Polypeptide with alpha-spiral bundle structure and application of polypeptide in treatment of Alzheimer's disease

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a polypeptide with an alpha-spiral bundle structure and application of the polypeptide in treatment of Alzheimer's disease, and the amino acid sequence of the polypeptide is shown as SEQ ID NO: 1. The invention designs a novel protein skeleton sequence based on an alpha-spiral bundle structure, and the novel protein skeleton sequence can be specifically combined with and inhibit a key hydrophobic core region (KLVFF, 16-21 amino acids) of an A beta 42 peptide fragment in Alzheimer's disease (AD). The sequence is stably combined with a key site of an Abeta42 peptide fragment through optimization design, so that Abeta42 aggregation is effectively inhibited, the pathological progress of the Alzheimer's disease is expected to be slowed down, and a potential clinical application prospect is provided for early intervention and treatment of AD (Alzheimer's Disease).

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to an α-helical bundle structure polypeptide and its application in the treatment of Alzheimer's disease. Background Art

[0002] Alzheimer's disease (AD) is a progressive neurodegenerative disease that mainly affects brain neurons, leading to a gradual decline in patients' memory, thinking, and behavior. Although its pathogenesis has not been fully understood, existing research has proposed various hypotheses, such as the amyloid protein deposition hypothesis and the neurofibrillary tangle hypothesis, etc. However, so far, there is no conclusive evidence to determine its root cause, which makes the precise treatment of Alzheimer's disease still full of challenges.

[0003] Currently, the treatment of Alzheimer's disease mainly focuses on alleviating symptoms rather than curing the disease. Common drugs include cholinesterase inhibitors (such as donepezil) and NMDA receptor antagonists (such as memantine). These drugs can slow down the progression of the disease to a certain extent, but they cannot prevent or reverse the fundamental process of the disease. And drug research and development face the dilemmas of long cycle, high cost, and failure in clinical trials, resulting in the relatively slow emergence of new drugs.

[0004] In addition, the occurrence of Alzheimer's disease is affected by multiple factors, such as genetic susceptibility, environmental factors, and lifestyle, etc. Therefore, researchers are looking for more comprehensive treatment methods, including early diagnosis techniques, personalized treatment strategies, and innovative drugs targeting the disease mechanism. Although some progress has been made currently, a large amount of work still needs to be completed in future research to bring more effective treatment options for patients. Summary of the Invention

[0005] The purpose of the present invention is to overcome the deficiencies of the above-mentioned prior art and provide an α-helical bundle structure polypeptide, which is designed to specifically bind to and inhibit the key hydrophobic core region (KLVFF, amino acids 16 - 21) in the Aβ42 peptide segment, thereby inhibiting the aggregation of Aβ42 and further slowing down the pathological progression of Alzheimer's disease.

[0006] To achieve the above purpose, the technical solutions adopted by the present invention include:

[0007] In the first aspect, the present invention provides a polypeptide, and the amino acid sequence of the polypeptide is as shown in (a) or (b):

[0008] (a) The amino acid sequence is as shown in SEQ ID NO: 1;

[0009] (b) An amino acid sequence obtained by substituting, deleting, or adding at least one amino acid to the sequence shown in SEQ ID NO: 1 and having the same function as the polypeptide described in (a).

[0010] Preferably, the amino acid sequence of the polypeptide is as shown in SEQ ID NO: 1; the C-terminus of the polypeptide is amidated, and the structural formula of the polypeptide is as follows:

[0011]

[0012] Preferably, the polypeptide has an α-helical bundle structure.

[0013] The present invention proposes a polypeptide sequence based on the α-helical bundle structure, aiming to specifically bind to and inhibit the aggregation process of the key pathological factor Aβ42 in Alzheimer's disease (AD), thereby slowing down the progression of the disease. The Aβ42 peptide segment contains a key hydrophobic core region (the KLVFF sequence, amino acids 16-21), which is widely regarded as the core driving factor for Aβ42 to form toxic oligomers and fibrils. Therefore, specific binding to this region has become the core of a new treatment strategy.

