A dual-functional glycopeptide targeting immunomodulation and anti-tumor

Abstract

The present application relates to a kind of targeting immunoregulation and anti-tumor morchella bifunctional glycopeptide, belong to the field of biotechnology.The molecular sequence of morchella glycopeptide provided by the present application is LPS (Fuc) PMLLPQ, and the fucose of morchella glycopeptide molecule is connected to the serine hydroxyl of peptide chain by O-connection glycosylation mode with α-glycosidic bond.The morchella glycopeptide of the present application is obtained from the morchella extract extracted by pectinase with morchella as substrate, is derived from edible fungi, and is safe to use;Can effectively bind TLR4 (Toll-like receptor 4), block inflammatory response, effectively bind and inhibit VEGFR2 (vascular endothelial cell growth factor receptor-2), and has antitumor activity;It can also inhibit the release of macrophage cytokines induced by LPS, further indicating that the morchella glycopeptide of the present application has immunoregulatory and antitumor activity.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a Morchella bifunctional glycopeptide targeting immune regulation and anti-tumor. BACKGROUND

[0002] Morchella esculenta (L.) Pers., also known as Morchella, Morchella mushroom, and Morchella, belongs to the Ascomycota order Morchella genus, and is a rare medicinal and edible fungus. Morchella is recorded in Compendium of Materia Medica, which has the effects of benefiting the intestines and stomach, promoting digestion and food, reducing phlegm and regulating qi, tonifying the kidney and absorbing qi, and nourishing the brain and refreshing the mind. Morchella is rich in protein, polysaccharide, polypeptide, vitamins, minerals and other nutrients, and has broad application prospects in food, medicine, health products and other fields.

[0003] Glycopeptides are composed of peptide molecules and sugar molecules connected by glycopeptide bonds, and have more complex structures. Glycopeptides developed into drugs mainly focus on glycopeptide antibiotics (such as vancomycin, etc.), which are mainly used for the treatment of gram-positive bacterial infections, especially drug-resistant bacteria (such as methicillin-resistant Staphylococcus aureus, MRSA). Glycopeptide drugs on the market or in clinical use are mainly derived from natural products of microorganisms (such as actinomycetes and streptomycetes), and some are improved in properties by chemical modification (semisynthesis).

[0004] TLR4 (Toll-like receptor 4) is a key pattern recognition receptor of the innate immune system, which mainly recognizes pathogen-associated molecular patterns (PAMPs, such as bacterial lipopolysaccharide LPS) and damage-associated molecular patterns (DAMPs). The molecular recognition of TLR4 mainly depends on its interaction with MD-2 protein. MD-2 is a cofactor of TLR4, and MD-2 has a hydrophobic pocket which is the core region of molecular binding. If combined with the acidic residues (such as Asp, Glu) of TLR4, it can promote the dimerization of TLR4 and the activation of signal, and can play the role of agonist. If the MD-2 pocket is occupied, it can competitively inhibit the binding of LPS and block excessive inflammatory response, and play the role of antagonist.

[0005] VEGFR2 (vascular endothelial cell growth factor receptor-2) is an important target for tumor treatment. VEGFR2 belongs to the tyrosine kinase receptor, and the ATP binding pocket of the kinase domain of VEGFR2 is the core region of catalytic phosphotransfer reaction. Its catalytic key residue Lys868 is involved in the positioning of the gamma-phosphate group of ATP, stabilizing the transition state, and mutation of the residue will significantly reduce the kinase activity; Asp1046 is located in the catalytic loop (Catalytic Loop), which can stabilize the transition state of ATP hydrolysis, and mutation of the residue will significantly reduce the kinase activity. 2+Ion coordination, coordination of ATP triphosphate group; Cys919 is located at the entrance of ATP binding pocket, which is the target of covalent inhibitors (such as apatinib) and can form reversible / irreversible covalent bond. Hinge region residues Glu885 and Val888 form hydrogen bonds with ATP adenine ring, which are the binding sites of most ATP competitive inhibitors. DFG motif Asp1046-Phe1047-Gly1048 (D-F-G) determines the activity state of the kinase, and after binding to the hydrophobic pocket in the DFG-out conformation (such as regorafenib), the kinase activation can be blocked. Therefore, the ATP binding pocket of VEGFR2 is the core target of antitumor drugs, and the key sites such as hinge region (Glu885 / Val888), catalytic residues (Lys868 / Asp1046) and Cys919 provide a structural basis for inhibitor optimization.

