Oyster polypeptide with anti-protein aggregation function and application thereof

Abstract

The application discloses an oyster polypeptide with protein aggregation resistance and application thereof, and belongs to the field of small molecule polypeptides. The oyster is used as raw material, and a pure natural oyster polypeptide is extracted, separated and identified through steps of enzymatic separation, ultrafiltration purification and LC-MS / MS identification. The amino acid sequence of the oyster polypeptide is shown as SEQ ID NO:1, the molecular formula is C 68 H 98 N 20 O 21 , the average relative molecular mass is about 1531.62 Da, the theoretical isoelectric point is pH=6.0, and the oyster polypeptide is a hydrophilic polypeptide. The oyster polypeptide has functions of antioxidation, protein aggregation resistance and nerve protection, and can be further used for development of food, medicine and health care products, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of small molecule peptides, specifically relating to an oyster peptide with anti-protein aggregation function and its application. Background Technology

[0002] Protein aggregation refers to the process by which proteins, under biological or experimental conditions, transform from their originally soluble form into insoluble polymers or fibrous structures. This process usually stems from abnormal changes in the spatial conformation of proteins, rendering them unable to maintain their normal folding state or structural stability, thus causing them to tend to aggregate. The cytotoxicity caused by protein aggregation induces a state of physiological stress in the body. On the one hand, aggregated proteins lose their original three-dimensional structure, leading to impaired normal biological functions and interfering with normal cellular metabolism and life activities; on the other hand, the aggregation process itself gradually accumulates to form specific protein aggregates.

[0003] Neurodegenerative diseases are a class of diseases characterized by the gradual decline of neuronal structure and function, ultimately leading to cell death. One common pathological feature of these diseases is the formation of aggregates from abnormal protein aggregation, such as extracellular amyloid fibrillary deposits or intracellular abnormal protein fibrillary inclusions. Common neurodegenerative diseases include Huntington's disease, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), and prion diseases. Taking Alzheimer's disease as an example, one of its typical pathological factors is Aβ oligomers. Aβ is a fragment produced by enzymatic cleavage of amyloid precursor protein and readily aggregates in the brains of patients. Aβ oligomers are composed of 2 to 12 Aβ monomers and form earlier than long fibrous amyloid plaques; they are currently considered one of the key early toxic factors in the disease. These oligomers can interfere with normal neuronal function, induce oxidative stress, inflammatory responses, and synaptic damage, thereby promoting cognitive decline and the neurodegenerative process. These toxic effects are generally believed to originate from the abnormal aggregation of proteins.

[0004] In general, the toxicity of protein aggregates manifests primarily in cytotoxicity, inflammatory responses, and intracellular toxicity. These toxic effects are closely related to various neurodegenerative diseases (such as Alzheimer's and Parkinson's) and other diseases (such as Huntington's disease). Missense mutations, oxidative modifications, and amino acid pairing errors during translation that occur during aging lead to changes in protein structure and weakened cellular repair capabilities, resulting in protein aggregation. Aging is not only a key risk factor for these diseases but also accelerates disease progression by exacerbating functional decline and weakening intrinsic repair mechanisms. Current research focuses on exploring novel therapeutic strategies that can effectively intervene in abnormal protein aggregation and inhibit its toxicity, addressing this core pathology.

[0005] Bioactive peptides are protein fragments composed of short-chain amino acids with a variety of biological functions. They can be extracted from food, microorganisms, or cells and exert antioxidant, anti-inflammatory, blood pressure-lowering, and immunomodulatory effects in vivo. Bioactive peptides play an important role in anti-protein aggregation. Protein aggregation is a key process in the development of many diseases (such as Alzheimer's disease and Parkinson's disease), and bioactive peptides can intervene in protein aggregation through the following mechanisms: (1) Inhibiting the aggregation of abnormal proteins: Some peptides can directly bind to abnormally folded proteins, inhibiting them from forming aggregates. (2) Promoting normal protein folding: Bioactive peptides can help maintain the correct folding of proteins, thereby reducing the occurrence of misfolding and aggregation. (3) Clearing aggregates: Some peptides can promote intracellular autophagy and protein degradation pathways, helping to clear abnormal aggregates accumulated in the body. (4) Protecting cells: Through antioxidant and anti-inflammatory effects, bioactive peptides can protect cells from damage caused by protein aggregation. These mechanisms make bioactive peptides an important direction for researching potential treatments for diseases caused by protein aggregation.

