A method for extracting synaptic vesicles

Abstract

The present invention provides a method for extracting synaptic vesicles, comprising the following steps: Step 1): Homogenize the mouse brain with a homogenization buffer; Step 2): Perform a first centrifugation to collect the first supernatant; Step 3): Perform a second centrifugation to collect the second supernatant and the precipitate; Step 4): Add ddH2O to the precipitate and homogenize; Step 5): Perform a third centrifugation to collect the third supernatant to obtain the supernatant; Step 6): Collect the second and third supernatants and perform a fourth centrifugation to collect the fourth supernatant; Step 7): Purify the fourth supernatant by Optiprep density gradient centrifugation; Step 8): Select the layer with the highest vesicle content and elute through a column. Through this extraction method, synaptic vesicle samples can be efficiently extracted from ex vivo mammalian brains. The synaptic vesicles have a high content in proteins, above 25%, even above 30%, and a high purity. The present invention also provides the synaptic vesicles extracted by the above extraction method.

Description

Technical Field

[0001] This invention patent relates to the field of biological extraction technology, and in particular to a method for extracting mammalian synaptic vesicles. Background Art

[0002] Synaptic Vesicles (SVs) are small vesicles at the synaptic terminals of neurons, which are used to store neurotransmitters and are important organelles for neurotransmitter release. Through membrane fusion and secretion, synaptic vesicles release the stored neurotransmitters into the synaptic cleft, where they bind to receptors on the postsynaptic terminal, thus triggering the transmission of nerve signals. Synaptic vesicles store and protect various neurotransmitters, such as acetylcholine, glutamate, and GABA, etc., and release them into the synaptic cleft when needed. By regulating the release rate and quantity of synaptic vesicles, neurons can adjust the intensity and frequency of nerve signals. In addition, the release of synaptic vesicles is also involved in the regulation of synaptic plasticity, which is the ability of neurons in the nervous system to regulate the strength and function of connections, including long-term potentiation and long-term depression, etc.

[0003] Synaptic vesicles play an important role in the nervous system. They are sac-like structures at the neuron terminals, specifically responsible for storing and releasing neurotransmitters. Synaptic vesicles are crucial for the transmission of nerve signals. There are several situations related to diseases associated with synaptic vesicles: Firstly, there are neurotransmitter imbalance diseases. Synaptic vesicles are responsible for storing neurotransmitters such as dopamine and glutamate, and the imbalance of neurotransmitters is related to various associated diseases, such as Parkinson's disease and Alzheimer's disease, etc. Secondly, if the transport of synaptic vesicles is blocked or damaged, it may lead to the occurrence of neurotransmitter transport disorders. For example, genetic defects in synaptic vesicle proteins may cause abnormal neuron functions, ultimately leading to diseases related to movement, cognition, etc. Finally, the abnormal functions of synaptic vesicles are related to some mental diseases, such as schizophrenia and bipolar disorder. These diseases may be related to the abnormal regulation of neurotransmitter release by synaptic vesicles. The pathogenesis of these diseases is very complex and is closely related to the abnormal functions of synaptic vesicles. Studying the structure, function, and regulatory mechanisms of synaptic vesicles helps to deeply understand the occurrence and development of diseases and provides a theoretical basis for developing relevant treatment methods.

[0004] At present, the commonly used methods for extracting synaptic vesicles mainly include ultracentrifugation, antibody passive adsorption method, differential centrifugation method, and soluble protein method. The ultracentrifugation method separates cell tissues through the centrifugation process and separates synaptic vesicles at different centrifugation speeds. However, some small particles, membrane fragments, or vesicles cannot be removed, which will contaminate the vesicles. The antibody passive adsorption method uses specific antibodies to bind to vesicle proteins, and after adsorption on a solid-phase carrier, it is eluted to extract synaptic vesicles. The operation is simple, but there are specific problems and vesicle aggregation damage. The differential centrifugation method separates cell components with different densities through multiple centrifugations. The region with a lower density usually contains synaptic vesicles, but the operation is complex and the vesicle purity is low. The soluble protein method uses specific proteins to bind to synaptic vesicles for purification. For example, thioetherase can improve the vesicle purity, but it still needs further optimization and verification. The above methods have disadvantages such as complex operation, vesicle damage and contamination, low purity, and insufficient specificity.