[0006] Macrophages play an important role in many diseases and are the main participants of the body's innate immune response. In vitro cell experiments, LPS is used to induce the establishment of a macrophage inflammation model. LPS promotes the activation of macrophages through the TLR4 pathway and stimulates macrophages to secrete various inflammatory factors and inflammatory mediators. NO, as an inflammatory mediator secreted by macrophages, is a common indicator for observing the immune activity of macrophages. Macrophages are divided into M1 and M2 types according to the type of immune response. TNF-alpha, IL-1 beta, IL-6, IL-12, NO are the marker cytokines of M1 type macrophages. LPS is used to treat RAW264.7 macrophages, and by analyzing the decrease in the secretion level of the above marker cytokines after intervention of the active substance, the role of the active substance in immune regulation and anti-tumor can be determined.

[0007] At present, there is no related report on food-derived glycopeptide molecules with immune regulation and anti-tumor activity. SUMMARY

[0008] Therefore, the purpose of the present application is to provide a Morchella glycopeptide, Morchella extract and preparation method and application thereof. The Morchella glycopeptide identified in the Morchella extract obtained by the preparation method can effectively bind to TLR4 and VEGFR2, block the inflammatory response, and has immune regulation and anti-tumor activity.

[0009] The present application provides a Morchella glycopeptide, the molecular sequence of which is LPS(Fuc)PM LLPQ. In the Morchella glycopeptide molecule, the fucose is connected to the hydroxyl group of the serine of the peptide chain by an O-linked glycosylation mode through an alpha-glycosidic bond.

[0010] The present application also provides a Morchella extract containing the Morchella glycopeptide.

[0011] The present application also provides a preparation method of the Morchella extract, comprising the following steps:

[0012] The Morchella is mixed with water at 1g:(10-30)mL, and pectinase is used for enzymolysis at 50-70 DEG C for 18-100 min; the mass ratio of the pectinase to the Morchella is (3-8):100.

[0013] Preferably, the particle size of the Morchella is less than 100 mesh.

[0014] The present application also provides application of the Morchella glycopeptide, the Morchella extract or the preparation method in preparation of a product for reducing secretion levels of macrophage NO, IL-6, IL-12, IL-1beta or TNF-alpha.

[0015] The present application also provides application of the Morchella glycopeptide, the Morchella extract or the preparation method in preparation of an anti-inflammatory product.

[0016] The present application also provides application of the Morchella glycopeptide, the Morchella extract or the preparation method in preparation of a TLR4 protein inhibitor.

[0017] The present application also provides application of the Morchella glycopeptide, the Morchella extract or the preparation method in preparation of a VEGFR2 protein inhibitor.

[0018] The present application also provides application of the Morchella glycopeptide, the Morchella extract or the preparation method in preparation of an immune-regulating product.

[0019] The present application also provides application of the Morchella glycopeptide, the Morchella extract or the preparation method in preparation of an anti-tumor product.

[0020] The present application has the following beneficial effects:

[0021] The present application identifies the Morchella glycopeptide in the obtained Morchella extract by enzymolysis of Morchella, and the Morchella glycopeptide is derived from edible fungi and is safe to use; can effectively combine TLR4, block inflammatory reactions, effectively combine and inhibit VEGFR2, has anti-tumor activity; and the Morchella glycopeptide of the present application can inhibit expression of LPS-induced macrophage cytokines, further indicating that the Morchella glycopeptide of the present application has immune-regulating and anti-tumor activity. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 It is a mass spectrum of the Morchella glycopeptide molecule.

