Mussel immunologically active peptides and their applications

Abstract

The present invention discloses a mussel immunologically active peptide and its application, belonging to the technical field of active peptides. The sequence of the immunologically active peptide is FPR, FRE, KFP, LLPK or PDRM. The acquisition of this immunologically active peptide is derived from the digestion of mussel meat under a static simulated digestion model, polypeptide sequence identification, prediction by bioinformatics tools, molecular docking, polypeptide synthesis and verification. The mussel immunologically active peptide of the present invention has good proliferative activity on mouse macrophage RAW264.7 and neutral red phagocytosis ability. The above-mentioned mussel immunologically active peptide screened can be used as an auxiliary immunomodulatory active ingredient in products with immunomodulatory functions such as immunomodulators, pharmaceutical compositions or nutritional products, which has important guiding significance for the development of new products.

Description

Technical Field

[0001] The present invention belongs to the field of bioactive peptides, and particularly relates to mussel immunomodulatory bioactive peptides, their screening methods and applications. Background Art

[0002] Immunity is a specific physiological reaction that occurs when the body comes into contact with "antigenic foreign substances" or "nonself molecules". The immune system works in coordination through three major components: immune organs, immune cells, and immune molecules to achieve the body's defense, surveillance, and homeostasis functions.

[0003] Mytilus edulis ( Mytilus coruscus ), also known as mussel, mussel meat, and blue mussel, is a bivalve mollusk widely distributed in temperate waters around the world and belongs to the family Mytilidae. It has high food therapy and medicinal values. Currently, there is no research on the immunomodulatory bioactive peptides in the simulated digestive products of mussels.

[0004] Virtual screening of bioactive peptides based on bioinformatics tools and molecular docking is based on the known amino acid sequences of bioactive peptides. Using database search and software analysis, it simulates and predicts the possible biological activities, safety, and structure-activity relationships of peptide segments to obtain target peptide segments. By further evaluating the interaction between ligands and receptors, it is a method for quickly targeting and screening target bioactive peptides. Compared with traditional preparation methods, virtual screening using bioinformatics tools and molecular docking can save a large amount of time and experimental costs. In actual operation, combining these two methods can greatly improve the effectiveness of screening and the accuracy of results. Screening natural mussel immunomodulatory bioactive peptides in mussels by this means has important significance for assisting the application of immunomodulatory active ingredients in functional products. Summary of the Invention

[0005] One of the objectives of the present invention is to provide a mussel immunomodulatory bioactive peptide.

[0006] Another objective of the present invention is to provide the application of the above-mentioned mussel immunomodulatory bioactive peptide.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows:

[0008] The present invention has screened 5 mussel immunomodulatory bioactive peptides with relatively high activities in mussels. The sequences of the mussel peptides are respectively FPR (Phe-Pro-Arg), FRE (Phe-Arg-Glu), KFP (Lys-Phe-Pro), LLPK (Leu-Leu-Pro-Lys, as shown in SEQ ID NO.1), and PDRM (Pro-Asp-Arg-Met, as shown in SEQ ID NO.2).

[0009] The screening method of the mussel immunologically active peptide, the method comprising the following steps:

[0010] (1) Preparation of mussel meat homogenate: Take a certain amount of mussels, wash and dry them, take out all the soft tissues and juices, and homogenize them 3 times in a high-speed tissue homogenizer for 30 s each time to obtain a homogenate;

[0011] (2) INFOGEST in vitro static simulation digestion model: First, add simulated saliva in a volume ratio of 1:1, and digest in a constant temperature oscillator at 37 °C and 100 rpm for 2 min to simulate oral digestion; then, adjust the pH to 3.0 with 6 mol / L hydrochloric acid solution to terminate the oral digestion process, add simulated gastric juice in a volume ratio of 1:1, mix evenly, and digest with constant temperature oscillation for 2 h to simulate gastric digestion. During the experiment, control the pH of the digestive juice at 3.0 with 1 mol / L hydrochloric acid solution; finally, adjust the pH to 6.0 with 5 mol / L sodium hydroxide solution to terminate the gastric digestion process, add simulated intestinal juice in a volume ratio of 1:1, mix evenly, and then adjust the pH to 7.0 with 1 mol / L sodium bicarbonate solution, and digest with constant temperature oscillation for 2 h to simulate intestinal digestion;

[0012] (3) Identification of mussel protein peptide sequence: Perform peptide mapping identification on the mussel protein simulated digestion product through the Thermo Fisher EASY-nLC1200 Q Exactive liquid chromatography-mass spectrometry system;