[0006] The application of biopeptides in anti-protein aggregation research faces several limitations, which affect the feasibility of their large-scale production. The main limitations include: (1) High production costs: The synthesis of biopeptides typically requires complex technologies and equipment, especially when high purity and specific modifications are required. These costs increase significantly during large-scale production. (2) Complex production processes: The production of biopeptides involves multiple steps, such as peptide synthesis, purification, folding, and modification. Each step may introduce instability or efficiency issues, making large-scale production difficult. (3) Stability issues: Many biopeptides are prone to degradation or loss of activity during storage and handling. Maintaining their stability requires specific conditions and processing methods, which increases the complexity and cost of production. (4) Purification difficulties: Purifying biopeptides from complex mixtures is a technical challenge, especially during large-scale production, where purification efficiency may decrease, leading to product quality and consistency issues. These factors combined make the production of biopeptides face many challenges in large-scale applications. Although biopeptides have potential in anti-protein aggregation, further research and optimization are needed to overcome these limitations. Summary of the Invention

[0007] In order to overcome the shortcomings and deficiencies of the prior art, the primary objective of this invention is to provide an oyster polypeptide with aging function, which has the functions of prolonging life, reducing the content of aging pigments, and resisting oxidative stress.

[0008] Another object of the present invention is to provide a method for preparing the above-mentioned oyster polypeptide.

[0009] Another object of the present invention is to provide the application of the above-mentioned oyster polypeptide.

[0010] The objective of this invention is achieved through the following technical solution: An oyster polypeptide with anti-protein aggregation function, the amino acid sequence of which is shown in SEQ ID NO:1: NENPPVFGQNPYR.

[0011] The molecular formula of the oyster polypeptide is C 68 H 98 N 20 O 21 It has an average relative molecular mass of approximately 1531.62 Da and a theoretical isoelectric point of approximately pH=6.0, making it a hydrophilic polypeptide.

[0012] The method for preparing the oyster polypeptide with anti-protein aggregation function includes the following steps: (1) The above-mentioned oyster polypeptide with anti-protein aggregation function is prepared directly by solid-phase synthesis, or by using oyster as raw material and obtaining the above-mentioned oyster polypeptide with anti-protein aggregation function through enzymatic hydrolysis, separation and extraction and purification.

[0013] The enzymatic hydrolysis is preferably performed by sequentially using trypsin and papain.

[0014] The preferred specific operation for the enzymatic hydrolysis is as follows: Mix oyster freeze-dried powder with water, adjust the pH of the system to 7.5-8.0, add trypsin for enzymatic hydrolysis; then adjust the pH of the system to 6.5-7.0, add papain for enzymatic hydrolysis; after enzymatic hydrolysis, inactivate the enzyme to obtain oyster protein hydrolysate.

[0015] The preferred dosage of trypsin is 10,000 to 20,000 U of trypsin per gram of oyster freeze-dried powder.

[0016] The preferred dosage of papain is 10,000 to 20,000 U of papain per gram of oyster freeze-dried powder.

[0017] The preferred specific operation for the separation and extraction is as follows: After cooling the oyster protein hydrolysate obtained by enzymatic hydrolysis, 95% ethanol was added to bring the ethanol concentration of the solution to 60-80%, and the solution was allowed to stand. Then, the precipitate was removed by vacuum filtration and centrifugation. The supernatant was then evaporated under reduced pressure to remove ethanol, and the solution was freeze-dried to obtain crude oyster polypeptide powder.

[0018] The purification is preferably at least one of ultrafiltration purification and gel chromatography column purification.

[0019] The ultrafiltration purification is preferably performed using an Ultracel-3 membrane to collect components with a molecular weight of less than 3 kDa.

[0020] The preferred specific procedure for the gel chromatography column purification is as follows: The ultrafiltration purified components were desalted and then further purified using a Sephadex G-25 dextran gel chromatography column.

[0021] The purification process preferably further includes using reversed-phase high-performance liquid chromatography or size exclusion chromatography to further separate the components into individual peptides.

[0022] The application of the oyster polypeptide with anti-protein aggregation function in the preparation of products with anti-protein aggregation or anti-oxidation function.

[0023] The application of the oyster polypeptide with anti-protein aggregation function in the preparation of products with neuroprotective activity.

[0024] The application of the oyster polypeptide with anti-protein aggregation function in the preparation of products for the prevention and treatment of neurodegenerative diseases.

