Method for extracting high-purity pelteobagrus fulvidraco liver cell membrane

Abstract

The invention discloses a method for extracting high-purity liver cell membranes of pelteobagrus fulvidraco. The method comprises the following steps: firstly, cutting the liver into pieces to obtain large liver tissues; digesting the large liver tissue for multiple times by using isopyknic protease liquid; adding an extraction reagent 1 containing a protease inhibitor and a phosphatase inhibitor into the digested small tissues and cell precipitates, and ultrasonically crushing to obtain homogenate; performing low-speed centrifugation on the homogenate for multiple times to collect supernate, and performing high-speed centrifugation for multiple times to collect clear liquid; carrying out ultracentrifugation to collect the precipitate, and resuspending; obtaining a coarse cell membrane suspension; adding the coarse cell membrane suspension into a centrifugal tube, then adding a sucrose density gradient buffer solution until the centrifugal tube is filled with the coarse cell membrane suspension, and carrying out ultracentrifugation; and resuspending the cell membrane precipitate to obtain the pelteobagrus fulvidraco liver cell membrane. Immunoblotting verification on the cell membrane and other organelles extracted by the method shows that the cell membrane extracted by the method conforms to the expected effect of high purity, and other organelles contained in the cell membrane have few impurities.

Description

Technical Field

[0001] This invention relates to the field of animal biotechnology, specifically to a method for extracting high-purity liver cell membranes from yellow catfish. Background Technology

[0002] The cell membrane, also known as the plasma membrane, is a vital biological membrane system constituting the cell boundary. It serves as a channel coordinating extracellular signals and intracellular responses, linking intracellular processes with intercellular interactions and tissue organization. Its structural integrity and functional specificity are indispensable for maintaining cellular homeostasis and life activities. Current research has revealed that the cell membrane plays a crucial role in various biological functions, including ion transport, signal transduction, intercellular communication, and transmembrane transport of substances. Therefore, whether exploring the specific transport mechanisms of related transport proteins in substance transport, measuring their distribution in the cell membrane and other organelles, or detecting differences in cell membrane-related receptors among different cells, it is necessary to establish a reliable extraction and purification technique for cell membranes and other organelles.

[0003] Yellow catfish ( Pelteobagrus fulvidraco Yellow catfish (Pelteobagrus fulvidraco) is a freshwater bony fish widely distributed both domestically and internationally, and is a renowned and high-quality freshwater aquaculture species in my country. Currently, under intensive aquaculture conditions, yellow catfish exhibit high lipid accumulation, and its whole genome sequence has been obtained, making it an attractive model for studying hepatic lipid metabolism. Since the cell membrane is a crucial transport and reception carrier in lipid metabolism, a reliable and feasible method for cell membrane extraction is needed for more in-depth mechanistic research. Current methods for extracting cell membranes both domestically and internationally mainly utilize ultracentrifugation at different gradient densities using different separation media, and these methods primarily focus on mammalian and thiazide-related cell lines. However, no publicly published patents have been found regarding methods for extracting cell membranes from fish.

[0004] Therefore, in order to further explore the changes in cell membrane function and structure in yellow catfish, it is necessary to establish a reliable and feasible method for the extraction and purification of yellow catfish liver cell membranes. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for extracting high-purity liver cell membranes from yellow catfish. This method is simple to operate and the extracted cell membranes have intact structures and high purity, laying the foundation for further research on the structure and function of fish cell membranes.