[0013] (4) Screening of mussel immunologically active peptide assisted by bioinformatics tools: Query the novelty of the peptide through the BIOPEP database, predict the biological activity of the peptide through the Peptide Ranker tool, predict the digestion resistance characteristics and cell membrane permeability of the peptide through the Peptide Cutter tool and CPP pred tool, evaluate the stability of the peptide in the blood through the PLiePred tool, and predict the potential toxicity and physicochemical properties of the peptide through the Toxin Pred tool;

[0014] (5) Molecular docking: Take the screened mussel immunologically active peptide as the ligand and TLR4-MD2 as the receptor, and analyze the binding sites and interaction forces between the mussel immunomodulatory peptide and TLR4-MD2 through molecular docking technology;

[0015] (6) Determination of immunomodulatory activity: Perform in vitro immunological activity detection, including the effects of immunologically active peptides on the proliferation activity, NO release amount and neutral red phagocytosis ability of mouse macrophages RAW264.7, and find the peptide segment with the strongest immunomodulatory activity.

[0016] Preferably, for the operation in step (1), after homogenization, the homogenate needs to be placed in an experimental freezer or immediately proceed with the subsequent experiments to avoid degradation of protein and polypeptide components caused by long-term storage at room temperature.

[0017] Preferably, for the operation in step (2), after each stage of simulated digestion is completed, if the next stage of simulated digestion is not required, the simulated digestive juice needs to be immediately placed in an ice bath to terminate the digestive reaction of this stage.

[0018] Preferably, for the operation in step (3), after separation using the Nano-HPLC liquid phase system EASY-nLC 1200 (ThermoFisher Scientific), the components obtained in the above step (2) are subjected to mass spectrometry analysis using a Q-Exactive mass spectrometer (Thermo Fisher Scientific); the obtained mass spectrometry data is analyzed using software MAXQUANT, and the resulting polypeptide sequences are compared with the Uniprot-Acipenser database to obtain the protein sources of the peptide sequences.

[0019] Preferably, for the operation in step (4), a Peptide Ranker score > 0.4 and the absence of toxicity are used as the basic screening conditions.

[0020] Preferably, for the operation in step (5), the results of molecular docking are expressed as binding energy values, and a screening condition is that the docking binding energy with TLR4-MD2 is lower than -5.9 kcal / mol.

[0021] The mussel immunologically active peptides of the present invention can be synthesized and produced by solid-phase synthesis methods, including but not limited to Fmoc-SPPS method, BOC-SPPS method, fragment condensation and ligation method, and can be carried out using all synthesizable methods, so as to obtain mussel multifunctional peptides more efficiently and provide materials for the further utilization of the screened polypeptides.

[0022] Advantages of the present invention:

[0023] The present invention discloses a mussel immunologically active peptide and its applications. The mussel immunologically active peptide of the present invention is mainly obtained through simulated digestion, LC-MS / MS mass spectrometry identification, analysis and prediction by bioinformatics tools, molecular docking, polypeptide synthesis and activity verification and screening. A total of 99 sequences were obtained through peptide spectrum identification, among which 41 active sequences have been reported. Among the remaining 58 unreported sequences, 21 have a Peptide Ranker score exceeding 0.4. According to the comprehensive screening of Peptide Ranker score, stability, digestion resistance characteristics and cell membrane penetrability, 8 peptide sequences were selected for molecular docking, and 5 peptide sequences have a docking binding energy with TLR4-MD2 lower than -5.9 kcal / mol. After comprehensively considering potential activity, safety and bioaccessibility, 5 sequences were selected for synthesis and activity verification. Through immunological activity experiments, it was proved that the polypeptides FPR, FRE, KFP, LLPK, and PDRM all have a certain degree of immunomodulatory activity. The above-mentioned peptide segments can be added as auxiliary immunomodulatory active ingredients to functional products such as immunomodulators, pharmaceutical compositions or nutritional products. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is the preparation technical route diagram of the mussel immunologically active peptide in the embodiment of the present invention;

[0025] Figure 2 It is the overall three-dimensional structure diagram of the molecular docking of polypeptide FPR and TLR4-MD2;

[0026] Figure 3 It is the two-dimensional schematic diagram of the interaction of the active site residues of the molecular docking of polypeptide FPR and TLR4-MD2;

[0027] Figure 4 It is the overall three-dimensional structure diagram of the molecular docking of polypeptide FRE and TLR4-MD2;

[0028] Figure 5 It is the two-dimensional schematic diagram of the interaction of the active site residues of the molecular docking of polypeptide FRE and TLR4-MD2;

