A nereid antioxidant peptide, its preparation method and application

Abstract

The present invention belongs to the technical field of polypeptide products, and particularly relates to a nereis antioxidant peptide, a preparation method thereof and an application. The present invention prepares a nereis antioxidant peptide, and its sequence is shown as SEQ ID NO: 1. The nereis antioxidant peptide prepared by the present invention has no toxic effect on cells, has good scavenging ability for hydroxyl radicals and superoxide anion radicals, and can reduce the ROS level increased by LPS induction. The present invention provides a preparation method of a nereis antioxidant peptide, comprising the following steps: pulping nereis, enzymolysis, ultrafiltration separation, and chromatographic purification to obtain. The nereis antioxidant peptide prepared by the present invention can be widely used in the preparation of antioxidant cosmetics, antioxidant health products and antioxidant drugs.

Description

Technical Field

[0001] The present invention belongs to the technical field of polypeptide products, and particularly relates to a nereis antioxidant peptide, a preparation method thereof, and an application thereof. Background Art

[0002] There is a certain relationship between many human diseases and fatigue and the oxidation in the human body, and the oxidation in the human body is related to the existence of excessive free radicals in the body. Therefore, appropriate intake of some antioxidant substances can effectively reduce the content of free radicals in the body and prevent lipid peroxidation. At present, antioxidant substances include chemically synthesized drugs and extracts of natural products. Chemically synthesized drugs have good antioxidant effects but have certain side effects, while antioxidant substances derived from natural products are safe and non-toxic and have high antioxidant activity. In particular, antioxidant peptides extracted from organisms have stronger antioxidant persistence and stability compared with traditional natural antioxidants Vc and Ve, can inhibit the peroxidation of biological macromolecules and scavenge excessive free radicals in the body, and have a certain correlation with other biological activities, including immunomodulation, anti-tumor, cholesterol reduction, etc. Therefore, it is of great significance to extract antioxidant peptides from organisms. Summary of the Invention

[0003] The purpose of the first aspect of the present invention is to provide a nereis polypeptide or a salt thereof.

[0004] The purpose of the second aspect of the present invention is to provide a conjugate.

[0005] The purpose of the third aspect of the present invention is to provide a fusion protein.

[0006] The purpose of the fourth aspect of the present invention is to provide a biomaterial.

[0007] The purpose of the fifth aspect of the present invention is to provide a preparation method.

[0008] The purpose of the sixth aspect of the present invention is to provide an application.

[0009] The purpose of the seventh aspect of the present invention is to provide a product.

[0010] In order to achieve the above purposes of the present invention, the technical solutions adopted by the present invention are as follows:

[0011] In the first aspect of the present invention, there is provided a nereis polypeptide or a salt thereof, characterized in that: the sequence of the nereis polypeptide includes SEQ ID NO: 1.

[0012] Preferably, the nereis includes Perinereis aibuhitensis.

[0013] In the second aspect of the present invention, there is provided a conjugate, comprising a modifying part and the nereis polypeptide of the first aspect of the present invention.

[0014] Preferably, the modification part is located at the N-terminus and / or C-terminus of the nereid polypeptide.

[0015] Preferably, the modification part comprises at least one of chemical modification, targeting part, fluorescent dye and protein tag.

[0016] Preferably, the chemical modification comprises at least one of amidation, acetylation, amination, methylation, phosphorylation, glycosylation, ubiquitination.

[0017] Preferably, the targeting part comprises at least one of ligand, receptor, antibody.

[0018] Preferably, the fluorescent dye comprises FITC.

[0019] Preferably, the protein tag comprises at least one of His, Flag, GST, MBP, HA, Myc, GFP, biotin.

[0020] In the third aspect of the present invention, there is provided a fusion protein, which is characterized in that it comprises another polypeptide and the nereid polypeptide of the first aspect of the present invention.

[0021] Preferably, the other polypeptide is located at the N-terminus and / or C-terminus of the nereid polypeptide.

[0022] Preferably, the other polypeptide and the polypeptide of the first aspect of the present invention are connected to the N-terminus and / or C-terminus of the nereid polypeptide through a linker.

