An immune modulating peptide from euphausia superba

Abstract

The application provides an Euphausia superba immunomodulatory peptide, which is prepared by alkaline protease and flavour protease for enzymolysis, and then the enzymolysis product is purified by cation exchange chromatography, Sephadex G25 gel chromatography and C18 reverse phase high performance liquid chromatography. The amino acid sequence of a polypeptide is SEQ ID NO: 1 or SEQ ID NO: 2. The Euphausia superba is used as raw material, Euphausia superba peptides are obtained by enzymolysis, and the amino acid sequences of the Euphausia superba peptides are analyzed and identified by LC-MS / MS after step-by-step purification. With the help of an activity prediction tool and molecular docking, a target peptide with good immunomodulatory activity potential is screened. The target peptide is artificially synthesized, the immunomodulatory activity is verified, and the action mechanism is further clarified. Data support is provided for the prediction screening and structure-activity relationship research of Euphausia superba source high-efficiency immunomodulatory peptides.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of functional biological products, and particularly relates to a Euphausia superba immunomodulatory peptide. BACKGROUND

[0002] In recent years, with the increasing proportion of sub-health population, more and more people are facing the situation of low immunity. Low immunity can cause illness, fatigue, weak stomach and intestines, and easy to be infected with diseases, which are not conducive to human health. The immune system is a defense mechanism against pathogenic bacteria and other invasions, and plays a crucial role in maintaining the health of the body. A large number of immunomodulatory drugs have been applied in clinic to solve the problem of low immunity of human body, but these immunomodulatory drugs have chemical, microbial and other side effects on human body. Natural immunomodulatory peptides have the advantages of non-toxicity and side effects compared with most existing immunomodulatory drugs. Natural immunomodulatory peptides are expected to replace clinical immunomodulatory drugs and provide new effective strategies for the prevention and treatment of chronic diseases, and have broad utilization value.

[0003] Euphausia superba is a major fishing species in the Antarctic Ocean, with a large population and fast breeding speed, and is regarded as one of the largest biological reserves in the world today. However, it has not been fully developed and utilized, and increasing its development and application has far-reaching significance for promoting the progress of new marine industries. A large number of studies have proved that Euphausia superba is rich in protein, has a high proportion of essential amino acids, and has a reasonable amino acid composition, which is a high-quality marine protein with broad development value. The active peptides prepared from Euphausia superba have multiple biological activities such as antioxidant, antibacterial, metal chelation, antihypertensive, immunomodulatory, etc., and have wide application prospects in the fields of nutrition and health care, food science, and beauty and skin care. SUMMARY

[0004] The present application provides a Euphausia superba immunomodulatory peptide and a preparation method thereof. The enzyme product prepared by enzymolysis is separated, purified and structurally identified to characterize the fine active structure of the immunopeptide and perform functional research.

[0005] The present application first provides a Euphausia superba immunomodulatory peptide, which is prepared by enzymolysis of alkaline protease and flavor protease, and then purified by cation exchange chromatography, Sephadex G25 gel chromatography and C18 reverse phase high performance liquid chromatography.

[0006] Further, the mass ratio of the alkaline protease and the flavor protease is 1:1.

[0007] The enzymolysis is carried out at pH 6-9 and 50-55 DEG C for 3-6 hours.

[0008] Preferably, the enzymolysis is performed at 55℃ for 4 hours.

[0009] Further, the Euphausia superba immunomodulatory peptide has an amino acid sequence of FDYGAN (SEQ ID NO: 1) or ESDLYDYPWQ (SEQ ID NO: 2).

[0010] The application also provides a use of the Euphausia superba immunomodulatory peptide, which is used in the preparation of an immunomodulatory product.

[0011] The product can be an immunomodulatory drug or a health product.

[0012] The application has the following advantages and effects over the prior art:

[0013] 1) The Euphausia superba is used as a raw material to obtain Euphausia superba peptides through enzymolysis, and the amino acid sequences of the Euphausia superba peptides are analyzed and identified by LC-MS / MS after step-by-step purification. With the help of an activity prediction tool and molecular docking, target peptides with good immunomodulatory activity potential are screened out. The target peptides are artificially synthesized, and their immunomodulatory properties are verified, and their action mechanisms are further clarified. The application provides data support for the prediction and screening of Euphausia superba-derived high-efficiency immunomodulatory peptides and the study of structure-activity relationship.

