A polypeptide for preventing and treating tau disease and its application

Abstract

The present invention relates to a polypeptide for preventing and treating tau disease and its application. The present invention discovered that a 38-mer peptide with a sequence of amino acids 120-157 in the V-ATPase subunit ATP6V1B2 protein can competitively block the binding of hyperphosphorylated tau protein to ATP6V1B2, restoring the assembly of the V-ATPase complex and lysosomal acidification levels in cells with pathological tau protein overload. Compared to traditional tau-targeted therapies, this invention intervenes in the tau pathology cascade at the source by reestablishing intracellular degradation homeostasis, providing a new target for breaking through existing treatment bottlenecks and possessing broader potential clinical application value.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a polypeptide for preventing and treating tau disease and an application thereof. Background Art

[0002] Tauopathies are a class of neurodegenerative diseases caused by abnormalities in the neuronal microtubule-associated protein tau. They include over 20 clinical subtypes, including Alzheimer's disease (AD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), Pick's disease (PiD), chronic traumatic encephalopathy (CTE), and frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17). In typical tauopathy, such as AD, pathological tau protein loses its microtubule-stabilizing function, accumulating to form neurofibrillary tangles (NFTs), driving neuronal degeneration and leading to progressive cognitive decline. Senile plaques (SPs) formed by the deposition of NFTs and β-amyloid protein (Aβ) are specific pathological changes in the AD brain. Progressive memory loss, cognitive decline and behavioral changes are the clinical characteristics of AD.

[0003] There is currently no effective cure for tau disease, and current treatments are mainly symptomatic interventions such as cholinesterase inhibitors, which cannot reverse the disease process. In recent years, therapies targeting tau protein (such as monoclonal antibodies, antisense oligonucleotides, and tau aggregation inhibitors) have entered clinical trials, but most have been terminated due to insufficient efficacy or side effects. The core bottlenecks include: 1) The pathological mechanism of tau is complex, involving isoform diversity, post-translational modifications (such as phosphorylation and acetylation), and "prion-like" transmission characteristics, which makes precise targeting difficult; 2) The blood-brain barrier limits the efficiency of drug delivery, and the lack of sensitive biomarkers hinders early intervention; 3) The disease is highly heterogeneous, and tau abnormalities often interact with multiple factors such as Aβ and inflammation, making it difficult for a single therapy to be effective; 4) Preclinical models (such as transgenic animals) cannot fully simulate the pathological characteristics of human tau, resulting in a high rate of drug conversion failure.

[0004] Lysosomes serve as intracellular recycling stations, receiving inactivated macromolecules, aging organelles, and extracellular materials transported through autophagy and endocytosis into their lumen for degradation by nearly 60 acidic hydrolases. Recent studies have indicated that lysosomal dysfunction, characterized by abnormal acidification, is an early event in the pathogenesis of AD and other tau disorders, severely impacting cellular metabolism and function. Lysosomal acidification is maintained by the vacuolar-type ATPase proton pump (V-ATPase), and most lysosomal enzymes are optimally active under these conditions (around pH 4.5). V-ATPase is a large protein complex, of which ATP6V1B2 is a subunit crucial for its formation and stability. Existing strategies primarily focus on directly clearing tau aggregates or inhibiting their phosphorylation, but have failed to effectively address this upstream pathological link, abnormal lysosomal acidification. Summary of the Invention

[0005] The present invention aims to provide a peptide drug with the function of preventing and treating tau disease. The peptide is amino acids 120-157 of the ATP 6V1B2 protein, with the sequence RTPVSEDMLGRVFNGSGKPIDRGPVV LAEDFLDIMGQP (SEQ ID NO: 1), designated as 38-peptide. Its molecular weight is 4.2 kDa, and it has the ability to penetrate the blood-brain barrier and can be delivered to the central nervous system in a targeted manner. Because ATP6V1B2 is highly expressed in the central nervous system, the 38-peptide can be effectively enriched in neurons.

