Abalone-derived polypeptide with anti-oxidation effect and preparation method of abalone-derived polypeptide

Abstract

The invention relates to the technical field of biological agents, in particular to abalone-derived polypeptide with antioxidant activity and a preparation method thereof. According to the invention, Haliotis discus hannai Ino is taken as a research object, a small peptide library is established by using a virtual enzymolysis technology from an important anti-oxidation pathway of cells, namely a Keap1-Nrf2 pathway, an abalone-derived anti-oxidation peptide DEDEDEDK with relatively strong binding capacity with Keap1 is screened out by using a molecular docking technology, and then, the anti-oxidation activity of the abalone-derived anti-oxidation peptide DEDEDEDK is verified by using a cell oxidative damage model, so that the abalone-derived anti-oxidation peptide DEDEDEDK can be used for preparing the anti-oxidation peptide DEDEDEDK with relatively strong binding capacity with Keap1. And a theoretical basis is provided for research and application of the antioxidant peptide.

Description

Technical Field

[0001] The present invention relates to the technical field of biological preparations, and in particular to an abalone polypeptide with antioxidant activity and a preparation method thereof. Background Art

[0002] Oxidative stress is the imbalance of the oxidation-antioxidation system when the body is stimulated by oxidative damage, resulting in excessive accumulation of reactive oxygen species (ROS), which in turn damages biological macromolecules such as nucleic acids, proteins and lipids, causing diseases such as atherosclerosis, liver disease, cancer, and aging. Improving the body's antioxidant capacity is one of the important ways to maintain good health. At present, most of the common antioxidants on the market are chemically synthesized substances, but these antioxidants may have certain toxicity to the human body and bring adverse effects. Therefore, it is of great significance to develop non-toxic and effective natural antioxidants. With the abundance of protein resources, the increase in the types of proteases, and the advancement of separation and purification technology, more and more antioxidant peptides are being discovered.

[0003] Antioxidant peptides are often obtained from food proteins by simulating the digestion process of the human gastrointestinal tract with pepsin, trypsin and chymotrypsin to degrade the proteins, and then screen out the target peptides with antioxidant activity through separation, purification and sequence identification. However, this method is time-consuming, labor-intensive, and costly, and there is a serious loss of peptide sequences during the separation and purification process, and the final result may not achieve the expected effect. Computer-aided design (CAD) has the potential to accelerate the development of bioactive peptides from the laboratory to the market. First, the amino acid sequence of the raw protein is retrieved from protein databases such as NCBI, PDB and UniProt. After obtaining its primary structure, virtual enzymatic hydrolysis is performed using online tools such as BIOPEP, Enzyme Predictor and PeptideRanker. When the 3D structure of the target receptor protein is clear, the interaction between the receptor protein and the ligand small molecule can be detected by molecular docking and molecular dynamics simulation, and their binding affinity can be evaluated. Therefore, virtual enzymatic hydrolysis combined with molecular docking technology can predict and discover a large number of peptides under simulated conditions, thereby efficiently screening out bioactive peptides with novel structures, which not only effectively reduces the workload, reduces labor costs and shortens the R&D cycle, but also improves the success rate of identifying active peptides. With the increasing application of CAD in the research of active peptides, researchers have screened out peptides with antioxidant activity from protein resources such as fish, star worms, and soybeans.

[0004] In the body's antioxidant process, the Keap1-nuclear factor-related factor 2 (Nrf2) signaling pathway is a crucial antioxidant pathway. This pathway can be activated by inhibiting the interaction between Keap1 and Nrf2, thereby promoting the expression of a series of protein-related genes with antioxidant and cytoprotective effects, thereby improving the body's antioxidant capacity. As a key transcription factor that regulates cellular oxidative stress, Nrf2 is bound by Keap1 and rapidly ubiquitinated and degraded under normal circumstances. However, when cells are subjected to oxidative stress, Nrf2 can escape Keap1-mediated degradation, enter the cell nucleus and bind to the antioxidant response element (ARE), activating the expression of a series of antioxidant and cytoprotective genes, thereby enhancing the body's antioxidant defense ability. Therefore, if we start from the Keap1-Nrf2 pathway, screening antioxidant peptides is more targeted.