[0023] In the fourth aspect of the present invention, there is provided a biological material related to the nereid polypeptide of the first aspect of the present invention, the conjugate of the second aspect of the present invention, or the fusion protein of the third aspect of the present invention, and the biological material comprises any one of a1) to a8):

[0024] a1) A nucleic acid molecule encoding the nereid polypeptide of the first aspect of the present invention, the conjugate of the second aspect of the present invention, or the fusion protein of the third aspect of the present invention;

[0025] a2) An expression cassette comprising the nucleic acid molecule described in a1);

[0026] a3) A vector comprising the nucleic acid molecule described in a1);

[0027] a4) A vector comprising the expression cassette described in a2);

[0028] a5) A transgenic cell line comprising the nucleic acid molecule described in a1);

[0029] a6) A transgenic cell line comprising the expression cassette described in a2);

[0030] a7) A transgenic cell line containing the vector described in a3);

[0031] a8) A transgenic cell line containing the vector described in a4);

[0032] The transgenic cell line does not contain propagation materials.

[0033] In the fifth aspect of the present invention, a method for preparing the nereid polypeptide of the first aspect of the present invention is provided, which is any one of (a) to (c):

[0034] (a) Extracted from nereids as raw materials;

[0035] (b) Synthesized by liquid-phase or solid-phase synthesis methods;

[0036] (c) Obtained by culturing the transgenic cells described in claim 5.

[0037] Preferably, the preparation method of (a) extracted from nereids as raw materials includes: pulping nereids, enzymatic hydrolysis, ultrafiltration separation, and chromatographic purification to obtain.

[0038] Preferably, the enzymes for enzymatic hydrolysis include at least one of alkaline protease, papain, trypsin, and pepsin.

[0039] Preferably, the enzyme for enzymatic hydrolysis includes alkaline protease.

[0040] Preferably, the ratio of the alkaline protease to the nereids after pulping is 2000 - 4000 U / g.

[0041] Preferably, the time for enzymatic hydrolysis is 3 - 12 h.

[0042] Preferably, the temperature for enzymatic hydrolysis is 40 - 60 °C.

[0043] Preferably, the step of inactivating the enzyme is further included after enzymatic hydrolysis.

[0044] Preferably, the temperature for inactivating the enzyme is 90 - 100 °C.

[0045] Preferably, the time for inactivating the enzyme is 5 - 15 min.

[0046] Preferably, the ultrafiltration separation yields nereid antioxidant peptides with a molecular weight less than 8 KD.

[0047] Preferably, the ultrafiltration separation yields nereid antioxidant peptides with a molecular weight less than 5 KD.

[0048] Preferably, the chromatographic purification includes gel permeation chromatography and semi-preparative chromatography.

[0049] Preferably, the chromatographic purification specifically uses gel filtration chromatography to separate nereis antioxidant peptides with a molecular weight less than 5 KD, collect four elution peaks of G1-G4, use semi-preparative chromatography to separate and purify the G4 elution peak, obtain four elution peaks of S1-S4, and retain the S2 elution peak.

[0050] In a sixth aspect of the present invention, there is provided the use of the nereis polypeptide of the first aspect of the present invention, the conjugate of the second aspect of the present invention, the fusion protein of the third aspect of the present invention, or the biological material of the fourth aspect of the present invention in the preparation of antioxidant products.

[0051] In a seventh aspect of the present invention, there is provided a product comprising at least one of b1) to b4):

[0052] b1) the polypeptide of the first aspect of the present invention or a salt thereof;

[0053] b2) the conjugate of the second aspect of the present invention;

[0054] b3) the fusion protein of the third aspect of the present invention;

[0055] Preferably, the product includes at least one of pharmaceuticals, health products, and cosmetics.

[0056] Preferably, the product includes at least one of antioxidant pharmaceuticals, antioxidant health products, and antioxidant cosmetics.

[0057] The beneficial effects of the present invention are as follows:

[0058] The present invention has prepared a nereis antioxidant peptide, the sequence of which is shown in SEQ ID NO: 1. The nereis antioxidant peptide prepared by the present invention has no toxic effect on cells, has good scavenging ability for hydroxyl radicals and superoxide anions, and can reduce the ROS level increased by LPS induction. The present invention provides a preparation method of nereis antioxidant peptide, which includes the following steps: pulping nereis, enzymolysis, ultrafiltration separation, and chromatographic purification to obtain. The nereis antioxidant peptide prepared by the present invention can be widely used in the preparation of antioxidant cosmetics, antioxidant health products, and antioxidant drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 Shows the scavenging ability of nereis enzymolysis solutions with different molecular weights for hydroxyl radicals.

[0060] Figure 2 Is the IC of nereis enzymolysis solutions with different molecular weights for scavenging hydroxyl radicals 50 value.