[0029] Figure 6 It is the overall three-dimensional structure diagram of the molecular docking of polypeptide KFP and TLR4-MD2;

[0030] Figure 7 It is the two-dimensional schematic diagram of the interaction of the active site residues of the molecular docking of polypeptide KFP and TLR4-MD2;

[0031] Figure 8 It is the overall three-dimensional structure diagram of the molecular docking of polypeptide LLPK and TLR4-MD2;

[0032] Figure 9Two-dimensional schematic diagram of the interaction between the active site residues of the polypeptide LLPK and the TLR4-MD2 molecule;

[0033] Figure 10 Overall three-dimensional structure diagram of the docking of the polypeptide PDRM with the TLR4-MD2 molecule;

[0034] Figure 11 Two-dimensional schematic diagram of the interaction between the active site residues of the polypeptide PDRM and the TLR4-MD2 molecule;

[0035] Figure 12 Liquid-phase detection result diagram of the synthetic polypeptide FPR in the embodiment of the present invention;

[0036] Figure 13 Liquid-phase detection result diagram of the synthetic polypeptide FRE in the embodiment of the present invention;

[0037] Figure 14 Liquid-phase detection result diagram of the synthetic polypeptide KFP in the embodiment of the present invention;

[0038] Figure 15 Liquid-phase detection result diagram of the synthetic polypeptide LLPK in the embodiment of the present invention;

[0039] Figure 16 Liquid-phase detection result diagram of the synthetic polypeptide PDRM in the embodiment of the present invention;

[0040] Figure 17 Mass spectrometry detection result diagram of the synthetic polypeptide FPR in the embodiment of the present invention;

[0041] Figure 18 Mass spectrometry detection result diagram of the synthetic polypeptide FRE in the embodiment of the present invention;

[0042] Figure 19 Mass spectrometry detection result diagram of the synthetic polypeptide KFP in the embodiment of the present invention;

[0043] Figure 20 Mass spectrometry detection result diagram of the synthetic polypeptide LLPK in the embodiment of the present invention;

[0044] Figure 21 Mass spectrometry detection result diagram of the synthetic polypeptide PDRM in the embodiment of the present invention;

[0045] Figure 22 Result diagram of the effect of the synthetic peptide on the proliferation activity of RAW264.7 cells in the embodiment of the present invention;

[0046] Figure 23 Result diagram of the effect of the synthetic peptide on the NO release amount of RAW264.7 cells in the embodiment of the present invention;

[0047] Figure 24This is the result graph of the effect of the synthetic peptide in the embodiments of the present invention on the neutral red phagocytosis activity of RAW264.7 cells. Detailed implementation manners

[0048] The present invention will be further described below in conjunction with specific embodiments. Unless otherwise specified, the technical means used in the embodiments are conventional means well-known to those skilled in the art. The test methods in the following embodiments are all conventional methods unless otherwise specified. Unless otherwise specified, the reagents and materials used can be obtained by purchasing from the market.

[0049] Example 1:

[0050] The technical route for preparing mussel immunologically active peptides is as Figure 1 shown, where in vitro simulated digestion of mussels means digesting mussel meat using the INFOGEST in vitro static simulated digestion model.

[0051] The specific steps include:

[0052] (1) Digesting mussel meat based on the INFOGEST in vitro static simulated digestion model: First, take a certain amount of mussels, wash and dry them, take out all the soft tissues and juices, and homogenize them 3 times in a high-speed tissue homogenizer for 30 s each time to obtain a homogenate. Secondly, add simulated saliva (final concentration of α-amylase EC 232-565-6 is 150 U / mL) in a volume ratio of 1:1, and digest in a constant temperature oscillator at 37 °C and 100 rpm for 2 min to simulate oral digestion; then, adjust the pH to 3.0 using 6 mol / L hydrochloric acid solution to terminate the oral digestion process, add simulated gastric juice (final concentrations of pepsin EC 3.4.23.1 and gastric lipase EC 3.1.1.3 are 4000 U / mL and 120 U / mL respectively) in a volume ratio of 1:1, mix evenly, and digest with constant temperature oscillation for 2 h to simulate gastric digestion (during the experiment, control the pH of the digestive fluid at about 3.0 with 1 mol / L hydrochloric acid solution); finally, adjust the pH to 6.0 using 5 mol / L sodium hydroxide solution to terminate the gastric digestion process, add simulated intestinal fluid (final concentration of trypsin EC3.4.21.4 is 200 U / mL, final concentration of bovine bile salt is 20 mmol / mL) in a volume ratio of 1:1, mix evenly, then adjust the pH to 7.0 using 1mol / L sodium bicarbonate solution, and digest with constant temperature oscillation for 2 h to simulate intestinal digestion (during the experiment, pay attention to controlling the pH of the digestive fluid at about 7.0).