[0025] The neurodegenerative diseases mentioned include Huntington's disease (HD), Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), or prions.

[0026] An antioxidant product comprising at least one of the following as an active ingredient: oyster polypeptide with anti-protein aggregation function, protease hydrolysate containing oyster polypeptide with anti-protein aggregation function, and hydrolysate containing oyster polypeptide with anti-protein aggregation function.

[0027] A drug for preventing and treating neurodegenerative diseases, comprising at least one of the following as active ingredients: oyster polypeptide with anti-protein aggregation function, protease hydrolysate containing oyster polypeptide with anti-protein aggregation function, and hydrolysate containing oyster polypeptide with anti-protein aggregation function.

[0028] The principle of this invention: This invention uses oysters as raw material and extracts, separates, and identifies a pure natural oyster polypeptide through enzymatic hydrolysis, ultrafiltration purification, and LC-MS / MS identification. The amino acid sequence of this oyster polypeptide is NENPPVFGQNPYR, and its molecular formula is C 68 H 98 N 20 O 21The average relative molecular mass is approximately 1531.62 Da, and the theoretical isoelectric point is approximately pH=6.0, classifying it as a hydrophilic polypeptide. This invention further utilizes the model organism *C. elegans* as a model organism and has discovered that this oyster polypeptide possesses anti-protein aggregation and protein homeostasis regulating functions, as well as neuroprotective activity, which can help prevent neurodegenerative diseases, as detailed below: (1) ASH neuron survival test: This test verifies that the anti-protein aggregation peptide can effectively inhibit the toxic aggregation of pathogenic proteins and protect ASH neurons from damage.

[0029] (2) PolyQ (polyglutamine) aggregation inhibition test: This test verifies that the anti-protein aggregation peptide has the effect of inhibiting polyQ aggregation.

[0030] (3) Paraquat oxidative stress test: This test verifies that the anti-protein aggregation peptide can improve the survival of nematodes under oxidative stress and enhance the antioxidant capacity of nematodes.

[0031] In summary, the present invention demonstrates through the above experiments that oyster polypeptides possess anti-protein aggregation function, meaning they can prevent or slow down the aggregation of proteins into insoluble deposits within cells or the body. The advantages of these anti-protein aggregation oyster polypeptides include potent antioxidant defense capabilities, good biocompatibility, low potential side effects, the ability to inhibit abnormal protein folding, helping proteins fold correctly and preventing aggregation, and effectively regulating protein homeostasis, thereby achieving a neuroprotective effect.

[0032] The present invention has the following advantages and effects compared with the prior art: (1) This invention uses natural oysters as the target, and obtains oyster polypeptide components with neuroprotective activity through enzymatic hydrolysis and separation methods. Then, it obtains a single polypeptide through sequence identification, sequence comparison and polypeptide synthesis methods. Its amino acid sequence is shown in SEQ ID NO:1, which are Asn, Glu, Asn, Pro, Pro, Val, Phe, Gly, Gln, Asn, Pro, Tyr and Arg in sequence, and its molecular formula is C 68 H 98 N 20 O 21 It has an average relative molecular mass of approximately 1531.62 Da and a theoretical isoelectric point of approximately pH 6.0, making it a hydrophilic polypeptide. This polypeptide can be isolated and purified from oysters or synthesized artificially.

[0033] (2) In this invention, the polyQ aggregation model AM141 nematode and the Huntington's disease model HA759 nematode were selected to conduct polyglutamine aggregation inhibition test and neuronal survival test, respectively, to study the neuroprotective effect of oyster polypeptide. The results showed that the oyster polypeptide can significantly inhibit the aggregation of abnormal proteins and polyQ, reduce neuronal damage, protect neurons, and improve neuronal survival rate. Therefore, the oyster polypeptide has a clear anti-protein aggregation effect and excellent neuroprotective activity.

[0034] (3) The oyster polypeptide provided by the present invention can prolong the survival time of nematodes under paraquat oxidative stress and enhance the antioxidant capacity of nematodes.

[0035] (4) The oyster polypeptide provided by the present invention is derived from the enzymatic hydrolysate of natural food and medicine oysters. It has a small molecular weight, is easy to absorb, and has a clear source. As a drug, it can improve the efficiency and accuracy of treatment, reduce the occurrence of immune reactions, and ensure its long-term efficacy and safety.

