucleic acid comprising a nucleic acid encoding the polypeptide of claim 3.

5. A composition comprising the giant salamander meat peptide extract of any one of claims 1-2 or the polypeptide of claim 3; Optionally, it may also include a pharmaceutically, food-, or health-acceptable carrier, optional excipients, and optional active ingredient.

6. The use of the giant salamander meat peptide extract according to any one of claims 1-2, or the polypeptide according to claim 3, or the composition according to claim 5 in the preparation of a medicament for enhancing immune response; Optionally, the enhanced immune response includes enhancing macrophage proliferative activity, increasing macrophage nitric oxide secretion levels, enhancing macrophage phagocytic capacity, enhancing macrophage antigen presentation capacity, enhancing immune organ function, regulating T cell subsets, improving cellular immune factor levels, or enhancing immunoglobulin levels.

7. The use of the giant salamander meat peptide extract of any one of claims 1-2, the polypeptide of claim 3, or the composition of claim 5 in enhancing immune responses.

8. The application according to claim 7, wherein, The enhanced immune response includes enhancing macrophage proliferation activity, increasing macrophage nitric oxide secretion levels, enhancing macrophage phagocytic capacity, enhancing macrophage antigen presentation capacity, enhancing immune organ function, regulating T cell subsets, improving cellular immune factor levels, or enhancing immunoglobulin levels.

9. A medicament for enhancing an immune response, comprising the giant salamander meat peptide extract of any one of claims 1-2, the polypeptide of claim 3, or the composition of claim 5.

10. The medicament according to claim 9, wherein, The enhanced immune response includes enhancing macrophage proliferation activity, increasing macrophage nitric oxide secretion levels, enhancing macrophage phagocytic capacity, enhancing macrophage antigen presentation capacity, enhancing immune organ function, regulating T cell subsets, improving cytokine levels, or enhancing immunoglobulin levels.

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