Rapid preparation method of multi-size alpha-synuclein aggregate

Abstract

The invention provides a rapid preparation method of a multi-size alpha-synuclein aggregate, and solves the problems of tedious steps and high cost of an existing preparation method of different-size alpha-synuclein aggregates. The method comprises the following steps: 1) dissolving a separated and purified alpha-synuclein monomer in a physiological buffer solution, and uniformly mixing to obtain a monomer protein solution; (2) putting the monomer protein solution into a centrifugal tube, shaking and incubating under a certain condition, and respectively sampling at different time points in the incubation process; (3) respectively adding samples into an ultrafiltration centrifugal tube, and carrying out centrifugal separation to remove small-size proteins; and 4) adding a physiological buffer solution into the ultrafilter of each ultrafiltration centrifuge tube, inversely placing each ultrafilter in a centrifuge tube matched with the ultrafilter, and centrifugally separating and collecting the protein aggregate on the inner membrane of each ultrafilter to obtain the protein aggregate with different morphological structures.

Description

Technical Field

[0001] The invention belongs to the field of biology and new medicine technology, and specifically relates to a method for rapidly preparing multi-sized alpha-synuclein aggregates. Background Art

[0002] Parkinson's disease (PD) is the second most common neurodegenerative disease, characterized by motor or non-motor symptoms such as bradykinesia, tremor, constipation, mental disorders and sleep disorders. These clinical symptoms have greatly affected the normal life of Parkinson's patients and brought tremendous pressure to society. Although remarkable progress has been made in the pathogenesis and epidemiology in recent years, the cause of Parkinson's disease remains a mystery, and no cure or preventive therapy has been found. The most important pathological feature of Parkinson's disease is the massive degeneration and death of dopaminergic neurons in the substantia nigra compacta of the brain, which further leads to a significant decrease in striatal dopamine content, which is associated with motor symptoms such as bradykinesia and tremor. During the course of the disease, obvious pathological features appear in the limbic system and cortex of the brain, and are associated with a series of non-motor features.

[0003] α-Synuclein (α-Syn) is a 140-residue protein that is widely expressed in the brain and can interact with many specific proteins involved in signal transduction, vesicle trafficking, synaptic behavior, oxidative stress regulation, and mitochondrial function. It is the main component of intracellular protein-rich inclusions, Lewy bodies, and Lewy neurites, which are precisely the hallmark lesions of Parkinson's disease (PD). Throughout the development of Parkinson's disease, α-synuclein monomers are transformed into proteins of different sizes, structures, and morphologies over time, and all these different α-synucleins coexist, making it challenging to identify toxic species in the human body. In recent years, more and more studies have shown that soluble oligomers of α-synuclein, namely α-synuclein oligomers, play a vital role in the development of Parkinson's disease. There is no clear definition of oligomers, including soluble aggregates that exist unstable between monomers and insoluble fibers. The mechanisms by which oligomers severely cause neuronal degeneration and death include cell membrane destruction, synaptic damage, mitochondrial dysfunction, and neuroinflammation.

[0004] Therefore, the precise preparation of α-synuclein oligomers of different sizes (i.e., different molecular weights and different morphological structures) is of great significance for the study of the occurrence and development of Parkinson's disease (PD). Lashuel et al. purified α-synuclein oligomers of different sizes using liquid chromatography and observed their morphological differences. Emin et al. used the mechanism that proteins accumulate in different concentrations of sucrose with different masses during centrifugation to prepare a series of α-synuclein aggregates of different sizes using the sucrose gradient method. However, the above-mentioned α-synuclein oligomer preparation and purification schemes are relatively cumbersome (for example, impurities such as sucrose are introduced, and subsequent steps for removing impurities such as sucrose are added), and expensive equipment such as protein liquid chromatography is required. At present, there is still a lack of low-cost, efficient, fast and simple α-synuclein aggregate preparation schemes.