[0014] To design a polypeptide that can effectively interfere with the aggregation of Aβ42, the present invention uses the α-helical bundle structure as the backbone. This structure forms a hydrophobic pocket in the sequence through hydrophobic and aromatic amino acid residues (such as leucine, alanine, etc.), enabling the polypeptide to efficiently embed and tightly bind to the hydrophobic core region (the KLVFF sequence) of Aβ42. Through the optimized design of this structure, the target sequence can stably bind to the key site of Aβ42, thereby inhibiting the aggregation of Aβ42, reducing the generation of toxic oligomers, and slowing down the pathological progression of AD.

[0015] The design of this polypeptide has undergone multiple rounds of computer simulation, structural optimization, and experimental verification to ensure its high affinity and specific binding to the Aβ42 peptide segment. The inventors also conducted comparative screening of a large number of candidate polypeptides, and after many debugging and optimizations, finally obtained this highly efficient and stable polypeptide sequence. This research not only provides new ideas for the treatment of AD but also lays a foundation for the development of protein structure-based targeted therapeutic molecules.

[0016] In a second aspect, the present invention provides the use of the polypeptide in the preparation of a drug for preventing or treating neurodegenerative diseases.

[0017] Preferably, the neurodegenerative disease is Alzheimer's disease.

[0018] The present invention evaluated the therapeutic effect on an Alzheimer's disease mouse model through pharmacological activity experiments and found that the novel α-helical bundle structure polypeptide can significantly slow down the memory decline of Alzheimer's disease mice, and thus can be used for the drug development and research of Alzheimer's disease.

[0019] Preferably, the polypeptide targets and inhibits the aggregation of Aβ42 and / or Aβ40.

[0020] Preferably, the polypeptide targets the central hydrophobic core region (KLVFF, amino acids 16 - 21) in the Aβ42 peptide segment, thereby inhibiting the aggregation of Aβ42.

[0021] Preferably, the effective dose of the polypeptide is 0.5 - 1.0 mg / kg.

[0022] Preferably, the effective dose of the polypeptide is 1.0 mg / kg.

[0023] Through dose - dependent studies, the present invention determines the effective dose range of the novel α - helix bundle - structured polypeptide. The results of pharmacological activity experiments show that within the dose range of 0.5 mg / kg to 1.0 mg / kg, the polypeptide can significantly improve the cognitive function of Alzheimer's disease mice, and the best effect is observed in the 1.0 mg / kg dose group.

[0024] In a third aspect, the present invention provides a drug for preventing or treating neurodegenerative diseases, and the drug comprises the polypeptide described above.

[0025] Preferably, the drug is a pharmaceutically acceptable lyophilized product and / or solvate.

[0026] In the present invention, the extracted novel α - helix bundle - structured polypeptide is lyophilized and then formulated into solutions with different concentrations for administration with solvents. The solvents include but are not limited to water, isopropanol, ethanol, methanol, dimethyl sulfoxide, ethyl acetate, acetic acid, and aminoethanol.

[0027] Preferably, the neurodegenerative disease is Alzheimer's disease.

[0028] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0029] The present invention designs a novel protein backbone sequence based on the α - helix bundle structure, which can specifically bind to and inhibit the key hydrophobic core region (KLVFF, amino acids 16 - 21) of the Aβ42 peptide segment in Alzheimer's disease (AD). Through optimization design, this sequence forms stable binding with the key sites of the Aβ42 peptide segment, thereby effectively inhibiting the aggregation of Aβ42. By reducing the aggregation of Aβ42, the α - helix bundle - structured polypeptide described by this method is expected to slow down the pathological progression of Alzheimer's disease and provide potential clinical application prospects for the early intervention and treatment of AD. Brief Description of the Drawings

[0030] Figure 1 For the mass spectrometry analysis chart ( Figure 1 A) and HPLC chromatogram ( Figure 1 B) of the novel α - helix bundle - structured polypeptide;

[0031] Figure 2 It is the circular dichroism (CD) spectrogram of the novel α-helical bundle structure polypeptide;

[0032] Figure 3 It is the inhibitory effect diagram of the novel α-helical bundle structure polypeptide on the aggregation of Aβ40 and Aβ42 at different concentrations (1 μM to 5 μM), with the percentage of fluorescence intensity as the index;

[0033] Figure 4 It is the result diagram of the inhibitory effect of the novel α-helical bundle structure polypeptide on the aggregation of Aβ40 or Aβ42 in HEK293T cells. Detailed implementation manners

[0034] To make the objectives, technical solutions and effects of the present invention clearer and more definite, the present invention will be further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0035] Unless otherwise specified, the experimental methods in the following embodiments are all conventional methods, and the test materials used in the following embodiments can all be obtained through conventional commercial channels unless otherwise specified.

