Recombinant plasmid and its application in extracting mitochondria

Abstract

The present invention discloses a recombinant plasmid and its application in extracting mitochondria. The recombinant plasmid is obtained by introducing a mitochondrial localization sequence into a backbone vector containing a TurboID sequence. The recombinant plasmid is labeled with adjacent protein biotin to realize efficient, rapid and high-purity extraction of mitochondrial proteins in mammalian cells, thereby providing a new experimental method for conducting mitochondrial protein-related research.

Description

Technical Field

[0001] The invention belongs to the technical field of biological separation, and specifically relates to a recombinant plasmid, and application of the recombinant plasmid in extracting mammalian cell mitochondria, and also relates to a method for extracting mammalian cell mitochondria. Background Art

[0002] Mitochondria are a type of organelles covered by two membranes that exist in most cells. They are structures that produce energy in cells and are the main place for cells to perform aerobic respiration. In addition to providing energy for cells, mitochondria are also involved in processes such as cell differentiation, cell information transmission, and cell apoptosis, and have the ability to regulate cell growth and cell cycle. Their main functions include participating in energy conversion, tricarboxylic acid cycle, oxidative phosphorylation, and storing calcium ions. In addition to being a "cell energy station", studies have shown that mitochondria also undertake many other physiological functions: such as regulating membrane potential and controlling programmed cell death; regulating cell proliferation and cell metabolism; synthesizing cholesterol and certain heme, etc. Therefore, extracting mitochondria from cells or tissues can provide effective support for mitochondrial dysfunction or mitochondrial-related diseases.

[0003] At present, common mitochondrial extraction methods include sucrose density gradient centrifugation or tissue mitochondrial separation and extraction kit method, such as the Chinese patent application with publication number CN116716292A, which provides a kit for extracting mitochondrial DNA and protein at the same time, which can extract mitochondrial DNA and protein at the same time for subsequent molecular biological analysis. Another example is the Chinese patent application with publication number CN114958719A, which discloses a method for quickly extracting high-concentration mitochondria from tissues, which has a high mitochondrial extraction rate and purity while being time-consuming and low-damage. However, most of the existing mitochondrial extraction kits use grinding methods to lyse cells, and different grinding methods need to be explored for different cells, and the grinding process is time-consuming and labor-intensive, and it is impossible to carry out mitochondrial extraction of multiple samples at the same time. For ordinary differential centrifugation, the purity of the separated mitochondria is not high, and there are pollution problems of multiple organelles. In addition, the centrifugation time of sucrose density gradient centrifugation is long, and an inert gradient medium solution needs to be prepared, and the operation process is strict and difficult to master. In general, the current existing mitochondrial extraction methods have the problems of high cost, low extraction yield, low purity, slow extraction speed, difficult operation, and susceptibility to contamination by other proteins and nuclear proteins in the cytoplasm.

[0004] The biotin-labeled plasmids for neighboring proteins play a vital role in screening and identifying protein-protein interactions and exploring related functions in organisms. TurboID is a new biotin ligase that can convert biotin into a reactive intermediate that covalently labels proximal proteins, thereby labeling neighboring proteins with biotin. In the presence of ATP and biotin, the TurboID fusion protein can biotin-label any exposed lysine residues within a radius of 10nm or 35nm, whether it is a directly interacting protein or a neighboring protein, depending on whether it retains the linker. Subsequently, the biotin-labeled proteins can be separated and purified from the cell lysate by Streptavidin magnetic beads or gel, and the interacting proteins can be identified by mass spectrometry (MS). In general, the biotin-labeled plasmids for neighboring proteins play a vital role in screening and identifying protein-protein interactions and exploring related functions in organisms.

[0005] However, there is currently no method for rapid and efficient extraction of mitochondria based on biotin labeling of neighboring proteins. Summary of the invention

[0006] Based on the above reasons, the primary purpose of the present invention is to provide a recombinant plasmid, and a method for rapid and efficient extraction of mitochondria using the recombinant plasmid, using the adjacent protein biotin to label the recombinant plasmid to achieve efficient, rapid and high-purity extraction of mitochondrial proteins in cells, minimize contamination of other organelles in the cell nucleus and cytoplasm, and provide an experimental method for conducting mitochondrial protein-related research.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] The first aspect of the present invention provides a recombinant plasmid, wherein the recombinant plasmid is obtained by introducing a mitochondrial localization sequence into a backbone vector, wherein the backbone vector contains a TurboID sequence as shown in SEQ ID NO.2.