[0005] Therefore, it is necessary to provide a method for extracting synaptic vesicles with little vesicle damage and high purity. Summary of the Invention

[0006] The present invention aims to solve at least one of the technical problems existing in the prior art to some extent. Thus, in the first aspect of the present invention, the present invention provides a method for extracting synaptic vesicles, including the following steps:

[0007] Step 1): Homogenize the mouse brain with a homogenization buffer.

[0008] Step 2): Perform the first centrifugation and collect the first supernatant.

[0009] Step 3): Perform the second centrifugation on the first supernatant obtained in Step 2), and collect the second supernatant and the precipitate.

[0010] Step 4): Add ddH2O to the precipitate obtained in Step 3) and homogenize.

[0011] Step 5): Perform the third centrifugation on the solution obtained in Step 4), and collect the third supernatant to obtain the supernatant.

[0012] Step 6): Perform the fourth centrifugation on the supernatant obtained in Step 5), and collect the fourth supernatant.

[0013] Step 7): Purify the fourth supernatant obtained in Step 6) by Optiprep density gradient centrifugation.

[0014] Step 8): Select the layer with the highest vesicle content, pass through the column and elute. The packing material of the chromatographic column is Controlled poreglass - BET specific surface area.

[0015] In one or more embodiments of the present invention, in step 1), the homogenization buffer comprises 320 mM sucrose, 4 mM HEPES, and the pH of the homogenization buffer is 7.4; the homogenization buffer further comprises protease inhibitors, and the protease inhibitors include PMSF and pepstatin A.

[0016] In one or more embodiments of the present invention, in step 2), the centrifugal force for the first centrifugation is 900 - 1100 g, and the centrifugation time is 8 - 12 min.

[0017] Preferably, the centrifugal force for the first centrifugation is 1000 g, and the centrifugation time is 10 min.

[0018] In one or more embodiments of the present invention, in step 3), the centrifugal force for the second centrifugation is 14000 - 16000 g, and the centrifugation time is 12 - 18 min.

[0019] Preferably, the centrifugal force for the second centrifugation is 15000 g, and the centrifugation time is 15 min.

[0020] In one or more embodiments of the present invention, in step 5), the centrifugal force for the third centrifugation is 16000 - 18000 g, and the centrifugation time is 12 - 18 min; step 5) further includes combining the third supernatant with the second supernatant to obtain the supernatant.

[0021] Preferably, the centrifugal force for the third centrifugation is 17000 g, and the centrifugation time is 15 min;

[0022] In one or more embodiments of the present invention, in step 6), the centrifugal force for the fourth centrifugation is 47000 - 49000 g, and the centrifugation time is 20 - 30 min.

[0023] Preferably, the centrifugal force for the fourth centrifugation is 48000 g, and the centrifugation time is 25 min.

[0024] In one or more embodiments of the present invention, in step 7), the fourth supernatant obtained in step 6) is purified by Optiprep density gradient centrifugation, including: preparing Optiprep centrifugation solutions with gradient concentrations, adding the Optiprep centrifugation solutions with gradient concentrations into the centrifuge tube respectively, adding the fourth supernatant to the uppermost layer of the Optiprep centrifugation solutions with gradient concentrations, and performing the fifth centrifugation.

[0025] In one or more embodiments of the present invention, in step 7), the mass concentrations of the Optiprep centrifugation solutions in the centrifuge tube from top to bottom are successively: 10%, 18%, 22 - 27%, 30%.

[0026] Preferably, in step 7), the mass concentrations of Optiprep centrifugation liquid in the centrifuge tube from top to bottom are: 10%, 18%, 23.5%, and 30% in sequence.