[0036] Example 1

[0037] This example provides a preparation method of the α-helical bundle structure polypeptide, and the specific method is as follows:

[0038] 1. Plasmid construction and transfection of Escherichia coli

[0039] Construct a plasmid vector expressing the novel α-helical bundle structure polypeptide by standard molecular biology techniques, and transfect the plasmid into BL21(DE3) Escherichia coli competent cells.

[0040] 1.1 Plasmid vector construction

[0041] 1. Selection of plasmid vector: Select the pET-28a plasmid, which contains the T7 promoter and the 6xHis tag and is suitable for expressing fusion proteins in Escherichia coli.

[0042] 2. Gene insertion:

[0043] (a) Synthesize the DNA sequence encoding the novel α-helical bundle polypeptide and optimize its codons. The nucleotide sequence of the DNA is shown in SEQ ID NO: 2;

[0044] (b) Add NdeI and XhoI restriction enzyme recognition sites to both ends of the sequence;

[0045] (c) Double digest the pET-28a plasmid and the polypeptide gene fragment with NdeI and XhoI;

[0046] (d) Use T4 DNA ligase to ligate the digested polypeptide gene with the linearized plasmid overnight at 16°C.

[0047] 1.2 Transformation of competent cells

[0048] 1. Transform the ligation product into BL21(DE3) competent cells using the heat shock method (heat shock at 42°C for 45 seconds and cool on ice for 2 minutes);

[0049] 2. Add 1 mL of LB medium to the transformed cells and recover at 37°C for 1 hour;

[0050] 3. Spread the recovered bacterial solution on an LB agar plate containing kanamycin (50 μg / mL) and culture overnight at 37°C.

[0051] 1.3 Screening of positive clones

[0052] 1. Pick a single colony and inoculate it into 5 mL of LB medium (containing 50 μg / mL kanamycin), and culture it overnight with shaking at 37°C and 250 rpm;

[0053] 2. Use a plasmid extraction kit to extract the plasmid, and verify the correctness of the inserted fragment by digestion with NdeI and XhoI to confirm the successful construction of the plasmid.

[0054] 2. Protein expression and purification

[0055] Induce the expression of the novel α-helical bundle structure polypeptide in E. coli by IPTG and purify it by nickel affinity chromatography.

[0056] 2.1 Protein expression

[0057] 1. Inoculate the positive strain into 50 mL of LB medium containing kanamycin (50 μg / mL), and culture it with shaking at 37°C and 250 rpm until the OD600 reaches 0.6 - 0.8;

[0058] 2. Add IPTG to a final concentration of 0.5 mM, lower the culture temperature to 16°C, and continue to culture for 16 hours to induce protein expression;

[0059] 3. After the culture is completed, centrifuge at 4°C and 4000 rpm for 10 minutes to collect the bacterial cells.

[0060] 2.2 Cell lysis

[0061] 1. Resuspend the cell pellet in 10 mL of lysis buffer. The lysis buffer formulation is: 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10 mM Imidazole, 1 mM PMSF (protease inhibitor);

[0062] 2. Use an ultrasonic disruptor to perform ultrasonic lysis in an ice bath. Sonicate for 10 seconds each time, with a 20-second interval, and repeat 20 times to ensure complete cell lysis;

[0063] 3. After lysis, centrifuge at 12,000 g for 30 minutes at 4 °C and collect the supernatant for protein purification.

[0064] 2.3 Protein Purification

[0065] 1. Pass the supernatant through a pre-equilibrated Ni-NTA affinity chromatography column and equilibrate it with an equilibration buffer containing 10 mM Imidazole;

[0066] 2. Wash away non-specifically bound proteins with a wash buffer (50 mM Tris-HCl, pH 8.0, 300 mM NaCl, 20 mM Imidazole);

[0067] 3. Elute the target polypeptide with an elution buffer (50 mM Tris-HCl, pH 8.0, 300 mM NaCl, 250 mM Imidazole) and collect the eluted fractions.