[0009] In some specific embodiments of the present invention, the recombinant plasmid has a backbone vector with a sequence as shown in SEQ ID NO.1, and is obtained by recombination in any one of the following ways (1) to (3):

[0010] (1) replacing the multiple cloning site fragment of SEQ ID NO.3 in the backbone vector with the TOM20 sequence of SEQ ID NO.5, and inserting a linker of SEQ ID NO.8 after the TOM20 sequence;

[0011] (2) deleting the multi-cloning site fragment of the backbone vector sequence such as SEQ ID NO.3, and replacing the HA tag of the backbone vector sequence such as SEQ ID NO.4 with a linker sequence such as SEQ ID NO.8 and a MAO sequence such as SEQ ID NO.6;

[0012] (3) The multiple cloning site fragment of the backbone vector sequence such as SEQ ID NO.3 is deleted, and the HA tag of the backbone vector sequence such as SEQ ID NO.4 is replaced with a linker sequence such as SEQ ID NO.8 and a MAVS sequence such as SEQ ID NO.7.

[0013] The second aspect of the present invention provides a recombinant lentivirus, which is a packaging body obtained by co-transfecting a host cell with the recombinant plasmid described in the first aspect of the present invention and a lentiviral auxiliary packaging plasmid.

[0014] The third aspect of the present invention provides the use of the recombinant plasmid or the recombinant lentivirus as described above in extracting mitochondria from mammalian cells.

[0015] A fourth aspect of the present invention provides a method for extracting mitochondria from mammalian cells.

[0016] A fifth aspect of the present invention provides a kit for extracting mitochondria from mammalian cells.

[0017] Beneficial effects of the present invention:

[0018] The invention constructs a recombinant plasmid, which contains a biotinidase TurboID sequence and a mitochondrial localization sequence. By using the recombinant plasmid and adopting a method of biotin labeling of adjacent proteins, efficient extraction of mammalian cell mitochondria can be achieved, and the extracted mitochondria have high purity and complete functions.

[0019] The present invention also provides a method for extracting mitochondria from mammalian cells, which has the following advantages:

[0020] (1) Rapid: Excluding the preparation stage, the extraction step of this method takes less than 30 minutes, which greatly improves the time required for mitochondrial isolation and opens up a new method for related metabolic research.

[0021] (2) Preserving the integrity of mitochondrial function and structure: This method does not damage mitochondria, preserves the integrity of mitochondria, and provides conditions for the study of mitochondrial function.

[0022] (3) The extracted mitochondria are of high purity: This method innovatively uses the biotin labeling method of adjacent proteins to separate mitochondria, effectively avoiding contamination of the cell nucleus and other organelles.

[0023] (4) Simple operation: The entire extraction process requires very low technical skills from the experimenter, and the reagents used are inexpensive and easy to obtain.

[0024] In summary, the method for extracting mitochondria from mammalian cells in the present invention adopts specific labeling, which can efficiently, quickly and highly pure extract mitochondrial proteins from cells, minimize the contamination of other organelles in the nucleus and cytoplasm, and the extracted mitochondria are of high purity, complete function and structure, which promotes mitochondrial-related research. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a map of the vector plasmid pLV3-CMV-3×FLAG-MCS-TurboID-HA-Puro in Example 1 of the present invention.

[0026] Figure 2 This is a map of the recombinant plasmid PLV3-CMV-3×FLAG-TOM20-TurboID-Puro-HA in Example 1 of the present invention.

[0027] Figure 3 This is a map of the recombinant plasmid PLV3-CMV-3×FLAG-TurboID-MAO-Puro-HA in Example 1 of the present invention.

[0028] Figure 4 This is a map of the recombinant plasmid PLV3-CMV-3×FLAG-TurboID-MAVS-Puro-HA in Example 1 of the present invention.

[0029] Figure 5 These are the Western Blot characterization and analysis results of mitochondrial extraction in Examples 2-4 of the present invention.

[0030] Figure 6 This is an immunofluorescence image of HCCLM3 cells transfected with the recombinant plasmid PLV3-CMV-3×FLAG-TOM20-TurboID-Puro-HA in Example 5 of the present invention.

