Synaptic vesicle-nanodisk fusion system and application thereof in drug screening

Abstract

The invention provides a synaptic vesicle-nanodisk fusion system which comprises a synaptic vesicle and a nanodisk, and the volume ratio of the synaptic vesicle to the nanodisk is (7-9): 1. The invention also provides an application of the synaptic vesicle-nanodisk fusion system in drug screening. According to the synaptic vesicle-nanodisk fusion system provided by the invention, direct detection and quantitative analysis of neurotransmitter release are realized through the fusion process of the synaptic vesicles and the nanodisk simulating the presynaptic membrane. Compared with the prior art, the detection time is shortened and is controlled within 5 minutes. The synaptic vesicle-nanodisk fusion system provided by the invention can also be applied to screening and action mechanism research of neural active substances, new neural active substances can be screened and found by evaluating the influence of different drug molecules or protein factors on synaptic vesicle fusion and neurotransmitter release, and the action mechanism of the new neural active substances can be deeply discussed, so that the new neural active substances can be found. And new research tools and ideas are provided for the development of medicines for treating nervous system diseases.

Description

Technical Field

[0001] The invention patent relates to the field of biotechnology, and in particular to a synaptic vesicle-nanodisc fusion system and its application in drug screening. Background Art

[0002] In the field of neurobiology, in vitro fusion experiments are a commonly used technique to simulate the fusion of synaptic vesicles and presynaptic membranes in vivo. They are widely used because they provide an open platform for researchers to introduce various proteins or drugs that regulate the fusion of synaptic vesicles and presynaptic membranes. The current mainstream in vitro fusion experiments are based on the fusion of two liposomes representing synaptic vesicles and presynaptic membranes, respectively.

[0003] The liposomes used in fusion experiments are similar in shape to synaptic vesicles, usually spherical with a diameter of about 50nm, composed of phospholipids and transmembrane proteins. The types of phospholipids and proteins vary according to the target they simulate. For example, liposomes that simulate synaptic vesicles are synthesized from a phospholipid mixture containing a higher proportion of acidic phospholipids, while liposomes that simulate presynaptic membranes contain a lower proportion of acidic phospholipids. In terms of proteins, liposomes that simulate synaptic vesicles usually carry the core protein VAMP2 that is involved in the fusion of synaptic vesicles and presynaptic membranes. This protein is also a signature protein of synaptic vesicles, while liposomes that simulate presynaptic membranes carry the core proteins SNAP25 and Syntaxin-1A that are involved in the fusion. These three proteins have high affinity, and when combined together, they form a fusion machine called the SNARE complex, which can shorten the distance between synaptic vesicles and presynaptic membranes and promote membrane fusion. VAMP2 is also called v-SNARE in this complex because it exists on the vesicle membrane, and SNAP25 and Syntaxin-1A are called t-SNAREs in this complex because they exist on the presynaptic membrane (also called target membrane). This complex is the core protein complex involved in fusion, the smallest unit for studying the fusion process, and the target of many proteins and drugs. By affecting the function of this core protein complex, the fusion process of the two membranes can be directly affected, and ultimately the release of neurotransmitters can be affected.