[0006] To achieve the above objectives, the technical solution designed by the present invention is as follows: This invention provides a method for extracting high-purity liver cell membranes from yellow catfish. The method is carried out entirely on ice or at a temperature of 4°C, and includes the following steps: 1) Take a yellow catfish of suitable size, crush the marrow, cut off the gills, bleed out the liver, remove the gallbladder, and wash it multiple times with extraction reagent 1 to remove impurities. Then cut the washed liver into pieces and wash it again with extraction reagent 2 (to remove excess oil contained in the liver); large pieces of liver tissue are obtained. 2) Digest large liver tissue pieces multiple times using an equal volume of protease solution, based on the volume of the liver tissue block; to obtain small liver tissue pieces and cell precipitates; 3) Add extraction reagent 1 containing protease inhibitor and phosphatase inhibitor to the digested small tissue pieces and cell precipitate, slowly homogenize with a glass homogenizer until there are no obvious tissue pieces, centrifuge and then sonicate to obtain a homogenate. 4) Centrifuge the homogenate multiple times at low speed (to remove undisturbed cells and impurities such as cell nuclei), and collect the supernatant; 5) The supernatant above was centrifuged at high speed multiple times to remove mitochondria, and the supernatant was collected. 6) The above supernatant was subjected to ultracentrifugation to collect the precipitate, and then resuspended using extraction reagent 1 to obtain a crude cell membrane suspension; 7) Add sucrose to the sucrose density gradient buffer to prepare three sucrose gradient solutions with different mass fractions; and add the three different concentrations of sucrose gradient solutions to centrifuge tubes in descending order of mass fraction; 8) Add the crude cell membrane suspension to the centrifuge tube from step 7), then add sucrose density gradient buffer until the centrifuge tube is full, and centrifuge at high speed; 9) Collect the cell membrane components between the 55% sucrose gradient solution and the 45% sucrose gradient solution, dilute with extraction reagent 1 to reduce microsome and some cytoplasmic contamination, and centrifuge at 95,000g for 1 h; discard the supernatant, resuspend the cell membrane precipitate with 50µl of extraction reagent 1 to obtain the yellow catfish liver cell membrane.

[0007] Furthermore, in step 1), the extraction reagent 1 contains 225 mM mannitol, 75 mM sucrose and 30 mM Tris-HCl.

[0008] Furthermore, in step 1), the extraction reagent 2 is the extraction reagent 1 containing BSA and EGTA, wherein the mass fraction of BSA in the extraction reagent 2 is 0.5% and the concentration of EGTA is 0.5mM.

[0009] Furthermore, in step 2), the protease solution is a mixture of collagenase II and trypsin in a volume ratio of 1:1; the digestion is performed 3 to 4 times, with each digestion lasting 5 minutes.

[0010] Furthermore, in step 3), the extraction reagent 1 containing protease inhibitor and phosphatase inhibitor contains PMSF at a concentration of 0.5% (v / v) and phosphatase inhibitor at a concentration of 1% (v / v).

[0011] Furthermore, in step 3), the ultrasonic amplitude is 10-15% and the ultrasonic time is 2 minutes.

[0012] Furthermore, in step 4), the centrifugal force for low-speed centrifugation is 850g, and the centrifugation time is 10min. Furthermore, in step 5), the centrifugal force of the high-speed centrifugation is 12000g and the centrifugation time is 10min; and 850g, 10min low-speed centrifugation is interspersed between the high-speed centrifugation of 12000g and 10min (to improve extraction efficiency and reduce impurity contamination).

[0013] Furthermore, in step 6), the centrifugal force of the ultracentrifugation is 28000g, and the centrifugation time is 20min.

[0014] Furthermore, in step 7), the sucrose density gradient buffer solution is prepared by the following method: Dissolve 0.523 g Bis-Tris in 400 ml of double-distilled water and add 1 ml of 100 mM EDTA (pH 7.4). Mix the solution with the double-distilled water to bring the final volume to 500 ml and store at 4°C.

[0015] The 38% sucrose gradient solution was obtained by dissolving 19 g of sucrose in 31 g of sucrose density gradient buffer and mixing thoroughly. The 45% sucrose gradient solution was obtained by dissolving 22.5 g of sucrose in 27.5 g of sucrose density gradient buffer and mixing thoroughly. The 55% sucrose gradient solution was obtained by dissolving 27.5 g of sucrose in 22.5 g of sucrose density gradient buffer and mixing thoroughly.