[0031] Figure 7 This is an immunofluorescence image of HCCLM3 cells transfected with the recombinant plasmid PLV3-CMV-3×FLAG-TurboID-MAO-Puro-HA in Example 6 of the present invention.

[0032] Figure 8 This is an immunofluorescence image of HCCLM3 cells transfected with the recombinant plasmid PLV3-CMV-3×FLAG-TurboID-MAVS-Puro-HA in Example 7 of the present invention. DETAILED DESCRIPTION

[0033] Embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0034] The present invention provides a method for extracting mitochondria from mammalian cells, which mainly uses a specially constructed recombinant plasmid to achieve mitochondrial extraction through adjacent protein biotin labeling. The following is a detailed description of each step or process in the method.

[0035] <Construction of recombinant plasmid and recombinant lentivirus>

[0036] The recombinant plasmid of the present invention is obtained by introducing a mitochondrial localization sequence into a backbone vector containing biotin ligase TurboID and then recombining the vector.

[0037] In some specific implementation cases of the present invention, the mitochondrial localization sequence is a TOM20 sequence, a MAO sequence or a MAVS sequence, and a linker sequence may be inserted according to specific needs. In the present invention, the recombinant plasmid has a backbone vector with a sequence as shown in SEQ ID NO.1, and is obtained by recombining in any one of the following (1) to (3):

[0038] (1) replacing the multiple cloning site fragment of SEQ ID NO.3 in the backbone vector with the TOM20 sequence of SEQ ID NO.5, and inserting a linker of SEQ ID NO.8 after the TOM20 sequence;

[0039] (2) deleting the multi-cloning site fragment of the backbone vector sequence such as SEQ ID NO.3, and replacing the HA tag of the backbone vector sequence such as SEQ ID NO.4 with a linker sequence such as SEQ ID NO.8 and a MAO sequence such as SEQ ID NO.6;

[0040] (3) The multiple cloning site fragment of the backbone vector sequence such as SEQ ID NO.3 is deleted, and the HA tag of the backbone vector sequence such as SEQ ID NO.4 is replaced with a linker sequence such as SEQ ID NO.8 and a MAVS sequence such as SEQ ID NO.7.

[0041] It is understandable that the construction of the recombinant plasmid in the present invention can be obtained by those skilled in the art through known methods and databases, designing primers and adopting corresponding PCR amplification or artificial synthesis methods. Those skilled in the art have such ability, so it will not be described in detail here.

[0042] The recombinant plasmid contains the biotin ligase TurboID, and subsequent use of adjacent protein biotin labeling can achieve efficient extraction of mitochondria, and the extracted mitochondria are of high purity and complete function and structure.

[0043] The recombinant lentivirus described in the present invention is a packaging body obtained by co-transfecting the host cell with the recombinant plasmid described above and the lentivirus auxiliary packaging plasmid.

[0044] The method for constructing the recombinant lentivirus described herein can be a method well known in the art. Specifically, the recombinant plasmid of the present invention is co-transfected with a lentiviral envelope vector and a packaging vector into a cell line. The transfection method is not particularly limited and a conventional transfection method in the art can be used.

[0045] In some specific embodiments of the present invention, the lentiviral envelope vector is preferably pMD2.G; the packaging vector is preferably psPAX2; and the cell line is preferably 293T cells, but not limited thereto.

[0046] The transfection system used for cell transfection herein can be selected as stable transfection or transient transfection according to experimental needs. In some specific embodiments of the present invention, the stable transfection system is shown in Table 1, and the transient transfection system is shown in Table 2, but is not limited thereto.

[0047] Table 1 Stable transfection system

[0048] Plasmid and reagent names 24 holes 12 holes 6 holes 6cm dish psPAX2 200ng 400ng 800ng 2μg pMD2.G 100ng 200ng 400ng 1 μg Target plasmid 200ng 400ng 800ng 2μg Lipo Fiter 1μL 1.5μL 3μL 7.5μL Opti-MEM 50μL 100μL 200μL 400μL

[0049] Table 2 Transient transfection system

[0050] Plasmid and reagent names 24 holes 12 holes 6 holes 6cm dish Target plasmid 0.5 μg 1 μg 2μg 5μg Lipo Fiter 1μL 1.5μL 3μL 7.5μL Opti-MEM 50μL 100μL 200μL 400μL

[0051] Specifically, the stable transfection system can infect any cell line and obtain stable inheritance; the transient transfection system mainly transfects 293T cells, and the three recombinant plasmids described in this article are applicable to the above two transfection systems. It is understood that those skilled in the art can make specific selections for the transfection system according to the mitochondria of the specific cell line to be extracted, without any special restrictions.