[0068] 2.4 Purity Analysis

[0069] 1. Analyze the eluted protein sample using SDS-PAGE. Prepare a 10% polyacrylamide gel, load the sample, and perform electrophoresis;

[0070] 2. After electrophoresis, detect the protein bands using Coomassie Brilliant Blue staining to evaluate the purity of the polypeptide.

[0071] 3. Protein Qualitative Analysis

[0072] Perform qualitative analysis of the purified novel α-helical bundle structure polypeptide by HPLC and mass spectrometry to further confirm its correctness and quality.

[0073] 3.1 High Performance Liquid Chromatography (HPLC) Analysis

[0074] 1. HPLC conditions:

[0075] (a) Column: C18 reversed-phase column (4.6 mm × 250 mm, 5 μm particle size)

[0076] (b) Mobile phase:

[0077] Phase A: 0.1% trifluoroacetic acid (TFA) aqueous solution; Phase B: 0.1% TFA acetonitrile solution

[0078] (c) Gradient elution:

[0079] 0 - 5 minutes: 5% Phase B; 5 - 30 minutes: linear gradient of 5% - 60% Phase B; 30 - 50 minutes: linear gradient of 60% - 80% Phase B; 50 - 55 minutes: 80% Phase B

[0080] (d) Elution conditions:

[0081] Flow rate: 1 mL / min; Detection wavelength: 214 nm; Column temperature: 25 °C

[0082] 2. Sample preparation:

[0083] (a) Dissolve the lyophilized polypeptide in water / acetonitrile (1:1, containing 0.1% TFA) to prepare a concentration of 0.1 mg / mL;

[0084] (b) Inject 20 μL of the sample into the HPLC system to analyze the retention time and peak shape of the sample.

[0085] 3. Result analysis:

[0086] (a) Evaluate the purity and consistency of the polypeptide by comparing the retention time and the symmetry of the chromatographic peak;

[0087] (b) The retention time of the novel polypeptide should be consistent with the reference value, indicating its correct structure.

[0088] 3.2 Mass spectrometry analysis

[0089] 1. Sample preparation:

[0090] (a) Dissolve the lyophilized polypeptide in water / methanol solution (1:1, containing 0.1% formic acid) to prepare a concentration of 0.01 mg / mL;

[0091] (b) Inject the sample directly into the mass spectrometer for analysis after sampling.

[0092] 2. Mass spectrometry conditions:

[0093] (a) Mass spectrometry instrument: electrospray ionization mass spectrometry (ESI-MS)

[0094] (b) Mass spectrometry mode: positive ion mode

[0095] (c) Scanning range: 2050 - 2400 m / z

[0096] 3. Result analysis:

[0097] Ion peaks corresponding to the theoretical molecular weight of the polypeptide should appear in the mass spectrum to further confirm the molecular weight and correctness of the polypeptide.

[0098] 4. Protein Lyophilization and Storage

[0099] The extracted polypeptide is lyophilized for subsequent experiments and long-term storage.

[0100] 4.1 Buffer Replacement

[0101] 1. Dialyze the eluted protein through a dialysis bag (molecular weight cut-off 3.5 kDa), and change the buffer to 20 mM Tris-HCl (pH 7.5).

[0102] 2. After dialysis, aliquot and freeze the dialyzed protein solution.

[0103] 4.2 Lyophilization

[0104] 1. Place the aliquoted protein solution in an -80 °C refrigerator for pre-freezing for 4 hours;

[0105] 2. Use a lyophilizer to perform vacuum lyophilization on the frozen protein sample for 24 - 48 hours to ensure complete removal of moisture.

[0106] 4.3 Storage Conditions

[0107] The lyophilized protein powder is stored at -20 °C to ensure the activity and stability of the polypeptide.