[0023] Figure 2It is a 3D structure diagram of the Morchella sugar peptide molecule; wherein, yellow is a peptide molecule, orange is fucose, red is an oxygen atom, blue is a carbon atom, and gray is a hydrogen atom.

[0024] Figure 3 It is a diagram of the effect of the Morchella sugar peptide on the secretion of cytokines by RAW264.7 macrophages; wherein, **** represents P<0.0001.

[0025] Figure 4 It is a diagram of the docking of the LPS(Fuc)PMLLPQ of the Morchella sugar peptide molecule and the TLR4 molecule, wherein, in the Morchella sugar peptide molecule, red is a peptide molecule, and green is a fucose molecule.

[0026] Figure 5 It is a diagram of the docking of the LPS(Fuc)PMLLPQ of the Morchella sugar peptide molecule and the VEGFR2 molecule, wherein, in the Morchella sugar peptide molecule, red is a peptide molecule, and green is a fucose molecule. DETAILED DESCRIPTION

[0027] The present application provides a Morchella sugar peptide, the molecular sequence of which is LPS(Fuc)PMLLPQ; in the Morchella sugar peptide molecule, fucose is connected to the hydroxyl of serine of the peptide chain by an α-glycosidic bond in an O-linked glycosylation manner.

[0028] In the present application, in the molecular sequence LPS(Fuc)PMLLPQ of the Morchella sugar peptide, (Fuc) is located after serine (S), indicating that O-linked fucose glycosylation modification occurs on serine (S), resulting in an increase of 146.06 Da in the molecular weight. The molecular weight of the Morchella sugar peptide is preferably 1140 Da. The peptide sequence in the molecular of the Morchella sugar peptide is preferably LPSPMLLPQ (SEQ ID NO. 1).

[0029] Preferably, the structural formula of the Morchella sugar peptide is shown as formula I:

[0030]

[0031] The present application also provides a Morchella extract containing the Morchella sugar peptide. The Morchella sugar peptide can be identified in the Morchella extract.

[0032] The present application also provides a preparation method of the Morchella extract, comprising the following steps:

[0033] Morchella is mixed with water at 1g:(10-30)mL, pectinase is added, and enzymolysis is carried out at 50-70℃ for 18-100min; the mass ratio of the pectinase to Morchella is (3-8):100.

[0034] In the present application, the Morchella is preferably Morchella with a particle size of <100 mesh. The present application does not have special limitations on the way of controlling the particle size of Morchella, and the conventional method for controlling the particle size in the art can be used. In an embodiment, the Morchella with a particle size of <100 mesh can be obtained by crushing and sieving. The <100 mesh Morchella is mixed with water at a ratio of 1g:(10-30)mL. The mixing ratio of the Morchella and water is preferably 1g:10mL, 1g:15mL, 1g:20mL, 1g:25mL or 1g:30mL. After mixing, pectinase is added. The mass ratio of the pectinase to the Morchella is (3-8):100, preferably 3:100, 4:100, 5:100, 6:100, 7:100 or 8:100. Enzymatic hydrolysis is performed at 50-70°C for 18-100min. In an embodiment, the enzymatic hydrolysis can be performed at 50°C, 55°C, 60°C, 65°C or 70°C for 18min, 20min, 40min, 50min, 60min, 80min or 100min. In the present application, the preparation method preferably further comprises the steps of centrifugation and freeze-drying. The centrifugation is preferably performed after enzymatic hydrolysis. The present application does not have special limitations on the conditions of the centrifugation, and the conventional centrifugation conditions in the art can be used. In an embodiment, the centrifugation can be performed at a speed of 6000-8000rpm for 10-20min, such as at a speed of 6000rpm, 7000rpm or 8000rpm for 10min, 15min or 20min. After centrifugation, the supernatant is preferably taken for freeze-drying. The freeze-drying is preferably performed at -68- -80°C for 24-50h. In an embodiment, the freeze-drying can be performed at -68°C, -70°C, -75°C or -80°C for 24h, 30h, 36h, 40h, 48h or 50h.