[0004] The existing in vitro simulation of the fusion of synaptic vesicles and presynaptic membranes during neurotransmitter release is mainly based on the synthetic artificial liposome scheme, which synthesizes two liposomes in vitro, one is v-liposome (v-liposome) and the other is t-liposome (t-liposome). Among them, v-liposome refers to a liposome with a protein such as VAMP2 (referred to as v-SNARE) inserted, which simulates synaptic vesicles; the other t-liposome refers to a liposome with the addition of protein t-SNAREs (t-SNAREs are composed of SNAP25 and Syntaxin-1A in a molar ratio of 1:1), which simulates the presynaptic membrane. The synthesis method of v-liposomes is to mix POPC, DOPS, NBD-DOPE and Rhodamine-DOPE in a molar ratio of 72% in chloroform, vortex to mix and dry the phospholipids under nitrogen for 15 minutes, then vacuum dry for 1-2 hours, and resuspend the dried phospholipids in buffer (1M HEPES, 2M KCl, 0.2mMTCEP, 10% glycerol and 1.5% OG), and then add phospholipid mixture to protein at a molar ratio of 1:1. v-SANRE was added at a ratio of 100:1, and after thorough mixing, 1.5% OG was removed by dialysis overnight. The next day, the liposomes were purified by density gradient centrifugation (30% Optiprep, 20% Optiprep, and the sample to be purified from bottom to top) (55000rpm, 5.5h). Finally, a clear white liposome layer can be found in the 20% Optiprep layer, which can be obtained by aspirating 200 microliters with a long needle. Similarly, the preparation of t-liposomes is similar to that of v-liposomes, except that the phospholipid composition becomes POPC with a molar ratio of 85% and DOPS with a molar ratio of 15%, and the added protein becomes t-SNAREs (the molar ratio of SNAP25 to Syntaxin-1A is 1:1). After the two liposomes are prepared, they can be used. Mix v-liposomes and t-liposomes in a volume ratio of 1:9, incubate at 4°C in the dark for 2 hours, add them to a black 96-well plate, put them into an ELISA reader, adjust the kinetic parameters (the excitation wavelength of the ELISA reader is set to 460nm, and the detection wavelength is set to 535nm) to measure the results. Figure 1As shown, v-liposomes with v-SNARE inserted and t-liposomes with t-SNAREs inserted will form SNARE complexes together. The complex protein is the core component that mediates the fusion of synaptic vesicles and presynaptic membranes in neurons, and mediates the fusion of v-liposomes and t-liposomes here. In this scheme, the fusion is detected using a pair of fluorescent dyes NBD-RhoB. The principle is: within a certain distance range, the stimulation of excitation light of a suitable wavelength (460nm) will cause the donor (NBD) to produce emission light, which can become the excitation light of the receptor (RhoB) and be absorbed by the receptor. Beyond this distance, the donor's emission light (535nm) cannot be absorbed by the receptor and is detected by us. As Figure 1 As shown, one of the two liposomes carries the pair of fluorescent dyes, while the other does not. When fusion occurs, the distance between the donor and the acceptor increases and energy transfer cannot be completed, which results in an increase in the donor emission light (535 nm) detected, thus showing Figure 5 The right figure shows the curve change of fusion, the horizontal axis represents time, and the vertical axis represents the percentage of fusion. This scheme is often used to simulate the fusion process of synaptic vesicles and presynaptic membranes in vitro, and further research can be carried out by adding proteins of interest in the preparation of liposomes or during the fusion process. However, it has obvious shortcomings, mainly manifested in: ① Spherical liposomes with higher curvature cannot objectively simulate the low curvature presynaptic membrane in vivo. In other words, the contact between two spheres cannot well simulate the contact effect between a sphere and a plane, and many studies have shown that curvature is an important physical parameter affecting synaptic release. ② Even if the two liposomes fuse, that is, the fluorescence increases, it cannot directly indicate that the neurotransmitter is released, and the release of neurotransmitters is the ultimate goal of fusion. Because the kiss and run phenomenon will occur both in vivo and in vitro. In other words, the fusion occurs in a very short time, completing part of the phospholipid exchange and increasing the distance between the NBD-RhoB fluorescent dyes, and the fusion signal is enhanced, but there is no time to release the neurotransmitter, so this scheme can only study the fusion, and cannot directly reflect the release of neurotransmitters. ③It is not enough to insert only VAMP2 protein. The present invention found that the fusion degree of vesicles derived from mouse brain is higher and faster. If the protein required for fusion is further inserted into the liposome, the workload of protein extraction may also change the original structure and size of the liposome. Moreover, there is no neurotransmitter in the liposome that simulates synaptic vesicles, and the neurotransmitter of interest needs to be packed into it in advance, so directly using synaptic vesicles extracted from mouse brain becomes the most appropriate choice.

[0005] Therefore, it is necessary to provide a more suitable in vitro system for simulating the fusion of synaptic vesicles and presynaptic membranes during neurotransmitter release. Summary of the invention

[0006] Nanodisc is a synthetic disc-like membrane structure. Compared with liposomes, its curvature is very low, which can more realistically simulate the existence of the presynaptic membrane. Nanodiscs are also composed of phospholipids and proteins, but the difference from liposomes is that in order to maintain its disc structure, it is necessary to add an amphiphilic membrane scaffold protein (such as MSP) to surround the periphery of the phospholipid bilayer and use it to laterally bind the phospholipid bilayer, thereby forming a disc-like structure with extremely low curvature. The diameter of the disc is determined by adjusting the molar ratio of the membrane scaffold protein and phospholipids. Similar to liposomes, nanodiscs can also be used as carriers of the above-mentioned transmembrane proteins and membrane interaction proteins, such as t-SNAREs, thereby simulating the presynaptic membrane in terms of morphology and function.