[0005] To this end, the present invention explores a method for rapidly preparing multi-sized α-synuclein aggregates. Summary of the invention

[0006] The purpose of the present invention is to solve the shortcomings of the existing methods for preparing α-synuclein aggregates of different sizes, such as complicated steps and high cost, and to provide a method for rapidly preparing α-synuclein aggregates of multiple sizes, which is simple and efficient.

[0007] To achieve the above purpose, the technical solution provided by the present invention is:

[0008] The rapid preparation method of multi-sized α-synuclein aggregates is special in that it comprises the following steps:

[0009] 1) Dissolve the isolated and purified α-synuclein monomer in a 2-6° C. PBS buffer solution (only physiological buffer solution can be used, otherwise other factors will be introduced to affect protein aggregation) according to a concentration standard of 0.8-1.2 mg / ml and mix well (vortexing, inversion, pipette blowing, etc. can be used to assist in dissolving the α-synuclein monomer and mixing well) to obtain a monomer protein solution;

[0010] 2) placing the monomeric protein solution obtained in step 1) in a centrifuge tube, shaking and incubating at 300-700 rpm at human physiological temperature (shaking and incubating can accelerate the rate of protein aggregation and make the size of the generated aggregates more uniform), and sampling at different time points during the incubation process (here, several time points can be selected according to the required protein size and the law of particle size change, that is, different sampling times can be selected for different needs), and each sampling is not less than 200 μL (considering that there will be protein loss during ultrafiltration centrifugation and re-centrifugation collection, if the sampling volume is too small, the final protein aggregate concentration will be low, so the sampling volume of this step is not less than 200 μL);

[0011] 3) adding the samples obtained in step 2) to 100 kDa ultrafiltration centrifuge tubes (because the molecular weight of α-synuclein monomer is 14 kDa, the use of this type of ultrafiltration centrifuge tube can ensure that α-synuclein monomer passes through the filter membrane, while the aggregates can be effectively retained on the filter membrane), centrifuging at 2-6° C. and 14000-16000 g for 3-10 min to separate the monomers from the aggregates. At this time, what remains in the ultrafiltration centrifuge tube is the protein aggregates;

[0012] 4) Add 50-100 μL of PBS buffer to the ultrafilter of each ultrafiltration centrifuge tube, and invert each ultrafilter into a matching centrifuge tube, centrifuge at 2-6°C and 300-600g for 3-10 min, and collect protein aggregates on the inner membrane of each ultrafilter. At this time, protein aggregates of different sizes (i.e., different morphological structures and different molecular weights) can be obtained and stored at 2-6°C for future use.

[0013] It can be seen that the present invention can obtain α-synuclein aggregates of different sizes after incubation of the same α-synuclein monomer for different times, and the entire preparation process can regulate the size of the protein aggregates by regulating the incubation time. The present invention avoids the introduction of protein aggregates of different sizes by separating at different incubation time points, reduces the difficulty of separation and purification, and the protein aggregates obtained by separation and purification at different incubation degrees have good size uniformity, and can respectively obtain protein aggregates of small, medium and large sizes and protein fibers of small, medium and large sizes. After characterization, it is also confirmed that the size of the aggregates prepared by the method of the present invention is relatively uniform rather than a mixture of various aggregates, which is enough to reflect the simplicity, rapidity and efficiency of the preparation method of the present invention.

[0014] Furthermore, in step 1), the isolated and purified α-synuclein monomer is dissolved in PBS buffer at 4° C. (relatively speaking, protein monomer is more stable at 4° C.) according to a concentration standard of 1 mg / ml, and vortexed to mix to obtain a monomer protein solution.

[0015] Furthermore, in step 2), the monomer protein solution obtained in step 1) is placed in a centrifuge tube, incubated with shaking at 37°C and 500rpm, and samples are taken after incubation for 6h, 15h, 24h, and 48h, respectively, and each sample is not less than 200μL; according to needs, sampling, separation and purification are performed at the above-mentioned incubation time points to obtain protein aggregates with significantly different sizes and different morphological structures.