[0061] Figure 3 Shows the scavenging ability of nereis enzymolysis solutions with different molecular weights for superoxide anions.

[0062] Figure 4 IC of nereid enzymatic hydrolysates with different molecular weights for scavenging superoxide anions 50 value.

[0063] Figure 5 Gel filtration chromatography separation results of nereid enzymatic hydrolysates with a molecular weight less than 5KD.

[0064] Figure 6 Effect of elution peaks G1 - G4 obtained by gel filtration chromatography separation of nereid enzymatic hydrolysates on RAW cell viability.

[0065] Figure 7 Effect of elution peaks G1 - G4 obtained by gel filtration chromatography separation of nereid enzymatic hydrolysates on the viability of LPS - induced RAW cells.

[0066] Figure 8 Fluorescence results of ROS levels in LPS - induced RAW cells.

[0067] Figure 9 Fluorescence results of different concentrations of elution peak G1 on ROS levels in LPS - induced RAW cells.

[0068] Figure 10 Fluorescence results of different concentrations of elution peak G2 on ROS levels in LPS - induced RAW cells.

[0069] Figure 11 Fluorescence results of different concentrations of elution peak G3 on ROS levels in LPS - induced RAW cells.

[0070] Figure 12 Fluorescence results of different concentrations of elution peak G4 on ROS levels in LPS - induced RAW cells.

[0071] Figure 13 Chromatogram results of elution peaks S1 - S4 obtained after semi - preparative chromatography purification of elution peak G1.

[0072] Figure 14 Effect of different concentrations of elution peaks S1 - S4 on RAW cell viability.

[0073] Figure 15 Effect of different concentrations of elution peaks S1 - S4 on the viability of LPS - induced RAW cells.

[0074] Figure 16 Fluorescence results of ROS levels in LPS - induced RAW cells.

[0075] Figure 17 Fluorescence results of different concentrations of elution peak S1 on ROS levels in LPS - induced RAW cells.

[0076] Figure 18Fluorescence results of different concentrations of S2 elution peaks on the ROS level of LPS-induced RAW cells.

[0077] Figure 19 Fluorescence results of different concentrations of S3 elution peaks on the ROS level of LPS-induced RAW cells.

[0078] Figure 20 High-performance liquid chromatography diagram of S2 elution peak.

[0079] Figure 21 First-level mass spectrometry diagram of S2 elution peak.

[0080] Figure 22 Second-level mass spectrometry diagram of S2 elution peak. Detailed implementation mode

[0081] The following will clearly and completely describe the concept and technical effects of the present invention in combination with embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative efforts belong to the scope of protection of the present invention.

[0082] Example 1 Preparation of Nereis Protein Hydrolysate (NPH)

[0083] Weigh 500 g of Nereis, homogenize it, add alkaline protease (Shanghai Yuanye Bio-Technology Co., Ltd., product number: S10154) at a ratio of 3000 U / g, the material-to-water ratio is 1:5, adjust the pH = 8.5, place it in a shaker at 50 °C for enzymatic hydrolysis for 7 h. After the enzymatic hydrolysis is completed, place it in boiling water at 100 °C to inactivate the enzyme for 10 minutes. After the enzyme inactivation is completed, cool it to room temperature at room temperature, and centrifuge at 8000 r / min to separate and take the supernatant.

[0084] Separate the Nereis enzymatic hydrolysate with three ultrafiltration membranes with different molecular weights of 8 kDa (product number C6MR12468-029), 5 kDa (product number C6JR77589-020) and 3 kDa (product number C6HR77579-010). First, use an ultrafiltration membrane with a cut-off molecular weight of 8 kDa to ultrafilter and separate the sample to obtain Nereis ultrafiltration components with >8 kDa (component 4) and <8 kDa. Then, use an ultrafiltration membrane with a cut-off molecular weight of 5 kDa to separate the <8 kDa ultrafiltrate to obtain two parts of 5 kDa - 8 kDa (component 3) and <5 kDa. Then, use an ultrafiltration membrane with a cut-off molecular weight of 3 kDa to separate the <5 kDa ultrafiltrate to obtain two parts of 3 kDa - 5 kDa (component 2) and <3 kDa (component 1), and a total of four groups of ultrafiltration components with different molecular segments are obtained.

[0085] The ultrafiltration components of nereis were concentrated by rotary evaporation, and the samples were dried by freeze-drying.