[0108] Figure 1 A is the mass spectrometry analysis chart obtained from the protein qualitative analysis of the newly obtained α-helical bundle structure polypeptide. The X-axis represents the mass-to-charge ratio (m / z), and the Y-axis represents the relative abundance (%). The peak value of the molecular ion peak in the figure, 2267.3860 m / z, corresponds to the theoretical mass-to-charge ratio of the newly obtained α-helical bundle structure polypeptide (M = 2266.3341), verifying the correct molecular weight of the polypeptide. Figure 1 B is the high-performance liquid chromatography (HPLC) separation chart of the newly obtained α-helical bundle structure polypeptide. The X-axis represents time (minutes), and the Y-axis represents the detector response (mV). The retention time of the target polypeptide is 9.980 minutes, showing its good separation effect and high purity in HPLC.

[0109] Example 2

[0110] In this example, the circular dichroism (CD) method was used to explore the structural characteristics of the α-helical bundle structure polypeptide prepared in Example 1. The specific experimental method is as follows:

[0111] 1. Sample Preparation

[0112] Take a certain amount from the polypeptide sample, dissolve it with deionized water and prepare a solution of about 0.1 mg / mL. The sample solution is filtered through a 0.22 μm filter to remove possible particulate matter.

[0113] 2. Instrument conditions

[0114] (a) Instrument: Use a CD spectrometer (Jasco J-1500) with a temperature control device.

[0115] (b) Optical path: Select a quartz cell with an optical path of 0.1 cm.

[0116] (c) Scanning range: 190 nm to 250 nm.

[0117] (d) Step size: 1 nm.

[0118] (e) Bandwidth: 1 nm.

[0119] (f) Scanning speed: 50 nm / min.

[0120] 3. Measurement procedure

[0121] (a) Place the sample solution in a quartz cell;

[0122] (b) Scan and record the CD signal to obtain the ellipticity ([θ], unit: deg·cm 2 / dmol) at different wavelengths;

[0123] (c) To ensure data accuracy, each sample needs to be scanned 3 times and the average value is taken.

[0124] 4. Baseline correction

[0125] Perform baseline correction using deionized water and subtract the solvent signal.

[0126] Figure 2 For the circular dichroism (CD) spectrogram of the polypeptide with an α-helix bundle structure, the picture shows the typical α-helix structure characteristics of the polypeptide in the range of 190 - 250 nm. Significant negative peaks appear at 208 nm and 222 nm, indicating a high α-helix content. Therefore, according to the CD spectrum, it is confirmed that the polypeptide has successfully formed the expected α-helix bundle structure, and this structural property is consistent with the design goal, further supporting its ability to bind Aβ42 and verifying the potential application value of the polypeptide in inhibiting Aβ42 aggregation.

[0127] Effect Example 1

[0128] This effect example explored the ThT inhibition experiment of the α-helix bundle structure polypeptide obtained in Example 1 on Aβ40 and Aβ42. The specific method is as follows:

[0129] 1. Experimental materials:

[0130] (a) Aβ40 peptide segment: Purchased from Shanghai Biocom Technology Co., Ltd., with a purity of ≥95%, and the final concentration used is 5 μM.

[0131] (b) Aβ42 peptide segment: Purchased from Beijing Biotech Co., Ltd., with a purity of ≥95%, and the final concentration is prepared to be 5 μM.

[0132] (c) Novel α - helix bundle structure polypeptide: The polypeptide prepared in Example 1 is formulated into solutions with different concentrations (1 μM, 2 μM, 3 μM, 4 μM, 5 μM) with sterile water or DMSO solvent.

[0133] (d) Thioflavin T (ThT): Purchased from Shanghai Yuanye Bio - technology Co., Ltd., with a final concentration of 20 μM.

[0134] (e) Dimethyl sulfoxide (DMSO): Purchased from Sinopharm Chemical Reagent Co., Ltd., analytical pure, used to dissolve Aβ40 and Aβ42 peptide segments.

[0135] 2. Experimental procedures:

[0136] (a) Dissolve Aβ40 and Aβ42 in DMSO respectively and dilute to a concentration of 5 μM.

[0137] (b) Prepare the novel α - helix bundle structure polypeptide into 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, and mix it with Aβ40 / Aβ42 and ThT solutions.

[0138] (c) Incubate at 37 °C for 96 hours, and detect the fluorescence intensity using an excitation wavelength of 430 nm and an emission wavelength of 485 nm.