[0005] Marine organisms have become an important resource of antioxidant peptides due to their diverse species and unique nutritional components. Currently, a variety of antioxidant peptides have been obtained from marine protein sources such as shellfish, fish, and algae. However, compared with the research on terrestrial antioxidant peptides, the research on marine antioxidant peptides is relatively small and still needs further exploration and development. Abalone, as a primitive marine shellfish, is rich in nutrients and has extremely high medicinal value. It is a high-quality resource for the development of marine bioactive peptides. Therefore, this study took the wrinkled abalone (Haliotis discus hannaiIno) as the research object, starting from the important antioxidant pathway of the cell, the Keap1-Nrf2 pathway, and used virtual enzymatic hydrolysis technology to establish a small peptide library. Abalone-derived antioxidant peptides with strong binding ability to Keap1 were screened out by molecular docking technology. Subsequently, their antioxidant activity was verified by a cell oxidative damage model, providing a theoretical basis for the research and application of antioxidant peptides. Summary of the invention

[0006] The purpose of the present invention is to provide an abalone polypeptide with antioxidant activity and a preparation method thereof.

[0007] To achieve the above object, the present invention adopts the following technical solution: The first aspect of the present invention provides an abalone-derived polypeptide having antioxidant activity, wherein the amino acid sequence of the abalone-derived polypeptide is: DEDEDEDK.

[0008] The second aspect of the present invention provides a method for preparing the above-mentioned abalone polypeptide having antioxidant activity, the method comprising the following steps: S1: Nine abalone proteins and their amino acid sequences of H. discushannaiIno were retrieved from the Uniprot database (https: / / www.uniprot.org / ); the abalone proteins were virtually enzymatically hydrolyzed using the ExPASy PeptideCutter program (http: / / web.expasy.org / peptidecutter / ), using proteases including pepsin (pH 1.3) (EC 3.4.23.1), trypsin (EC 3.4.21.4) and chymotrypsin (EC 3.4.21.1), to remove single peptides, dipeptides and repeated peptides, and obtain an abalone peptide library consisting of 363 small peptides; the abalone peptide library was optimized using the Discovery Studio software built-in function; S2: All PDB files reflecting human Keap1 protein were retrieved from the PDB database, and finally the receptor part in the PDB file 2FLU reflecting the binding of the Kelch region of Keap1 protein to 16mer Nrf2 was selected as the docking receptor file; the receptor file was optimized using the built-in function of Discoery Studio software; S3: The optimized abalone peptide library was used as the ligand file, the optimized Keap1 protein was used as the receptor file, and the designed site (x: 14.8387, y: 14.8842, z: 5.49218, radius: ) as the docking site for molecular docking, and the CDOCKER module in the Discovery Studio software was used for molecular docking to screen and obtain candidate abalone-derived polypeptide sequences with potential antioxidant activity; S4: The candidate abalone-derived polypeptide sequences are verified by solid phase chemical synthesis method and antioxidant activity verification experiment to obtain abalone-derived polypeptides with antioxidant activity.

[0009] The third aspect of the present invention provides the use of the above-mentioned abalone-derived polypeptide having antioxidant activity in the preparation of antioxidants.