[0016] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention is the first to propose a technique for extracting and purifying liver cell membranes from yellow catfish. The technique is simple and easy to operate, has high extraction efficiency, and produces cell membranes with few other impurities extracted from tissue. 2. This invention uses mixed proteases to digest the intercellular matrix of tissue cells, reducing cell media contamination and improving efficiency, and further increases extraction efficiency by using ultrasound to disrupt cells; 3. This invention compares sucrose separation solutions of different densities to fully remove cell debris contamination and extraction efficiency issues in existing methods, and selects the most suitable concentration for extraction.

[0017] The cell membrane and other organelles extracted by the method of the present invention were verified by immunoblotting, which showed that the cell membrane extracted by the present invention met the expected effect of high purity and contained few impurities in other organelles. Attached Figure Description

[0018] Figure 1 A flowchart illustrating the main steps in the extraction of liver cell membranes and other organelles from yellow catfish. Figure 2 A schematic diagram illustrating the process of extracting and screening the optimal ratio of digestive enzymes from the liver cell membrane of yellow catfish; Figure 3 A schematic diagram illustrating the verification of the purity of extracted cell membranes using Western blotting. In the diagram, H represents homogenate, MT represents mitochondria, ER represents endoplasmic reticulum, Cyto represents cytoplasm, and PM represents cell membrane; PM marker: Na + / K + -ATPase is located in the cell membrane and is a cell membrane marker protein; ER marker: Calnexin is associated with calcium ion transport in the endoplasmic reticulum and is mainly located on the endoplasmic reticulum membrane; Cyto marker: β-actin is a cytoplasmic marker protein that is highly expressed in the cytoplasm; MT marker: Tom20 is a mitochondrial marker protein located on the outer mitochondrial membrane; Figure 4 This diagram illustrates the purity of cell membranes extracted using different embodiments of the immunoblotting method. In the figure, PM marker: Na + / K + -ATPase is located in the cell membrane and is a cell membrane marker protein; Cyto marker: β-actin is a cytoplasmic marker protein that is highly expressed in the cytoplasm. Detailed Implementation

[0019] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can understand it.