[0007] Synaptic vesicles are a type of small organelles that exist in the animal nervous system and are used to store and release neurotransmitters. They are most densely distributed in the central nervous system, that is, the animal brain. When excitement is transmitted to the axon terminal, the voltage-gated calcium channel opens, calcium ions flow in, and the synaptic vesicles fuse with the presynaptic membrane, leading to the synchronous release of neurotransmitters. Synaptic vesicles isolated and purified from the brains of experimental animals can be used as ideal materials for studying fusion and release because they retain all their original functions, contain all proteins that are difficult to achieve membrane fusion in artificially prepared liposomes, and are also wrapped with various neurotransmitters.

[0008] The present invention relates to a method for fusing nanodisks with synaptic vesicles to detect the effect of target proteins or drugs on neurotransmitter release in vitro. The mainstream in vitro fusion experiments of artificial synthetic liposomes have many shortcomings. For example, the high-curvature liposome vesicles containing t-SNAREs cannot simulate the smooth surface of the presynaptic membrane with low curvature; the liposome vesicles containing v-SNAREs are difficult to replace the real synaptic vesicles, because the synaptic vesicles extracted from the body are small organelles containing all proteins and encapsulating neurotransmitters, and there are multiple drug response sites on them that regulate neurotransmitter release; liposomes can only be fused and detected, and neurotransmitter release cannot be detected, because even if the neurotransmitter is pre-encapsulated in the liposome, the two liposomes are still a closed space after fusion, and the neurotransmitter cannot be released into the external solution for detection by us. Therefore, in view of the shortcomings of the above-mentioned liposome in vitro fusion experiment, we used the fusion of synaptic vesicles and nanodiscs. First, nanodiscs can provide a smooth surface with low curvature, which can better simulate the presynaptic membrane in terms of morphology; secondly, the use of synaptic vesicles from mouse brain can simulate the fusion and release under physiological conditions to a great extent; thirdly, nanodiscs are used to release the original neurotransmitters in synaptic vesicles, which can open the synaptic vesicles and detect the neurotransmitters directly into the external solution, thereby realizing the direct detection of neurotransmitters; finally, we have also improved the composition of nanodiscs by adding a special phospholipid - phosphatidylinositol-4,5-bisphosphate (PIP2), which is the effector phospholipid of many synaptic vesicle proteins (such as the calcium sensor protein Synaptotagmin-1), which can assist the conformational change of Synaptotagmin-1 after binding to calcium ions and promote the occurrence of fusion. In addition, the openness of this fusion system makes it possible to directly detect the effects of different proteins or drugs on the fusion of synaptic vesicles and the release of neurotransmitters in synaptic vesicles, thereby enabling us to conduct a variety of in vitro studies such as target protein research, drug screening, and disease treatment.

[0009] The present invention aims to solve one of the technical problems existing in the prior art to at least a certain extent. Therefore, in a first aspect of the present invention, the present invention provides a synaptic vesicle-nanodisc fusion system, comprising synaptic vesicles and nanodiscs, wherein the volume ratio of the synaptic vesicles to the nanodiscs is 7-9:1.

[0010] In one or more embodiments of the present invention, the nanodisc raw material has a molar ratio of 55-65:0.5-1.5:1.5-2.5 of phospholipid mixture, MSP and t-SNAREs.

[0011] In one or more embodiments of the present invention, the raw materials of the phospholipid mixture include 78-83% POPC, 13-18% DOPS, 0.5-1.5% PIP2, 1-2% NBD-DOPE and 1-2% Rhodamine-DOPE in molar ratio.