[0027] In one or more embodiments of the present invention, in step 8), the steps for selecting the layer with the highest vesicle content include:

[0028] a: Establishment of the standard curve of VAMP2 vs. A450 absorbance. Dilute the VAMP2 protein into a concentration gradient proportionally, measure the A450 absorbance with an enzyme-linked immunosorbent assay (ELISA) reader, and establish the standard curve of VAMP2 vs. A450 absorbance;

[0029] b: Establishment of the standard curve of BSA vs. A562 absorbance. Dilute the BSA into a concentration gradient proportionally, measure the A562 absorbance with an ELISA reader, and establish the standard curve of total protein vs. A562 absorbance;

[0030] c: Determine the synaptic vesicle content of each layer by ELISA method. Calculate the ratio of VAMP2 concentration to total protein concentration according to the standard curves in steps a and b. The higher the ratio, the higher the content;

[0031] In one or more embodiments of the present invention, in step 8), the elution is isocratic elution, the flow rate of the eluent is 0.3 ml / min, the eluent contains 250 - 300 mM glycine and 3 - 7 mM HEPES, and the pH of the eluent is 7 - 8.

[0032] In the second aspect of the present invention, the present invention provides a synaptic vesicle obtained by the extraction method described in the first aspect of the present invention.

[0033] The beneficial effects of the present invention are as follows:

[0034] 1. The present invention provides a method for extracting mammalian brain synaptic vesicles. Through this method, synaptic vesicle samples can be efficiently extracted from mammalian brains, providing strong support for synaptic function research and the development of related fields.

[0035] 2. The present invention provides the synaptic vesicles extracted by the above extraction method. The content of these synaptic vesicles in proteins is high, above 25%, even above 30%. The purity of the synaptic vesicles is high, and their morphology is intact without damage.

[0036] 3. The present invention proposes a scheme for isolating SVs from mouse brains, using differential centrifugation, density gradient centrifugation, and size exclusion chromatography methods to maintain high purity without reducing vesicle yield.

[0037] 4. The present invention also provides a method for drug screening or toxicity assessment using the extracted mammalian brain synaptic vesicles. By contacting the substance to be studied with the extracted synaptic vesicle sample and analyzing its effect on the synaptic vesicles, the regulatory effect of the substance to be studied on synaptic function can be evaluated. This provides a rapid and reliable method for synaptic research, drug development, and toxicity assessment, and helps to screen out compounds with potential drug activity or evaluate the safety of drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 It is a flow chart for the extraction of mouse brain synaptic vesicles in Example 1;

[0039] Figure 2 It is a chromatogram of the separated vesicles in Comparative Example 1 and Example 1;

[0040] Among them, Figure 2 A is the chromatogram of the extracted vesicles in Comparative Example 1; Figure 2 B is the chromatogram of the extracted vesicles in Example 1;

[0041] Figure 3 It is for Figure 2 the electron micrographs corresponding to Peak I, Peak II, and Peak III;

[0042] Among them, Figure 3 I is the electron micrograph corresponding to Peak I; Figure 3 II is the electron micrograph corresponding to Peak II; Figure 3 III is the electron micrograph corresponding to Peak III;

[0043] Figure 4 It is the electron micrograph of separating synaptic vesicles by sucrose density gradient centrifugation in Comparative Example 2 and separating synaptic vesicles by Optiprep density gradient centrifugation in Example 1;

[0044] Among them, Figure 4 A is the electron micrograph of separating synaptic vesicles by sucrose density gradient centrifugation in Comparative Example 2; Figure 4 B is the electron micrograph of separating synaptic vesicles by Optiprep density gradient centrifugation in Example 1;

[0045] Figure 5 It is the standard curve of VAMP2 vs. A450 absorbance;

[0046] Figure 6 It is the standard curve of protein vs. A562 absorbance;

[0047] Figure 7 It is the graph of the ratio of VAMP2 concentration to total protein concentration in each layer of Example 1;

[0048] Figure 8 It is the transmission electron microscope (TEM) characterization diagram of Layer 7 in Example 1;

[0049] Figure 9 It is a graph of the ratio of VAMP2 concentration to total protein concentration for each layer in Example 2;