[0052] The target plasmid can be transfected by the liposome transfection method commonly used in the art. Specific operations can be performed according to different transfection systems, which will not be described in detail here.

[0053] After obtaining the recombinant lentivirus, it can be screened by resistance screening methods well known in the art. The antibodies used can be types that are relatively well known in the art. In some specific embodiments of the present invention, puromycin (Puro) is preferably used.

[0054] It is to be understood that the specific transfection and screening conditions can be determined by those skilled in the art through known methods or experiments, and therefore there is no particular limitation.

[0055] In addition, the screened cells need to be cultured to a density of 80% to 90% for subsequent mitochondrial extraction. The specific culture method is well known in the art, such as using DMEM culture medium, but is not limited to this. Those skilled in the art can select culture medium and culture conditions according to specific cells, and use culture medium and culture conditions suitable for the cells to be cultured.

[0056] <Biotin labeling>

[0057] In the present invention, the biotin labeling is achieved by replacing the original culture medium with a complete culture medium containing biotin and then culturing.

[0058] Among them, the concentration of biotin in the complete culture solution is 0.05-500 μM, for example, it can be any concentration of 0.05 μM, 0.1 μM, 0.5 μM, 1 μM, 2 μM, 5 μM, 10 μM, 30 μM, 50 μM, 100 μM, 200 μM or 500 μM, which can be selected according to needs, preferably 300-500 μM. It can be understood that the specific cell culture temperature can be selected according to the type or type of cells without special restrictions. Most cells are best cultured at 37°C, but some special cells need to be cultured at a specific temperature. Those skilled in the art can adjust according to cell needs; in addition, the labeling time of biotin can be extended or shortened according to experimental needs. Those skilled in the art can determine it according to known methods or experiments, so there is no special restriction. Generally speaking, the higher the concentration of biotin, the shorter the required culture time can be. In some specific embodiments of the present invention, the culture conditions are preferably cultured at 37°C for 10 min to 18 h. Preferably, it is best to culture 10 min at a concentration of 500 μM biotin.

[0059] <Cell Digestion>

[0060] In the present invention, cell digestion is achieved by adding a cell digestion solution to a cell culture container, wherein the cell digestion solution can be any type well known in the art, such as trypsin. In some specific embodiments of the present invention, 0.25% trypsin is used and digestion is performed at 37°C. However, it is understandable that the conditions described in the present invention are not limited thereto. Depending on the cell type, any cell digestion solution and culture conditions that can achieve the purpose of cell digestion can be used, and those skilled in the art can adjust or select according to actual needs.

[0061] In the specific operation, after observing that all cells have become round and completely detached from the bottom of the cell culture container (for example, it can be observed through a microscope), add a culture medium with a volume equal to that of the cell digestion solution to terminate the digestion.

[0062] In addition, it should be noted that before cell digestion, the step of washing the biotin-labeled cells is also included. In some specific embodiments of the present invention, the cells are washed at least twice with a PBS solution preheated at 37°C, and the remaining liquid is aspirated to wash away unlabeled biotin and residual culture medium and cell products.

[0063] <Resuspend cells>

[0064] In the present invention, cell resuspension is to resuspend the counted cells with cell washing solution or buffer to ensure uniform distribution of cells, which is conducive to normal growth and reproduction of cells, as well as subsequent analysis or observation. In some specific embodiments of the present invention, during the specific operation, 2 to 5 million cell suspensions are taken in a sterile container (such as an EP tube), centrifuged at 1000 to 1200 rpm for 4 to 5 minutes, the supernatant is discarded, and the cells are resuspended with 1 mL of pre-cooled PBS, centrifuged at 4°C, 550 to 650 g for 3 to 5 minutes, the supernatant is removed, and the precipitate is retained.