[0012] In one or more embodiments of the present invention, the preparation method of the phospholipid mixture includes: mixing POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), DOPS (1,2-dioleoyl-sn-glycero-3-phospho-L-serine (sodium salt)), PIP2 (1,2-dioleoyl-sn-glycero-3-phospho-(1'-inositol-4',5'-diphosphate) (ammonium salt)), NBD-DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazole-4-yl)) and Rhodamine-DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl)) to obtain the phospholipid mixture.

[0013] In one or more embodiments of the present invention, the preparation of the nanodisc comprises the following steps: mixing, standing, incubating, shaking, and purifying the phospholipid mixture, MSP, and t-SNAREs to obtain the nanodisc.

[0014] In one or more embodiments of the present invention, the purification comprises: gel filtration column incubation separation.

[0015] In one or more embodiments of the present invention, the method for preparing synaptic vesicles comprises:

[0016] Step 1): Soak the mouse brain in a buffer solution (the buffer solution consists of 320 mM sucrose and 4 mM HEPES pH=7.4), and homogenize the mouse brain using a homogenizer;

[0017] Step 2): first centrifugation, second centrifugation of the supernatant obtained by the first centrifugation, hypotonic disruption of the synaptosome precipitate obtained by the second centrifugation, and third centrifugation;

[0018] Step 3): Purify the supernatant obtained from the third centrifugation by Optiprep density gradient centrifugation, select the layer with the highest synaptic vesicle content and take it out.

[0019] In one or more embodiments of the present invention, in step 2), the centrifugal force of the first centrifugation is 950-1050g, and the centrifugation time is 8-12min; the centrifugal force of the second centrifugation is 14000-16000g, and the centrifugation time is 12-18min; the centrifugal force of the third centrifugation is 16000-18000g, and the centrifugation time is 20-30min.

[0020] In one or more embodiments of the present invention, in step 3), the mass concentrations of the Optiprep centrifuge solution in the centrifuge tube from top to bottom are: 0%, 16%, 23.5%, and 30%.

[0021] In a second aspect of the present invention, the present invention provides a use of the synaptic vesicle-nanodisc fusion system described in the first aspect of the present invention in drug screening.

[0022] The beneficial effects of the present invention are:

[0023] 1. The present invention provides a synaptic vesicle-nanodisc fusion system, constructs an in vitro synaptic vesicle fusion and neurotransmitter release detection system close to physiological conditions, and realizes direct detection and quantitative analysis of neurotransmitter release through the fusion process of synaptic vesicles and nanodiscs simulating postsynaptic membranes. Compared with the prior art, the detection time is shortened and controlled within 5 minutes.

[0024] 2. The present invention provides an application of the synaptic vesicle-nanodisc fusion system described in the first aspect of the present invention in drug screening.

[0025] 3. The present invention provides a synaptic vesicle-nanodisc fusion system that can be applied to the screening of neuroactive substances and the study of their mechanism of action. By using the constructed in vitro fusion detection system, by evaluating the effects of different drug molecules or protein factors on synaptic vesicle fusion and neurotransmitter release, new neuroactive substances can be screened and discovered, and their mechanism of action can be explored in depth, providing new research tools and ideas for the development of drugs for nervous system diseases.

[0026] 4. The present invention has important application value in basic research of neurobiology and drug development for nervous system diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is a schematic diagram of the fusion of synaptic vesicles and presynaptic membrane in the in vitro simulation of neurotransmitter release in the prior art;

[0028] Figure 2 This is a peak diagram of the nanodisc purification process in Example 1;

[0029] Figure 3 for Figure 2Protein gel image of the sample with peak 1;

[0030] Figure 4 This is a distribution diagram of synaptic vesicles and impurities after density gradient centrifugation in Example 2;

[0031] Figure 5 A comparison diagram of the fusion curves of the synaptic vesicles and nanodiscs of the present invention and the fusion curves of the liposomes in the background art;

[0032] Figure 6 Schematic diagram of neurotransmitter release by fusion of nanodiscs with synaptic vesicles;

[0033] Figure 7 This is a comparison chart of the fusion curves of Example 4 with the target protein added and Example 3 step (1) without the target protein added;

[0034] Figure 8 This is a comparison chart of dopamine (DA) release in Example 4 with the target protein added and in Example 3 step (1) without the target protein added in the fusion experiment;

[0035] Fig. 9 This is a comparison chart of the fusion curves of Example 5 with the addition of the agonist phorbol ester and Example 3 step (1) fusion experiment without the addition of the agonist phorbol ester. DETAILED DESCRIPTION

[0036] The present invention will be further described below in conjunction with specific examples and accompanying drawings, but the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. In the following examples, if specific conditions are not specified, they are carried out according to normal conditions or conditions recommended by the manufacturer. The methods used are conventional methods known in the art unless otherwise specified, and the consumables and reagents used are commercially available unless otherwise specified. Unless otherwise specified, 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 recorded content may also be applied to the present invention.