[0050] Figure 10 It is a transmission electron microscope (TEM) characterization graph of the 7th layer in Example 2;

[0051] Figure 11 It is a graph of the ratio of VAMP2 concentration to total protein concentration for each layer in Example 3;

[0052] Figure 12 It is a transmission electron microscope (TEM) characterization graph of the 9th layer in Example 3;

[0053] Figure 13 It is a comparison graph of the ratio of VAMP2 concentration to total protein concentration for each layer in Example 1, Example 2, and Example 3. Detailed implementation manners

[0054] The present invention will be further described below in conjunction with specific embodiments and the accompanying drawings. However, the following embodiments are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. For those not specified in the following embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. The methods used, unless otherwise specified, are all conventional methods well known in the art. The consumables and reagents used, unless otherwise specified, are all commercially available. Unless otherwise stated, the professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art. In addition, any method or material similar or equivalent to the described content can also be applied to the present invention.

[0055] Example 1

[0056] In Example 1, the flow chart of the extraction method of mammalian brain synaptic vesicles is as Figure 1 shown. Specifically, the extraction method of mammalian brain synaptic vesicles includes the following steps:

[0057] (1) Add 9 ml of homogenization buffer (9 μl of 200 mM PMSF and 9 μl of 1 mg / ml pepstatin A) to homogenize the excised mouse brain 10 times at a speed of 900 rpm / min. Since PMSF is unstable in aqueous solution, protease inhibitors should be immediately added to the homogenization buffer. Stir the solution while adding protease inhibitors to avoid precipitation. Homogenization buffer: 320 mM sucrose, 4 mM HEPES (pH 7.4).

[0058] (2) Centrifuge at 1,000 g for 10 min at 4 °C and collect the supernatant (S1). When collecting the supernatant, avoid sucking up the precipitate.

[0059] (3) Centrifuge at 4°C and 15,000 g for 15 min, collect the supernatant (S2) and store it on ice. Resuspend the pellet (P2) in 1 ml of homogenization buffer and wash the pellet.

[0060] (4) Add 9 ml of ddH2O to the pellet (P2) and homogenize 3 times at 2,000 rpm / min to release SVs from the synaptosomes. Immediately add 50 μl of 1 M HEPES and protease inhibitors (9 μl of 200 mM PMSF and 9 μl of 1 mg / ml pepstatin A).

[0061] (5) Centrifuge the lysate at 4°C and 17,000 g for 15 min to remove contaminants such as mitochondria, myelin, etc. Collect the supernatant (LS1) and combine it with S2.

[0062] (6) Centrifuge at 4°C and 48,000 g for 25 min, collect the supernatant (CS1). Homogenize 3 - 5 times at 900 rpm / min to avoid aggregation of SVs.

[0063] (7) Optiprep density gradient preparation: Slowly add Optiprep solutions with different concentrations into PET thin-walled Tubes centrifuge tubes in order from high to low density, place them symmetrically in the centrifuge to ensure the center of gravity balance during centrifugation; The Optiprep solutions from top to bottom are: 2 ml of 10%, 2 ml of 18%, 2 ml of 23.5%, 2 ml of 30% Optiprep density gradient centrifugation liquid, and also place it on ice during density extraction.

[0064] (8) Add the supernatant (CS1) after homogenization in step (6) to the top layer of the Optiprep density gradient centrifugation liquid, and centrifuge at 4°C and 32,000 rpm for 4 h.

[0065] (9) Select the layer with the highest content of synaptic vesicles:

[0066] a: Establishment of the standard curve of VAMP2 vs. A450 absorbance

[0067] VAMP2 is one of the main components on the synaptic vesicle membrane, mainly distributed on the synaptic vesicle membrane of neurons, and participates in the fusion process of synaptic vesicles and neuronal cell membranes by interacting with other membrane proteins (such as SNARE proteins). Since VAMP2 is an important protein on the synaptic vesicle membrane, the purity of synaptic vesicles can be judged by its ratio to the total protein.