[0065] <Cell membrane lysis>

[0066] In this step, the cell membrane of the cell precipitate obtained after centrifugation is lysed by adding a lysis solution. Preferably, in some specific embodiments of the present invention, the cell lysis solution used is Digitonin, and the lysis is carried out at 3-5°C for 2-5 min, preferably, at 4°C for 5 min. The specific temperature and time can be determined according to the specific situation, for example, adjusted according to the cell type and the number of cells.

[0067] Digitonin is a glycoside extracted from foxglove. It is a cholesterol-specific detergent with mild action and generally does not cause irreversible denaturation of proteins. Its structure is shown below:

[0068]

[0069] The cell membrane is rich in cholesterol. Digitonin can bind to the cholesterol on the cell membrane and make holes in the cell membrane, thus achieving the purpose of cell membrane lysis. Digitonin has the advantage of mild lysis, and its effect on mitochondrial structure and function is very small. It has a certain universality and can complete cell membrane lysis in a short time while ensuring the integrity of mitochondrial structure and function. It is also simple to operate, just add Digitonin, mix well and wait.

[0070] In some specific embodiments of the present invention, the amount of Digitonin added is 100 mg / mL Digitonin lysis solution per 2 to 5 million cells.

[0071] In the present invention, the step of lysing the cell membrane can be carried out in any of the following ways as required:

[0072] For example, in some specific embodiments of the present invention, the step of lysing the cell membrane comprises the steps of sequentially subjecting the cell precipitate obtained after centrifugation to hypotonic treatment, adding lysis solution and isotonic treatment.

[0073] Wherein, the hypotonic treatment is to add a certain amount of hypotonic buffer to the cell pellet after centrifugation to resuspend the cell pellet. In some specific embodiments of the present invention, the specific operation is to add the hypotonic buffer to the resuspended cells to resuspend the cell pellet, and let it stand at 4°C for 3 minutes. The specific type of hypotonic buffer can be a common type in the art. In some specific embodiments of the present invention, the composition of the hypotonic buffer is: 50mM HEPES (1M; pH = 7.0), 1mM EDTA (0.5M; pH = 8.0), 1mM Dithiothreitol (DTT), 1×Protease Inhibitor Cocktail, 1mg / mL Bovine Serum Albumin (BSA), wherein Cocktail and DTT are added immediately after use. But it is not limited to this.

[0074] Furthermore, the isotonic treatment is to avoid the damage of hypotonicity to the integrity of mitochondria. Specifically, the isotonic treatment in the present invention is achieved by adding an equal volume of hypertonic buffer to the cells after hypotonic lysis and then centrifuging at low temperature. In some specific embodiments of the present invention, the hypertonic buffer is 2×Isotonic buffer, which is composed of: 100mM HEPES (1M; pH=7.0), 2mM EDTA (0.5M; pH=8.0), 2mM Dithiothreitol (DTT), 2×Protease Inhibitor Cocktail, 2mg / mL Bovine Serum Albumin (BSA), 1.2M D-Sorbitol, wherein Cocktail and DTT are added immediately after use. Among them, the low-temperature centrifugation is at 4°C, 700g, and centrifugation for 5min, but it is not limited thereto.

[0075] In some other specific embodiments of the present invention, the step of lysing the cell membrane includes the steps of resuspending the cell pellet with KPBS and adding a lysis solution. This lysis method can release more mitochondria in the cell, and is particularly suitable for extracting mitochondria from some difficult-to-lyse cells. In the specific operation, add an appropriate amount of KPBS to resuspend the cell pellet, add 100 mg / mL Digitonin lysis solution, gently blow and lyse at 4°C for 5 minutes, and centrifuge (4°C, 1000g, 2 minutes).

[0076] KPBS: 136mM KCl, 10mM KH2PO4, 1mM Dithiothreitol (DTT), 1×ProteaseInhibitor Cocktail, pH = 7.25. (KOH adjusts the pH to 7.25 to avoid the introduction of Na + , filtered with a 0.22 μm filter head and stored at 4°C. KPBS is stable for several months).

[0077] <Extracting cytoplasm>

[0078] In the present invention, the cytoplasm is extracted by sucking the supernatant after centrifugation of the lysed cell membrane. It should be noted that when sucking, be careful not to suck up suspended matter. Preferably, in some specific embodiments of the present invention, the yield can be increased by the following steps:

[0079] That is, 200 μL of buffer (the corresponding buffer is selected according to the method of lysing the cell membrane) is added to the precipitate after the supernatant is removed to resuspend the precipitate, and after centrifugation, 300 μL of supernatant is taken and the two supernatants are combined. Among them, the centrifugation conditions vary according to the different buffers, and those skilled in the art can select them according to their needs. For KPBS, the centrifugation conditions are 4°C, 1000g, 2min; for isotonic treatment to lyse the cell membrane, the centrifugation conditions are 4°C, 750g, 4min.