[0014] 2) The application screens two new peptides FDYGAN and ESDLYDYPWQ with immunomodulatory activity from the identified peptide segments through virtual screening and molecular docking. They play an immunomodulatory role by activating macrophages to improve their phagocytic ability and NO secretion ability, and up-regulating the mRNA levels and protein expression levels of IL-1β, IL-6, IL-10 and TNF-α.

[0015] 3) The Euphausia superba peptides with immunomodulatory effects extracted by the application can be applied to nutritional and health food, and have good application and economic prospects. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 Figure 4 is a graph of the spleen lymphocyte proliferation activity of Euphausia superba enzymatic hydrolysates obtained by different single-enzyme enzymolysis,

[0017] Figure 2 Figure 6 is a graph of the spleen lymphocyte proliferation activity of Euphausia superba enzymatic hydrolysates obtained by different complex-enzyme enzymolysis,

[0018] Figure 3 Figure 8 is a graph of the verification of the immunomodulatory activity of RAW264.7 cells to Euphausia superba peptides,

[0019] Figure 4 Figure 10 is an elution spectrum of the separation and purification of Euphausia superba peptides and a graph of the effect of the peptide components on the proliferation of spleen lymphocytes.

[0020] Figure 5 The secondary mass spectrum of the immunopeptide FDYGAN and ESDLYDYPWQ,

[0021] Figure 6 The molecular docking simulation diagram of the immunopeptide with TLR-4 and MHC-II respectively,

[0022] Figure 7 The effect diagram of the immunopeptide on RAW264.7 cells. DETAILED DESCRIPTION

[0023] The identification, screening and application of the Antarctic krill immunomodulatory peptide of the application include the following steps:

[0024] 1) Pretreatment of Antarctic krill: Antarctic krill is thawed by flowing water, and the head and shell are removed, and then the Antarctic krill is ground by a meat grinder.

[0025] 2) Single enzyme hydrolysis reaction: a certain amount of water is added to the Antarctic krill paste so that the solid-liquid ratio is 1:2-1:5, then different proteases (single enzyme or complex enzyme) are added, the pH value of the system is adjusted to 6-9, and the system is incubated at 50-55°C for 3-6 hours; the enzyme is inactivated by boiling for 5-10 min to obtain an Antarctic krill enzyme hydrolysate; the enzyme hydrolysate is concentrated and dried to obtain Antarctic krill peptide dry powder;

[0026] The amount of protease added accounts for 0.5-3.0% of the mass of Antarctic krill meat;

[0027] The pH value of the system is adjusted to 8 by using 1 mol / L NaOH solution or HCl solution;

[0028] The centrifugation is performed at 8500 r / min for 15-20 min;

[0029] The protease includes commercial flavor protease, alkaline protease, trypsin, neutral protease, and papain, and the amount of enzyme added is 0.5-3%;

[0030] As a preferred scheme of the application, a certain amount of water is added to the Antarctic krill paste so that the solid-liquid ratio is 1:4, then alkaline protease: flavor protease = 1:1 is added, the amount of enzyme added is 1.5%, the pH value of the system is adjusted to 8, and the system is incubated at 55°C for 4 hours; the enzyme is inactivated by boiling for 10 min to obtain an Antarctic krill enzyme hydrolysate. The supernatant is reserved after centrifugation of the enzyme hydrolysate, and the Antarctic krill mixed peptide powder is obtained after freeze-drying.

[0031] 3) Screening of the enzyme hydrolysate: the immunomodulatory activity of different enzyme hydrolysates is evaluated by the proliferation activity of spleen lymphocytes, and the Antarctic krill peptide with the optimal activity is screened.