[0006] The present invention has identified the potential of the 38-peptide in preventing and treating AD in both AD cell and animal models. The 38-peptide can block the abnormal binding of ATP6V1B2 protein to phosphorylated tau protein, improve lysosomal acidification in cells with pathological tau overload, and enhance the degradation and clearance of intracellular waste products. Furthermore, animal model experiments showed significant expression of the 38-peptide in hippocampal neurons of treated mice, reduced ATP6V1B2 binding to tau, enhanced activity of key lysosomal hydrolases CTSB / CTSD (p<0.001), and restored lysosomal hydrolysis function. The average percentage time for novel object recognition increased by 8.57%, the average time to first reach the platform in the water maze decreased by 6.23 seconds, the average number of platform crossings increased by 1.3, the average percentage time spent in the platform quadrant increased by 8.83%, and Aβ and phosphorylated tau deposition decreased by 40%-50%. This significantly improved cognitive function in AD mice, effectively treating AD.

[0007] The 38-peptide of the present invention can be directly chemically synthesized, or a gene expression vector containing the corresponding amino acid sequence RTPVS EDMLGRVFNGSGKPIDRGPVVLAEDFLDIMGQP can be constructed and transformed into Escherichia coli for efficient expression, followed by purification by high-performance liquid chromatography to obtain the final product. The 38-peptide of the present invention is suitable for people diagnosed with tau disease at an early stage or at high risk of such a disease. Routes of administration include intravenous injection, transdermal patch, and nasal spray.

[0008] Specifically, the present invention provides a polypeptide for preventing and treating tau disease, whose sequence is RTPVSEDMLGRVFNGSGKPIDRGPVVLAEDFLDIMGQP (SEQ ID NO: 1).

[0009] The present invention also provides a nucleotide encoding the polypeptide, and an expression cassette, a recombinant vector or a cell containing the nucleotide.

[0010] The present invention also provides a composition containing the polypeptide or the nucleotide, an expression cassette, a recombinant vector, and cells.

[0011] Furthermore, the composition is a medicine or a vaccine, which contains the polypeptide as a main active ingredient and is supplemented with a medically acceptable carrier or adjuvant.

[0012] Furthermore, the composition includes an injection, a transdermal patch or a spray.

[0013] Furthermore, the preparation method of the injection solution is: dissolving the polypeptide in physiological saline to prepare an injection solution of not less than 1 mg / mL; the preparation method of the transdermal patch is: mixing the polypeptide and the penetration enhancer in a carboxymethyl cellulose gel matrix, coating it on a medical non-woven fabric and covering it with a film to prepare a patch; the preparation method of the spray is: using chitosan-PEG nanocarriers to encapsulate the polypeptide.

[0014] Furthermore, the concentration of the polypeptide is not less than 10 mg, the penetration enhancer is azone, and the weight ratio of the two is 5%.

[0015] The present invention also provides the use of the polypeptide or nucleotide, expression cassette, recombinant vector, cell or the composition in the preparation of drugs for preventing and treating tau diseases.

[0016] The present invention also provides the use of the polypeptide or nucleotide, expression cassette, recombinant vector, cell or the composition in preparing a phosphorylated tau protein inhibitor.

[0017] The present invention also provides a method for preparing the polypeptide, which comprises expressing the nucleotide, expression cassette and recombinant vector in a recombinant cell and obtaining the polypeptide after purification.

[0018] Beneficial effects:

[0019] This invention provides a peptide for the prevention and treatment of tau diseases. It competitively blocks the binding of pathological tau to ATP6V1B2, restoring V-ATPase complex function and lysosomal acidification, providing a new target for overcoming existing therapeutic bottlenecks. Compared to traditional tau-targeted therapies, this invention addresses the tau pathology cascade at its source by restoring intracellular degradation homeostasis, potentially offering broader clinical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0021] Figure 1 : The pH value of the lysosomal lumen of HEK293tau cells decreased after 38-peptide treatment;

[0022] Figure 2 : 38 peptides bind to phosphorylated tau protein;

[0023] Figure 3 : 38 peptides were injected into the tail vein and entered the brain tissue and expressed in neurons;

[0024] Figure 4 : 38 peptides blocked the binding of ATP6V1B2 protein to tau protein;

[0025] Figure 5 : 38 peptide improves the learning and memory ability of AD mice (3×Tg mice);

[0026] Figure 6 :38 peptide enhances the lysosomal hydrolysis capacity in the brain tissue of AD mice (3×Tg mice);

[0027] Figure 7 :38 peptide reduces the level of pathological proteins in the brain tissue of AD mice (3×Tg mice). DETAILED DESCRIPTION

[0028] The following examples are only used to more clearly illustrate the technical solutions of the present invention and are therefore only used as examples and are not intended to limit the scope of protection of the present invention. It should be noted that, unless otherwise stated, the technical terms or scientific terms used in this application should be the usual meanings understood by those skilled in the art to which the present invention belongs. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the field of this technology. Unless otherwise specified, the reagents and materials used in the following examples are all commercially available. The 38 peptides of the present invention were synthesized by Wuhan Shumi Brain Science and Technology Co., Ltd.