[0080] <Extracting Mitochondria>

[0081] In the present invention, the mitochondrial protein in the cytoplasm is enriched by coupling streptavidin to biotin, and purified to obtain extracted mitochondrial protein. Among them, the method of coupling streptavidin to biotin is to use streptavidin magnetic beads, and the magnetic beads need to be washed before use, generally after washing 2 to 4 times with a buffer solution, and then resuspended with a buffer solution for use. Among them, for the number of cells below 5 million, 50 μL of magnetic beads are usually used, and for the number of cells greater than 5 million or larger cells, the amount of magnetic beads can be appropriately increased as needed, and those skilled in the art have such ability. In some specific embodiments of the present invention, in the magnetic bead suspension, the volume ratio of magnetic beads to buffer is 1: 1. The choice of similar buffers in the cytoplasm part varies according to the different steps of lysing the cell membrane, for example, an isotonic buffer 1×isotonic buffer (containing cocktail) or KPBS is selected.

[0082] The present invention also provides a kit for extracting mammalian cell mitochondria, which contains the recombinant plasmid described above.

[0083] In a further embodiment, the kit further comprises a culture solution containing 0.05 to 500 μM biotin, and preferably, the kit further comprises a Digitonin lysate, wherein the concentration of the Digitonin lysate is preferably 100 mg / mL. It is understood that the kit can be used to perform the mitochondrial extraction method described in the present invention to achieve efficient and rapid extraction of mitochondria, and the kit can also comprise at least one of a hypotonic buffer, a hypertonic buffer, a washing solution, a cell digestion solution, etc. required in the extraction method, which can be selected according to actual needs.

[0084] The embodiments of the present invention are described in detail below, and unless otherwise defined, all technical and scientific terms used herein are identical with the meanings generally understood by the technicians belonging to the technical field of the present invention. The term used in the specification of the present invention herein is just for the purpose of describing specific embodiments, and is not intended to limit the present invention. In addition, if not otherwise specified, the method for not specifically recording conditions or steps is a conventional method, and the reagents and materials adopted can be obtained from commercial sources.

[0085] Example 1 Construction of recombinant plasmid

[0086] The vector plasmid pLV3-CMV-3×FLAG-MCS-TurboID-HA-Puro with the sequence shown in SEQ ID NO.1 was commissioned to be synthesized by General Biotechnology (Anhui) Co., Ltd. The vector plasmid map is shown in Figure 1 As shown in .

[0087] The vector plasmid contains a TurboID sequence as shown in SEQ ID NO.2 and an HA tag sequence as shown in SEQ ID NO.4.

[0088] The vector plasmid pLV3-CMV-3×FLAG-MCS-TurboID-HA-Puro is used as the backbone vector, and the mitochondrial localization sequence (MAO, TOM20 or MAVS) and the linker sequence are introduced into the appropriate multiple cloning site by deletion, replacement or insertion to obtain a recombinant plasmid. The recombinant plasmid is constructed by any of the following methods (1) to (3):

[0089] (1) The sequence after 3×Flag in the vector plasmid pLV3-CMV-3×FLAG-MCS-TurboID-HA-Puro, such as the multiple cloning site fragment of SEQ ID NO.3, was replaced with the TOM20 sequence (SEQ ID NO.5), and a linker sequence (SEQ ID NO.8) was inserted after the TOM20 sequence to obtain the recombinant plasmid PLV3-CMV-3×FLAG-TOM20-TurboID-Puro-HA. The plasmid map is shown in Figure 2 As shown in .

[0090] (2) The sequence after 3×Flag in the vector plasmid pLV3-CMV-3×FLAG-MCS-TurboID-HA-Puro, such as the multiple cloning site fragment of SEQ ID NO.3, was deleted, and the HA tag sequence (SEQ ID NO.4) in the vector plasmid was replaced with a linker sequence (SEQ ID NO.8) and a MAO sequence (SEQ ID NO.6) to obtain the recombinant plasmid PLV3-CMV-3×FLAG-TurboID-MAO-Puro-HA. The plasmid map is shown in FIG. Figure 3 As shown in .