[0032] 4) Isolation, purification and structure identification of mixed Euphausia superba peptide: Using the proliferation activity of spleen lymphocytes as the evaluation index, the Euphausia superba peptide was purified by SP Sephadex C25 cation exchange chromatography, Sephadex G25 gel chromatography and C18 reverse phase high performance liquid chromatography. The purified components were dissolved in ultrapure water to form a 1 mg / mL sample, and the peptide sequence was identified by mass spectrometry.

[0033] The peptide sequence was determined by UHPLC using an Agilent Advance-Bio Peptide Map C18 column (2.1x150mm, 2.7μm). The UHPLC parameters were as follows: mobile phase A: 0.1% formic acid-acetonitrile, mobile phase B: 0.1% formic acid-water, flow rate: 0.25 mL / min, column temperature:

[0034] 40℃, gradient elution program 0-2min (5% A), 2-27min (5% A-20% A), 27-37min (20% A-35% A), 37-39min (35% A-80% A). Scan range: 50-1500m / z, electrospray mode: electrospray positive ion, electrospray voltage 5500v.

[0035] 5) Screening of immunopeptides: The peptide sequence was input into the BIOPEP-UWM bioactive peptide database (https: / / biochemia.uwm.edu.pl / biopep-uwm / ) for activity prediction. Immunopeptide fragments with immune activity and regulatory activity were screened, and these peptide fragments were subjected to molecular docking with TLR4 and MHC-II protein receptors. According to the binding energy, the most highly immunoregulatory peptide fragments were screened.

[0036] After the mixed peptide was separated and purified by stages and structure identification, two peptide fragments FDYGAN and ESDLYDYPWQ with good immune regulation were screened by combining activity prediction and computational chemistry methods.

[0037] 6) Activity verification of immunopeptides: A RAW264.7 cell model was established to study the effects of immunopeptides on RAW264.7 cell activity, phagocytosis rate, NO and other factor secretion.

[0038] The Euphausia superba peptide (including mixed peptide, FDYGAN and ESDLYDYPWQ) prepared by the above method can be used for preparing immunoregulatory drugs or health products. When used, the Euphausia superba peptide can be compounded with active substances with immunoregulatory effects.

[0039] In the following examples, the method for determining the proliferation activity of spleen lymphocytes by colorimetry is as follows:

[0040] 1) Spleen lymphocyte extraction

[0041] Five-week-old ICR mice were fasted for 12 h and then sacrificed by cervical dislocation. The abdomen was cut open with scissors at the lower middle part, and the spleen (a deep red and long strip on the right side of the intestines) was removed and placed in a 2 mL sterile centrifuge tube. The surface of the spleen was sterilized with 1% double-antibiotic-containing PBS, and then washed with PBS to remove the double-antibiotic and transferred to a 200-mesh screen. 50 mL of the centrifuge tube was added to the bottom of the screen, and grinding was performed with a 1 mL syringe in reverse. During the grinding, 1 mL of PBS was added, and then 1 mL of PBS was added after the grinding was completed. The uniformly blown cells were centrifuged at 1200 rpm for 5 min. The cell precipitate was blown with 2 mL of sterile water, and 2 mL of 2x PBS was added within 20 s to neutralize the sterile water. Then, 4 mL of PBS was added for blowing, and at this time, a flocculent precipitate appeared, which was removed. The cell liquid was centrifuged at 1200 rpm for 5 min. Then, 2 mL of PBS was added to wash the cells (if there is still a significant flocculent precipitate in the centrifuge tube, it needs to be washed again with PBS). After the end, complete medium was added to resuspend the cells, which were transferred to a culture bottle and placed in a cell culture box for culture. After 4 h, the cell liquid was transferred to a 15 mL centrifuge tube, and the cells were resuspended with 1640 complete medium to obtain the spleen lymphocyte liquid.

[0042] 2) Cell culture

[0043] ① Cell plating

[0044] The cells were diluted to 2x10 6 In the 96-well plate, 100 μL of the cell suspension was added to each well, and PBS was added to the four surrounding wells to prevent evaporation of the culture medium.