[0029] Example 1 Chemical synthesis of peptide 38

[0030] 1. Solid-phase peptide synthesis

[0031] The target peptide (sequence: RTPVSEDMLGRVFNGSGKPIDRGPVVLAEDFLDIMGQP) was synthesized by Fmoc solid-phase synthesis.

[0032] 1.1 Rink amide resin (loading capacity 0.6 mmol / g) was loaded into a synthesis column, and sequentially deprotected (20% piperidine / DMF) and coupled with various amino acids (Fmoc protection, 4 equivalents, HBTU / HOBt / DIEA activation).

[0033] 1.2 After the synthesis is completed, the peptide is cleaved using a cleavage solution (TFA / water / TIS = 95:2.5:2.5) at room temperature for 2 hours to release the peptide.

[0034] 1.3 Purify by ether precipitation, collect the polypeptide by centrifugation, and lyophilize to obtain the crude product.

[0035] 2. High Performance Liquid Chromatography (HPLC) Purification

[0036] 2.1 A C18 reverse phase column (5 μm, 250×4.6 mm) was used, with a mobile phase consisting of 0.1% TFA aqueous solution (A) and 0.1% TFA acetonitrile solution (B), and a gradient elution (20%-60% B, 30 min).

[0037] 2.2 The target peak was collected, lyophilized, and then the molecular weight was verified by mass spectrometry (MALDI-TOF) (theoretical value: 4227.8 Da), and the purity was >95%.

[0038] 3. Preparation

[0039] The purified polypeptide was dissolved in physiological saline (pH 7.4) to prepare a 1 mg / mL injection solution, sterilized through a 0.22 μm filter membrane, and stored in aliquots at -80°C.

[0040] Example 2: Improvement of lysosomal acidification in HEK293tau cells by peptide 38

[0041] 1. Experimental Materials

[0042] HEK293wt cells, HEK293tau cells (overexpressing human full-length tau).

[0043] Lipofectamine 3000 liposome transfection reagent (Thermo Corporation), LysoSensor Yellow / Blue DND-160 probe (Thermo Corporation).

[0044] Antibodies: Flag (Sigma), pS262-tau (phosphorylated tau, Thermo), anti-CTSB / CTSD (lysosomal hydrolases, Cell Signaling Technology).

[0045] 38-peptide expression vector: pCDH-ATP6V1B2(120-157)-3×Flag (constructed by Wuhan Shumi Brain Science Technology Co., Ltd.).

[0046] 2. Experimental Procedure

[0047] 2.1 Cell grouping and processing

[0048] Normal group: HEK293 wt cells were transfected with empty vector.

[0049] Model group: HEK293 tau cells were transfected with empty vector.

[0050] Treatment group: HEK293 tau cells were transfected with 38-peptide expression vector.

[0051] 2.2 Lysosomal pH determination

[0052] Cells were seeded in 96-well plates (5×10 4 / well), and 48 hours after transfection, the culture medium containing LysoSensor probe (1μM) was replaced and incubated for 10 minutes. After PBS washing, the fluorescence intensity at 490nm (pH sensitive) and 550nm (pH insensitive) was detected by microplate reader, and the lysosomal pH value was calculated by standard curve ( Figure 1 ), and the pH standard curve was determined based on the 550 nm / 490 nm fluorescence intensity ratio.

[0053] 2.3 Immunofluorescence detection of the interaction between 38 peptides and tau protein

[0054] After cell fixation, the cells were labeled with anti-Flag (red) and anti-pS262-tau (green) antibodies, and the nuclei were stained with DAPI (blue). That is, the Flag antibody was combined with a fluorescent secondary antibody to label the 38-peptide (red fluorescence), and the pS262-tau antibody was combined with a fluorescent secondary antibody to label the phosphorylated tau protein (green fluorescence). The colocalization and interaction between the 38-peptide and tau protein were observed under an ultra-high resolution confocal microscope (Leica Sp8, Lightning mode).