[0037] Example 1: Preparation of Nanodisks

[0038] Nanodisk fabrication involves the following steps:

[0039] (1) Phospholipids POPC, DOPS, PIP2, NBD-DOPE, and Rhodamine-DOPE were mixed at a molar ratio of 81%:15%:1%:1.5%:1.5%, and the lipids were dried under N2 gas for 15 minutes and then vacuum dried for 1-2 hours.

[0040] (2) Use the pre-prepared nanodisc buffer (25mM HEPES, 140mM KCl, 10% glycerol, 0.2mMTCEP, 1mM MgCl2, 1% OG, pH 7.4) to resuspend the lipids in step (1) and mix them in the nanodisc buffer system according to the molar ratio of phospholipid mixture: MSP: t-SNARE = ​​60:1:2 (where t-SNAREs are composed of SNAP25 and Syntaxin1A in a molar ratio of 1:1). MSP (membrane scaffold protein), SNAP25 (synaptosome-associated protein 25kDa) and Syntaxin1A (synaptic fusion protein 1A) can all be purchased from the abcam official website. Mix in a vortex at room temperature for 15 minutes, then stand at 4°C for at least 4 hours with gentle shaking. Finally, transfer to 1g Bio-beads [Bio-beads TM SM2 (nonpolar polystyrene adsorbent)] and continue incubation at 4°C overnight with gentle shaking to remove 1% detergent OG in the buffer.

[0041] (3) The next day, the Akta protein purification system was used, and the Superdex 200 10 / 300GL gel filtration column was used to separate the samples incubated overnight, in order to separate the nanodisks from impurities. The specific operation method is to equilibrate the gel filtration column with PBS buffer in advance, and then load the nanodisks to be purified into the column. The detector is set to start collecting the liquid flowing through the column when the absorbance is greater than 5mAU, and stop collecting when it is less than 5mAU. The final peak shape and collection status are shown in Figure 2. Figure 2 As shown. In the figure, the ordinate represents the absorption value (UV value) of the protein at a wavelength of 280nm, in mAU, the abscissa represents the volume of the buffer solution flowing through, in ml, and the peak area represents the relative content of the protein flowing through the detector during this period of time. The first peak represents the nanodisc that has been successfully inserted with t-SNAREs, the second peak represents an empty nanodisc, and the third peak represents free protein. The liquid in the first peak is collected and retained, and it contains the nanodisc that has been successfully inserted with t-SNAREs. According to the principle of size exclusion, the larger the particles, the faster they elute and the sooner they flow out of the gel filtration column. The diameter of the nanodisc inserted with t-SNAREs is larger than that of the other impurities, so it flows out first, and this nanodisc contains a higher protein content than the other impurities, which is reflected in Figure 2 The peak value and peak area are the largest, so we can follow Figure 2 The peak graph collects the liquid effluent from the first peak.

[0042] (4) The liquid in peak 1 was collected, concentrated to an appropriate concentration, and then run on an SDS protein gel to verify whether it was the target nanodisc. Figure 3 This is the protein gel image of the sample of peak 1, with a sample loading of 5 μg. From bottom to top, the proteins are MSP, SNAP25, and Syntaxin1A (the latter two are collectively referred to as t-SNAREs proteins in a 1:1 molar ratio). This proves that the nanodisc containing t-SNAREs was successfully produced.