[0139] 3. Experimental results: According to the fluorescence intensity, record the inhibitory effects of polypeptides with different concentrations on the aggregation of Aβ40 or Aβ42. Figure 3 The results show that as the concentration of the polypeptide increases, the fluorescence intensity gradually decreases, indicating the effective inhibitory effect of the polypeptide on the aggregation of Aβ40 and Aβ42. And through dose - dependent experiments, it is confirmed that the preferred effective concentration of the novel α - helix bundle structure polypeptide is 1 μM to 5 μM. Within this range, the polypeptide can significantly inhibit the aggregation of Aβ40 and Aβ42, and the inhibitory effect is the best at a concentration of 5 μM.

[0140] Effect Example 2

[0141] This effect example explores the inhibitory effect of the α - helix bundle structure polypeptide obtained in Example 1 on the aggregation of Aβ40 and Aβ42 at the cellular level. The specific method is as follows:

[0142] 1. Experimental materials:

[0143] (a) HEK293T cells: Purchased from the China Center for Type Culture Collection (CCTCC), with the number CCTCC-HEK293T.

[0144] (b) DAPI dye: Purchased from Beyotime Biotechnology Co., Ltd., with a concentration of 1 μg / mL, used for nuclear staining.

[0145] (c) Novel α-helical bundle structure polypeptide: The α-helical bundle structure polypeptide obtained in Example 1, with the final concentrations set at 2 μM and 5 μM.

[0146] (d) Aβ40 gene vector: Purchased from Shanghai GeneChem Co., Ltd., formulated in the pEGFP-N1 vector, used for cell transfection.

[0147] (e) Aβ42 gene vector: Obtained from Vigene Biosciences (Nanjing) Co., Ltd. and expressed using the pEGFP-N1 vector.

[0148] 2. Experimental procedures:

[0149] (a) Cultured HEK293T cells in DMEM medium containing 10% fetal bovine serum until 80% confluence;

[0150] (b) Transfected HEK293T cells with Aβ40 and Aβ42 gene vectors respectively to express Aβ40 and Aβ42, then added the novel polypeptide with concentrations of 2 μM and 5 μM, and observed the staining after culturing for 24 hours;

[0151] (c) Conducted DAPI staining to show the cell nuclei, and observed the aggregation state of Aβ40 or Aβ42 in the cells through a confocal microscope.

[0152] 3. Experimental results: Figure 4 The fluorescence signal showed that with the increase in the polypeptide concentration, the aggregation of Aβ40 or Aβ42 in the cells decreased significantly, indicating that the novel polypeptide has an inhibitory effect on the aggregation of Aβ40 or Aβ42 at the cellular level and shows a concentration-dependent effect.

[0153] Effect Example 3

[0154] This effect example explored the pharmacological activity experiment of the α-helical bundle structure polypeptide obtained in Example 1. Eight-month-old male APP mice (body weight 30 ± 2 g) were purchased from the Nanjing Biomedical Research Institute of Nanjing University (NBRI). Different doses of the novel α-helical bundle structure polypeptide, 0.1 mg / kg, 0.5 mg / kg, and 1.0 mg / kg, were intraperitoneally injected into them once a day for 7 consecutive days. At the same time, Morris water maze test, Y maze test, and novel object recognition test were conducted. The specific test methods are as follows:

[0155] 1. Morris Water Maze Test (MWM)

[0156] 1. Experimental purpose: To test the spatial learning and memory ability of mice.

[0157] 2. Experimental method:

[0158] 2.1 Experimental grouping

[0159] (a) Blank group: Normal mice without any treatment.

[0160] (b) AD group: Alzheimer's disease model mice purchased from Nanjing University Model Animal Research Institute (NBRI).

[0161] (c) AD polypeptide treatment group: Mice treated with the novel α-helical bundle structure polypeptide described in Example 1 at different doses (0.1 mg / kg, 0.5 mg / kg, and 1.0 mg / kg) after establishing the Alzheimer's disease model.

[0162] 2.2 Equipment

[0163] (a) Water maze (1.2 meters in diameter, 30 centimeters in depth) filled with milky water, and the water temperature is maintained at 22 ± 1 °C.