[0045] ② Grouping and sample addition

[0046] NC group: 100 μL of 1640 complete medium was added to each well

[0047] PC group: 100 μL of 10 μg / mL LPS solution prepared from 1640 complete medium was added to each well, with a final concentration of 5 μg / mL

[0048] Sample high-dose group: 100 μL of 2 mg / mL peptide sample prepared from 1640 complete medium was added to each well, with a final concentration of 1 mg / mL

[0049] Sample low-dose group: 100 μL of 200 μg / mL peptide sample prepared from 1640 complete medium was added to each well, with a final concentration of 100 μg / mL

[0050] Six parallel samples were set for each group, and the sample addition method was recorded. The culture was performed for 32 h.

[0051] ③ Cell proliferation activity determination

[0052] After the end of cell culture, 20 μL of 5 mg / mL MTT was added to each well, and the culture was continued for 4 hours. Then the well plate was removed, and the cell solution was centrifuged to obtain a cell precipitate. The cell precipitate was dissolved with 200 μL of DMSO. Finally, all were transferred to an enzyme-labeled plate, and the absorbance was measured at 570 nm.

[0053] (3) Calculation formula

[0054]

[0055] In the formula:

[0056] A1 - absorbance of the sample group;

[0057] A2 - absorbance of the blank group

[0058] In the following examples, the experimental method of the effect of the peptide sample on RAW264.7 cells is as follows:

[0059] 1) RAW264.7 cell culture

[0060] ① Cell recovery

[0061] After the cells stored in liquid nitrogen were taken out, they were immediately thawed in a 37°C water bath, and after centrifugation to remove the freezing solution, an appropriate amount of DMEM complete medium was added to mix the cells, which were then transferred to a culture bottle and placed in a cell culture box for culture, and the culture medium was replaced in time.

[0062] ② Cell passage

[0063] When the cells grow to 80%-90% of the culture bottle, it indicates that the passage operation can be performed. The culture bottle was removed, and the culture medium was discarded. The culture medium was aspirated with a pipette to blow the cells off the bottle wall, mixed, and then divided into 2-3 parts and inoculated into new culture bottles for continuous culture.

[0064] ③ Cell freezing

[0065] After 6.2 mL of DMEM high-sugar medium, 2 mL of FBS, and 0.8 mL of DMSO were mixed, they were filtered through a 0.22 μm filter to remove bacteria to prepare a cell freezing solution. After centrifugation of the cell solution, 1 mL of the cell freezing solution was mixed and transferred to a freezing tube, which was placed in a -80°C refrigerator for slow freezing and then transferred to liquid nitrogen for freezing.

[0066] 2) Effect of Antarctic krill peptide on RAW264.7 cell activity

[0067] The cells were adjusted to 6 x 10 4Cells / mL. 200 μL of cell culture was added to each well of a 96-well plate and cultured overnight. Cells were then treated with culture media of different sample concentrations. After 24 hours, cells were treated with MTT assay, and absorbance was measured at 570 nm.

[0068] 3) Effect of Antarctic krill peptides on phagocytic rate of RAW264.7 cells

[0069] Adjust the cell count to 6×10 4 Cells / mL. 200 μL of cell culture was added to each well of a 96-well plate and cultured overnight. Cells were then treated with culture media of different sample concentrations. After 24 hours, cells were treated with 20 μL of neutral red staining solution for 2 hours, neutral red was removed, and neutral red lysis buffer was added for lysis. Absorbance was measured at 540 nm.

[0070] Phagocytosis rate (%) = (Absorbance of sample group / Absorbance of blank group) × 100%

[0071] 4) Effects of Antarctic krill peptides on NO secretion in RAW264.7 cells

[0072] Adjust the cell count to 6×10 4 Cells / mL. 200 μL of cell culture was added to each well of a 96-well plate and cultured overnight. Cells were then treated with culture media of different sample concentrations. After 24 hours, the NO content in the supernatant was determined using the Griess method.

[0073] 5) Group sampling

[0074] NC group: Add 200 μL of complete culture medium to each well;

[0075] PC group: Add 200 μL of LPS solution containing 5 μg / mL prepared from complete culture medium to each well;

[0076] Sample group: Add 200 μL of sample solution of different concentrations prepared from complete culture medium to each well;

[0077] Each group has 6 parallel groups.