[0043] Example 2: Extraction of synaptic vesicles

[0044] Synaptic vesicles were extracted by a combination of differential centrifugation and density gradient centrifugation using Optiprep, including the following steps:

[0045] (1) The mouse brain was removed and homogenized with a homogenization buffer (320 mM sucrose and 4 mM HEPES pH = 7.4) to obtain a mouse brain homogenate. The homogenate was centrifuged at 1000 g for 10 min to remove the cell nuclei, and then the supernatant was collected. The supernatant was centrifuged at 15000 g for 15 min to precipitate the mitochondria and synaptosomes, and the precipitate was resuspended with pure water and homogenization buffer (320 mM sucrose and 4 mM HEPES pH = 7.4) at a ratio of 9:1 to break the synaptosomes and release synaptic vesicles. The broken homogenate was centrifuged at 17000 g for 25 min to precipitate and remove impurities such as mitochondria, and then the supernatant was collected.

[0046] (2) Dilute 60% Optiprep to 30%, 23.5%, 16%, and 0% with homogenization buffer (320 mM sucrose and 4 mM HEPES pH = 7.4), add to the centrifuge tube from bottom to top, and load the supernatant of the previous step to the top of the Optiprep gradient. Centrifuge at 19000g for 5 h. Figure 1 The 7th layer in the left picture is the synaptic vesicle layer; the right picture is a concentration test of VAMP2 protein, a signature protein on synaptic vesicles, which confirms that the 7th layer is the synaptic vesicle layer.

[0047] (3) Use a long needle to remove the synaptic vesicle layer and detect the total protein concentration using the BCA method before use.

[0048] Example 3: Synaptic vesicle fusion experiment and neurotransmitter detection experiment

[0049] (1) Fusion experiment

[0050] The synaptic vesicles prepared in Example 2 were mixed with the nanodisks prepared in Example 1 at a volume ratio of 9:1, wherein the total protein concentration of the synaptic vesicles was 0.5 μg / μL, and the total volume of the two was 50 μL, and then detected in a microplate reader at 37°C for 10 minutes, and the program was set to detect one point every 30 seconds. Subsequently, 1.5% of the detergent OG (octyl-β-D-pyranoglucoside) was added to fully rupture the synaptic vesicles and nanodisks and reach the maximum fluorescence value of NBD-DOPE, and then detected in a microplate reader for 10 minutes. Finally, the formula: fusion degree = (fluorescence intensity detected at each time point - initial fluorescence intensity) / (maximum fluorescence intensity after adding detergent - initial fluorescence intensity) was used to calculate and make a fusion curve. The results are as follows. Figure 5 As shown, Figure 5 The fusion curves of the synaptic vesicles and nanodiscs of the present invention are compared with the fusion curves of the liposomes in the background art, wherein Figure 5 The left side is the fusion curve of the synaptic vesicle and nanodisc of the present invention; Figure 5 The right side is the fusion curve of liposome in the background technology. Figure 5 In the figure, the horizontal axis is the detection point, the fusion time between every two detection points is 30s, and the vertical axis is the fusion degree. Figure 5 It can be seen that compared with traditional liposome fusion, the fusion efficiency of synaptic vesicles and nanodiscs is higher, shortening the detection time by at least 40 times (because the fusion of synaptic vesicles and nanodiscs reached a plateau in 2 minutes, while the liposome fusion experiment detected it for 80 minutes and still did not reach a plateau). Under the reaction condition of 37°C, the longer the detection time, the greater the attenuation of the protein or drug, which is not conducive to studying the effect of drugs or target proteins on vesicle fusion, so the present invention provides an efficient and fast fusion solution.

[0051] (2) Neurotransmitter detection experiment

[0052] The synaptic vesicles prepared in Example 2 and the nanodiscs prepared in Example 1 were mixed at a volume ratio of 9:1, wherein the total protein concentration of the synaptic vesicles was 0.5 μg / μL, and the total volume of the two was 30 μL. After incubation in a 37°C incubator for 30 min, the mixture was centrifuged for 30 min using a 30 kMWCO 0.5 ml ultrafiltration tube to filter the neurotransmitters to the bottom of the tube, collected, and tested by Elisa or HPLC. Figure 6 This image shows that synaptic vesicles are packed with neurotransmitters and released through nanodiscs. In the image, the black dots represent neurotransmitters.