[0086] Determination of antioxidant activity of ultrafiltration components of nereis in Example 2

[0087] 1. Determination method and calculation of hydroxyl radical scavenging ability (o-phenanthroline method)

[0088] Add 1.2 mL of o-phenanthroline (5 mmol / L), 0.8 mL of phosphate buffer (0.2 mol / L, pH = 7.4), 2 mL of samples with different concentrations (concentration gradients of 2.5, 5, 7.5, 10, 12.5 mg / L), 1.2 mL of EDTA-2Na (15 mmol / mL), 1.2 mL of FeSO4 (5 mmol / mL), and 1.6 mL of H2O2 (0.1%) into the test tube in sequence, make up the volume to 10 mL and mix well. Incubate in a water bath at 37 °C for 90 min. After centrifugation, measure the absorbance at a wavelength of 536 nm with a UV spectrophotometer. Use ultrapure water to replace the sample in the damage tube, and the other steps are the same as those in the sample tube. Non-damage tube: Replace H2O2 with ultrapure water, and the other steps are the same as those in the damage tube. Do three parallels for each concentration. The experimental results are expressed by the scavenging rate E:

[0089] 2. Determination method and calculation of scavenging ability of superoxide anion radical (O 2- )

[0090] Add 1 mL of samples with different concentrations (0.5, 1, 1.5, 2, 2.5, 3, 3.5 mg / L) into the test tube, add 4.5 mL of Tris-HCL buffer (0.01 mol / L, pH = 8.2), keep it at a constant temperature of 25 °C for 20 min. After taking it out, immediately add 4.2 mL of ultrapure water and 0.3 mL of pyrogallol solution (3 mmol / L) preheated at 25 °C. Shake well quickly and pour it into the cuvette. Measure the absorbance A1 every 0.5 min at a wavelength of 325 nm for a total of 5 min. Calculate the change rate of absorbance of the control solution with time △A1. Use water with the same volume to replace the sample as the blank control, that is, the pyrogallol autoxidation rate △A0. The final result is expressed by the scavenging rate E:

[0091] 3. Experimental results

[0092] The results of the scavenging ability of NPH with different molecular weights on hydroxyl radicals are as Figure 1 shown. The polypeptides in each molecular segment of the nereis enzymolysis solution have the ability to scavenge hydroxyl radicals, and their scavenging ability is positively correlated with the concentration of the components. As Figure 2 can be seen, the IC 50The values are 5.52 mg / mL, 5.45 mg / mL, 6.2 mg / mL, and 7.01 mg / mL in sequence. Combining with the IC 50 value of the hydroxyl radical scavenging ability of each component and the significant difference analysis of the IC 50 value of the hydroxyl radical scavenging ability of each component, it can be known that the ability of polypeptides in each molecular segment to scavenge hydroxyl radicals decreases in the order of Component 1 = Component 2 > Component 3 > Component 4. The hydroxyl radical scavenging ability of Component 1 and Component 2 in the ultrafiltration components of nereid enzymatic hydrolysate is significantly higher than that of other components. Therefore, it can be concluded that nereid polypeptides with a molecular weight less than 5 kDa have a good ability to scavenge hydroxyl radicals.

[0093] Pyrogallol undergoes autoxidation under alkaline conditions, generating superoxide anions and releasing colored substances simultaneously. Moreover, the oxidation rate of pyrogallol is related to the generation concentration of superoxide anions, and the generated superoxide anions have a catalytic effect on the autoxidation of pyrogallol. The accumulation amount of colored substances within 5 minutes has a good linear relationship with time.

[0094] As Figure 3 shown, each ultrafiltration component has the effect of scavenging superoxide anion radicals at each concentration. Combining with the calculation formula of the scavenging rate, it can be known that under the action of nereid antioxidant peptides, the oxidation rate of pyrogallol is lower than that of the blank group, indicating that nereid polypeptides in each molecular segment have a certain ability to capture superoxide anions, thereby reducing the catalytic effect of superoxide anions on the autoxidation of pyrogallol and further reducing the generation rate of colored substances.

[0095] As Figure 4 shown, the IC 50 values of Component 1, Component 2, Component 3, and Component 4 for scavenging superoxide anions are 1.49 mg / mL, 1.77 mg / mL, 2.4 mg / mL, and 3.3 mg / mL in sequence. Combining with the results of the significant difference analysis, it can be concluded that the ability of each molecular segment to scavenge superoxide anions decreases in the order of: Component 1 = Component 2 > Component 3 > Component 4. It can be seen that the superoxide anion radical scavenging ability of Component 1 and Component 2 is significantly higher than that of other components. Therefore, it can be concluded that nereid polypeptides with a molecular weight less than 5 kDa have a good ability to scavenge superoxide anion radicals.

