Polypeptide for preventing and treating tauopathy and application thereof

Abstract

The invention relates to a polypeptide for preventing and treating tauopathy and application thereof. The invention finds that the 38 peptide of the 120-157 amino acid sequence in the V-ATPase subunit ATP6V1B2 protein can competitively block the combination of the excessively phosphorylated tau protein and the ATP6V1B2, and recover the assembly of the V-ATPase complex in the pathological tau protein overload cell and the lysosome acidification level. Compared with a traditional tau targeted therapy method, the tau targeted therapy method has the advantages that a new target spot is provided for breaking through existing treatment bottlenecks by reconstructing intracellular degradation homeostasis and intervening in tau pathological cascade reaction from the source, and the tau targeted therapy method has wider potential clinical application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, 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, including more than 20 clinical subtypes such as 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 tau diseases such as AD, pathological tau proteins lose their microtubule stabilization function, accumulate to form neurofibrillary tangles (NFTs), drive neuronal degeneration, and lead to progressive cognitive decline. Senile plaques (SPs) formed by the deposition of NFTs and β-amyloid (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 the current treatment is mainly symptomatic intervention such as cholinesterase inhibitors, but it 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 of them 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 failure rate of drug conversion.

[0004] Lysosomes are recycling stations in cells. They accept inactive macromolecules, aging organelles, and extracellular substances transported by autophagy and endocytosis into their lumen and degrade them through nearly 60 acid hydrolases. Recent studies have pointed out that lysosomal dysfunction characterized by abnormal acidification is an early event in the pathogenesis of AD and other tau diseases, which seriously affects cell metabolism and function. Lysosomal acidification depends on the maintenance of the vacuolar-type ATPase proton pump (V-ATPase). Most enzymes in lysosomes are most active under this condition (around pH 4.5). V-ATPase is a large protein complex, and ATP6V1B2 is one of its subunits, which is crucial for its formation and stability. Existing strategies mostly focus on directly clearing tau aggregates or inhibiting their phosphorylation, but have failed to effectively intervene in the upstream pathological link of abnormal lysosomal acidification. Summary of the invention

[0005] The purpose of the present invention is to provide a polypeptide drug with the function of preventing and treating tau disease. The polypeptide is 120-157 amino acids of ATP 6V1B2 protein, the sequence is RTPVSEDMLGRVFNGSGKPIDRGPVV LAEDFLDIMGQP (SEQ ID NO: 1), named 38 peptide, its molecular weight is 4.2kDa, has the effect of penetrating the blood-brain barrier, can be targetedly delivered in the central nervous system, and because ATP6V1B2 is highly expressed in the central nervous system, 38 peptide can be effectively enriched in neurons.

[0006] The present invention identified the function of the 38-peptide in preventing and treating AD at the AD cell model level and the AD animal model level: the 38-peptide can block the abnormal binding of ATP6V1B2 protein and phosphorylated tau protein, improve the insufficient lysosomal acidification of pathological tau overloaded cells, and enhance the degradation and removal of intracellular waste. In addition, animal model experiments showed that the expression of 38-peptide in neurons of the hippocampus of the treated mice was significant, the binding of ATP6V1B2 to tau was reduced, the activity of the key lysosomal hydrolase CTSB / CTSD was enhanced (p<0.001), and the lysosomal hydrolysis function was restored. The average time percentage of new object recognition increased by 8.57%, the average time of reaching the platform for the first time in the water maze decreased by 6.23s, the average number of crossing the platform area increased by 1.3 times, the average time percentage in the platform quadrant increased by 8.83%, and the deposition of Aβ and phosphorylated tau decreased by 40%-50%, that is, the cognitive function of AD mice was significantly improved, and AD was effectively treated.

[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, which is then transferred into Escherichia coli for efficient expression, and then purified 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 disease, and the administration routes 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 nucleotides encoding the polypeptide, and expression boxes, recombinant vectors or cells containing the nucleotides.