[0068] The VAMP2 protein was diluted proportionally into 7 concentration gradients (0, 0.01, 0.02, 0.05, 0.08, 0.1, 0.2 ng / μl), with 3 parallel samples for each concentration. The absorbance at A450 was measured using a microplate reader. With the concentration of VAMP2 as the abscissa and the absorbance values at each concentration under A450 as the ordinate, the standard curves for each of the above 7 concentration ranges were plotted. The results are as shown in Figure 5 shown. The VAMP2 protein concentration of the sample can be calculated based on the standard curve.

[0069] b: Establishment of the standard curve for BSA and the absorbance at A562

[0070] BSA (5 mg / mL) was diluted proportionally into 8 concentration gradients (0, 0.05, 0.1, 0.15, 0.2, 0.3, 0.4, 0.5 μg / μl), with 3 parallel samples for each concentration. The absorbance at A562 was measured using a microplate reader. With the concentration of BSA as the abscissa and the absorbance values at each concentration under A562 as the ordinate, the standard curves for each of the above 8 concentration ranges were plotted. The results are as shown in Figure 6 shown. The protein concentration of the sample can be calculated based on the standard curve.

[0071] c. Determination of the synaptic vesicle content in each fraction by ELISA

[0072] 10 fractions were collected separately from the top of the Optiprep gradient (as shown in Figure 1 , namely the sample layer, interface, 2 ml 10%, interface, 2 ml 18%, interface, 2 ml 23.5%, interface, 2 ml 30%, bottom of the tube). To determine which fractions were enriched in SVs, the synaptic vesicle content was quantified using ELISA. 100 μl of the liquid from each layer was taken into a 96-well plate, and the VAMP2 protein was gradually mixed from 0 to 0.2 ng / μl, 100 μl / well, and incubated overnight. The next day, after washing 3 times with 300 μl / well of PBST, it was blocked with 0.2% BSA in PBST for 1 hour. The VAMP2 antibody was diluted 1:2000 in 100 μl per well and incubated at room temperature for 1 h. After incubation, it was washed 3 times with PBST, 300 μl / well. Then it was incubated with HRP-labeled goat anti-rabbit (1:5000) for 1 hour. Finally, after incubating with the chromogenic solution for 15 min, 100 μl of the stop solution was added, and the absorbance at 450 nm was measured. From the absorbance obtained in the previous step, the VAMP2 concentration can be calculated. The ratio of the VAMP2 concentration to the total protein concentration in each layer is as shown in Figure 7 shown. The higher the ratio, the higher the purity of the synaptic vesicles in this layer.

[0073] d. Electron microscopy characterization of synaptic vesicles

[0074] The higher the VAMP2 content is in the total protein value, the higher the purity of synaptic vesicles in this layer. Therefore, the 7th layer was selected for TEM imaging. The specific process is as follows: 10 μL of synaptic vesicles were dropped onto the sealing film. The copper grid was placed in contact with the droplet of the nanomaterial solution on the front side to suspend it, and then incubated for 10 min. The excess solution on the copper grid was blotted off with filter paper. Then, 10 μL of uranyl acetate solution was dropped onto the sealing film on the copper grid. The copper grid was placed in contact with the droplet of uranyl acetate solution on the front side and incubated for 10 min. The excess dye solution on the copper grid was blotted off with filter paper, and the dye solution was allowed to evaporate completely. Transmission electron microscopy (TEM) was used to characterize the synaptic vesicles, and the results are as Figure 8 shown.

[0075] (10) The layer with the highest SVs content (the 7th layer) was loaded onto a chromatographic column (100 cm × 1 cm) packed with Controlled poreglass - BET specific surface area (MilliporeSigma, NIST2206) and equilibrated. The column was then rinsed with an eluent containing 300 mM glycine, 5 mM HEPES, pH 7.4. The device was connected to a peristaltic pump and operated at a flow rate of 0.3 ml / min. After the chromatographic column run was completed, the chromatogram was as Figure 2 shown in Figure B. The TEM images of the substances corresponding to peaks I, II, and III in the chromatogram are as Figure 3 shown; Figure 3 I is the TEM image corresponding to peak I; Figure 3 II is the TEM image corresponding to peak II;

[0076] Figure 3 III is the TEM image corresponding to peak III. As can be seen from the figure, the first peak contains larger membrane fragments (in the range of 100 - 400 nm); the second peak contains microvesicles (about 100 nm); the SVs (50 nm in diameter) should be concentrated in the third peak. The signal change at 280 nm of the eluent was monitored by UV. An eluent collector was used to collect the eluent passing through the UV monitor. To obtain the best separation effect, the size of each fraction was 1 ml.