[0096] Example 3 Influence of Ultrafiltration Components on Cell Activity and Antioxidant Ability

[0097] 1. Sephadex G-15 Gel Filtration Chromatography

[0098] Sephadex G25 dextran gel was swollen with 20% ethanol for 24 h and then packed into a column with a specification of 1.6 cm × 90 cm. After sample injection, ultrapure water was used for elution. The sample loading volume was 2 mL, the concentration was 200 mg / mL, and the flow rate was controlled at 1.5 mL / min. The eluate was monitored and collected at an absorbance of 280 nm. The collected fractions were directly freeze-dried and stored in a -80 °C refrigerator for later use.

[0099] According to the separation range of different gels, Sephadex G-25 was used to separate and purify the <5 kDa ultrafiltration fraction of nereid protease hydrolysate. The results are as Figure 5 shown. After gel filtration chromatography, four fractions, namely G1 - G4, could be eluted from the nereid protease hydrolysate. The eluates of the four peaks were collected for subsequent experiments.

[0100] 2. Effects of gel chromatography separated fractions on the viability of RAW cells

[0101] 1) Cryopreservation, resuscitation and culture of RAW cells

[0102] Prepare cell cryopreservation solution (volume ratio: 90% fetal bovine serum + 10% DMSO) in advance and store it in a 4 °C refrigerator for pre-cooling and later use. Observe RAW cells in the cell flask under a microscope. When the cell area in the flask reaches more than 80% of the cell flask, cryopreservation can be carried out. Aspirate the cell culture medium, then digest the cells, add 1 mL of 0.25 (w / v)% trypsin, and let it act at room temperature for about 30 s. To prevent over-digestion, the trypsin must be aspirated in time. Add basal medium (DMEM) and pipette the cells on the wall to disperse the cells, then transfer them to a 15 mL centrifuge tube. Set the centrifuge at 1000 rpm / min for 5 min and aspirate the supernatant. Add 1 mL of cryopreservation solution, gently disperse the cells with a pipette and transfer them into a cryopreservation tube. Tighten the tube cap and clearly label the name of the cryopreserved cells and the cryopreservation date. After sealing with a sealing film, place the cryopreservation bottle in a cell gradient cryopreservation box and store it in a -80 °C refrigerator.

[0103] Thaw DMEM and trypsin at room temperature for later use, and at the same time, sterilize the cell laboratory and the laminar flow hood with ultraviolet light for 30 min. The RAW cell cryopreservation tube taken out from the -80 °C refrigerator should be immediately placed in warm water at 37 °C, and the cryopreservation tube should be shaken quickly and in the same direction for 1 - 2 min until it is completely melted. On the sterile operating table, transfer the cell suspension to a 15 mL centrifuge tube, slowly add 4 mL of basal culture medium, set the centrifuge at 1000 rpm / min and centrifuge for 5 min. After aspirating the supernatant, add 4 - 6 mL of complete medium, gently mix the cells and then transfer them to a 25 cm 2 cell culture flask, label the cell name and the resuscitation date, and place it in an incubator (37 °C) for 4 h.

[0104] All operations were carried out on a sterile operating table. Complete medium was prepared in advance, and the formula was 10% (V / V) fetal bovine serum + 90% (V / V) DMEM. After the RAW cells were resuscitated, 4 - 6 mL of complete medium was added. The culture conditions were 5% CO2, 95% air, 37 °C, and saturated humidity. The medium was changed once a day, and subculture was carried out once every two days. Cells in the logarithmic growth phase with excellent status were selected for the experiment.

[0105] 2) RAW cell cytotoxicity assay

[0106] The cultured RAW cells were inoculated into 96 - well plates at a cell density of 1X10 4 / well, and incubated overnight. After 4 h of cell attachment, the medium was changed and the drug was added. A normal control group (without adding nereis polypeptide, with the same other treatments) and a sample group were set up, with at least 3 replicate wells in each group. After treating macrophages with 2 μL of the test samples (elution samples of G1 - G4 peaks) at different concentrations (0.0001, 0.001, 0.01, 0.1, 1 mg / mL) for 24 h, CCK8 reagent was added, and then the cells were incubated in the incubator for 1 - 4 h. Then, the absorbance at 450 nm was measured with an enzyme - linked immunosorbent assay (ELISA) reader, once every hour. Taking the average absorbance of the normal control group as 100%, the cell viability was calculated to determine whether the test samples at different concentrations were safe and non - toxic.