[0036] (5) The oyster polypeptide provided by the present invention has antioxidant, anti-protein aggregation and neuroprotective functions, and can be further used in the development of food, pharmaceutical and health products, with broad application prospects. Attached Figure Description

[0037] Figure 1 This is a graph showing the results of the analysis of the effect of the oyster polypeptide Cg#2907 of the present invention on the survival rate of neurons in HA759 of *C. elegans*. Figure 2 This is a statistical chart showing the number of polyQ aggregation points in *C. elegans* AM141 after treatment with the oyster polypeptide Cg#2907 of this invention. Figure 3 This is a graph showing the distribution of polyQ aggregation points in *C. elegans* after 72 h of treatment with oyster polypeptide Cg#2907 according to the present invention. Figure 4 This invention describes the survival-improving effect of 50mM oyster polypeptide Cg#2907 on oxidative stress in wild-type nematodes. Figure 5 This invention describes the survival-improving effect of 100mM oyster polypeptide Cg#2907 on oxidative stress in wild-type nematodes. Detailed Implementation

[0038] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0039] The concentrated NA22 bacterial solution in the example is food for nematodes. The preparation method is as follows: activated Escherichia coli NA22 is inoculated into LB liquid medium and cultured with shaking. Then, the bacterial cells are collected by centrifugation to obtain the concentrated NA22 bacterial solution.

[0040] The C. elegans models of N2, AM141, and HA759 are all from the Caenorhabditis Genetics Center at the University of Minnesota, USA.

[0041] Example 1. Isolation and Identification of Oyster Polypeptides 1. Enzymatic Hydrolysis and Separation (1) Fresh oysters were cleaned, chopped, and homogenized. After freeze-drying the homogenate, 5 g of oyster freeze-dried powder was weighed, deionized water was added at a solid-liquid ratio of 1:4, and it was stirred evenly with a glass rod and preheated in a constant temperature water bath at 45 °C. Then, the pH of the solution was adjusted to 7.5 with NaOH, 1 mL of trypsin (15000 U / mL) was added, and the reaction was carried out at 45 °C for 4 h. After that, the pH of the solution was adjusted to 6.5 with HCl, 1 mL of papain (15000 U / mL) was added, and the reaction was carried out at 65 °C for 4 h. It was inactivated by boiling to obtain the oyster protease hydrolysate.

[0042] (2) After the oyster protease hydrolysate prepared in step (1) was naturally cooled to room temperature, 170 mL of 95% ethanol by volume was slowly added under stirring with a glass rod. After standing overnight at room temperature, it was subjected to vacuum filtration and centrifugation (4 °C, 8000 rpm, 5 min) to remove the precipitate. The supernatant was evaporated to remove ethanol by vacuum rotary evaporation and then freeze-dried to obtain the oyster crude polypeptide dry powder.

[0043] 2. Ultrafiltration Purification and Gel Chromatography Column Purification (1) The oyster crude polypeptide dry powder prepared in step 1 was formulated into a 20 mg / mL solution, and ultrafiltration was carried out using a centrifugal filter of Amicon Ultra-2 equipped with Ultracel-3, Ultracel-5, and Ultracel-10 membranes of Millipore. The retention components of the oyster protease hydrolysate with MW > 10 kDa, 5 kDa < MW < 10 kDa, 3 kDa < MW < 5 kDa, and MW < 3 kDa were obtained by membrane ultrafiltration. The fraction with a molecular weight less than 3 kDa was collected, then passed through a 0.45 μm filter membrane to remove impurities, and the salts in the polypeptide sample were removed using a desalting column.

[0044] (2) The polypeptide fraction after desalting in step (1) was further separated using a packed Sephadex G-25 dextran gel chromatography column. It was eluted with deionized water at a flow rate of 1 mL / min, and monitored at 280 nm with a 785 UV / VIS detector while collecting the fractions of each absorption peak. A total of 5 polypeptide fractions were obtained, and these 5 polypeptide fractions were freeze-dried separately to obtain the corresponding oyster polypeptide freeze-dried powders.

[0045] 3. LC-MS / MS identification The polypeptide composition and amino acid sequence of the oyster polypeptide freeze-dried powder obtained in step 2 were identified by LC-MS / MS.