[0035] The present application does not have special limitations on the source of the Morchella and pectinase, and the conventional commercially available products in the art can be used.

[0036] The Morchella extract containing the Morchella glycopeptide of the present application is obtained for the first time by the preparation method of the present application. The Morchella glycopeptide of the present application can effectively bind to TLR4, block inflammatory response, effectively bind to VEGFR2, and further inhibit VEGFR2, thereby having anti-tumor activity. Meanwhile, the Morchella glycopeptide of the present application can inhibit the expression of LPS-induced macrophage cytokines, further indicating that the Morchella glycopeptide of the present application has immunoregulatory and anti-tumor activity.

[0037] The present application also provides the use of the Morchella glycopeptide, the Morchella extract or the preparation method in the preparation of a product for reducing the secretion level of macrophage NO, IL-6, IL-12, IL-1β or TNF-α, an anti-inflammatory product, an immunoregulatory product or an anti-tumor product. In the present application, the product preferably comprises a drug.

[0038] The efficacy component of the product preferably comprises the Morchella glycopeptide, and the product preferably further comprises a carrier commonly used in the art, including but not limited to a filler, a diluent, a disintegrant, a colorant and / or a flavoring agent.

[0039] The present application also provides use of the Morchella glycopeptide, the Morchella extract or the preparation method in preparation of a TLR4 protein inhibitor or a VEGFR2 protein inhibitor. The efficacy component of the inhibitor preferably comprises the Morchella glycopeptide, and the inhibitor preferably further comprises a carrier commonly used in the art, including but not limited to a filler, a diluent, a disintegrant, a colorant and / or a flavoring agent.

[0040] The technical solutions provided by the present application are described in detail below in conjunction with the examples, but they should not be understood as limiting the scope of protection of the present application.

[0041] In the following examples, all the conventional methods are used unless otherwise specified.

[0042] In the following examples, all the materials and reagents used are commercially available unless otherwise specified.

[0043] In the following examples, the pectinase used is purchased from Beijing Solabio Technology Co., Ltd., wherein the enzyme activity of the pectinase is 100,000 U / g.

[0044] Example 1

[0045] Preparation of a Morchella extract containing Morchella glycopeptide molecules

[0046] Dry Morchella is selected as the raw material, crushed and sieved to obtain Morchella powder with a particle size of <100. The Morchella powder is mixed with water at a solid-liquid ratio of 1:30 (g / mL), 7% (w / w, enzyme / Morchella powder) pectinase is added, and the enzyme solution is obtained by enzymolysis at 50°C for 50 min. The enzyme solution is centrifuged at 8000 rpm for 15 min, and the supernatant is collected and freeze-dried at -70°C for 48 h to obtain the Morchella extract.

[0047] Example 2

[0048] Preparation of a Morchella extract containing Morchella glycopeptide molecules

[0049] Dry Morchella is selected as the raw material, crushed and sieved to obtain Morchella powder with a particle size of <100. The Morchella powder is mixed with water at a solid-liquid ratio of 1:10 (g / mL), 8% (w / w, enzyme / Morchella powder) pectinase is added, and the enzyme solution is obtained by enzymolysis at 60°C for 100 min. The enzyme solution is centrifuged at 7000 rpm for 10 min, and the supernatant is collected and freeze-dried at -80°C for 24 h to obtain the Morchella extract.

[0050] Example 3

[0051] Preparation of a Morchella extract containing Morchella glycopeptide molecules

[0052] Dry Morchella was used as raw material, and was ground and sieved to obtain Morchella powder with a particle size of <100 mesh. The Morchella powder was mixed with water at a solid-liquid ratio of 1:20 (g / mL), and 3% (w / w, enzyme / Morchella powder) pectinase was added. Enzymatic hydrolysis was carried out at 70°C for 18 min to obtain an enzymatic hydrolysate. The enzymatic hydrolysate was centrifuged at 6000 rpm for 20 min, and the supernatant was collected and freeze-dried at -68°C for 50 h to obtain a Morchella extract.