[0016] Furthermore, in step 3), the samples obtained in step 2) are added into 100 kDa ultrafiltration centrifuge tubes respectively, and centrifuged at 4° C. and 15300 g for 5 min to separate monomers from aggregates.

[0017] Furthermore, in step 4), protein aggregates with different morphological structures on the inner membrane of the ultrafilter are collected by centrifugation at 4°C and 450 g for 4 min, and stored at 4°C for later use.

[0018] At the same time, the present invention adopts the above-mentioned rapid preparation method to prepare samples at different incubation times to obtain multi-sized α-synuclein aggregates with uniform morphology.

[0019] The multi-sized α-synuclein aggregates obtained by the rapid preparation method of the present invention can be applied to the study of α-synuclein disease pathology, such as Parkinson's disease; the specific study method of Parkinson's disease pathology is to differentiate neuroblastoma cells (SH-SY5Y cells) into mature dopaminergic neuron cells and induce Parkinson's disease pathology, and the specific method of differentiation is to differentiate SH-SY5Y cells into mature dopaminergic neurons by using a combination of retinoic acid (RA) and brain-derived neurotrophic factor (BDNF); or, by observing the degree of damage to the blood-brain barrier by α-synuclein aggregates with different morphological structures in a Parkinson's disease chip model with blood-brain barrier function. Based on the above application, the α-synuclein aggregates obtained by the above rapid preparation method can also be used to induce α-synuclein disease, such as Parkinson's disease, in the cultured mature dopaminergic neuron cells, add exogenous α-synuclein monomers or aggregates with different morphological structures, and reproduce the abnormal aggregation of α-synuclein, oxidative stress and mitochondrial dysfunction in Parkinson's disease.

[0020] The advantages of the present invention are:

[0021] 1. The preparation method of the present invention only needs to use ultrafiltration centrifuge tubes, combined with shaking incubation time regulation, to achieve the preparation and extraction of α-synuclein aggregates of different sizes, greatly reducing the preparation cost of α-synuclein aggregates, the preparation method is simple and efficient, and the obtained protein aggregates are stable and uniform. At the same time, based on this scheme, the neuronal toxicity analysis of α-synuclein aggregates can also be achieved, confirming that medium-sized α-synuclein aggregates have the strongest neuronal and mitochondrial toxicity, providing a powerful tool for the study of the occurrence and development of Parkinson's disease.

[0022] 2. The method of the present invention can be used to adjust the incubation time to prepare protein aggregates of different sizes. The entire preparation method is ingeniously conceived, fast and simple, which greatly simplifies the preparation process. The prepared protein aggregates are relatively stable and uniform. No expensive equipment is used in the preparation process, which greatly reduces the preparation cost.

[0023] 3. The present invention also characterizes and confirms the target product through a variety of physical and biological means. The prepared α-synuclein aggregates can be used not only for the pathological study of Parkinson's disease and the establishment of in vitro disease models, but also for the pathological study and model establishment of other different α-synuclein diseases such as Lewy body dementia and multiple system atrophy. In addition, the prepared α-synuclein aggregates can be used in organ chips to explore the mutual influence between diseases and other organs, such as exploring Parkinson's damage to the blood-brain barrier; the above applications can further confirm the practicality of α-synuclein aggregates of different morphological structures prepared by the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a schematic diagram of the process of extracting and purifying α-synuclein monomers of the present invention;

[0025] Figure 2 Schematic diagram of the preparation of α-synuclein aggregates with different morphological structures of the present invention;

[0026] Figure 3 TEM characterization images of α-synuclein monomers and α-synuclein aggregates of different morphological structures of the present invention; a. α-synuclein monomer (M); b. oligomer 1 (O1, 6h); c. oligomer 2 (O2, 15h); d. short fibers (SF, 24h); e. long fibers (LF, 48h); partial enlarged images in fc;

[0027] Figure 4 The AFM characterization images of the α-synuclein monomer and α-synuclein aggregates of different morphological structures of the present invention; a. α-synuclein monomer (M); b. oligomer 1 (O1, 6h); c. oligomer 2 (O2, 15h); d. short fiber (SF, 24h); e. long fiber (LF, 48h);