[0020] Example 1 like Figure 1 The method for extracting high-purity cell membranes from yellow catfish liver tissue, as shown, includes the following specific steps: 1) Take a yellow catfish of appropriate size, crush the marrow, cut off the gills, bleed out the liver, and remove the gallbladder. First, wash it with PBS 3 times to remove impurities. After washing, transfer the liver into extraction reagent 1, cut it into pieces, and then rinse it with extraction reagent 2 3 times to remove excess oil contained in the liver; large pieces of liver tissue are obtained. 2) The liver tissue blocks were digested with an equal volume of protease for 5 minutes each time, for a total of 3 times. After digestion was stopped, small pieces of liver tissue and cell precipitate were obtained. The protease was a mixture of collagenase II and trypsin in a 1:1 volume ratio. 3) Add 3 ml of extraction reagent 1 containing protease inhibitor and phosphatase inhibitor per gram of tissue (small pieces of liver tissue and cell precipitate), and slowly and gently homogenize with a glass homogenizer until there is no obvious liver tissue. After centrifugation, sonicate for 2 min under ultrasonic amplitude of 10-15% to obtain homogenate. 4) Centrifuge the homogenate after disruption at 850 g for 10 min to remove the precipitate and keep the supernatant; centrifuge the supernatant twice more under the above conditions (to basically remove unbroken cells and cell debris), and finally collect the supernatant. 5) Centrifuge the supernatant at 12000 g for 10 min, collect the supernatant, and centrifuge again at 850 g for 10 min. Repeat twice (to remove mitochondria) to obtain the supernatant. 6) Centrifuge the above supernatant at 28,000 g for 20 min to collect the precipitate. Resuspend it in extraction reagent 1 and reuse it at 28,000 g to fully remove contamination from cytoplasm and other organelles, obtaining a crude cell membrane precipitate. Gently resuspend the crude cell membrane precipitate in 1.5 ml of extraction reagent 1. 7) Create a discontinuous sucrose gradient solution, as follows: Preparation of sucrose density gradient buffer: Dissolve 0.523 g Bis-Tris in 400 ml of double-distilled water and add 1 ml of 100 mM EDTA (pH 7.4). Mix the solution with the double-distilled water to make a final volume of 500 ml and store at 4°C. The 38% sucrose gradient solution was prepared by dissolving 19 g of sucrose in 31 g of sucrose density gradient buffer. A 45% sucrose gradient solution is prepared by dissolving 22.5 g of sucrose in 27.5 g of sucrose density gradient buffer. A 55% sucrose gradient solution is prepared by dissolving 27.5 g of sucrose in 22.5 g of sucrose density gradient buffer. 8) Add 3 ml of 55% sucrose gradient solution to the bottom of a 13 ml thin-walled ultracentrifuge tube. Then, place 4 ml of 45% sucrose solution on top of the 3 ml 55% sucrose gradient solution. Finally, place 4 ml of 38% sucrose gradient solution on top of the 4 ml 45% sucrose gradient solution. 9) The collected crude cell membrane suspension was layered and placed on top of an 11 ml discontinuous sucrose gradient in an ultracentrifuge tube. Then, the sucrose density gradient buffer was gently placed on top of the crude cell membrane suspension to fill the centrifuge tube, and the tube was centrifuged at 95,000 g for 2.5 h. 10) After ultracentrifugation, cell membrane components were collected between 55% and 45% sucrose gradient solutions using a Pasteur pipette. The mixture was diluted with Extraction Reagent 1 to reduce microsomal and partial cytoplasmic contamination. The mixture was then centrifuged at 95,000g for 1 h. The supernatant was discarded, and the cell membrane precipitate was resuspended in 50µl of Extraction Reagent 1.

[0021] Preparation of Extraction Reagent 1: Dissolve 20.5 g mannitol and 13 g sucrose in 400 ml double-distilled water and add 15 ml Tris-HCl (1 M, pH 7.4). Cool the buffer solution at 40°C for approximately 30 minutes. Check the pH of the buffer solution and adjust it with KOH (if too low) or HCl (if too high) if necessary. Mix the solution with double-distilled water to achieve a final volume of 500 ml and store at 4°C. Preparation of extraction reagent 1 containing protease inhibitors and phosphatase inhibitors: Before use, extraction reagent 1 for homogenization should contain a mixture of PMSF (15µl / 3 ml, Beyotime) and phosphatase inhibitor (Beyotime) to prevent sample degradation. The concentration of protease inhibitor in extraction reagent 1 containing protease inhibitor and phosphatase inhibitor is 0.5% (v / v) and the concentration of phosphatase inhibitor is 1% (v / v). Preparation of extraction reagent 2: Dissolve 0.75 g BSA in 150 ml of extraction reagent 1, add 750 µl of 100 mM MEGTA (pH 7.4), and store at 4 °C.

[0022] Example 2 The preparation method in this embodiment is basically the same as that in Example 1, except that: The protease is a mixture of collagenase II and trypsin in a volume ratio of 1:2.

[0023] Example 3 The preparation method in this embodiment is basically the same as that in Example 1, except that: The protease is a mixture of collagenase II and trypsin in a volume ratio of 2:1.

[0024] Example 4 The preparation method in this embodiment is basically the same as that in Example 1, except that: The three sucrose gradient solutions of different concentrations are 38%, 39%, and 55% from top to bottom.

[0025] Example 5 The preparation method in this embodiment is basically the same as that in Example 1, except that: The three sucrose gradient solutions of different concentrations are 38%, 41%, and 55% from top to bottom.

[0026] Example 6 The preparation method in this embodiment is basically the same as that in Example 1, except that: The three sucrose gradient solutions of different concentrations, from top to bottom, are 38%, 43%, and 55%.