[0046] 4. Database comparison and filtering Based on the identified peptide sequences with antioxidant activity provided in the BIOPEP-UWM database, the oyster peptide sequences obtained in step 3 were compared with this database for virtual activity screening. The specific steps were as follows: Log in to the BIOPEP-UWM website, select the Bioactive peptides database, click the Analysis option, select Profile of potential biological activity in the tab, and then enter the oyster peptide sequences obtained in step 3 by liquid chromatography-mass spectrometry (LC-MS) for comparison. Oyster peptide sequences with potential antioxidant and neuroprotective activities were selected for the following experiments. After screening, the oyster peptide with the amino acid sequence shown in SEQ ID NO:1 was obtained and named Cg#2907. Its amino acids are, in order, Asn, Glu, Asn, Pro, Pro, Val, Phe, Gly, Gln, Asn, Pro, Tyr, and Arg. The molecular formula was calculated using a specialized peptide calculator as Cg#2907. 68 H 98 N 20 O 21 The average relative molecular mass of the peptide was calculated to be approximately 1531.62 Da using the online tool Expasy Compute pI / Mw. The theoretical isoelectric point is pH=6.0, indicating that it is a hydrophilic peptide. The peptide provided by this invention has an isoelectric point close to 7, making it more widely applicable.

[0047] Example 2. Synthesis of oyster polypeptides using the Fmoc solid-phase method Oyster polypeptide Cg#2907 was synthesized by Shanghai Taopu Biotechnology Co., Ltd., using the following specific method: 1. Weigh out chlorotriphenylmethyl chloride resin and mix it with DCM (dichloromethane) solution, shake for 30 min to allow the resin to fully swell.

[0048] 2. Remove DCM solvent by core filtration. Add 3 molar excess of Fmoc- (the first amino acid at the C-terminus)-OH, 10 molar excess of N,N-diisopropylethylamine, and a small amount of dimethyl fumarate to dissolve the resin. Shake for 1 h to allow the first amino acid to bind to the resin. Then, wash 6 times alternately with dimethyl fumarate and DCM. Wash with a 20% (v / v) solution of piperidine dimethyl fumarate for 5 min to remove the protective effect of Fmoc-. Repeat this process for 15 min.

[0049] 3. Drain the solution, take out a dozen or so resin grains, wash them three times with ethanol, then add one drop each of ninhydrin, potassium cyanide and phenol solution in sequence, and heat at high temperature until a positive reaction occurs, turning a deep blue color.

[0050] 4. Add dimethyl fumarate, methanol, and dimethyl fumarate to the reaction tube in sequence according to the ratio. Wash twice with dimethyl fumarate. Then add 3 times the molar excess of Fmoc-(the second amino acid at the C-terminus)-OH, 3 times the molar excess of O-benzotriazole-tetramethylurea hexafluorophosphate, and a small amount of dimethyl fumarate to dissolve. Immediately afterward, add 10 times the molar excess of N,N-diisopropylethylamine and react for 40 min to carry out the amino acid condensation reaction.

[0051] 5. Repeat the above-described deprotection, ninhydrin detection, and condensation reaction procedures sequentially until all target amino acids are sequentially linked to the resin from right to left and deprotected from Fmoc-. Use ninhydrin to detect the reaction until a negative result indicates complete reaction. Finally, wash the resin three times with methanol solution and allow the liquid to evaporate at room temperature.

[0052] 6. Transfer the resin to a centrifuge tube, add the cleavage buffer to fully separate the peptide from the resin, and incubate at a constant temperature and shake for 120 min to cleave the peptide from the resin. The cleavage buffer was prepared according to the following ratio: 94.5% trifluoroacetic acid, 2.5% water, 2.5% 3,4-ethylenedioxythiophene, and 1% disulfide peptide (all volume fractions). Filter the above liquid using a sintering filter. Dry the resulting lysate as much as possible with nitrogen, then wash repeatedly with anhydrous diethyl ether 6 times, centrifuge at 4000 r / min for 3 min, collect the precipitate, and evaporate to dryness at room temperature to obtain crude oyster peptides. Purify the peptides by high performance liquid chromatography to obtain peptides with a purity of over 95%.