[0028] Figure 5 The bright field images of different morphological structures of SH-SY5Y cells before and after differentiation of the present invention (scale bar: 50 μm);

[0029] Figure 6 Characterization of mature dopaminergic neuronal cells differentiated by the present invention;

[0030] Figure 7 The differentiated mature dopaminergic neuron cells of the present invention are characterized, and the differentiated mature dopaminergic neuron cells significantly express dopamine transporter (DAT) and tyrosine hydroxylase (TH);

[0031] Figure 8 The changes in mitochondrial membrane potential level after the differentiated mature dopaminergic neuron cells are treated with α-synuclein aggregates of different morphological structures of the present invention;

[0032] Fig. 9 The changes in the expression level of DAT after the differentiated mature dopaminergic neuron cells were treated with α-synuclein aggregates of different morphological structures of the present invention (scale bar: 100 μm);

[0033] Fig.10 The fluorescence image morphological quantitative analysis of the DAT protein expression level of the present invention; a. average area; b. average branches; c. average fluorescence intensity; d. average number of networks;

[0034] Fig.11 The changes in TH expression levels after differentiated mature dopaminergic neuronal cells were treated with α-synuclein aggregates of different morphological structures of the present invention (scale bar: 100 μm);

[0035] Fig.12 This is the quantitative analysis of the fluorescence image morphology of the TH protein expression level of the present invention; a. average area; b. average branches; c. average fluorescence intensity; d. average number of networks;

[0036] Fig.13 The level of reactive oxygen species (ROS) produced by differentiated mature dopaminergic neuronal cells after being treated with α-synuclein aggregates of different morphological structures of the present invention (scale bar: 200 μm);

[0037] Fig.14 This is the morphological quantitative analysis of the fluorescence image of ROS production level in the present invention, a. average area; b. average branches; c. average fluorescence intensity; d. average number of networks. DETAILED DESCRIPTION

[0038] The present invention is further described in detail below with reference to the accompanying drawings and specific embodiments:

[0039] The present invention first extracts α-synuclein monomers, and then uses shaking incubation and ultrafiltration separation methods to prepare a series of α-synuclein aggregates with different morphological structures for inducing Parkinson's disease. The prepared α-synuclein aggregates act on differentiated and mature dopaminergic neuronal cells, and the most neurotoxic α-synuclein aggregates are determined by detecting the expression level of marker proteins, the level of reactive oxygen production, and the level of damage to mitochondrial membrane potential. An α-synuclein aggregate with the greatest impact on nerve cells is determined through experimental verification at the cell level.

[0040] The specific preparation method is as follows:

[0041] 1) Extraction and purification of α-synuclein monomers, such as Figure 1 Shown

[0042] 1.1) The recombinant α-synuclein Escherichia coli strain was constructed by the research group in the early stage and cultured at 37°C with constant temperature shaking until the OD600 reached 0.6-0.8.

[0043] 1.2) After adding IPTG (isopropyl-β-D-thiogalactoside), the culture was induced at 37°C for 4 hours, and the fermentation broth was centrifuged at 15000 rpm for 10 minutes to collect the bacteria.

[0044] 1.3) The bacterial cells were disrupted by ultrasonic disruptor and then centrifuged, the supernatant was collected, and the impure protein solution was purified using a nickel column to obtain α-synuclein with a purity of more than 95%.

[0045] 1.4) The purified α-synuclein was freeze-dried in a freeze dryer and stored at -20°C for a long term.

[0046] 2) Preparation of α-synuclein aggregates with different morphological structures, the specific preparation process is as follows Figure 2 As shown:

[0047] 2.1) The obtained α-synuclein monomer was dissolved to 1 mg / ml in 4° C. PBS buffer and vortexed to obtain a monomer protein solution.

[0048] 2.2) The obtained monomer protein solution was placed in a 2 ml centrifuge tube and incubated with shaking at 37° C. and 500 rpm.

