Mussel immunoactive peptide and application thereof

Abstract

The invention discloses a mussel immune active peptide and application thereof, and belongs to the technical field of active peptides, the sequence of the immune active peptide is FPR, FRE, KFP, LLPK or PDRM, and the acquisition of the immune active peptide is derived from digestion of mussel meat under a static simulation digestion model, polypeptide sequence identification, bioinformatics tool prediction, molecular docking and polypeptide synthesis and verification. The mussel immune active peptide disclosed by the invention has relatively good proliferation promoting activity and neutral red phagocytic ability of mouse macrophages RAW264.7. The screened mussel immune active peptide can be used as an auxiliary immune regulation active component to be applied to products with an immune function improving function, such as an immunomodulator, a pharmaceutical composition or a nutritional product, and has important guiding significance for 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] The mussel ( Mytilus coruscus ), also known as the common mussel, blue mussel, or edible mussel, is a bivalve mollusk widely distributed in temperate waters around the world and belongs to the family Mytilidae. It has high nutritional and medicinal value. There is currently 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 rapidly 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: Five mussel immunomodulatory bioactive peptides with relatively high activities were screened out from mussels. The sequences of the mussel peptides are 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).

[0008] The screening method of the above-mentioned mussel immunomodulatory bioactive peptide, the method comprises the following steps: (1) Preparation of mussel meat homogenate: Take a certain amount of mussels, wash and dry them, take out all the soft tissues and juice, and homogenize them 3 times in a high-speed tissue homogenizer for 30 s each time to obtain the homogenate; (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 well, and digest in a constant temperature oscillator 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 well, and then adjust the pH to 7.0 with 1 mol / L sodium bicarbonate solution, and digest in a constant temperature oscillator for 2 h to simulate intestinal digestion; (3) Identification of mussel protein peptide sequence: Identify the peptide spectrum of the mussel protein simulated digestion product through the Thermo Fisher EASY-nLC1200 Q Exactive liquid chromatography-mass spectrometry system; (4) Screening of mussel immunomodulatory peptides assisted by bioinformatics tools: Query the novelty of peptides through the BIOPEP database, predict the biological activity of peptides through the Peptide Ranker tool, predict the digestion resistance characteristics and cell membrane permeability of peptides through the Peptide Cutter tool and CPP pred tool, evaluate the stability of peptides in the blood through the PLiePred tool, and predict the potential toxicity and physicochemical properties of peptides through the Toxin Pred tool; (5) Molecular docking: Take the screened mussel immunomodulatory 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; (6) Determination of immunomodulatory activity: Conduct in vitro immunological activity detection, including the effects of immunomodulatory 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.

[0009] Preferably, for the operation in step (1), after homogenization, the homogenate needs to be placed in an experimental freezer or immediately proceed with subsequent experiments to avoid degradation of protein and polypeptide components due to long-term storage at room temperature.

[0010] 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 digestion reaction of this stage.

[0011] 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 protein sources of the peptide sequences are obtained by comparing the resulting polypeptide sequences with the Uniprot-Acipenser database.

[0012] 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.

[0013] Preferably, for the operation in step (5), the results of molecular docking are represented by the binding energy value, and a binding energy with TLR4-MD2 lower than -5.9 kcal / mol is used as the screening condition.

[0014] The mussel immunologically active peptide 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 any synthetic method, so as to obtain mussel multifunctional peptides more efficiently and provide materials for the further utilization of the screened polypeptides.

[0015] Advantages of the present invention: The present invention discloses a mussel immunologically active peptide and its application. The mussel immunologically active peptide of the present invention is mainly obtained through simulated digestion, LC-MS / MS mass spectrometry identification, bioinformatics tool analysis and prediction, 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 Peptide Ranker score, stability, digestion resistance characteristics and cell membrane permeability, 8 peptide sequences were comprehensively screened for molecular docking, and 5 peptide sequences have a binding energy with TLR4-MD2 lower than -5.9 kcal / mol. After comprehensively considering potential activity, safety and bioaccessibility, 5 sequences were screened 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 peptide segments can be added as auxiliary immunomodulatory active ingredients to functional products such as immunomodulators, pharmaceutical compositions or nutritional products. Description of the Drawings