[0055] 3. Results

[0056] The results showed that the pH value of the lysosomal cavity was 4.52±0.03 in the normal group, increased to 5.7±0.125 in the model group, and decreased to 4.85±0.05 in the 38-peptide treatment group ( Figure 1 ), the restoration of lysosomal acidity suggests an enhancement of lysosomal hydrolysis capacity. Secondly, immunofluorescence showed that the 38-peptide co-localized with phosphorylated tau protein ( Figure 2 ), confirming its ability to directly bind to phosphorylated tau protein.

[0057] Example 3 Therapeutic Effects of 38 Peptide on AD Mouse Model

[0058] 1. Experimental Materials

[0059] Ten-month-old male C57BL / 6J and 3×Tg mice (provided by the Experimental Animal Center of Tongji Medical College, Huazhong University of Science and Technology) weighing 27±2g were randomly divided into two groups: a model group and a 38-peptide treatment group, with 10 mice in each group.

[0060] AAV-PHP.eB-EF1α-ATP6V1B2(120-157)-3×Flag-EGFP (packaged by Wuhan Shumi Brain Science Technology Co., Ltd.): Construct an AAV viral vector containing the target gene, so that the target gene can be expressed in animals for several months to several years (or even lifelong). The PHP.eB serotype can enhance the ability of the vector to cross the blood-brain barrier. 3×Flag is fused with the target gene to facilitate biochemical detection, and EGFP green fluorescence facilitates the display of infection efficiency.

[0061] Antibodies: 4G8 (Aβ, Biolegend), AT100 (phosphorylated tau, Thermo Fisher Scientific), anti-CTSB / CTSD (lysosomal hydrolases, Cell Signaling Technology)

[0062] 2. Experimental Procedure

[0063] 2.1 Virus injection and grouping

[0064] The virus concentration was 1×10 13vg / ml, injection volume was 100nl.

[0065] Normal group: C57BL / 6J mice were injected with control AAV via tail vein.

[0066] Model group: 3×Tg mice were injected with control AAV.

[0067] Treatment group: 3×Tg mice were injected with 38-peptide AAV.

[0068] 2.2 Behavioral assessment (4 weeks after injection)

[0069] (1) Novel object recognition: used to evaluate the memory and object recognition abilities of mice. The experiment is divided into three phases: adaptation phase, training phase, and testing phase. Adaptation phase: The mice were placed in a 60cm×60cm×40cm test box and allowed to explore freely for 5 minutes. Training phase: Two identical cylinders A and B were placed in the lower left and lower right corners of the test box. The mice were placed in the test box with their backs facing the two objects and allowed to explore freely for 5 minutes. Testing phase: 1-24 hours after the completion of the training phase, object A in the lower left corner was removed and replaced with a new cube C. The mice were placed in the test box and the time they explored objects B and C was recorded. The test time was 5 minutes. The behavior of the mice in the test box was recorded using a camera. The time the mice explored object B (old object) (TB) and the time they explored object C (new object) (TC) were recorded. When the tip of the mouse's nose was directed toward or touched about 2 cm within the range of the object, the time the mouse explored the object was recorded. The time mice spent exploring new / old objects was recorded, and the novel object recognition preference (%) of mice was calculated by TC / (TC+TB)×100. The higher the value, the higher the recognition rate of mice for novel objects, and the better their memory, learning ability and cognitive function.

[0070] (2) Morris water maze: used to evaluate the spatial learning and memory ability of mice. The experiment is divided into two phases: training and testing. The water maze consists of a circular pool with a diameter of 2m. The water is dyed opaque white by titanium dioxide and the water temperature is maintained at 22℃. The pool is divided into 4 quadrants and has different graphic markings on the pool wall. One of the quadrants hides an escape platform 1cm below the water surface. Training phase: The mouse is placed in the water facing the maze pool wall and allowed to explore freely for 1min. Through 5 days of training in 4 quadrants, the mouse learns to find the location of the platform. Testing phase: On the 7th day, the platform is removed and the mouse is allowed to explore freely for 1min. A camera is used to record the behavior of the mouse in the test box, and the time (s) when the mouse first reaches the platform area, the time it stays in the platform quadrant, and the number of times it crosses the platform are recorded for further analysis. The preference (%) in the target quadrant is obtained by the time it stays in the platform quadrant / 60 (s) × 100. The shorter the time it takes for the mouse to reach the platform for the first time, the more times it crosses the platform, and the higher its preference value in the target quadrant, indicating that the mouse has better spatial learning and memory ability.