[0010] The significant advantages of the present invention are: The human body will produce reactive oxygen free radicals (ROS) during normal physiological metabolism or under external stimulation (such as high temperature, radiation, photolysis, chemicals, etc.). A small amount of ROS can participate in the immune and signal transduction processes and play an important role in cell proliferation, differentiation and apoptosis. However, excessive ROS will cause damage to proteins, DNA and membrane lipids in the body, causing an imbalance between the body's oxidase system and non-enzymatic oxidants, and being in a state of oxidative stress, thereby causing cardiovascular, hypertension, cancer, neurodegeneration and other diseases. Although the body itself can use endogenous antioxidant defense mechanisms to remove oxidants in the body, with the increase of age, the occurrence of diseases or other traumas, the body's oxidation-reduction equilibrium state will be destroyed. Therefore, in order to reduce or inhibit the excessive accumulation of ROS, synthetic antioxidants such as tert-butylhydroquinone (TBHQ), butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) are widely used in medicine, cosmetics and food industries, but with the deepening of research, the toxicity and side effects of the body itself are becoming increasingly prominent. Therefore, it is of great significance to seek non-toxic and effective natural antioxidants from food-derived proteins. The present invention takes Haliotis discus hannai Ino as the research object, starts from the Keap1-Nrf2 pathway, an important antioxidant pathway of cells, and uses virtual enzymolysis technology to establish a small peptide library, and screens out abalone-derived antioxidant peptides with strong binding ability to Keap1 through molecular docking technology. Subsequently, its antioxidant activity is verified by a cell oxidative damage model, providing a theoretical basis for the research and application of antioxidant peptides. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 : Optimized abalone peptide library.

[0012] Figure 2 :PDB file 2FLU structure.

[0013] Figure 3 :2FLU binding site.

[0014] Figure 4 :Effects of different concentrations of abalone peptide DEDEDEDK on oxidative stress levels of HUVEC cells damaged by AAPH (A) ROS content; (B) GSH content; (C) SOD activity; (D) CAT content. *P<0.05, **P<0.01 vs. model group. DETAILED DESCRIPTION

[0015] In order to make the contents of the present invention easier to understand, the technical solution of the present invention is further described below in conjunction with specific implementation methods, but the present invention is not limited thereto.

[0016] Embodiment 1: 1. Construction and optimization of small peptide library Nine proteins and their amino acid sequences of H. discushannai Ino were retrieved from the Uniprot (https: / / www.uniprot.org / ) database (Table 1). The abalone proteins were virtually enzymatically hydrolyzed using the ExPASy PeptideCutter (http: / / web.expasy.org / peptidecutter / ) program. The proteases used included pepsin (pH 1.3) (EC 3.4.23.1), trypsin (EC 3.4.21.4) and chymotrypsin (EC 3.4.21.1). Single peptides, dipeptides and repeated peptides were removed to obtain an abalone peptide library consisting of 363 small peptides. At the same time, the abalone peptide library was optimized using the Discovery Studio software's built-in function.

[0017] Table 1 Abalone proteins in the Uniprot database 2. Receptor file selection and optimization All PDB files reflecting human Keap1 protein were retrieved from the PDB database, and finally the receptor part in the PDB file 2FLU reflecting the binding of the Kelch region of Keap1 protein to 16mer Nrf2 was selected as the docking receptor file. On this basis, the receptor file was optimized using the built-in functions of the Discoery Studio software.

[0018] 3. Molecular docking: The optimized abalone peptide library was used as the ligand file, the optimized Keap1 protein was used as the receptor file, and the designed site (x: 14.8387, y: 14.8842, z: 5.49218, radius: ) was used as the docking site for molecular docking, and the CDOCKER module in the Discovery Studio software was used for molecular docking to screen out a candidate abalone-derived polypeptide sequence DEDEDEDK with potential antioxidant activity.

[0019] 4. Solid Phase Synthesis and Antioxidant Activity Verification The abalone polypeptide DEDEDEDK with a purity of more than 98% was synthesized by solid phase synthesis.

[0020] Set up the model group and the experimental group, and set the density to 5×10 4HUVEC cells with a concentration of 100 μL per well were inoculated in a 96-well plate. After the cells adhered to the wall, the normal control group was added with culture medium, and the model group and the experimental group were added with 10 μL of 2,2'-azobisisobutylamidine dihydrochloride (AAPH) solution to make its final concentration 6 mmol / L. After incubation in a CO2 incubator (37°C, 5% CO2) for 24 h, the experimental group was added with 10 μL of abalone peptide DEDEDEDK to make the final concentration of the peptide 250, 500, and 1000 μg / mL. The normal control group and the model group were added with equal amounts of culture medium, respectively. After incubation in a CO2 incubator (37°C, 5% CO2) for 24 h, ROS detection was performed according to the instructions of the reactive oxygen species detection kit.