[0027] Example 7 The preparation method in this embodiment is basically the same as that in Example 1, except that: The three sucrose gradient solutions of different concentrations are 38%, 47%, and 55% from top to bottom.

[0028] Example 8 The preparation method in this embodiment is basically the same as that in Example 1, except that: The three sucrose gradient solutions of different concentrations, from top to bottom, are 38%, 49%, and 55%.

[0029] I. Comparison of extraction efficiency of cell membranes from different proteases like Figure 2 As shown: In the cell membrane extracted by the protease used in Example 2, the separation of the cell membrane layer was relatively blurred, indicating poor extraction effect. The ratio of trypsin and collagenase II was biased towards trypsin, which may have led to insufficient action of collagenase and failure to fully decompose the cell membrane, resulting in low extraction efficiency.

[0030] In Example 1, the cell membrane extracted using the protease showed the best extraction effect. The figure shows clear cell membrane separation, indicating that trypsin and collagenase II exerted optimal synergistic effects, achieving the best extraction efficiency. The balanced action of both effectively decomposes the extracellular matrix and cell membrane, resulting in the highest extraction efficiency.

[0031] In Example 3, the excessive use of trypsin in the protease-extracted cell membrane resulted in poor extraction efficiency, and the separation of the cell membrane was not as pronounced as in Example 1. In this case, excessive trypsin may have overly aggressively decomposed the cell membrane and extracellular matrix, causing damage to the cell membrane structure and hindering effective extraction.

[0032] As can be seen from the above, in Example 1, the protease was a mixture of collagenase II and trypsin in a volume ratio of 1:1. The effects of the two enzymes were in the best balance, and their respective advantages were fully utilized, resulting in the best extraction effect.

[0033] II. Comparison of extraction efficiency of cell membranes extracted in Examples 1 and 4-8 like Figure 4 As shown: Sucrose gradient centrifugation is used to separate cell membranes and other organelles. In sucrose solutions of different concentrations, the cell membrane separates from other components in the solution according to its density. Sucrose concentration has a significant impact on the extraction efficiency of cell membranes. When the concentration is too low, the cell membrane may precipitate along with other intracellular components, while when the concentration is too high, the cell membrane may not be able to be separated sufficiently. Therefore, at a sucrose concentration of 45%, the cell membrane can usually be separated well from other intracellular components (such as cytoplasm, mitochondria, etc.). When the sucrose concentration is too low, the separation of the cell membrane from other components may not be effective, while when the concentration is too high, the separation effect of the cell membrane from other substances may be insignificant. Therefore, a concentration of 45% is a relatively ideal balance point.

[0034] At this concentration, the band for Na+ / K+-ATPase (a cell membrane marker protein) is strong, while the band for β-actin (a cytoplasmic marker protein) is weak, indicating high purity of cell membrane extraction. The high enrichment of Na+ / K+-ATPase marker proteins and low cell membrane contamination indicate that this concentration provides the most efficient membrane protein extraction.

[0035] Analysis of the band intensity of β-actin showed that cytoplasmic contamination was minimal at a 45% sucrose concentration. With varying concentrations, excessively low sucrose concentrations may lead to increased cytoplasmic contamination, while excessively high concentrations may result in incomplete cell membrane separation, leading to decreased cell membrane purity. The results at a 45% concentration indicate that cell membrane extraction is relatively pure with minimal interference from other cellular components.

[0036] As shown above, a 45% sucrose concentration provides the best extraction effect for cell membrane purity. This concentration can effectively separate cell membrane from cytoplasmic components, reduce contamination, and maintain a high concentration of membrane proteins.