[0164] (b) Hidden platform (10 centimeters in diameter), placed 1 centimeter below the water surface.

[0165] 2.3 Experimental steps

[0166] (a) Adaptation period: Each mouse swims freely in the maze for 2 minutes without a platform.

[0167] (b) Training period (5 days in total, 4 trials per day): Place the mice at different positions on the edge of the maze, and record the latency and path length required for them to find the hidden platform. The maximum time for each trial is 60 seconds. If the mouse does not find the platform, guide it to the platform and let it stay for 10 seconds.

[0168] (c) Probe test (on the 6th day): Remove the platform and record the residence time (60 seconds) of the mouse in the quadrant where the previous platform was located. This test is used to evaluate the retention of spatial memory.

[0169] 2.4 Data recording and analysis

[0170] (a) Latency: The time required for the mouse to find the platform in each training. The shorter the time, the stronger the learning ability.

[0171] (b) Path length: The total length of the path the mouse takes to reach the platform.

[0172] (c) Target quadrant residence time: The time that a mouse stays in the target quadrant during the exploration test.

[0173] 2. Y-Maze Test

[0174] 1. Experimental purpose: To test the short-term memory and spontaneous alternation behavior of mice.

[0175] 2. Experimental method:

[0176] (a) Control group: Normal mice without any treatment.

[0177] (b) AD group: Alzheimer's disease model mice purchased from the Model Animal Research Center of Nanjing University (NBRI).

[0178] (c) AD polypeptide treatment group: Mice treated with the novel α-helical bundle structure polypeptide described in Example 1 at different doses (0.1 mg / kg, 0.5 mg / kg, and 1.0 mg / kg) after establishing the Alzheimer's disease model.

[0179] 2.2 Equipment

[0180] Y-shaped maze (each arm is 40 cm long, 10 cm wide, and 12 cm high), and the angle between the three arms is 120°.

[0181] 2.3 Experimental procedure

[0182] (a) Adaptation period: Each mouse freely explores in the Y-maze for 5 minutes, and the exploration behavior is recorded.

[0183] (b) Test period: The mouse is placed at the end of one of the arms and allowed to freely explore for 5 minutes. Record the order in which the mouse enters different arms each time.

[0184] 2.4 Data recording and analysis

[0185] (a) Spontaneous alternation rate: Calculate the proportion of the mouse continuously selecting different arms among the three arms. Spontaneous alternation rate = number of spontaneous alternations / (total number of arm entries - 2) × 100%.

[0186] (b) Number of arm entries: Record the number of times the mouse enters each arm during the test period.

[0187] 3. Novel Object Recognition (NOR)

[0188] 1. Experimental purpose: To test the recognition memory of mice.

[0189] 2. Experimental method:

[0190] 2.1 Experimental grouping

[0191] (a) Blank group: Normal mice without any treatment.

[0192] (b) AD group: Alzheimer's disease model mice purchased from the Model Animal Research Center of Nanjing University (NBRI).

[0193] (c) AD polypeptide treatment group: Mice treated with the novel α-helical bundle structure polypeptide described in Example 1 at different doses (0.1 mg / kg, 0.5 mg / kg, and 1.0 mg / kg) after establishing the Alzheimer's disease model.

[0194] 2.2 Equipment

[0195] Open field (40 cm × 40 cm × 40 cm) and two objects (different in shape, size, and color).

[0196] 2.3 Experimental procedures

[0197] (a) Adaptation period: Mice freely explored the empty open field for 10 minutes to reduce unfamiliarity with the environment.

[0198] (b) Training period: Two identical objects were placed in the open field, and mice explored the field for 10 minutes. The exploration time of mice for the objects was recorded.

[0199] (c) Testing period (24 hours later): One object was replaced with a new object, and mice were allowed to explore for 5 minutes. The exploration time of mice for the new object and the old object was recorded.

[0200] 2.4 Data recording and analysis

[0201] (a) Discrimination index: Calculate the discrimination index, Discrimination index = (New object exploration time - Old object exploration time) / (New object exploration time + Old object exploration time) × 100%.

[0202] (b) Exploration time: Record the exploration time of mice for each object.