[0053] Example 3. Verification of the neuroprotective activity of oyster polypeptide Cg#2907 The nematode model HA759 is a disease model of Huntington's disease (HD), capable of simulating the pathogenesis of HD. Simultaneously, through gene technology, it stably expresses green fluorescent protein (GFP), which not only facilitates the localization of ASH neurons but also allows for the assessment of neuronal survival status by observing GFP expression, providing an important observational window for studying the mechanisms of neuronal death in HD. Under strong drive from the osm-10 promoter, which is associated with osmotic sensing, this model specifically expresses the 150-glutamate-residue huntingtin protein Htn-Q150 in head ASH and ASI neurons, as well as tail PHA and PHB neurons. Htn-Q150 expression is most significant in ASH neurons, while expression is weaker in the other three types of neurons. Furthermore, the point-mutated pqe-1 gene enhances the sensitivity of ASH neurons to Htn-Q150 toxicity, accelerating the induction of Htn-Q150 toxicity. This results in the mass death of HA759 ASH neurons due to protein toxicity accumulation within approximately 3 days under 15°C culture conditions. When ASH neurons die, the GFP fluorescent spots attached to the ASH neurons near the nematode's head immediately disappear, decreasing from two spots to one or zero. Both of these cases are recorded as ASH neuron death. Only when the GFP fluorescence on two ASH neurons is normal simultaneously is the nematode considered alive. This invention uses HA759 as an example to study the neuroprotective activity of oyster polypeptide Cg#2907. The specific method is as follows: 1. Culture HA759 nematodes to L4 stage using conventional methods; wash L4 stage HA759 nematodes three times with S. Medium solution to remove NA22 bacterial culture; then adjust the nematode density to 40-50 nematodes / 10 μL with S. Medium solution.

[0054] 2. Using a 96-well plate, add 10 μL of concentrated NA22 bacterial culture, 1.5 μL of 5 mg / mL 5-FUdR, 2 μL of 5 mg / mL AMP solution, 10 μL of insect culture, and peptide stock solution to each well. The final peptide concentrations were set at 0, 0.5, 1, 2, and 4 mM. An equal volume of S. Medium solution was added as a control. Each well was then brought to a final volume of 100 μL with S. Medium solution. Three replicates were performed for each concentration. The outermost ring was sealed with S. Medium solution to reduce water evaporation, and the plate was finally sealed with sealing film. The 96-well plate was incubated at 15°C and 120 rpm for 72 h using a constant temperature shaker.

[0055] 3. After 72 hours of culture, aspirate the nematodes and place them on an agarose mat. Fix and paralyze them, then photograph them using a fluorescence microscope, taking pictures from top to bottom and left to right. Use a microscope with 20x magnification and the blue fluorescence channel to obtain images showing green GFP fluorescent dots within the nematodes.

[0056] 4. To verify the neuroprotective function of oyster polypeptide Cg#2907, we observed its effect on the survival rate of ASH neurons in *C. nematode*. Experimental data showed ( Figure 1 The baseline neuronal survival rate in the control group was approximately 35%-40%. In contrast, all treatment groups treated with Cg#2907 peptide showed a significant increase in neuronal survival rate (p < 0.0001). Particularly at a 2 mM concentration, neuronal survival rate peaked (approximately 65%), representing an absolute increase of about 20% compared to the control group. These results confirm that Cg#2907 peptide can effectively intervene in the abnormal aggregation process of pathogenic proteins within neurons, inhibiting their toxic damage to cells, thereby exerting a neuroprotective effect and improving cell survival.

[0057] Example 4. Verification of the inhibitory efficacy of oyster polypeptide Cg#2907 polyQ (polyglutamine) on aggregation. The degree of protein aggregation can be evaluated by the number of aggregation points; an increase in the number of aggregation points indicates a deeper degree of aggregation. This experiment used the *C. elegans* polyQ aggregation model AM141 as the research subject. Immediately after the L1 stage, soluble Q40::YFP distribution was observed in the body wall muscle cells of AM141 nematodes. As they grow and develop, polyQ is expressed in the body wall muscle cells of AM141, gradually forming aggregation points from a diffuse state. When they reach adulthood, the AM141 nematode model will exhibit a phenotype of complete Q40::YFP aggregation. The effect of oyster polypeptide Cg#2907 on protein aggregation can be reflected by the change in the number of polyQ aggregation points. To determine the detection time of polyQ aggregation points, AM141 nematodes were cultured from the L1 stage to 24 h, 48 h, and 72 h, and the number of polyQ fluorescent aggregation spots in their bodies was measured. All nematode fluorescence images at 72 h were processed, and the distribution of polyQ protein aggregation points in the nematodes at this time was analyzed to evaluate the polyQ aggregation inhibitory effect of oyster polypeptide Cg#2907. The specific method is as follows: 1. The nematode AM141 was synchronized using conventional methods to obtain L1 stage larvae. The larvae were washed three times with S. Medium solution to remove NA22. The nematode density was adjusted to 30-40 larvae / 10 μL by adding S. Medium solution.