[0037] III. Comparison of the cell membrane prepared in Example 1 with other substances prepared by different methods The homogenate, mitochondria (the homogenate and mitochondria are intermediate products obtained by the method in Example 1), endoplasmic reticulum, and other cytoplasmic organelles (the endoplasmic reticulum and other cytoplasmic organelles were prepared by the method disclosed in publication number CN114591886B, entitled "A High-Purity Method for Coupled Extraction of Mitochondria and Endoplasmic Reticulum from Yellow Catfish Liver") were compared, and the characteristic proteins of different organelles were detected by Western blot, thereby verifying the purity of the cell membrane.

[0038] Cell membrane purity assessment primarily relies on the expression of marker proteins. The relative expression levels of marker proteins in extracts from different organelles can reflect the purity of that organelle. According to... Figure 3 The results of the Western blot are as follows: a. The homogenate contains all cellular components, and the expression levels of marker proteins represent the overall distribution of various organelles. As shown in the figure, various marker proteins are expressed to some extent in the homogenate, indicating that proteins from various organelles (including the cell membrane, endoplasmic reticulum, mitochondria, cytoplasm, etc.) are present in the homogenate.

[0039] b. High expression of the mitochondrial marker protein Tom20 indicates high purity of mitochondria in the mitochondrial extract. Compared with other organelle marker proteins (such as β-actin, Na+ / K+-ATPase, and Calnexin), Tom20 expression is more prominent, while other marker proteins are almost absent, indicating high mitochondrial purity.

[0040] c. The significant expression of the endoplasmic reticulum marker protein Calnexin in the endoplasmic reticulum extract further validated the purity of the endoplasmic reticulum. Compared with marker proteins in mitochondria and cytoplasm, Tom20 (mitochondrial marker protein) and β-actin (cytoplasmic marker protein) were almost not expressed in the endoplasmic reticulum extract, indicating that the purity of the endoplasmic reticulum was high.

[0041] d. The high expression of the cytoplasmic marker protein β-actin indicates that the cytoplasmic extract contains a large amount of cytoplasmic protein. The expression of other organelle marker proteins, such as Na+ / K+-ATPase (cell membrane marker protein) and Tom20 (mitochondrial marker protein), is extremely low or almost non-existent, further indicating that the cytoplasm is of high purity and that there is little contamination from other organelles.

[0042] e. Cell membrane marker protein Na+ / K+-ATPase was highly expressed in the cell membrane extract, while marker proteins from mitochondria, endoplasmic reticulum, and cytoplasm were expressed at extremely low levels. There was almost no expression of β-actin, Tom20, and Calnexin, indicating that the cell membrane extract was of high purity and almost free from contamination from other organelles.

[0043] Therefore, the high expression level of Na+ / K+-ATPase in the cell membrane is the most critical marker, proving that there are a large number of cell membrane proteins in the cell membrane extract, and that other organelle marker proteins (such as mitochondrial Tom20, endoplasmic reticulum Calnexin, and cytoplasmic β-actin) are expressed at almost no or very little, indicating that the cell membrane extract is very pure and basically free from other organelle contamination.

[0044] High expression of Na+ / K+-ATPase was detected by Western blot, and other marker proteins were almost absent, which is consistent with the standards for cell membrane extraction purity in the literature, indicating that the extraction method is free from impurities in the extraction of yellow catfish liver cell membranes.

[0045] As can be seen from the above, the method in Example 1 is highly efficient and can effectively avoid contamination of the cytoplasm, mitochondria and endoplasmic reticulum, resulting in high-purity yellow catfish liver cell membranes.