[0107] Cell survival rate (%) = Ai / A0 * 100%.

[0108] Where: Ai represents the absorbance value at 450 nm after adding ultrafiltration components at different concentrations; A0 represents the average absorbance value of the normal control group at 450 nm.

[0109] 3) Experimental results

[0110] As Figure 6 shown, there were no significant differences between the four separated peaks G1 - G4 of nereis protease hydrolysate and the blank control group in the five concentration ranges of 1.0 - 0.0001 mg / mL. It had no toxic side effects on RAW cells and could continue with the subsequent experiments.

[0111] 3. Effects of gel chromatography separation components on the intracellular ROS level of LPS - induced RAW cells

[0112] 1) Determination of ROS production in LPS - induced RAW cells

[0113] DCFHDA itself has no fluorescence and can freely penetrate the cell membrane. After entering the cell membrane, it can be hydrolyzed into DCFH under the action of cellular lipase, and DCFH cannot penetrate the cell membrane. Therefore, the probe is easily accumulated in the cell. Reactive oxygen species (ROS) in the cell can oxidize non-fluorescent DCFH to generate fluorescent DCF, and the intensity of green fluorescence is proportional to the level of ROS. At the maximum excitation wavelength of 480 nm and the maximum emission wavelength of 525 nm, a fluorescence microscope, flow cytometer, laser confocal microscope, etc. are used to detect the fluorescence signal.

[0114] In this example, LPS-induced RAW 264.7 mouse macrophages were used as a research model. The cultured RAW cells were inoculated into a 96-well plate (5×10 5 / well) and then placed in an incubator. After 4 h of cell attachment, the medium was changed and drugs were added. A normal control group, an LPS-stimulated group (1 μg / ml LPS), and a sample + LPS co-treatment group were set up, with 5 replicate wells in each group. After adding different concentrations of the sample to be tested to the sample + LPS co-treatment group and acting on the macrophages for 24 h, LPS (1 μg / mL) was added to stimulate each group except the normal control group. After 24 h, the old medium was discarded, 20 μL of DCFH was added to each well, and the cells were incubated in the dark in the incubator for 25 min. Then the cells were washed 3 times with DMEM, and then Hoechst33342 was added to each well and incubated for 15 min. The cells were washed 3 times with DMEM, and a microplate reader was used to measure DCFH and Hoechst33342. The excitation wavelength and emission wavelength of DCFH were 485 nm and 535 nm, respectively, and the excitation wavelength and emission wavelength of Hoechst33342 were 350 nm and 460 nm, respectively. The ratio of the measured DCFH fluorescence value to the Hoechst33342 fluorescence value is the content of ROS produced by RAW cells. Subsequently, the above operations of cell inoculation, drug addition, and LPS addition were repeated. The old medium was discarded, DCFHDA was added, and the cells were incubated in the dark in the cell culture incubator for 25 min. Then the well plate was washed three times with DMEM until the external medium of the cells no longer showed green fluorescence, and a Zessi fluorescence microscope was used to take pictures.

[0115] 2) Experimental results

[0116] The inhibitory effects of the 4 elution peaks obtained by gel filtration chromatography on the generation of ROS in LPS-induced Raw cells are as Figure 7 shown. LPS can stimulate cells to produce oxidative stress, and the content of reactive oxygen species increases significantly. All 4 gel chromatography peaks can inhibit the generation of ROS in LPS-induced RAW cells, and the content of reactive oxygen species in RAW cells is reduced to a certain extent. Among them, G1 shows a highly significant effect (P<0.05) at concentrations from 1 to 0.001 mg / mL, and G1 shows the best anti-LPS stimulation effect among the four preparative chromatography peaks.

[0117] The effects of each gel chromatography peak on ROS generated by LPS induction in RAW cells were investigated using the DCFHDA method. As Figure 7 shown, the fluorescence intensity of the positive group was greater than that of the blank group, and the ROS content increased, indicating that LPS indeed had an oxidative stress effect on RAW cells. As Figures 8 - 12 shown, the fluorescence intensity of each group added with G1-G4 elution samples (concentrations 1-5 were 1, 0.1, 0.01, 0.001, 0.0001 μg / mL respectively) was significantly lower than that of the positive group, and the ROS content decreased, indicating that the gel chromatography peaks had an obvious effect of slowing down the LPS stimulation on RAW cells. The 4 separated peaks obtained by gel chromatography separation of the <5 kDa ultrafiltration fraction of nereis can all inhibit the generation of ROS in RAW cells induced by LPS. Among them, the inhibition of peak G1 is better than the other 3 peaks. Therefore, G1 was determined for further purification.