[0021] Set up the model group and the experimental group, and set the density to 5×10 4 HUVEC cells / mL were inoculated in 6-well plates at 2mL per well. After the cells adhered to the wall, 2mL of AAPH solution was added to the model group and the test group respectively to make the final concentration of 6mmol / L, and the normal control group was added with the same amount of culture medium. After incubation in a CO2 incubator (37°C, 5% CO2) for 24h, 200μL of abalone peptide DEDEDEDK was added to the test group to make the final concentration of the peptide 250, 500, 1000μg / mL, and the normal control group and model group were added with the same amount of culture medium. After incubation in a CO2 incubator (37°C, 5% CO2) for 24h, the cells were collected. The protein content was measured by referring to the BCA kit, and the activity of SOD and CAT and the content of GSH were detected by referring to the methods of GSH, SOD, and CAT kits.

[0022] like Figure 4 As shown in a, compared with the normal control group, the ROS content in the model group was extremely significantly increased (P<0.01), indicating that AAPH treatment can cause oxidative stress in HUVEC cells and produce excessive reactive oxygen free radicals. Compared with the model group, the ROS content in the abalone peptide DEDEDEDK (250μg / mL, 500μg / mL) treatment group did not change significantly (P>0.05), and the ROS content in the abalone peptide DEDEDEDK (1000μg / mL) treatment group was significantly reduced (P<0.05), indicating that high concentrations of abalone peptide DEDEDEDK can significantly reduce the ROS content produced by AAPH damage.

[0023] Depend on Figure 4 b It can be seen that the GSH content in the normal control group was 13.79±0.36 mg GSH / L, while the content in the model group was extremely significantly reduced (P<0.01). After treatment with 250μg / mL, 500μg / mL and 1000μg / mL of abalone peptide DEDEDEDK, the intracellular GSH content increased significantly (P<0.01).

[0024] like Figure 4 c and Figure 4 As shown in Figure d, the SOD and CAT activities of the normal control group were 2.41±0.02U / mg and 12.24±0.37U / mg, respectively, while those of the model group were 1.69±0.03U / mg and 10.95±0.21U / mg, respectively, with extremely significant differences (P<0.01). After treatment with 250μg / mL, 500μg / mL and 1000μg / mL of abalone peptide DEDEDEDK, the intracellular SOD and CAT activities were significantly increased (P<0.05, P<0.01).

[0025] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. An abalone polypeptide having antioxidant activity, characterized in that: The amino acid sequence is: DEDEDEDK.

2. The method for preparing abalone polypeptide according to claim 1, characterized in that: The following steps are involved: S1: Obtain the protein sequence of Haliotis discus hannai from the UniProt protein database, perform virtual enzymatic hydrolysis on the Haliotis discus hannai protein using the online tool ExPASyPeptideCutter, remove single peptides, double peptides and repeated peptides, and obtain the abalone peptide library; use Discovery Studio software to optimize the abalone peptide library; S2: Obtain all PDB files reflecting human Keap1 protein from the PDB protein structure database, select the receptor part in the PDB file 2FLU reflecting the binding of the Kelch region of Keap1 protein to 16mer Nrf2 as the docking receptor file; optimize the receptor file using Discoery Studio software; S3: Using the optimized abalone peptide library as the ligand file and the optimized Keap1 protein as the receptor file, the CDOCKER module in the Discovery Studio software was used for molecular docking to screen candidate abalone peptide sequences with potential antioxidant activity; S4: The candidate abalone-derived polypeptide sequences are verified by solid phase chemical synthesis method and antioxidant activity verification experiment to obtain abalone-derived polypeptides with antioxidant activity.

3. The preparation method according to claim 2, characterized in that: The proteases used in the virtual enzymolysis include pepsin EC 3.4.23.1, trypsin EC 3.4.21.4 and chymotrypsin EC 3.4.21.

1.

4. The preparation method according to claim 2, characterized in that: The docking site of the molecular docking is x: 14.8387, y:14.8842, z:5.49218, radius:12.3Å.

5. Use of the abalone-derived polypeptide as claimed in claim 1 in the preparation of an antioxidant.

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