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

[0011] Furthermore, the composition is a medicine or a vaccine, which has 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 preparing a drug for preventing and treating tau disease.

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

[0017] The present invention also provides a method for preparing the polypeptide, wherein the nucleotide, expression cassette and recombinant vector are expressed in a recombinant cell, and the polypeptide is obtained after purification.

[0018] Beneficial effects:

[0019] The present invention provides a polypeptide for preventing and treating tau diseases, which can competitively block the binding of pathological tau to ATP6V1B2, restore the function of the V-ATPase complex and the acidification level of lysosomes, and provide a new target for breaking through the existing treatment bottleneck. Compared with traditional tau targeted therapy, the present invention has a wider potential clinical application value by reconstructing the intracellular degradation homeostasis and intervening in the tau pathological cascade reaction from the source. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without 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 peptides enhance the hydrolysis capacity of lysosomes in 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 cannot be used to limit the scope of protection of the present invention. It should be noted that, unless otherwise specified, the technical terms or scientific terms used in this application should be the usual meanings understood by technicians in the field 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 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 polypeptide (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 deprotected (20% piperidine / DMF) and coupled with each amino acid (Fmoc protection, 4 times equivalent, HBTU / HOBt / DIEA activation) in sequence.

[0033] 1.2 After the synthesis is completed, the peptide is cleaved with 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 obtain the crude product after freeze-drying.

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

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

[0037] 2.2 The target peak was collected, and after freeze-drying, 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 The improving effect of peptide 38 on lysosomal acidification in HEK293tau cells

[0041] 1. Experimental Materials

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

[0043] Lipofectamine 3000 liposome transfection reagent (Thermo Corporation), LysoSensor Yellow / BlueDND-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 treatment

[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 the culture medium containing LysoSensor probe (1μM) was replaced 48 hours after transfection, and incubated for 10 minutes. After PBS washing, the fluorescence intensity at 490nm (pH sensitive) and 550nm (pH insensitive) was detected by an enzyme-labeled instrument, and the lysosomal pH value ( Figure 1 ), the pH standard curve was determined based on the 550nm / 490nm fluorescence intensity ratio.

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

[0054] After the cells were fixed, they 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). Super-high resolution confocal microscopy (Leica Sp8, Lightning mode) was used to observe the co-localization and interaction between the 38-peptide and tau protein.

[0055] 3. Results

[0056] The results showed that the pH value of the lysosomal cavity in the normal group was 4.52±0.03, which 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 effect of 38 peptide on AD mouse model

[0058] 1. Experimental Materials

[0059] 10-month-old 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, male. Among them, 3×Tg mice were randomly divided into two groups: model group and 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 for life). 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 the EGFP green fluorescence is convenient for displaying the infection efficiency.

[0061] Antibodies: 4G8 (Aβ, Biolegend), AT100 (phosphorylated tau, Thermo), 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 injected with control AAV.

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

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

[0069] (1) Novel object recognition: used to evaluate the memory and object recognition abilities of mice. The experiment was divided into three phases: adaptation phase, training phase, and test phase. Adaptation phase: mice were placed in a 60 cm × 60 cm × 40 cm test box and allowed to explore freely for 5 min. Training phase: two identical cylinders A and B were placed in the lower left and lower right corners of the test box, and the mice were placed in the test box with their backs facing the two objects and allowed to explore freely for 5 min. Test phase: 1-24 hours after 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 min. The behavior of the mice in the test box was recorded using a camera, and 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 facing or touching the object within 2 cm, the time the mouse explored the object was recorded. The time the mice spent exploring new / old objects was recorded, and the mice's new object recognition preference (%) was calculated by TC / (TC+TB)×100. The higher the data, the higher the recognition rate of mice for new 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 was divided into two phases: training and testing. The water maze consisted of a circular pool with a diameter of 2 m. The water was dyed opaque white by titanium dioxide and the water temperature was maintained at 22°C. The pool was divided into 4 quadrants and had different graphic markings on the pool wall. One of the quadrants concealed an escape platform 1 cm below the water surface. Training phase: The mouse was placed in the water facing the maze pool wall and allowed to explore freely for 1 min. Through 5 days of training in 4 quadrants, the mice learned to find the location of the platform. Testing phase: On the 7th day, the platform was removed and the mice were allowed to explore freely for 1 min. A camera was used to record the behavior of the mice in the test box, and the time (s) when the mouse first reached the platform area, the time it stayed in the platform quadrant, and the number of times it crossed the platform were recorded for further analysis. The preference (%) in the target quadrant was obtained by the time it stayed 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 for the target quadrant, indicating that the mouse has better spatial learning and memory ability.