[0016] Figure 1 This is the preparation technical roadmap of mussel immunologically active peptides in the embodiments of the present invention; Figure 2 This is the overall three-dimensional structure diagram of the docking of polypeptide FPR with TLR4-MD2 molecule; Figure 3 This is the two-dimensional schematic diagram of the interaction of the active site residues in the docking of polypeptide FPR with TLR4-MD2 molecule; Figure 4 This is the overall three-dimensional structure diagram of the docking of polypeptide FRE with TLR4-MD2 molecule; Figure 5 This is the two-dimensional schematic diagram of the interaction of the active site residues in the docking of polypeptide FRE with TLR4-MD2 molecule; Figure 6 This is the overall three-dimensional structure diagram of the docking of polypeptide KFP with TLR4-MD2 molecule; Figure 7 This is the two-dimensional schematic diagram of the interaction of the active site residues in the docking of polypeptide KFP with TLR4-MD2 molecule; Figure 8 This is the overall three-dimensional structure diagram of the docking of polypeptide LLPK with TLR4-MD2 molecule; Figure 9 This is the two-dimensional schematic diagram of the interaction of the active site residues in the docking of polypeptide LLPK with TLR4-MD2 molecule; Figure 10 This is the overall three-dimensional structure diagram of the docking of polypeptide PDRM with TLR4-MD2 molecule; Figure 11 This is the two-dimensional schematic diagram of the interaction of the active site residues in the docking of polypeptide PDRM with TLR4-MD2 molecule; Figure 12 This is the liquid phase detection result diagram of the synthetic polypeptide FPR in the embodiments of the present invention; Figure 13 This is the liquid phase detection result diagram of the synthetic polypeptide FRE in the embodiments of the present invention; Figure 14 This is the liquid phase detection result diagram of the synthetic polypeptide KFP in the embodiments of the present invention; Figure 15 This is the liquid phase detection result diagram of the synthetic polypeptide LLPK in the embodiments of the present invention; Figure 16 This is the liquid phase detection result diagram of the synthetic polypeptide PDRM in the embodiments of the present invention; Figure 17 This is the mass spectrometry detection result diagram of the synthetic polypeptide FPR in the embodiments of the present invention; Figure 18 This is the mass spectrometry detection result diagram of the synthetic polypeptide FRE in the embodiments of the present invention; Figure 19Mass spectrometry detection result graph of synthetic polypeptide KFP in the embodiment of the present invention; Figure 20 Mass spectrometry detection result graph of synthetic polypeptide LLPK in the embodiment of the present invention; Figure 21 Mass spectrometry detection result graph of synthetic polypeptide PDRM in the embodiment of the present invention; Figure 22 Effect result graph of synthetic peptide on the proliferation activity of RAW264.7 cells in the embodiment of the present invention; Figure 23 Effect result graph of synthetic peptide on the NO release amount of RAW264.7 cells in the embodiment of the present invention; Figure 24 Effect result graph of synthetic peptide on the neutral red phagocytosis activity of RAW264.7 cells in the embodiment of the present invention. Specific implementation manners

[0017] 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.

[0018] Example 1: 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.

[0019] The specific steps include: (1) Digest mussel meat based on the INFOGEST in vitro static simulation 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. Second, 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, use 6 mol / L hydrochloric acid solution to adjust the pH to 3.0 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 well, 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, use 5 mol / L sodium hydroxide solution to adjust the pH to 6.0 to terminate the gastric digestion process, add simulated intestinal fluid (final concentration of pancreatin EC 3.4.21.4 is 200 U / mL, final concentration of bovine bile salt is 20 mmol / mL) in a volume ratio of 1:1, mix well, and then use 1 mol / L sodium bicarbonate solution to adjust the pH to 7.0, 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).

[0020] (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. The 10 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 values 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).

[0021] (3)Bioinformatics tools assisted in screening mussel immunologically active peptides: First, the novelty of the peptides was queried through the BIOPEP database, and sequences with previously reported activities were not subjected to subsequent screening; the Peptide Ranker tool was used to predict the biological activities of the peptides, and sequences with scores exceeding 0.4 were considered to have potential activities; the Peptide Cutter tool was used to predict the digestive resistance characteristics of the peptides. If there were no cleavage sites that could 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), the sequence was considered to have the potential for anti-gastrointestinal digestion; the CPP pred tool was used to predict the cell membrane permeability of the peptides, and sequences with scores exceeding 0.5 were considered to have the potential for complete transmembrane absorption; the PLiepred tool was used to evaluate the stability of the peptides in the blood, and peptide sequences with half-life values higher than 800 s were considered to have a certain stability in the blood; the Toxin Pred tool was used to predict the potential toxicity and physicochemical properties of the peptides, and sequences considered to have no potential toxicity were eligible for subsequent synthesis and verification.

[0022] A total of 99 sequences were identified by peptide fingerprinting. Among them, 41 active sequences have been reported. Among the remaining 58 unreported sequences, 21 have a Peptide Ranker score exceeding 0.4. Eight peptide sequences were selected for molecular docking based on the comprehensive screening of Peptide Ranker score, stability, digestion resistance, and cell membrane permeability.

[0023] (4)Virtual screening of mussel immunologically active peptides by molecular docking: The peptides screened in the previous process were used as ligands, and TLR4-MD2 was used as the receptor. The binding sites and interaction forces between mussel immunologically active peptides and TLR4-MD2 were analyzed by molecular docking technology. The three-dimensional structure of TLR4-MD2 (3FXI) was obtained from the PDB database (http: / / www.rcsb.org / ). The three-dimensional structure of the polypeptide was constructed using the Pymol program. The semi-flexible docking of mussel immunologically active peptides with TLR4-MD2 was performed using the Autdock software. The results of the molecular docking were represented by the binding energy value, and the conformation with the minimum binding energy was selected as the best binding site.