[0046] All other parts not described in detail are existing technologies. Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for extracting high-purity liver cell membranes from yellow catfish, characterized in that: The method is carried out entirely on ice or at a temperature of 4°C, and includes the following steps: 1) Take a yellow catfish of appropriate size, crush the marrow, cut off the gills, bleed out the liver, and remove the gallbladder; use extraction reagent 1 to wash it several times to remove impurities, then cut the washed liver into pieces, and then use extraction reagent 2 to wash it again to obtain large pieces of liver tissue; 2) Digest large liver tissue pieces multiple times using an equal volume of protease solution, based on the volume of the liver tissue block; to obtain small liver tissue pieces and cell precipitates; 3) Add extraction reagent 1 containing protease inhibitor and phosphatase inhibitor to the digested small tissue pieces and cell precipitate, slowly homogenize with a glass homogenizer until there are no obvious tissue pieces, centrifuge and then sonicate to obtain a homogenate. 4) Centrifuge the homogenate multiple times at low speed and collect the supernatant; 5) The supernatant above was centrifuged at high speed multiple times to remove mitochondria, and the supernatant was collected. 6) The above supernatant was subjected to ultracentrifugation to collect the precipitate, and then resuspended using extraction reagent 1; the centrifugation and resuspending were repeated to obtain a crude cell membrane suspension; 7) Add sucrose to the sucrose density gradient buffer to prepare three sucrose gradient solutions with different mass fractions; and add the three different concentrations of sucrose gradient solutions to centrifuge tubes in descending order of mass fraction; 8) Add the crude cell membrane suspension to the centrifuge tube from step 7), then add sucrose density gradient buffer until the centrifuge tube is full, and centrifuge at high speed; 9) Collect the cell membrane components between the 55% sucrose gradient solution and the 45% sucrose gradient solution, dilute with extraction reagent 1 to reduce microsome and some cytoplasmic contamination, and centrifuge at 95,000g for 1 h; discard the supernatant, resuspend the cell membrane precipitate with 50µl of extraction reagent 1 to obtain the yellow catfish liver cell membrane.

2. The extraction method according to claim 1, characterized in that: In step 1), the extraction reagent 1 contains 225 mM mannitol, 75 mM sucrose and 30 mM Tris-HCl.

3. The extraction method according to claim 1 or 2, characterized in that: In step 1), the extraction reagent 2 is the extraction reagent 1 containing BSA and EGTA, wherein the mass fraction of BSA in the extraction reagent 2 is 0.5% and the concentration of EGTA is 0.5mM.

4. The extraction method according to claim 1, characterized in that: In step 2), The protease solution is a mixture of collagenase II and trypsin in a 1:1 volume ratio; the digestion is performed 3 to 4 times, with each digestion lasting 5 minutes.

5. The extraction method according to claim 1, characterized in that: In step 3), the extraction reagent 1 containing protease inhibitor and phosphatase inhibitor contains PMSF as the protease inhibitor, with a PMSF concentration of 0.5% and a phosphatase inhibitor concentration of 1%.

6. The extraction method according to claim 1, characterized in that: In step 3), The ultrasonic amplitude is 10-15%, and the ultrasonic time is 2 minutes.

7. The extraction method according to claim 1, characterized in that: In step 4), the centrifugal force of low-speed centrifugation is 850g, and the centrifugation time is 10min.

8. The extraction method according to claim 1, characterized in that: In step 5), The centrifugal force of the high-speed centrifugation was 12000g, and the centrifugation time was 10min; and the high-speed centrifugation of 12000g for 10min was interspersed with the low-speed centrifugation of 850g for 10min.

9. The extraction method according to claim 1, characterized in that: In step 6), The centrifugal force for ultracentrifugation was 28,000 g, and the centrifugation time was 20 min.

10. The extraction method according to claim 1, characterized in that: In step 7), the sucrose density gradient buffer solution is prepared by the following method: Dissolve 0.523 g Bis-Tris in 400 ml of double-distilled water and add 1 ml of 100 mM EDTA. Mix the solution with the double-distilled water to make a final volume of 500 ml and store at 4°C. The 38% sucrose gradient solution was obtained by dissolving 19 g of sucrose in 31 g of sucrose density gradient buffer and mixing thoroughly. The 45% sucrose gradient solution was obtained by dissolving 22.5 g of sucrose in 27.5 g of sucrose density gradient buffer and mixing thoroughly. The 55% sucrose gradient solution was obtained by dissolving 27.5 g of sucrose in 22.5 g of sucrose density gradient buffer and mixing thoroughly.

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