[0058] 2. Add 10 μL of concentrated NA22 bacterial culture to each well of a 96-well plate (final OD per well). 570Add 2 μL of 5 mg / mL AMP solution (between 0.5 and 0.6 μg / mL), 30 μL of the insect fluid prepared in step 1, and the peptide sample stock solution for drug administration. The treatment group received 2 mM oyster peptide Cg#2907, while the control group received an equal volume of S. Medium solution. Then, each well was brought to a final volume of 100 μL with S. Medium solution. At least nine replicates were set up for each group. 100 μL of S. Medium solution was added around each well for liquid sealing. The edge of the plate cap was sealed with sealing film, and the cap was marked. The 96-well plate was incubated at 20°C and 120 rpm in a constant-temperature shaker until the appropriate time point.

[0059] 3. At 24 h, 48 h, and 72 h of culture, respectively, three wells of culture medium from each group were transferred from a 96-well plate to 1.5 mL centrifuge tubes. The nematodes were washed with M9 buffer until the supernatant was clear and thoroughly mixed. The nematodes were then aspirated onto an agarose pad and anesthetized with sodium azide. Fluorescence images were taken using a high-content imaging system, and the number of fluorescent spots around the nematodes was counted.

[0060] The inhibitory effect of oyster peptide Cg#2907 on polyglutamine aggregation at different time points was further evaluated, and the results are as follows: Figure 2 As shown in the figure, in the initial treatment phase (24 h), the 2 mM peptide did not exhibit significant inhibitory activity. This is presumably because this time point had not yet entered the period of massive polyQ protein accumulation in the AM141 nematode model, making exogenous administration difficult to intervene in its aggregation process during this period. With the extension of the culture time to 48 h, polyQ aggregation began to be inhibited to some extent; by 72 h, the inhibitory effect of the peptide reached a significant level. Compared with the control group, the Cg#2907 peptide reduced the amount of polyQ protein aggregation in somatic cells by approximately 20%, indicating that it can effectively alleviate the neurodegenerative pathological process induced by protein aggregation.

[0061] This study further performed fluorescence imaging and quantitative analysis on nematode samples 72 h after drug administration, and statistically analyzed the distribution of polyQ protein aggregation points among different individuals. The results are as follows: Figure 3As shown in the figure, in the control group, more than half of the nematode individuals (>50%) had a high concentration of fluorescent aggregation points in the range of 90–110; while only 3% of the individuals had a low concentration of 50–70 aggregation points, i.e., only one nematode. After treatment with 2 mM oyster peptide Cg#2907, the distribution of aggregation points in the nematode population changed significantly: the proportion of the high aggregation load population (90–110 aggregation points) decreased sharply to 12.5%; at the same time, the proportion of the low aggregation load population (50–70 aggregation points) increased significantly to 32.5%; and the proportion of the medium aggregation load population (70–90 aggregation points) remained stable at 45%–55%. This distribution shift indicates that Cg#2907 peptide can effectively inhibit the excessive aggregation of polyQ protein, thereby reducing the protein toxicity effect on the body by maintaining more individuals at medium or low aggregation load levels. This experiment confirms that this oyster bioactive peptide can reduce the risk of neurodegenerative diseases induced by polyQ deposition by intervening in the abnormal protein aggregation process.

[0062] Example 5. Verification of the antioxidant activity of oyster polypeptide Cg#2907 Some antioxidants exhibit antioxidant activity in in vitro experiments, but this cannot be verified in in vivo experiments. This is because in vitro experiments cannot fully simulate the metabolic processes, intercellular interactions, and complex physiological regulatory mechanisms within a living organism. Therefore, the antioxidant activity exhibited by certain substances in vitro may not achieve the expected effect in vivo due to low bioavailability, metabolic transformation, or interactions with other physiological components. Therefore, evaluating the antioxidant performance of samples should not be limited to in vitro experiments but should also incorporate in vivo antioxidant experiments using animal models to comprehensively assess the strength of the sample's antioxidant capacity.

[0063] Paraquat is a commonly used oxidative damage inducer, widely applied in modeling oxidative damage in the model organism *C. elegans*. At high doses, it can rapidly induce nematode death within a short period. Adding antioxidants before using paraquat to model oxidative stress and observing their effects on oxidative stress in *C. elegans* is also a common experimental method. To investigate whether oyster polypeptide Cg#2907 provides better protection against oxidative stress damage in nematodes under high-concentration oxidative stress modeling conditions, this experiment set up oxidative stress experiments with paraquat concentration gradients of 50 mM and 100 mM.