[0024] A total of 5 peptide sequences with eligible binding energies 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 the polypeptide. The 5 selected peptide sequences are: FPR, FRE, KFP, LLPK, and PDRM. The potential mussel polypeptide sequences screened and their related properties are shown in Table 1. The binding energies of the polypeptides FPR, FRE, KFP, LLPK, and PDRM to TLR4-MD2 are shown in Table 2.

[0025] Among them, the interactions between FPR, FRE, KFP, LLPK, and PDRM and TLR4-MD2 are as Figure 2 - 11 shown.

[0026] Among them, FPR, FRE, KFP, LLPK, and PDRM interact with TLR4-MD2. Seven hydrophobic interactions are formed between FPR and I32, V48, I52, L61, F121, C133, F151, and I153 ( Figure 2 , Figure 3 ); Five hydrophobic interactions are formed between FRE and I44, Y65, F76, V135, and F147 ( Figure 4 , Figure 5 ); Five hydrophobic interactions are formed between KFP and I32, I52, F121, F126, and F151 ( Figure 6 , Figure 7); Eleven hydrophobic interactions are formed between LLPK and I32, V48, I52, L61, F76, F119, F126, Y131, I135, F151 and I153 ( Figure 8 , Figure 9 ); One hydrogen bond is formed between PDRM and C133, and five hydrophobic interactions are formed between I52, L54, F126, V135 and F151 ( Figure 10 , Figure 11 ).

[0027] Table 1 Potential mussel polypeptide sequences screened and related properties

[0028] Table 2 Molecular docking results

[0029] As can be seen from Table 1 and Table 2, the polypeptides FPR, FRE, KFP, LLPK, and PDRM screened by bioinformatics tools and molecular docking all have certain immunomodulatory activities. Next, their activities will be verified after synthesis.

[0030] Example 2: Chemical synthesis and activity verification of polypeptide sequences Entrust Sangon Biotech (Shanghai) Co., Ltd. to chemically synthesize the polypeptides FPR, FRE, KFP, LLPK, and PDRM. The liquid phase detection results are respectively as Figure 12 - 16 shown, and the mass spectrometry detection results are respectively as Figure 17 - 21 shown.

[0031] The immunomodulatory abilities of the synthesized polypeptides FPR, FRE, KFP, LLPK, and PDRM were evaluated by RAW264.7 cell proliferation activity experiment and neutral red phagocytosis experiment.

[0032] (1) Effects of different concentrations of FPR, FRE, KFP, LLPK, and PDRM on the relative proliferation rate of RAW264.7 cells The effects of different concentrations of FPR, FRE, KFP, LLPK, and PDRM on the relative proliferation rate of RAW264.7 cells were determined by the CCK-8 method. The results are 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).

[0033] (2) Effects of FPR, FRE, KFP, LLPK, and PDRM at Different Concentrations on the NO Release Capacity of RAW 264.7 Cells The effects of FPR, FRE, KFP, LLPK, and PDRM at different concentrations on the NO release amount of macrophages were evaluated through a NO release experiment. The results are as Figure 23 shown. Although FPR, FRE, KFP, LLPK, and PDRM were not as effective as the positive control (lipopolysaccharide), they also exhibited strong immunological activity.

[0034] (3) Effects of FPR, FRE, KFP, LLPK, and PDRM at Different Concentrations on the Neutral Red Phagocytosis Capacity of RAW264.7 Cells The effects of FPR, FRE, KFP, LLPK, and PDRM at different concentrations on the phagocytosis capacity of macrophages were evaluated through a neutral red phagocytosis experiment. The results are as Figure 24 shown. Except for LLPK, all of them could significantly improve the neutral red phagocytosis capacity of RAW264.7 cells. When the concentration of FPR was 400 μg / mL, it reached the highest level of 222.97%.

[0035] The above experiments once again demonstrated that the bioactive peptides FPR, FRE, KFP, LLPK, and PDRM of the present invention all have strong immunological activity and can be used to prepare products with enhanced immune function.

[0036] The above-described embodiments are only 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 through replacement or change according to the technical content disclosed in this specification. All changes and improvements made on the principle of the present invention should be included within the protection scope of the present invention.

Claims

1. A mussel immune active peptide, characterized in that: The amino acid sequence of the immunoactive peptide is FPR, FRE, KFP, LLPK or PDRM.

2. Use of the mussel immune active peptide according to claim 1 in preparing a product having an immune function enhancing effect.

3. The use according to claim 2, characterized in that: The products for improving immune function include immunomodulators, pharmaceutical compositions or nutritional products.

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