[0078] The methods for isolating, purifying, and structurally identifying Antarctic krill peptides are as follows in the examples below:

[0079] 1) SP Sephadex C25 cation exchange column chromatography

[0080] The SP Sephadex C25 cationic filler was activated by soaking in pure water and then loaded into a 2.6 cm x 45 cm column. A 0.02 mol / L acetic acid solution was prepared and adjusted to pH 4.0 to serve as the buffer solution during the purification process. A 0.8 mol / L NaCl solution was prepared using the buffer solution to serve as the eluent during the purification process. A gradient mixer was used to slowly increase the concentration of the eluent from 0 to a final concentration of 0.4 mol / L. The flow rate was set to 0.8 mL / min, and one tube was collected every 6 minutes for a total of 100 tubes.

[0081] 2) Sephadex G25 gel column chromatography

[0082] The Sephadex G25 gel chromatography filler was activated by soaking in pure water and then loaded into a 1.6 cm x 80 cm column. Ultra-pure water was used as the buffer solution during the purification process. The flow rate was set to 0.5 mL / min, and one tube was collected every 6 minutes for a total of 70 tubes.

[0083] 3) Reverse phase high performance liquid chromatography

[0084] The HPLC used a Zorbax SB-C18 semi-preparative column (9.4 x 250 mm). The UHPLC parameters were: column temperature 35°C, flow rate 1 mL / min, UV detection wavelength 220 nm, mobile phase A ultra-pure water, mobile phase B 100% acetonitrile, and elution conditions 0-5 min, 5% B; 5-25 min, 5-20% B; 25-40 min, 20% B; 40-45 min, 20-5% B; 45-50 min, 5% B.

[0085] 4) Structural identification of immunologically active peptides

[0086] The UHPLC used an Agilent Advance-Bio Peptide Map C18 column (2.1 x 150 mm, 2.7 μm). The UHPLC parameters were: mobile phase A: 0.1% formic acid-acetonitrile, mobile phase B: 0.1% formic acid-water, flow rate: 0.25 mL / min, column temperature: 40°C, gradient elution program 0-2 min (5% A), 2-27 min (5% A-20% A), 27-37 min (20% A-35% A), 37-39 min (35% A-80% A). Scanning range: 50-1500 m / z, electrospray mode: electrospray positive ion, electrospray voltage 5500 v

[0087] In the following examples, the activity screening and molecular docking of the immunopeptides were performed as follows:

[0088] 1) Activity prediction

[0089] Enter Biopep (https: / / biochemia.uwm.edu.pl / biopep-uwm / ) and input the peptide fragments identified in 2.2.6 into the BIOPEP-UWM bioactive peptide database

[117] Activity prediction is performed. Specifically, enter the Biopep homepage, select Bioactive peptides, then enter the Analysis page, select Calculations, then click For you sequence, input the specific peptide sequence, and click Report to screen out immune peptide fragments with immune activity and regulatory activity according to the reported results. The peptide fragments are subjected to molecular docking, and the peptide fragment with the highest immune regulatory activity is screened out according to the binding energy.

[0090] 2) Molecular docking

[0091] TLR4 is selected according to the literature

[118] and MHC-II

[119] as a protein receptor. The molecular docking crystal structure of TLR4 (ID: 5IJD) and MHC-II (ID: 6BIX_B) is obtained from the RCSB PDB database (http: / / www.rcsb.org), and PYMOL software is used for processing to remove water molecules and their own small molecule ligands. The 3D structure of the selected peptide segment is drawn using Chem 3D software. Finally, the Auto Dock software is used to perform molecular docking of the screened peptide segment and the receptor protein, and the potential activity of the screened peptide segment is predicted according to the docking binding energy.

[0092] The application will be described in detail below in combination with specific examples and drawings.

[0093] Example 1: Preparation of Euphausia Superba Enzymatic Polypeptide

[0094] Enzymatic reaction: a certain amount of water is added to Euphausia Superba meat paste to make the solid-liquid ratio 1:

[0095] 4, then add protease, adjust the pH value of the system to 8, and incubate at 55 DEG C for 4 hours; boil for 10 min to inactivate the enzyme; obtain Euphausia Superba enzymatic solution; concentrate and dry the enzymatic solution to obtain Euphausia Superba peptide dry powder.