[0064] Table 1. Statistical analysis of experimental data on the oxidative survival of wild-type nematodes using 50mM oyster polypeptide Cg#2907. Depend on Figure 4As shown in Table 1, under oxidative stress induced by paraquat at a concentration of 50 mM, the oyster peptide Cg#2907 experimental group significantly prolonged the survival time of nematodes after 100 h, and the mortality rate of nematodes in the peptide-treated group was significantly slower than that in the experimental group after 100 h. The mean survival time and median nematode survival also indicate that Cg#2907 can prolong the survival time of nematodes at a paraquat concentration of 50 mM. In terms of mean survival time, oyster peptide Cg#2907 can extend the survival rate by 9.211%.

[0065] Table 2. Statistical analysis of experimental data on the oxidative survival of wild-type nematodes using 100mM oyster polypeptide Cg#2907. Depend on Figure 5 As shown in Table 2, under oxidative stress induced by ultra-high concentration (100 mM) paraquat, oyster peptide Cg#2907 also showed excellent effects in prolonging the survival time of nematodes, slowing down the mortality rate from the outset. Furthermore, its overall antioxidant effect was more significant than that under lower concentration (50 mM) paraquat oxidative stress. In terms of average survival time, oyster peptide Cg#2907 extended the survival rate by 22.922%.

[0066] Therefore, it can be concluded that the selected marine peptides can enhance the antioxidant capacity of nematodes under oxidative stress, and their antioxidant capacity is even stronger under high modeling agent concentrations.

[0067] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. An oyster polypeptide with anti-protein aggregation function, characterized in that: Its amino acid sequence is shown in SEQ ID NO:

1.

2. The method for preparing the oyster polypeptide with anti-protein aggregation function according to claim 1, characterized in that: It includes the following steps: (1) Oyster polypeptides with anti-protein aggregation function can be prepared directly by solid-phase synthesis, or oyster polypeptides with anti-protein aggregation function can be obtained by enzymatic hydrolysis, separation and extraction and purification using oysters as raw materials.

3. The method for preparing oyster polypeptide with anti-protein aggregation function according to claim 2, characterized in that: The enzymatic hydrolysis is preferably performed by sequentially using trypsin and papain.

4. The method for preparing oyster polypeptide with anti-protein aggregation function according to claim 3, characterized in that: The specific operation of the enzymatic hydrolysis is as follows: Mix oyster freeze-dried powder with water, adjust the pH of the system to 7.5-8.0, add trypsin for enzymatic hydrolysis; then adjust the pH of the system to 6.5-7.0, add papain for enzymatic hydrolysis; after enzymatic hydrolysis, inactivate the enzyme to obtain oyster protein hydrolysate.

5. The method for preparing oyster polypeptide with anti-protein aggregation function according to claim 2, characterized in that: The specific operations for separation and extraction are as follows: After cooling the oyster protein hydrolysate obtained by enzymatic hydrolysis, 95% ethanol was added to bring the ethanol concentration of the solution to 60-80%, and the solution was allowed to stand. Then, the precipitate was removed by vacuum filtration and centrifugation. The supernatant was then evaporated under reduced pressure to remove ethanol, and the solution was freeze-dried to obtain crude oyster polypeptide powder.

6. The method for preparing oyster polypeptide with anti-protein aggregation function according to claim 2, characterized in that: The purification is at least one of ultrafiltration purification and gel chromatography column purification; The ultrafiltration purification uses Ultracel-3 membrane separation to collect components with a molecular weight less than 3 kDa.

7. The use of the oyster polypeptide with anti-protein aggregation function as described in claim 1 in the preparation of products with anti-protein aggregation or anti-oxidation function.

8. The use of the oyster polypeptide with anti-protein aggregation function as described in claim 1 in the preparation of products for the prevention and treatment of neurodegenerative diseases.

9. The application according to claim 8, characterized in that: The neurodegenerative diseases mentioned are Huntington's disease, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, or prions.

10. A drug for preventing and treating neurodegenerative diseases, characterized in that: The active ingredient comprises at least one of the following: the oyster polypeptide with anti-protein aggregation function as described in claim 1, the protease hydrolysate containing the oyster polypeptide with anti-protein aggregation function as described in claim 1, and the hydrolysate containing the oyster polypeptide with anti-protein aggregation function as described in claim 1.

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