[0049] 2.3) Samples were taken at 6h, 15h, 24h, and 48h of incubation, and each sample was no less than 200 μL.

[0050] 2.4) The sampled solutions were added to 100 kDa ultrafiltration centrifuge tubes, and centrifuged at 4°C and 15300 g for 5 min to separate monomers from aggregates and remove small-sized proteins;

[0051] 2.5) Add 100 μL of PBS buffer to the ultrafilter of each ultrafiltration centrifuge tube, and invert each ultrafilter into the corresponding centrifuge tube, centrifuge at 4°C and 450g for 4 min to collect protein aggregates on the inner membrane of each ultrafilter, and finally obtain protein aggregates with different morphological structures, which are stored at 4°C for future use.

[0052] 3) Characterization of α-synuclein aggregates with different morphological structures

[0053] 3.1) Transmission electron microscopy (TEM) characterization

[0054] Transmission electron microscopy (TEM) was used to characterize the morphology of the prepared α-synuclein aggregates. The preparation method of TEM samples is as follows:

[0055] ① Dilute the sample to 0.1-1μM with ultrapure water;

[0056] ② Drop the sample solution directly onto the copper mesh with the support film and let it stand for 5 minutes;

[0057] ③ Use filter paper strips to absorb excess liquid from the edge of the droplet and dry it slightly;

[0058] ④ Add 5% phosphotungstic acid dye solution for negative staining and let stand for 3 minutes;

[0059] ⑤ Use filter paper strips to absorb excess dye, dry at 37°C for 8 hours, and then observe using a transmission electron microscope.

[0060] TEM images of α-synuclein aggregates with different morphological structures prepared by this method are shown in Figure 2. Figure 3 According to the TEM image, it can be seen that with the increase of shaking incubation time, the particle size of the protein aggregates gradually increased. Two α-synuclein oligomers with different particle sizes were formed at 6h and 15h of shaking incubation, short fibers were observed at 24h, and obvious long fibers were visible at 48h, with obvious size changes.

[0061] 3.2) Atomic force microscopy (AFM) characterization

[0062] Atomic force microscopy (AFM) was used to characterize the morphology of the prepared α-synuclein aggregates. The AFM sample preparation method is as follows:

[0063] ① Use ultrapure water to dilute the concentration of α-synuclein to about 50 μg / ml;

[0064] ②Pipette 40-50μL of sample solution and drop it onto a clean mica sheet and let it stand for 5-10 minutes to wait for the sample to settle on the surface of the mica sheet;

[0065] ③ Add a small amount of ultrapure water to clean the PBS contained in the sample;

[0066] ④ Dry at room temperature until there is no obvious water mark on the surface and then it can be used for imaging analysis.

[0067] AFM images of α-synuclein aggregates with different morphological structures prepared by this method are shown in Figure 2. Figure 4 As shown, the AFM morphology images of different α-synuclein aggregates are Figure 3TEM is highly consistent. Before the formation of fibers, as the shaking incubation time increases, the particle size of α-synuclein aggregates tends to increase, and two oligomers with different particle sizes are obtained at 6h and 15h of shaking incubation. Similarly, the samples incubated with shaking for 24h and 48h showed the morphology of short fibers and long fibers, respectively. In addition, it is worth mentioning that with the increase of shaking incubation time, AFM results show that the longitudinal height of α-synuclein also tends to increase.

[0068] 4) Inducing Parkinson's disease

[0069] 4.1) Specific implementation method of dopaminergic neuron differentiation:

[0070] SH-SY5Y cells were first differentiated in a culture flask in DMEM complete medium (10% FBS, 1% P / S) containing 20 μM RA for 3 days, then the cells were collected and resuspended in low-serum DMEM medium (2.5% FBS, 1% P / S) containing BDNF, and the suspension was inoculated into the well plate or chip channel to continue differentiation for 3 days. Finally, SH-SY5Y cells were successfully differentiated into dopaminergic neurons, and the morphology of the differentiated cells was observed using a fluorescence microscope. Figure 5 As shown in the figure (the morphology of the cells shows that SH-SY5Y cells have successfully differentiated into dopaminergic neurons), and the bright field images of SH-SY5Y cells before and after differentiation were quantitatively analyzed for morphology. Figure 6 The analysis results showed that the branch length of the differentiated cells was significantly longer than that before differentiation. In addition, after RA and BDNF combined induced differentiation, immunofluorescence staining and laser confocal microscopy showed that SH-SY5Y cells significantly expressed dopamine transporter (DAT) and tyrosine hydroxylase (TH), which are markers of dopaminergic neuron cells. Figure 7 , which also proves that SH-SY5Y cells successfully differentiated into dopaminergic neurons.

[0071] 4.2) Specific methods of α-synuclein-induced Parkinson's disease

[0072] For the differentiated dopaminergic neuron cells, the medium was replaced with low serum (2.5% FBS) DMEM medium containing 4.2 μg / ml α-synuclein aggregates of different morphological structures to induce Parkinson's disease pathology. The medium was replaced every 24 hours for 48 hours, and the Parkinson's disease pathology induction was completed.

[0073] 4.3) Characterization of induced Parkinson's disease

[0074] 4.3.1) Specific implementation method of mitochondrial membrane potential probe:

[0075] JC-10 is a fluorescent probe that can quickly and sensitively detect changes in mitochondrial membrane potential in cells or tissues and is often used for early apoptosis detection. When the mitochondrial membrane potential is high, JC-10 aggregates in the mitochondrial matrix to form polymers that can produce red fluorescence; when the mitochondrial membrane potential is low, JC-10 cannot aggregate in the mitochondrial matrix. At this time, JC-10 is a monomer and can produce green fluorescence. The JC-10 fluorescent probe was used to detect the changes in mitochondrial membrane potential of cells before and after treatment with α-synuclein aggregates of different morphological structures to evaluate their mitochondrial dysfunction. After cells were stained with 2.5μM JC-10, they were detected by flow cytometry and the red-green fluorescence ratio obtained by the detection was calculated.

[0076] The results are as follows Figure 8 As shown, compared with the control group, in cells treated with α-synuclein aggregates with different morphological structures, the red fluorescence of mitochondria weakened and the green fluorescence enhanced, indicating that the mitochondrial activity of the cells was reduced, indicating that α-synuclein aggregates damaged the mitochondrial function of the cells. The red-green fluorescence ratio was significantly reduced after treatment with oligomers, among which oligomer 2 (O2) seriously damaged the mitochondrial membrane potential.

[0077] 4.3.2) Specific implementation method of immunofluorescence staining to detect protein expression level:

[0078] First, the cells were fixed with 4% paraformaldehyde fixative, permeabilized and blocked, and then the differentiated SH-SY5Y cells were incubated with primary antibodies for dopaminergic neuron marker proteins DAT and TH. Goat anti-rabbit IgG (Alexa 488) was used as the secondary antibody for incubation, and finally, the images were taken using a laser confocal microscope. After the induction of Parkinson's pathology, the most toxic α-synuclein aggregates were determined by observing the decrease in the expression levels of the dopaminergic neuron marker proteins DAT and TH. Fig. 9 and Fig.11 It can be seen that the expression of DAT and TH was significantly weakened after treatment with oligomer 1 (O1) and oligomer 2 (O2). Then, the obtained fluorescence images were subjected to morphological quantitative analysis, in which four indicators, namely, average area, average branches, average fluorescence intensity and average number of networks, were selected for analysis. Fig.10 and Fig.12 The results showed that α-synuclein oligomer 2 (O2) was the most neurotoxic.