[0053] Comparative Example 1

[0054] Preparation of a Morchella extract

[0055] Dry Morchella was used as raw material, and was ground and sieved to obtain Morchella powder with a particle size of <100 mesh. The Morchella powder was mixed with water at a solid-liquid ratio of 1:20 (g / mL), and 3% (w / w, enzyme / Morchella powder) pectinase was added. Enzymatic hydrolysis was carried out at 70°C for 18 min to obtain an enzymatic hydrolysate. The enzymatic hydrolysate was centrifuged at 6000 rpm for 20 min, and the supernatant was collected and freeze-dried at -68°C for 50 h to obtain a Morchella extract.

[0056] Test Example 1

[0057] Structural identification of Morchella glycopeptides

[0058] The peptide molecules in the extract of Morchella obtained in Example 1 and Comparative Example 1 were identified by mass spectrometry. The extract was desalted and pretreated by using a ZipTip C18 micro chromatography column (Merck-Millipore, Shanghai Anpex Scientific Technology Co., Ltd.). The desalting method was as follows: 1.0 mg of the extract of Morchella was accurately weighed, dissolved in 10 μL of 0.1% (v / v) trifluoroacetic acid (TFA) to obtain a dissolved solution of the extract of Morchella; 50 μL of a solution containing 60% (v / v) acetonitrile (ACN) and 0.1% TFA in water was used to rinse the chromatography column for 10 times; 10 μL of 0.1% TFA was used to rinse the chromatography column for 10 times; the dissolved solution of the extract of Morchella was pumped into and discharged from the chromatography column for 20 times; 10 μL of 0.1% TFA was used to rinse the chromatography column for 5 times; 10 μL of a solution containing 60% ACN and 0.1% TFA in water was used to elute the chromatography column, and the eluate was collected, transferred to a polypropylene centrifuge tube, and dried under vacuum to obtain a dry powder. The dry powder was dissolved in 20 μL of a dissolving solution (0.1% (v / v) formic acid in water), vortexed, centrifuged at 17,000 rpm and 4°C for 20 min, and the supernatant was collected and transferred to a sample tube. The sample was injected in an amount of 3 μL, and LC-MS / MS sequence analysis was performed. The mobile phase A was 0.1% formic acid in water, and the mobile phase B was 0.1% formic acid in acetonitrile. The LC-MS / MS parameter settings are shown in Table 1. The PEAKS software was used for sequence database search.

[0059] Table 1. LC-MS / MS parameter settings

[0060]

[0061] By LC-MS / MS identification, a glycopeptide molecule with high mass abundance (peak area 1.22 x 10 5 ) was obtained in the extract of Morchella obtained in Example 1. The mass spectrum of the glycopeptide molecule is shown in Figure 1 ; the glycopeptide sequence is LPS(Fuc)PMLLPQ, the molecular weight is 1140 Da; the structural formula is shown as Formula I; in the glycopeptide molecule, the fucose is connected to the hydroxyl group of serine (Ser) in the peptide chain by O-linked glycosylation through an α-glycosidic bond. The glycopeptide molecule is named Morchella glycopeptide, and the 3D structure of the Morchella glycopeptide is shown in Figure 2 . The same Morchella glycopeptide molecule was not identified in the extract of Morchella obtained in Comparative Example 1.

[0062]

[0063] Test Example 2

[0064] Analysis of the efficacy of the Morchella glycopeptide

[0065] The Morchella glycopeptide molecule LPS(Fuc)PMLLPQ used in this test example was synthesized by Gilon Biochemical (Shanghai) Co., Ltd. according to the structure identified in Test Example 1, and the purity of the synthesized sample was greater than 98%.