[0053] (2)LC-MS / MS identification of polypeptide sequences: After separation using the Nano-HPLC liquid system EASY-nLC 1200 (ThermoFisher Scientific), the Q-Exactive mass spectrometer (Thermo Fisher Scientific) was used. Analysis was performed on a C18 capture column (inner diameter 100 μm, length 2 cm, 5-μm C18; SC001, Thermo Fisher Scientific) and a C18 analytical column (inner diameter 75 μm, length 10 cm, 3-μm C18; SC2003, Thermo Fisher Scientific). The two mobile phases were buffer A (0.1% formic acid / 99.9% water) and buffer B (80% acetonitrile / 1% formic acid / 19% water). The proportion of buffer B was increased from 4% to 100% over 90 min at a flow rate of 250 nL / min. Scanning was performed at a resolution of 70000 at m / z 200, and the scanning range was set to 400 to 1700 m / z. Ten of the most abundant MS1 features were selected and fragmented by high-energy collision dissociation at a resolution of 17500 at m / z 200. The ion injection time and ion target value were set to 20 ms and 3E6 (measurement scan) and 60 ms and 5E5 (mass spectrometry / mass spectrometry scan), respectively. Data were acquired using Xcalibur software (Thermo Scientific).

[0054] (3)Bioinformatics tool-assisted screening of mussel immunologically active peptides:

[0055] First, query the novelty of the peptides through the BIOPEP database, and sequences with reported activities will not be subject to subsequent screening; use the Peptide Ranker tool to predict the biological activities of the peptides, and sequences with scores exceeding 0.4 are considered to have potential activities; use the Peptide Cutter tool to predict the digestion resistance characteristics of the peptides. If there are no cleavage sites that can be cleaved by pepsin (Pepsin pH 1.3 and pH>2.0, EC 3.4.23.1), trypsin (Trypsin, EC3.4.21.4), and chymotrypsin (Chymotrypsin, EC 3.4.21.1) in the sequence, then the sequence is considered to have the potential for anti-gastrointestinal digestion; use the CPP pred tool to predict the cell membrane permeability of the peptides, and sequences with scores exceeding 0.5 are considered to have the potential for complete transmembrane absorption; use the PLiepred tool to evaluate the stability of the peptides in the blood, and peptide sequences with half-life values higher than 800 s are considered to have a certain stability in the blood; use the Toxin Pred tool to predict the potential toxicity and physicochemical properties of the peptides, and sequences considered to have no potential toxicity can be subject to subsequent synthesis and verification.

[0056] A total of 99 sequences were obtained through peptide mapping identification. Among them, 41 active sequences have been reported. Among the remaining 58 unreported sequences, 21 have Peptide Ranker scores exceeding 0.4. According to the comprehensive screening of Peptide Ranker scores, stability, digestion resistance characteristics, and cell membrane permeability, 8 peptide sequences were selected for molecular docking.

[0057] (4)Virtual screening of mussel immunomodulatory peptides by molecular docking:

[0058] Use the peptides screened in the previous process as ligands and TLR4-MD2 as receptors, and analyze the binding sites and interaction forces between mussel immunomodulatory peptides and TLR4-MD2 through molecular docking technology. The three-dimensional structure of TLR4-MD2 (3FXI) was obtained from the PDB database (http: / / www.rcsb.org / ). Use the Pymol program to construct the three-dimensional structure of the polypeptide. Use the Autdock software to perform semi-flexible docking of mussel immunomodulatory peptides and TLR4-MD2. The results of the molecular docking are represented by the binding energy value, and the conformation with the minimum binding energy is selected as the best binding site.

[0059] A total of 5 peptide sequences with binding energies meeting the criteria were obtained through molecular docking. After comprehensively considering potential activity, safety, and bioaccessibility, 5 peptide sequences were selected for visualization analysis to further explore the molecular mechanism of the virtual prediction of the interaction between TLR4-MD2 and polypeptides. The 5 selected peptide sequences are: FPR, FRE, KFP, LLPK, and PDRM. The potential mussel polypeptide sequences and related properties obtained are shown in Table 1, and the binding energies of the polypeptides FPR, FRE, KFP, LLPK, and PDRM to TLR4-MD2 are shown in Table 2.

[0060] Among them, the interactions of FPR, FRE, KFP, LLPK, and PDRM with TLR4-MD2 are as Figure 2 - 11 shown.