[0077] Example 2

[0078] The difference between Example 2 and Example 1 is only that in step (7), the Optiprep density gradient from top to bottom is: 2 ml of 10%, 2 ml of 18%, 2 ml of 22%, and 2 ml of 30% Optiprep density gradient centrifugation solution.

[0079] As a result, in step (9) of Example 2, the ratio of the VAMP2 concentration to the total protein concentration in each layer is as Figure 9 shown. Transmission electron microscopy (TEM) was used to characterize the synaptic vesicles, and the results are as Figure 10 shown.

[0080] Example 3

[0081] The difference between Example 3 and Example 1 is only that in step (7), the Optiprep density gradient from top to bottom is: 2 ml of 10%, 2 ml of 18%, 2 ml of 27%, and 2 ml of 30% Optiprep density gradient centrifugation solution.

[0082] As a result, in step (9) of Example 3, the ratio of the VAMP2 concentration to the total protein concentration in each layer is as Figure 11 shown. Transmission electron microscopy (TEM) was used to characterize synaptic vesicles, and the results are as Figure 12 shown. The comparison chart of the ratio of the VAMP2 concentration to the total protein concentration in each layer of Example 1, Example 2, and Example 3 is as Figure 13 shown.

[0083] Comparing Example 1, Example 2, and Example 3, it can be seen that when the mass concentrations of the Optiprep centrifugation solution from top to bottom in the centrifuge tube are 10%, 18%, 23.5%, and 30% in turn, the extraction effect is the best, the vesicle morphology is complete, the size is about 50 nm, the vesicle purity is higher, and there are no impurities such as cell debris.

[0084] Comparative Example 1

[0085] The difference between Comparative Example 1 and Example 1 is only that in step (10), the filler Controlledpore glass - BET specific surface area (MilliporeSigma, NIST2206) in the chromatographic column is replaced with Sepharose CL - 4B (Youningwei, 17015001).

[0086] After the chromatographic column operation ended, the chromatogram was as Figure 2 shown in A. Comparing the example and the comparative example, it can be seen that the separation products of the Controlledpore glass - BET specific surface area chromatographic column are concentrated in peak three, indicating that the Controlledpore glass - BET specific surface area chromatographic column has a better separation effect.

[0087] Comparative Example 2

[0088] The difference between Comparative Example 2 and Example 1 is only that in step (7), sucrose density gradient centrifugation is used instead of Optiprep density gradient centrifugation.

[0089] The inventors explored the purification by sucrose density gradient centrifugation with different concentration gradients and found that when the mass concentrations of sucrose in the centrifuge tube from bottom to top were 30%, 23.5%, 18%, and 10% respectively, the purification effect was the best. Then, this sucrose concentration gradient was used for comparison with Example 1. The specific operations of steps (7) and (8) in Comparative Example 2 are as follows:

[0090] (7) Slowly add sucrose solutions with different concentrations into the PET thin-walled Tubes centrifuge tubes in order from high density to low density, and place them symmetrically in the centrifuge to ensure the center of gravity balance during centrifugation; the addition amount and mass concentration from bottom to top in the centrifuge tube are as follows: 2 ml of 30%, 2 ml of 23.5%, 2 ml of 18%, 2 ml of 10%;

[0091] (8) Add the supernatant (CS1) homogenized in step (6) to the top layer of the sucrose density gradient centrifugation solution, and centrifuge at 32000 rpm for 4 h at 4°C.