[0096] In the application, five proteases (flavor protease, alkaline protease, trypsin, neutral protease, and papain) are selected for Euphausia Superba enzymolysis, and five enzyme addition gradients (0.5%, 1%, 1.5%, 2%, and 3%) are set to optimize the enzymolysis conditions by taking spleen lymphocyte proliferation activity as the screening index.

[0097] Figure 1It is shown that, among 5 enzymes and 5 enzyme addition amounts, the enzymatic products obtained by adding alkaline protease and flavor protease at an enzyme addition amount of 1.5% exhibit better proliferation activity, which is 108.05% and 104.32%, respectively.

[0098] The alkaline protease and the flavor protease are compounded in the application, 7 ratios (5:0, 4:1, 3:2, 1:1, 2:3, 1:4, 0:5) are set, the spleen lymphocyte proliferation activity is taken as a screening index, and the enzymolysis conditions of the compound enzyme are optimized.

[0099] Figure 2 It is shown that, when the enzyme addition amount is 1.5%, the spleen lymphocyte activity of the enzymatic product obtained by adding alkaline protease and flavor protease at a ratio of 1:1 for enzymolysis is the best, and the proliferation rates at 100 μg / mL and 1000 μg / mL are 182.70% and 227.28%, respectively.

[0100] As Figure 3 shown, the enzymatic product obtained by adding alkaline protease and flavor protease at a ratio of 1:1 for enzymolysis at an enzyme addition amount of 1.5% is subjected to activity verification by using a RAW264.7 cell model. Figure 3 A is the influence of Antarctic krill peptide (AKP) on the activity of RAW264.7 cells, and the results show that, within the concentration range of 10-400 μg / mL of AKP, the activity of RAW264.7 cells gradually increases from 123.45% to 137.94% in a dose-dependent manner, and the activity gradually decreases with the continuous increase of the sample concentration, but AKP still has no toxic effect on RAW264.7 cells; Figure 3 B is the influence of AKP on the phagocytosis rate of RAW264.7 cells, and the results show that AKP has a significant enhancing effect on the phagocytosis rate of RAW264.7 cells, and when the concentration is 400 μg / mL, the phagocytosis activity of RAW264.7 is increased by 141.29% compared with the blank group; Figure 3 C is the influence of AKP on the NO secretion amount of RAW264.7 cells, and the NO secretion amount of RAW264.7 cells increases in a dose-dependent manner with the increase of the peptide concentration, and the NO secretion amount reaches 11.92 μM at a concentration of 400 μg / mL. The results show that the Antarctic krill peptide can play an immunoregulatory activity by regulating macrophages.

[0101] Example 2: Isolation, purification and structure identification of the polypeptide with immunoregulatory activity

[0102] With the spleen lymphocyte proliferation activity as the evaluation index, the Antarctic krill peptide is purified by SP Sephadex C25 cation exchange chromatography, Sephadex G25 gel chromatography and C18 reverse phase high performance liquid chromatography three technologies in stages, and R-F1 with the best activity is screened out, the R-F1 is dissolved into 1 mg / mL sample by ultrapure water, and 73 peptide segments with ALC≥95% are obtained by mass spectrometry.

[0103] As shown in Figure 4 , the Antarctic krill peptide in the application is separated and collected into four components by SP Sephadex C-25 cation exchange chromatography column, wherein the C-F1 has the highest spleen lymphocyte proliferation activity. The C-F1 component is separated and collected into five components by Sephadex G-25 gel filtration chromatography, and the G-F4 shows the highest spleen lymphocyte proliferation activity. The G-F4 component is further purified by C18 reverse phase high performance liquid chromatography, and five components are obtained. Among them, the R-F1 component shows the highest spleen lymphocyte proliferation activity.

[0104] The peptide sequence of the R-F1 component is identified by HPLC-MS / MS in the application, 73 peptide segments are screened out by setting ALC≥95%, and 13 immune peptide segments with potential immune activity and regulation activity are screened out by Biopep (as shown in Table 1).