[0071] (3) Conditioned fear experiment: 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 0.5mA, duration 2 seconds), with an interval of 1min and a cycle of stimulation for 3 times for a total of 4 minutes to establish conditioned reflexes. 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 electric shock, with an interval of 1min and a cycle of stimulation for 3 times for a total of 4 minutes. The behavior of the mice in the test box is recorded with 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, and the gray value of the band is divided by the gray value of the internal reference GAPDH to obtain the relative expression of the protein, which is standardized to 1 with the normal control group 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 injected into the tail vein in brain neurons

[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. The 38-peptide was observed under a confocal microscope and found to be able to pass through the blood-brain barrier to reach the central part and enter the neuronal cells. The blue color shows the cell nucleus stained with DAPI ( Figure 3 ).

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

[0080] Immunoprecipitation experiments were performed to extract proteins related to ATP6V1B2 binding in fresh hippocampal tissue of mice. The normal group basically did not contain phosphorylated tau, and there was no tau-ATP6V1B2 binding. Protein immunoblotting experiments showed 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 the hippocampus of mice

[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 ), indicating that the acidic environment in the lysosomal cavity was improved and the hydrolysis capacity was enhanced.

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

[0084] The test results of novel object recognition showed that the exploration time of novel objects in the 38-peptide treatment group was significantly increased; the test results of Morris water maze showed that compared with the model group, the latency of finding the platform in the 38-peptide treatment group was significantly shortened, the number of crossing the platform area was significantly increased, and the time and distance in the target quadrant where the platform was located were also significantly increased; the test results of physical examination fear showed that the freezing time in the 38-peptide treatment group was significantly increased ( 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 marked abnormally processed β-amyloid protein (Aβ), and the AT100 antibody combined with the secondary antibody marked 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 expression of 38 peptides in the hippocampus of mice in the treatment group was significant, 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 of new object recognition increased by 8.57%, the average time of the first arrival at the platform in the water maze decreased by 6.23s, the average number of crossing the platform area increased by 1.3 times, the average time percentage in the platform quadrant increased by 8.83%, and Aβ and phosphorylated tau deposition decreased by 40%-50%, indicating that the 38 peptides of the present invention significantly improved the learning and memory ability of AD mice, can effectively treat AD, and have a wide range of clinical application value.

[0088] Example 4 Preparation of 38-peptide pharmaceutical preparation

[0089] 1. Preparation of Transdermal Patch

[0090] 38 peptides (10 mg) and a penetration enhancer (azone, 5% w / w) were mixed in a carboxymethylcellulose gel matrix. The mixture was applied to medical nonwoven 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 by chitosan-PEG nanocarrier (encapsulation efficiency>90%) and delivered via the olfactory nerve pathway, with the brain targeting efficiency increased by 3.2 times compared with 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 in the above embodiments. Within the scope of the claims and technical concept of the present invention, the technical solution of the present invention can be modified and changed in many simple ways, and these simple modifications all belong to the protection scope 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: RTPVSE DMLGRVFNGSGKPIDRGPVVLAEDFLDIMGQP.

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 prepare 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 or 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 tau disease.

9. Use of the polypeptide according to claim 1 or 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 phosphorylated tau protein inhibitor.

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

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