[0071] (3) Conditioned fear test: used to evaluate the environmental association memory ability of mice. Determine the freezing time. The experiment is divided into three stages: adaptation period, training period and test period. Adaptation period: The mice are placed in a 30cm×30cm×25cm test box and allowed to explore freely for 5 minutes. Training period: After the adaptation period, the mice continue to stay in the test box and begin to receive conditioned stimulation (auditory cue: 70-80 decibel sound) and unconditioned stimulation (mild foot shock: intensity of 0.5mA, duration of 2 seconds), with an interval of 1 minute and a cycle of stimulation for 4 minutes in total, to establish a conditioned reflex. Test period: 24 hours after the training period, the mice are returned to the test box and only the same auditory cue as in the training period is applied, without the electric shock, with an interval of 1 minute and a cycle of stimulation for 4 minutes in total. The behavior of the mice in the test box is recorded using a camera, and the freezing time (FT) of the mice is recorded. The novel object recognition preference (%) of the mice is calculated by FT / 240 (s)×100. The higher the data, the better the environmental association memory ability of the mice.

[0072] 2.3 Organizational Analysis

[0073] Immunoprecipitation: hippocampal tissue lysate was taken, the complex was captured with anti-ATP6V1B2 antibody, and the bound tau level was detected by western blotting ( Figure 4 ).

[0074] Lysosomal enzyme activity: Western blotting analysis of the active forms of CTSB and CTSD ( Figure 6), the protein band marked at the position of act-CTSB / CTSD is the active form. The gray value of this band is divided by the gray value of the internal reference GAPDH to obtain the relative expression of the protein. The normal control group is normalized to 1 to obtain the relative value, such as Figure 6 As shown in the picture on the right.

[0075] Pathological protein detection: Immunofluorescence quantification of Aβ (4G8) and phosphorylated tau (AT100) deposition ( Figure 7 ).

[0076] 2.4 Results

[0077] 1) Expression of 38-peptide in brain neurons after tail vein injection

[0078] Four weeks after the tail vein injection, the mice were perfused and the brains were removed. The brain tissues were fixed and frozen into sections with a thickness of 30 μm. The Flag antibody was combined with a fluorescent secondary antibody to label the 38-peptide. Confocal microscopy revealed that the 38-peptide could penetrate the blood-brain barrier to reach the central nervous system and enter the neurons. The blue color shows the nuclei stained with DAPI ( Figure 3 ).

[0079] 2) 38 peptides can block the binding of tau protein and ATP6V1B2 in mouse hippocampus tissue

[0080] Immunoprecipitation experiments extracted proteins related to ATP6V1B2 binding from fresh hippocampal tissue of mice. The normal group basically did not contain phosphorylated tau, and there was no tau-ATP6V1B2 binding. Western blotting experiments found that the 38-peptide treatment group had significantly less tau protein binding to ATP6V1B2 than the model group, and the pathological tau protein was significantly reduced (the right band of tau5 in the input (treatment group) was lighter than the left band (model group)) Figure 4 ), suggesting that the 38-peptide can block the abnormal binding of the two.

[0081] 3) Peptide 38 can improve the lysosomal hydrolysis capacity in mouse hippocampus tissue

[0082] Western blotting results showed that the activity levels of lysosomal cathepsin B (CTSB) and cathepsin D (CTSD) in the hippocampus of the 38-peptide treatment group were significantly increased compared with the model group ( Figure 5 ), suggesting that the acidic environment in the lysosomal cavity was improved and the hydrolysis capacity was enhanced.

[0083] 4) 38 peptide improves learning and memory abilities of 3×Tg mice

[0084] The results of the novel object recognition test showed that the 38-peptide treatment group had a significantly longer exploration time for novel objects; the results of the Morris water maze test showed that compared with the model group, the 38-peptide treatment group had a significantly shorter latency to find the platform, a significantly increased number of crossings into the platform area, and a significantly longer time and distance in the target quadrant where the platform was located; the results of the physical examination fear test showed that the 38-peptide treatment group had a significantly longer freezing time ( Figure 6 ), indicating that the 38-peptide improved the learning and memory abilities of 3×Tg mice.