[0105] Table 1: Immune peptide segment activity prediction table

[0106]

[0107] TLR4 and MHC-Ⅱ are selected as receptors, and molecular docking is performed on the 13 peptides to obtain the binding energy (as shown in Table 1), and the greater the absolute value of the binding energy, the more stable the combination. By comparing the binding energy, two peptide segments FDYGAN and ESDLYDYPWQ are screened out, and the two synthesized peptide segments are further verified for activity. The secondary mass spectrum of FDYGAN and ESDLYDYPWQ is shown in Figure 5 .

[0108] The molecular docking of FDYGAN and ESDLYDYPWQ with TLR4 (ID: 5IJD) is shown in Figure 6 (A) and (B). Among the binding force of FDYGAN with TLR4, the role of hydrogen bond is very small, so no yellow hydrogen bond and red amino acid residue related to hydrogen bond action are shown in Figure 6 A-(b), Figure 6A-(c) shows that the binding of FDYGAN and TLR4 mainly relies on van der Waals force. The binding between ESDLYDYPWQ and TLR4 is mainly stabilized by forming four hydrogen bonds of GLU122, HIS96, LYS263 and TYR102. The molecular docking of FDYGAN and ESDLYDYPWQ with MHC-Ⅱ (ID: 6BIX_B) is shown in Figure 6 (C) and (D). FDYGAN is stabilized by forming seven hydrogen bonds of THR93, TRP178, TYR102, ASP181, HIS112, PRO103 and LYS105 with MHC-Ⅱ; ESDLYDYPWQ is stabilized by forming nine hydrogen bonds of LYS105, HIS112, ASP181, VAL97, HIS177, VAL91, THR93, GLU179 and TRP178 with MHC-Ⅱ.

[0109] The immunomodulatory activity of the synthesized pure peptide was verified by establishing a RAW264.7 cell model, as shown in Figure 7 FDYGAN and ESDLYDYPWQ at a concentration of 10 μg / mL, the RAW264.7 cell viability was 121.34% and 109.43%, respectively. It is proved that they have no toxic effect on RAW264.7 cells and have a certain proliferation effect; when the concentration is 1 mg / mL, the phagocytic ability of macrophages is increased by 64.25% and 33.42%, respectively; the NO secretion capacity is increased from 2.36 μM to 14.96 μM and 8.97 μM, respectively. It is proved that they can play an immunomodulatory role by activating macrophages to improve their phagocytic rate and NO secretion rate.

[0110] In summary, the Antarctic krill is used as raw material in the present application, and Antarctic krill peptides with high immunomodulatory activity are prepared by enzymatic optimization. It is verified that Antarctic krill peptides can play an immunomodulatory role by regulating macrophages. After step-by-step purification, the amino acid sequence of Antarctic krill peptides is analyzed and identified by LC-MS / MS. With the help of active prediction tools and molecular docking, target peptides with good immunomodulatory activity potential are screened. The target peptides are artificially synthesized, and their immunomodulatory activity is verified. It is found that they can play an immunomodulatory role by activating macrophages to improve their phagocytic rate and NO secretion rate. Therefore, the immunopeptides in the present application, including FDYGAN, ESDLYDYPWQ and mixed peptides, can be used for preparing immunomodulatory drugs or health products, and will have good application prospect. At the same time, the present application provides strong data support for the prediction and screening of Antarctic krill-derived high-efficiency immunomodulatory peptides and the study of structure-activity relationship.

Claims

1. An Euphausia superba immunomodulatory peptide, characterized in that, The Euphausia superba immune-regulating peptide is prepared by alkaline protease and flavor protease for enzymolysis, and then the enzymolysis product is purified by cation exchange chromatography, Sephadex G25 gel chromatography and C18 reverse phase high performance liquid chromatography; the amino acid sequence of the Euphausia superba immune-regulating peptide is SEQ ID NO: 1 or SEQ ID NO:

2.

2. Application of the Euphausia superba immune-regulating peptide in claim 1 in the preparation of health care products for improving immunity.

3. A health product for immunomodulation, characterized by, The health care product contains an effective concentration of the Euphausia superba immune-regulating peptide in claim 1.

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