[0066] 1. Analysis of Morchella glycopeptide activity at the cellular level

[0067] Macrophages play an important role in many diseases and are the main participants in the body's innate immune response. In in vitro cell tests, a macrophage inflammation model is established by using LPS induction. LPS promotes the activation of macrophages through the TLR4 pathway and stimulates macrophages to secrete various inflammatory factors and inflammatory mediators. NO, as an inflammatory mediator secreted by macrophages, is a common indicator for observing the immune activity of macrophages. Macrophages are divided into M1 and M2 types according to the type of immune response. TNF-α, IL-1β, IL-6, NO, etc. are the marker cytokines of M1 type macrophages. RAW264.7 macrophages were treated with LPS, and by analyzing the decrease in the secretion level of the above marker cytokines after the intervention of Morchella glycopeptide, the role of Morchella glycopeptide in immune regulation and anti-tumor was determined.

[0068] RAW264.7 macrophages were cultured using complete culture medium, which was composed of DMEM high glucose culture medium containing 10% fetal bovine serum and 100 U / mL penicillin, 100 μg / mL streptomycin. Cells in the logarithmic growth phase were inoculated in a 96-well plate (1×10 6 The blank group (normal cultured RAW264.7 cells), the LPS-induced cultured RAW264.7 cell group (LPS group), and the RAW264.7 cell group cultured after induction with Morchella glycopeptide LPS(Fuc)PMLLPQ (200 μM) (glycopeptide group) were set. The LPS group was cultured by adding LPS (2 μM) to the RAW264.7 cells and incubating at 37°C for 24 h. The glycopeptide group was cultured by discarding the cell culture medium after 24 h of LPS induction, adding complete culture medium containing 200 μM Morchella glycopeptide LPS(Fuc)PMLLPQ, and incubating at 37°C for 24 h. The cell culture medium obtained from each group was collected and centrifuged at 1500 r / min for 5 min to collect the supernatant. The Griess kit (Hangzhou Gaosheng Biological Technology Co., Ltd.) was used to detect the level of nitric oxide (NO) secreted by macrophages. The ELISA kit (Shanghai Genechem Co., Ltd.) was used to determine the content of inflammatory factors IL-6, IL-12, IL-1β, and TNF-α secreted by cells.

[0069] The results of the effect of Morchella glycopeptide LPS(Fuc)PMLLPQ on the secretion of cytokines by RAW264.7 are shown in Table 1.Figure 3 As shown in the results, the secretion levels of NO, IL-6, IL-12, IL-1β and TNF-α were significantly increased in the LPS group, indicating that LPS induced an inflammatory response in RAW264.7 macrophages. The amount of NO released, the secretion levels of IL-6, IL-12, IL-1β and TNF-α were significantly reduced in the Morchella glycopeptide LPS(Fuc)PMLLPQ treatment group, among which the amount of NO released decreased by 49.75%, and the secretion amounts of IL-6, IL-12, IL-1β and TNF-α decreased by 42.11%, 48.57%, 47.44% and 46.03%, respectively, indicating that Morchella glycopeptide regulates macrophage function, inhibits inflammatory response, and significantly down-regulates the levels of pro-inflammatory factors (significance analysis P value < 0.0001), and has immunoregulatory and anti-tumor activities.

[0070] 2. Analysis of Morchella glycopeptide activity by molecular docking

[0071] The crystal structures of immune receptor protein TLR4 (PDB: 3FXI) and tumor treatment target receptor protein VEGFR2 (PDB ID: 3VHE) were downloaded from the RCSB database (https: / / www.rcsb.org / ). https: / / www.rcsb.org / The crystal structure of the receptor protein was optimized by MOE 2019 molecular docking software, and the water molecules were removed and the hydrogen atoms were supplemented. The 3D structure of Morchella glycopeptide was constructed by MOE software, and the molecular energy minimization process was performed. The Site Finder module of MOE was used to determine the active site of the amino acid residues of the receptor protein. The docking score, the number of bonds and the binding energy of the glycopeptide and the receptor protein were used as screening indicators to select the tight binding complex of the glycopeptide and the receptor protein. The MOE software was used to analyze the binding site and action mode between the glycopeptide molecule and the receptor protein in the complex.