[0085] 5) 38 peptide can reduce pathological related proteins in neurons of brain tissue of 3×Tg mice

[0086] The 4G8 antibody combined with the secondary antibody labeled abnormally processed β-amyloid protein (Aβ), and the AT100 antibody combined with the secondary antibody labeled phosphorylated tau protein. The results of immunofluorescence showed that the pathological Aβ and tau in the cortex and hippocampal neurons of the brain tissue of mice treated with 38 peptides were significantly reduced ( Figure 7 ).

[0087] In summary, the 38-peptide was significantly expressed in the hippocampus of treated mice, and the binding of ATP6V1B2 to tau was reduced. CTSB / CTSD activity was increased (p<0.001), lysosomal hydrolysis function was restored, the average time percentage for novel object recognition increased by 8.57%, the average time to first reach the platform in the water maze decreased by 6.23 seconds, the average number of platform crossings increased by 1.3 times, the average percentage of time spent in the platform quadrant increased by 8.83%, and Aβ and phosphorylated tau deposition decreased by 40%-50%. This indicates that the 38-peptide of the present invention significantly improved the learning and memory abilities of AD mice, is effective in treating AD, and has broad clinical application value.

[0088] Example 4 Preparation of 38-peptide Pharmaceutical Formulation

[0089] 1. Preparation of Transdermal Patch

[0090] 38 peptides (10 mg) were mixed with a penetration enhancer (azone, 5% w / w) in a carboxymethylcellulose gel matrix. The mixture was applied to medical non-woven fabrics and covered with a polyethylene film to form a 1 cm 2 Patch (containing peptide 1mg / cm 2 In vitro transdermal permeation experiments (Franz diffusion cell) showed that the cumulative transdermal permeation rate reached 35% within 24 hours.

[0091] 2. Preparation of Nasal Spray

[0092] The 38-peptide was encapsulated in chitosan-PEG nanocarriers (encapsulation efficiency > 90%) and delivered via the olfactory nerve pathway, with brain targeting efficiency increased by 3.2 times compared to intravenous injection.

[0093] The above specific embodiments describe the implementation of the present invention in detail, but the present invention is not limited to the specific details of the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

Claims

1. A polypeptide for preventing and treating tau disease, the sequence of which is shown in SEQ ID NO: 1, specifically: RTPVSEDMLGRVFNGSGKPIDRGPVVLAEDFLDIMGQP.

2. A nucleotide encoding the polypeptide according to claim 1, an expression cassette, a recombinant vector or a cell containing the nucleotide.

3. A composition comprising the polypeptide according to claim 1 or the nucleotide according to claim 2, an expression cassette, a recombinant vector, and cells.

4. The composition according to claim 3, characterized in that The composition is a medicine or a vaccine, which contains the polypeptide according to claim 1 as a main active ingredient and is supplemented with a medically acceptable carrier or adjuvant.

5. The composition according to any one of claims 3-4, characterized in that The composition includes an injection, a transdermal patch or a spray.

6. The composition according to claim 5, characterized in that The preparation method of the injection solution is: dissolving the polypeptide in physiological saline to prepare an injection solution of not less than 1 mg / mL; the preparation method of the transdermal patch is: mixing the polypeptide and the penetration enhancer in a carboxymethyl cellulose gel matrix, coating it on a medical non-woven fabric and covering it with a film to make a patch; the preparation method of the spray is: using chitosan-PEG nanocarriers to encapsulate the polypeptide.

7. The composition according to claim 6, characterized in that The concentration of the polypeptide is not less than 10 mg, the penetration enhancer is azone, and the weight ratio of the two is 5%.

8. Use of the polypeptide according to claim 1, the nucleotide, expression cassette, recombinant vector, cell according to claim 2, or the composition according to any one of claims 3 to 7 in the preparation of a drug for preventing and treating Alzheimer's disease.

9. The method for preparing the polypeptide according to claim 1, characterized in that: The method comprises expressing the nucleotide, expression cassette and recombinant vector according to claim 2 in a recombinant cell, and obtaining the polypeptide after purification.

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