[0118] Example 4 Effects of components separated by semi-preparative chromatography on cell oxidation activity

[0119] 1. Quiksep-50 type semi-preparative liquid chromatography

[0120] Purification was carried out using a QuikswP50D semi-preparative liquid chromatograph and a C18 column (Xterra MS 5 μM, 4.6×250 mm). The column temperature was 25 °C, the sample injection volume was 100 μL, the sample concentration was 50 mg / mL, the flow rate was 1.5 mL / min, and the mobile phase included A and B. Among them, mobile phase A was ultrapure water containing 0.005% trifluoroacetic acid, and mobile phase B was 100% acetonitrile. The elution time was gradient washed for 20 min, and the washing gradient was that acetonitrile increased from 3% to 23%, and water decreased from 97% to 77%.

[0121] The results of separating and purifying peak G1 using semi-preparative chromatography are as Figure 13 shown. Four components can be eluted from peak G1, namely S1 to S4. The S1-S4 elution peaks were collected, and their antioxidant activities were further verified by cell experiments.

[0122] 2. Effects of components separated by semi-preparative chromatography on the viability of RAW cells

[0123] The experimental method was the same as in Example 3.

[0124] As Figure 14 shown, the four separated peaks S1-S4 of the semi-preparative chromatography had no significant difference compared with the blank control group at five concentrations (0.002, 0.02, 0.2, 2, 20 μg), and had no toxic side effects on RAW cells.

[0125] 3. Effects of Components Separated by Semi-Preparative Chromatography on ROS Generation in LPS-Stimulated RAW Cells

[0126] The inhibitory effects of the four elution peaks S1 - S4 obtained by semi-preparative chromatography on ROS generation in LPS-stimulated RAW cells are shown as follows Figure 15 All four chromatographic peaks S1 - S4 obtained by semi-preparative chromatography inhibited the generation of ROS in LPS-stimulated RAW cells to a certain extent. Among them, S2 showed highly significant effects (P < 0.001) at concentrations from 20 to 0.002 μg / ml, while S1 showed highly significant effects (P < 0.001) at concentrations from 20 to 0.02 μg / mL, peak S3 showed highly significant effects (P < 0.001) at concentrations from 20 to 0.02 μg / mL, and significant effects (P < 0.05) at a concentration of 0.002 μg / mL. The inhibitory effect of peak S2 on ROS generation in LPS-stimulated RAW cells was better than that of peaks S1 and S3

[0127] The DCFHDA method was used to explore the effects of each preparative peak on ROS generated by LPS in RAW cells. Since the inhibitory effect of S4 on ROS generation in LPS-stimulated RAW cells was not obvious, the intracellular fluorescence value of S4 was not measured. As shown below Figures 16 - 19 The fluorescence intensity of the positive group was greater than that of the blank group, and the ROS content increased, indicating that LPS indeed induced oxidative stress in RAW cells. The fluorescence intensities of the groups added with preparative peaks were significantly lower than that of the positive group, and the ROS content decreased, indicating that the preparative peaks significantly alleviated the LPS stimulation of RAW cells. The four separated peaks obtained by preparative chromatography of G1 could all inhibit the generation of ROS in LPS-stimulated RAW cells. Considering that the ROS fluorescence value and the number of fluorescence in RAW cells were lower after adding S2 than those of the other three peaks, S2 was selected for mass spectrometry identification to determine its peptide sequence

[0128] Example 5 Identification of the Structure of Antioxidant Peptides from Nereis

[0129] 1. Liquid Chromatography - Mass Spectrometry Identification Method

[0130] LC-20AD Shimadzu High Performance Liquid Chromatography: First, freeze-dry S2 under vacuum, and dissolve it ultrasonically with 50 μl of 25 mM ammonium bicarbonate. Centrifuge at 10000 g for 5 min and take the supernatant for injection. Separate the peptide solution through a nano-liquid chromatography instrument of the LC-20AD model produced by Shimadzu Corporation. The columns used include a Trap column and an analytical column. The separation procedure is as follows: Inject the sample at a flow rate of 8 μL / min for 4 min first; then wash it with a gradient at a flow rate of 300 nl / min for 40 min, and the washing gradient is that buffer B (95% ACN containing 0.1% FA) rises from 2% to 35%; then linearly elute from 35% to 80% for 5 min. Finally, wash the column with 80% buffer B for 4 min and wash the column with buffer A (5% ACN containing 0.1% FA) for 1 min.