[0053] Example 4

[0054] The only difference between Example 4 and step (1) of Example 3 is that 5 nM of the target protein γ-synuclein is added while the synaptic vesicles are mixed with the nanodiscs. After the target protein is added, the fusion curve is as follows: Figure 7 Shown as the yellow curve in the middle. Figure 7 This is a comparison chart of the fusion curves of Example 4 with the target protein added (+Protein) and Example 3 step (1) fusion experiment without the target protein added (Ctrl). Figure 8 This is a comparison chart of dopamine (DA) release in Example 4 with the target protein added and in Example 3 step (1) fusion experiment without the target protein added, wherein Ctrl is the dopamine (DA) release in Example 3 step (1) fusion experiment without the target protein added, and +Pr is the dopamine (DA) release in Example 4 with the target protein added.

[0055] Example 5

[0056] The only difference between Example 5 and the fusion experiment in step (1) of Example 3 is that 30 nM of the agonist Phorbol 12-myristate 13-acetate is added while the synaptic vesicles are mixed with the nanodiscs. The fusion curve of the 30 nM agonist Phorbol 12-myristate 13-acetate is as follows: Fig. 9 Shown as the yellow curve in the middle. Fig. 9 This is a comparison of the fusion curves of Example 5 with the addition of agonist phorbol ester (+Drug) and Example 3 step (1) fusion experiment without the addition of agonist phorbol ester (Ctrl). As can be seen from the figure, the agonist phorbol ester can promote the fusion of synaptic vesicles and nanodiscs, which is consistent with the conclusion that multiple studies have shown that the agonist can promote fusion and neurotransmitter release by activating protein kinase C (PKC).

[0057] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention, which shall all be included in the scope of protection of the present invention.

Claims

1. A synaptic vesicle-nanodisc fusion system, characterized in that: The invention comprises synaptic vesicles and nanodiscs, wherein the volume ratio of the synaptic vesicles to the nanodiscs is 7-9:

1.

2. The synaptic vesicle-nanodisc fusion system according to claim 1, characterized in that: The nanodisc raw material includes a phospholipid mixture, MSP and t-SNAREs in a molar ratio of 55-65:0.5-1.5:1.5-2.

5.

3. The synaptic vesicle-nanodisc fusion system according to claim 2, characterized in that: The raw materials of the phospholipid mixture include 78-83% POPC, 13-18% DOPS, 0.5-1.5% PIP2, 1-2% NBD-DOPE and 1-2% Rhodamine-DOPE in terms of molar ratio.

4. The synaptic vesicle-nanodisc fusion system according to claim 3, characterized in that: The preparation method of the phospholipid mixture comprises: mixing POPC, DOPS, PIP2, NBD-DOPE and Rhodamine-DOPE, drying and resuspending to obtain the phospholipid mixture.

5. The synaptic vesicle-nanodisc fusion system according to claim 2, characterized in that: The preparation of the nanodisc comprises the following steps: mixing the phospholipid mixture, MSP and t-SNAREs, standing, incubating, shaking and purifying to obtain the nanodisc.

6. The synaptic vesicle-nanodisc fusion system according to claim 5, characterized in that: The purification comprises: gel filtration column incubation separation.

7. The synaptic vesicle-nanodisc fusion system according to claim 1, characterized in that: The preparation method of the synaptic vesicle comprises: Step 1): soak the mouse brain in a buffer solution and homogenize the mouse brain; Step 2): first centrifugation, second centrifugation of the supernatant obtained by the first centrifugation, hypotonic disruption of the synaptosome precipitate obtained by the second centrifugation, and third centrifugation; Step 3): Purify the supernatant obtained from the third centrifugation by Optiprep density gradient centrifugation, select the layer with the highest synaptic vesicle content and take it out.

8. The synaptic vesicle-nanodisc fusion system according to claim 7, characterized in that: In the step 2), the centrifugal force of the first centrifugation is 950-1050g, and the centrifugal time is 8-12min; the centrifugal force of the second centrifugation is 14000-16000g, and the centrifugal time is 12-18min; the centrifugal force of the third centrifugation is 16000-18000g, and the centrifugal time is 20-30min.

9. The synaptic vesicle-nanodisc fusion system according to claim 7, characterized in that: In the step 3), the mass concentrations of the Optiprep centrifuge solution in the centrifuge tube from top to bottom are: 0%, 16%, 23.5%, and 30%.

10. Use of the synaptic vesicle-nanodisc fusion system according to any one of claims 1 to 9 in drug screening.

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