[0203] 4. Experimental results

[0204] The specific results of the Morris water maze test, Y maze test, and novel object recognition test are shown in Tables 1 - 3. Among them, the target quadrant residence time is the time that mice stay in the target quadrant during the exploration experiment.

[0205] Table 1 Results of the Morris water maze experiment

[0206]

[0207] Table 1 results showed that the mice in the blank group exhibited the strongest learning and memory abilities, with significantly shortened latency and path length, and the longest residence time in the target quadrant. The mice in the AD group had severely impaired learning and memory abilities, manifested as the slowest latency and the longest path length, and the shortest residence time in the target quadrant. The mice in the AD polypeptide treatment groups (doses of 0.5 mg / kg and 1.0 mg / kg) showed significant improvement in learning and memory, with the latency and path length approaching those of the blank group, a significantly increased residence time in the target quadrant, and the best effect was observed in the 1.0 mg / kg dose group.

[0208] Table 2 Results of the Y-maze experiment

[0209]

[0210] Table 2 results showed that the mice in the blank group exhibited the best short-term memory function, with the highest spontaneous alternation rate and the most arm entries. The mice in the AD group had impaired short-term memory, with a significantly reduced spontaneous alternation rate and fewer arm entries. The short-term memory ability of the mice in the AD polypeptide treatment group was significantly restored, and the spontaneous alternation rate and arm entries were significantly higher than those in the AD group, and the effect was close to that of the blank group at a dose of 1.0 mg / kg.

[0211] Table 3 Results of the novel object recognition experiment

[0212]

[0213] Table 3 results showed that the mice in the blank group showed the strongest preference for the novel object, with the highest discrimination index, indicating normal recognition memory function. The discrimination ability of the mice in the AD group was severely impaired, with a significantly reduced discrimination index, indicating severely impaired recognition memory. The discrimination index of the mice in the AD polypeptide treatment group was significantly higher than that in the AD group, indicating that the novel α-helical bundle structure polypeptide has a powerful restorative effect on recognition memory function.

[0214] All of the above pharmacological activity experiments showed that the α-helical bundle structure polypeptide described in the present invention has significant effects and can greatly improve the cognitive function of Alzheimer's disease model mice, indicating the potential application value of the polypeptide in the treatment of Alzheimer's disease. And through dose-dependent studies, it was found that within the dose range of 0.5 mg / kg to 1.0 mg / kg, the α-helical bundle structure polypeptide can significantly improve the cognitive function of Alzheimer's disease mice, and the effect is the best at a dose of 1.0 mg / kg. Therefore, the dose of 0.5 - 1.0 mg / kg can be used as the preferred treatment dose of the polypeptide described in the present invention for Alzheimer's disease.

[0215] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A polypeptide, characterized in that The amino acid sequence of the polypeptide is shown in (a) or (b): (a) the amino acid sequence is shown in SEQ ID NO: 1; (b) An amino acid sequence having the same function as the polypeptide described in (a) obtained by substituting, deleting or adding at least one amino acid in the sequence shown in SEQ ID NO:

1.

2. The polypeptide according to claim 1, characterized in that The amino acid sequence of the polypeptide is shown in SEQ ID NO: 1; the C-terminus of the polypeptide is amidated with amino group, and the structural formula of the polypeptide is as follows:

3. The polypeptide according to claim 1 or 2, characterized in that The polypeptide is an α-helical bundle structure.

4. Use of the polypeptide according to any one of claims 1 to 3 in the preparation of a medicament for preventing or treating a neurodegenerative disease.

5. The use according to claim 4, characterized in that The neurodegenerative disease is Alzheimer's disease.

6. The use according to claim 4, characterized in that The polypeptide targets and inhibits the aggregation of Aβ42 and / or Aβ40.

7. The use according to claim 4, characterized in that The effective dose of the polypeptide is 0.5-1.0 mg / kg.

8. The use according to claim 7, characterized in that The effective dose of the polypeptide is 1.0 mg / kg.

9. A drug for preventing or treating neurodegenerative diseases, characterized in that: The drug comprises the polypeptide according to any one of claims 1 to 3.

10. The drug according to claim 9, characterized in that The drug is a pharmaceutically acceptable lyophilized agent and / or solvate.

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