[0092] In step (9), transmission electron microscopy (TEM) was used to characterize synaptic vesicles, and the results are as Figure 4 shown in A. In Example 1 Figure 8 corresponding to Figure 4 B. As can be seen from the figure, the recovery rate of Optiprep density gradient centrifugation is higher, the vesicle morphology is more complete, and the purity is also higher ( Figure 4 B). This may be because the osmotic pressure of sucrose is relatively high, which will affect the vesicle morphology and the size is not uniform.

[0093] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention, and all of them should be included in the protection scope of the present invention.

Claims

1. A method for extracting synaptic vesicles, characterized in that: The steps include: Step 1): Homogenize the mouse brain with homogenization buffer; Step 2): first centrifugation, collecting the first supernatant; Step 3): performing a second centrifugation on the first supernatant obtained in step 2), and collecting the second supernatant and the precipitate; Step 4): Add ddH2O to the precipitate obtained in step 3) and homogenize; Step 5): performing a third centrifugation on the solution obtained in step 4), collecting a third supernatant, and obtaining a supernatant; Step 6): performing a fourth centrifugation on the supernatant obtained in step 5) to collect the fourth supernatant; Step 7): Purify the fourth supernatant obtained in step 6) by Optiprep density gradient centrifugation; Step 8): Select the layer with the highest vesicle content and elute it through a column. The column filler is Controlled pore glass- BET specific surface area NIST2206; In the step 7), the fourth supernatant obtained in step 6) is purified by Optiprep density gradient centrifugation, comprising: preparing Optiprep centrifugal solutions of gradient concentrations, adding Optiprep centrifugal solutions of gradient concentrations to centrifuge tubes respectively, adding the fourth supernatant to the top layer of the Optiprep centrifugal solutions of gradient concentrations, and performing a fifth centrifugation; In step 7), the mass concentrations of the Optiprep centrifuge solution in the centrifuge tube from top to bottom are: 10%, 18%, 22-27%, and 30%; In the step 8), the elution is isocratic elution, the eluent contains 250-300 mM glycine, 3-7 mM HEPES, and the pH of the eluent is 7-8.

2. The method for extracting synaptic vesicles according to claim 1, characterized in that: In the step 1), the homogenization buffer comprises 320 mM sucrose and 4 mM HEPES, the pH value of the homogenization buffer is 7.4, and the homogenization buffer further comprises protease inhibitors, wherein the protease inhibitors comprise PMSF and pepstatin A.

3. The method for extracting synaptic vesicles according to claim 1, characterized in that: In the step 2), the centrifugal force of the first centrifugation is 900-1100 g, and the centrifugation time is 8-12 min.

4. The method for extracting synaptic vesicles according to claim 1, characterized in that: In step 3), the centrifugal force of the second centrifugation is 14000-16000 g, and the centrifugation time is 12-18 min.

5. The method for extracting synaptic vesicles according to claim 1, characterized in that: In the step 5), the centrifugal force of the third centrifugation is 16000-18000 g, and the centrifugation time is 12-18 min; the step 5) further includes combining the third supernatant with the second supernatant to obtain the supernatant.

6. The method for extracting synaptic vesicles according to claim 1, characterized in that: In the step 6), the centrifugal force of the fourth centrifugation is 47000-49000g, and the centrifugation time is 20-30 min.

7. The method for extracting synaptic vesicles according to claim 1, characterized in that: In step 8), selecting a layer with the highest vesicle content comprises the following steps: a: Establishment of the standard curve of VAMP2 and A450 absorbance: VAMP2 protein was diluted into a concentration gradient in proportion, and the A450 absorbance was measured with an ELISA instrument to establish the standard curve of VAMP2 and A450 absorbance; b: Establishment of the standard curve of BSA and A562 absorbance: BSA was diluted proportionally into a concentration gradient, and the absorbance of A562 was measured with an ELISA instrument to establish a standard curve of total protein and A562 absorbance; c: The synaptic vesicle content of each layer was determined by ELISA, and the ratio of VAMP2 concentration to total protein concentration was calculated according to the standard curves of step a and step b. The higher the ratio, the higher the content; In the step 8), the eluent flow rate is 0.3 ml / min.

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