[0079] 4.3.3) Specific implementation method of reactive oxygen level detection:

[0080] DCFH-DA itself has no fluorescence and can freely pass through the cell membrane. After entering the cell, it can be hydrolyzed into DCFH by esterase. DCFH cannot pass through the cell membrane, so it can be easily loaded into the cell. When there is reactive oxygen in the cell, the non-fluorescent DCFH can be oxidized to DCF with green fluorescence. By detecting the fluorescence of DCF, the level of reactive oxygen in the cell can be known. The DCFH-DA probe is used to detect the level of reactive oxygen (ROS) in the cell, and the fluorescence graph ( Fig.13 ) It can be seen that the active oxygen content in cells treated with oligomer 1 (O1) and oligomer 2 (O2) increased significantly. The obtained fluorescence images were also subjected to morphological quantitative analysis, in which four indicators were selected for analysis: average area, average branches, average fluorescence intensity, and average number of networks. Fig.14 The results also showed that oligomer 2 was the most neurotoxic. Thus, the usability of the α-synuclein aggregates prepared by the preparation method of the present invention was further verified.

[0081] In addition, the present invention successfully prepared α-synuclein aggregates of various sizes by adjusting the process parameters within the process range described above and sampling, separation and purification at different incubation time points (for example, incubating for 4h, 10h, 16h, 25h, 36h, and 47h according to the required size) in the same way. The whole process is simple and efficient, and the target product is stable and uniform; the prepared α-synuclein aggregates can not only be used for the pathological study of Parkinson's disease and the establishment of an in vitro disease model, but also can be used for the pathological study and model establishment of other different α-synuclein diseases such as Lewy body dementia and multiple system atrophy.

Claims

1. A method for rapidly preparing multi-sized α-synuclein aggregates, characterized in that: The following steps are involved: 1) Dissolving the isolated and purified α-synuclein monomer in a 2-6° C. PBS buffer solution at a concentration of 0.8-1.2 mg / ml and mixing well to obtain a monomer protein solution; 2) placing the monomeric protein solution obtained in step 1) in a centrifuge tube, shaking and incubating at 300-700 rpm at physiological temperature, and taking samples at different time points during the incubation process, and each sample is not less than 200 μL; 3) adding the samples obtained in step 2) into 100 kDa ultrafiltration centrifuge tubes respectively, and centrifuging at 2-6° C. and 14000-16000 g for 3-10 min to separate monomers from aggregates; 4) Add 50-100 μL of PBS buffer solution to the ultrafilter of each ultrafiltration centrifuge tube, and invert each ultrafilter into a matching centrifuge tube, centrifuge at 2-6°C and 300-600g for 3-10 min, collect protein aggregates on the inner membrane of each ultrafilter, obtain protein aggregates of different sizes, and store them at 2-6°C for future use.

2. The method for rapidly preparing multi-sized α-synuclein aggregates according to claim 1, characterized in that: In step 1), the isolated and purified α-synuclein monomer is dissolved in PBS buffer at 4° C. according to a concentration standard of 1 mg / ml, and vortexed to mix well to obtain a monomer protein solution.

3. The method for rapidly preparing multi-sized α-synuclein aggregates according to claim 2, characterized in that: In step 2), the monomer protein solution obtained in step 1) is placed in a centrifuge tube, incubated with shaking at 37°C and 500 rpm, and samples are taken after incubation for 6h, 15h, 24h, and 48h, respectively, and each sample is not less than 200 μL.

4. The method for rapidly preparing multi-sized α-synuclein aggregates according to claim 3, characterized in that: In step 3), the samples obtained in step 2) are added into 100 kDa ultrafiltration centrifuge tubes respectively, and centrifuged at 4° C. and 15300 g for 5 min to separate monomers from aggregates.

5. The method for rapidly preparing multi-sized α-synuclein aggregates according to any one of claims 1 to 4, characterized in that: In step 4), add 100 μL of PBS buffer solution to the ultrafilter of each ultrafiltration centrifuge tube, and invert each ultrafilter into a matching centrifuge tube, centrifuge at 4°C and 450 g for 4 min to collect protein aggregates of different sizes on the inner membrane of the ultrafilter, and store them at 4°C for later use.

6. Multi-sized α-synuclein aggregates, characterized in that The rapid preparation method according to any one of claims 1 to 5 is used to prepare the sample by taking samples at different incubation times.

Images