[0061] Among them, for the interactions of FPR, FRE, KFP, LLPK, and PDRM with TLR4-MD2, 7 hydrophobic interactions are formed between FPR and I32, V48, I52, L61, F121, C133, F151, and I153 ( Figure 2 , Figure 3 ); 5 hydrophobic interactions are formed between FRE and I44, Y65, F76, V135, and F147 ( Figure 4 , Figure 5 ); 5 hydrophobic interactions are formed between KFP and I32, I52, F121, F126, and F151 ( Figure 6 , Figure 7 ); 11 hydrophobic interactions are formed between LLPK and I32, V48, I52, L61, F76, F119, F126, Y131, I135, F151, and I153 ( Figure 8 , Figure 9 ); 1 hydrogen bond interaction is formed between PDRM and C133, and 5 hydrophobic interactions are formed between I52, L54, F126, V135, and F151 ( Figure 10 , Figure 11 ).

[0062] Table 1 Potential mussel polypeptide sequences and related properties obtained

[0063]

[0064] Table 2 Molecular docking results

[0065]

[0066] As can be seen from Table 1 and Table 2, the polypeptides FPR, FRE, KFP, LLPK, and PDRM screened through bioinformatics tools and molecular docking all have certain immunomodulatory activities, and their activities will be verified after further synthesis in the next step.

[0067] Example 2: Chemical Synthesis and Activity Verification of Polypeptide Sequences

[0068] Entrusted Sangon Biotech (Shanghai) Co., Ltd. to chemically synthesize polypeptides FPR, FRE, KFP, LLPK, and PDRM. The liquid phase detection results are shown respectively as Figure 12 - 16 shown, and the mass spectrometry detection results are shown respectively as Figure 17 - 21 shown.

[0069] Evaluate the immunomodulatory ability of synthetic polypeptides FPR, FRE, KFP, LLPK, and PDRM through RAW264.7 cell proliferation activity experiments and neutral red phagocytosis experiments.

[0070] (1) Effects of different concentrations of FPR, FRE, KFP, LLPK, and PDRM on the relative proliferation rate of RAW264.7 cells

[0071] Determine the effects of different concentrations of FPR, FRE, KFP, LLPK, and PDRM on the relative proliferation rate of RAW264.7 cells by the CCK-8 method. The results are shown as Figure 22 shown. FPR, FRE, KFP, LLPK, and PDRM can all increase the relative proliferation rate of RAW264.7 cells. Among them, LLPK can significantly enhance the proliferation activity of RAW264.7 cells, up to 180.05% (concentration 600 μg / mL); the effect of KFP is slightly inferior, and the highest proliferation rate is 133.38% (concentration 400 μg / mL).

[0072] (2) Effects of different concentrations of FPR, FRE, KFP, LLPK, and PDRM on the NO release ability of RAW 264.7 cells

[0073] Evaluate the effects of different concentrations of FPR, FRE, KFP, LLPK, and PDRM on the NO release amount of macrophages through the NO release experiment. The results are shown as Figure 23 shown. Although FPR, FRE, KFP, LLPK, and PDRM are not as good as the positive control (lipopolysaccharide), they also show strong immunological activity.

[0074] (3) Effects of different concentrations of FPR, FRE, KFP, LLPK, and PDRM on the neutral red phagocytosis ability of RAW264.7 cells

[0075] Evaluate the effects of different concentrations of FPR, FRE, KFP, LLPK, and PDRM on the phagocytosis ability of macrophages through the neutral red phagocytosis experiment. The results are shown as Figure 24As shown, except for LLPK, all of them can significantly improve the neutral red phagocytosis ability of RAW264.7 cells. When the concentration of FPR is 400 μg / mL, it reaches the highest at 222.97%.

[0076] The above experiments once again prove that the active peptides FPR, FRE, KFP, LLPK and PDRM of the present invention all have strong immunological activities and can be used to prepare products with improved immune functions.

[0077] The above-described embodiments are only the preferred embodiments of the present invention, which are only used to explain the present invention and do not limit the scope of implementation of the present invention. For those skilled in the art of this technology, other implementation manners can be easily made by means of substitution or change according to the technical content disclosed in this specification. Any changes and improvements made on the principle of the present invention should be included within the protection scope of the present invention.

Claims

1. Use of a mussel immunologically active peptide in the preparation of a product with enhanced immune function, characterized in that, The amino acid sequence of the immunologically active peptide is FPR.

2. The application according to claim 1, wherein, The products for enhancing immune function include immunomodulators and nutritional products.

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