[0131] MALDI-TOF Mass Spectrometry: The peptides separated by liquid chromatography enter a tandem ESI mass spectrometer: Q-EXACTIVE (ThermoFisher Scientific, San Jose, CA). The resolution of the first-stage mass spectrometry is set to 70000 (mass-to-charge ratio / full width at half maximum). Screen the peptides in the HCD (High energe Collision Dissociation) mode with a collision energy of 27, and detect the secondary fragments in Orbi with a resolution of 17500. For each precursor ion with a peak intensity exceeding 20000, 15 secondary spectra are acquired, and the first-stage scan and the second-stage scan are alternated. The dynamic exclusion is set to: the same precursor ion will not be scanned for the second time more than 2 times within 15 seconds. The ion source voltage is set to 1.6 kV. AGC (Automatic gain control) is achieved through Orbi, and its setting is: perform secondary scan identification on ions with the controlled aggregation amount in Orbi between 1e 5 to 3e 6 . The mass-to-charge ratio range of the scan is 350 - 2000 Da.

[0132] Data Processing: The data obtained from the instrument is a file with a.raw suffix. Use Proteome Discoverer (ver.1.3.0.339; ThermoFisher Scientific, San Jose, CA) software to integrate and convert it into a file with a.mgf suffix, and then use the Mascot search engine (ver.2.3.0; Matrix Science, London, United Kingdom) to identify peptides and proteins. The database search settings are: the digestion method is blank; the fixed modification and variable modification are blank; the error tolerance rate is 20 ppm for the first stage and 0.1 Da for the second stage. Import the identified information into Excel for viewing and comparison.

[0133] 2. Experimental Results

[0134] The results of the high-performance liquid chromatography are as Figure 20 shown. Using a tandem ESI mass spectrometer to perform first-order and second-order scans on H2, the nereis antioxidant decapeptide: LFTKLRASNT (SEQ ID NO: 1, from N-terminus to C-terminus) was identified, with a molecular weight of 1150.6346.

[0135] Table 1 Amino Acid Sequence Identification Table

[0136]

Claims

1. A nereid polypeptide or a salt thereof, characterized in that: The sequence of the lugworm polypeptide is shown in SEQ ID NO:

1.

2. A conjugate comprising a modified portion and the neriworm polypeptide of claim 1.

3. The conjugate according to claim 2, characterized in that: The modified portion is located at the N-terminus and / or C-terminus of the neriworm polypeptide; The modification moiety comprises at least one of a chemical modification, a targeting moiety, a fluorescent dye and a protein tag.

4. The conjugate according to claim 3, characterized in that: The chemical modification comprises at least one of amidation, acetylation, amination, methylation, phosphorylation, glycosylation, and ubiquitination; The targeting moiety comprises at least one of a ligand, a receptor, and an antibody; The fluorescent dye comprises FITC; The protein tag comprises at least one of His, Flag, GST, MBP, HA, Myc, GFP, and biotin.

5. A biological material related to the neriworm polypeptide according to claim 1 or the conjugate according to any one of claims 2 to 4, wherein the biological material comprises any one of a1) to a8): a1) a nucleic acid molecule encoding the polypeptide according to claim 1 or the conjugate according to any one of claims 2 to 4; a2) an expression cassette comprising the nucleic acid molecule described in a1); a3) a vector comprising the nucleic acid molecule described in a1); a4) a vector comprising the expression cassette described in a2); a5) a transgenic cell line comprising the nucleic acid molecule described in a1); a6) a transgenic cell line comprising the expression cassette described in a2); a7) a transgenic cell line comprising the vector described in a3); a8) a transgenic cell line comprising the vector described in a4); The transgenic cell line contains no reproductive material.

6. Use of the neriworm polypeptide or its salt according to claim 1, the conjugate according to any one of claims 2 to 4, and the biomaterial according to claim 5 in the preparation of antioxidant products.

7. A product comprising at least one of b1) to b2): b1) the polypeptide or a salt thereof according to claim 1; b2) the conjugate according to any one of claims 2 to 4; The product includes at least one of medicines, health products and cosmetics.

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