[0072] Eighteen hydrogen bond interaction forces were formed between the Morchella glycopeptide molecule LPS(Fuc)PMLLPQ and the TLR4 molecule, with a binding energy of -33.3 kcal / mol, in which the binding energy of the peptide molecule binding to TLR4 was -30.6 kcal / mol, and the binding energy of the sugar molecule binding to TLR4 was -2.7 kcal / mol. In the Morchella glycopeptide molecule, the peptide molecule formed a binding bond with the acidic residue GLU439 of TLR4. The oxygen atom in the fucose molecule binds to GLN505, which is also a key amino acid residue that can block the binding of LPS to TLR4. In summary, the Morchella glycopeptide molecule LPS(Fuc)PMLLPQ binds to TLR4 to block the inflammatory response (Table 2, Figure 4 ).

[0073] Table 2 Docking results of Morchella glycopeptide molecule LPS(Fuc)PMLLPQ and TLR4 molecule

[0074]

[0075]

[0076] The LPS(Fuc)PMLLPQ of the Morchella sugar peptide molecule forms 11 hydrogen bonds and 1 H-pi interaction force with the VEGFR2 molecule, and the binding energy is-21.3 kcal / mol, in which the binding energy of the peptide molecule binding with VEGFR2 is-12.6 kcal / mol, and the binding energy of the sugar molecule binding with VEGFR2 is-8.7 kcal / mol. In the Morchella sugar peptide molecule, the peptide molecule targets and binds with CYS919, forming a hydrogen bond interaction force with a binding energy of-8.5 kcal / mol, and this residue is also the binding site of the covalent inhibitor, so that binding with this residue can enhance the persistence of the molecule. In the Morchella sugar peptide molecule, the oxygen atom in the fucose forms a hydrogen bond interaction force with the catalytic key amino acid residues ASP1046 and LYS868 in the form of an ATP competitive inhibitor, and forms an H-pi interaction force with PHE1047, thereby blocking the kinase activation. In summary, the LPS(Fuc)PMLLPQ of the Morchella sugar peptide molecule targets and binds with the ATP binding pocket of the tumor-related receptor protein VEGFR2, thereby significantly inhibiting the activity of VEGFR2 (Table 3, Figure 5 ).

[0077] Table 3: Docking results of the Morchella sugar peptide molecule LPS(Fuc)PMLLPQ and the VEGFR2 molecule

[0078]

[0079]

[0080] The above results show that the Morchella sugar peptide of the present application can effectively bind to the immune receptor protein TLR4 and the tumor treatment target receptor protein VEGFR2, and has a good inhibitory effect thereon, thereby indicating that the Morchella sugar peptide of the present application has immunomodulatory and anti-tumor activity.

[0081] The above only describes the preferred embodiments of the present application, and it should be noted that those skilled in the art can make several improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A Morchella glycopeptide, characterized in that, The molecular sequence of the Morchella sugar peptide is LPS(Fuc)PMLLPQ; the fucose (Fuc) in the Morchella sugar peptide molecule is connected to the hydroxyl of the serine of the peptide chain by an O-linked glycosylation mode through an α-glycosidic bond.

2. A Morchella extract containing the Morchella sugar peptide of claim 1.

3. The method of claim 2, wherein the Morel extract is prepared by, The method comprises the following steps: The Morchella is mixed with water at a ratio of 1 g:30 mL, and pectinase is used for enzymolysis at 50°C for 50 min; the mass ratio of the pectinase to the Morchella is 7:

100.

4. The production method according to claim 3, characterized by, The particle size of the Morchella is less than 100 mesh.

5. Use of the Morchella sugar peptide of claim 1, the Morchella extract of claim 2, or the preparation method of any one of claims 3-4 in the preparation of an anti-inflammatory product.

Images