[0091] (3) The sequence after 3×Flag in the vector plasmid pLV3-CMV-3×FLAG-MCS-TurboID-HA-Puro, such as the multi-cloning site fragment of SEQ ID NO.3, was deleted, and the HA tag sequence (SEQ ID NO.4) in the vector plasmid was replaced with a linker sequence (SEQ ID NO.8) and a MAVS sequence (SEQ ID NO.7) to obtain the recombinant plasmid PLV3-CMV-3×FLAG-TurboID-MAVS-Puro-HA. The plasmid map is shown in FIG. Figure 4 As shown in .

[0092] It is understandable that the construction of the above recombinant plasmids is obtained by polymerase chain reaction (PCR) amplification well known in the art. The specific primer design, PCR reaction system and reaction conditions can be determined by those skilled in the art by known methods and will not be repeated here.

[0093] Example 2

[0094] This example provides a method for extracting mammalian mitochondria using the recombinant plasmid PLV3-CMV-3×FLAG-TOM20-TurboID-Puro-HA, including the following steps:

[0095] 1. Lentivirus infection: The recombinant plasmid is infected with lentivirus through cell transfection and screening for subsequent mitochondrial extraction. The specific cell transfection and screening steps are as follows:

[0096] (1) 293T cells (CL-0005, Pronocell) were transferred to a cell culture dish in advance and cultured to a cell density of 60% to 80%. Fresh DMEM complete medium was replaced before transfection. The DMEM complete medium was composed of DMEM basal medium (gibco, Lot: 6124064) containing 10% fetal bovine serum and 1% penicillin-streptomycin.

[0097] (2) Select a transient transfection system (as shown in Table 2) and prepare the transfection system according to the corresponding target plasmid transfection method.

[0098] (3) Plasmid transfection: Liposome transfection was used to mix the target plasmid with half of Opti-MEM (31985070, Thermo Fisher Scientific). The mixture was added to a sterile EP tube, gently blown to mix, and placed in a 37°C incubator for 5 min.

[0099] (4) Add Lipo Fiter reagent (HB-TRLF-1000, Hanheng Biotechnology HB-TRLF-1000) and the remaining half of Opti-MEM medium into a sterile EP tube, blow gently to mix, and place in a 37°C incubator for 5 min.

[0100] (5) Add the plasmid-Opti-MEM mixture to the Lipo Fiter-Opti-MEM mixture, pipette to mix, and let stand at room temperature for 20 min.

[0101] (6) Add the prepared transfection mixture dropwise into the cell culture dish and place in a 37°C, 5% CO2 incubator for cell transfection.

[0102] (7) 8 to 12 hours after transfection, replace the culture medium with fresh one and continue culturing for 24 to 36 hours.

[0103] (8) Add appropriate amount of puromycin (Puro) (the final concentration depends on the sensitivity of the cell line, and a concentration gradient pre-experiment can be performed in advance) to the experimental group and control group cells (in transient transfection, use the same batch of cells, the same conditions, and transfect a fluorescent protein plasmid with the same vector) for resistance screening. When all the cells in the control group die, remove the screening culture medium, wash once with PBS, and replace with fresh culture medium for continued culture.

[0104] 2. Extraction of mitochondria. The specific steps are as follows:

[0105] (1) Cell culture: The cells screened in the above article are cultured to a density of 80% to 90%.

[0106] (2) Biotin labeling: Replace the original culture medium with fresh DMEM complete culture medium containing 500 μM biotin and culture in a 37°C incubator for 10 min.

[0107] (3) Washing: Wash twice with PBS solution preheated at 37°C and aspirate the remaining liquid.

[0108] (4) Cell digestion: Add an appropriate amount of preheated 0.25% trypsin to the culture dish and place it in a 37°C incubator to wait for digestion; when it is observed under a microscope that all cells have become round and completely detached from the bottom of the culture dish, add a volume of culture medium equal to that of trypsin to terminate digestion; gently pipette and transfer the cell suspension to a sterile EP tube to count the cells.

[0109] (5) Cell preparation: Take 5 million cells after counting and place them in a sterile EP tube. Centrifuge at 1200 rpm for 5 min, discard the supernatant, resuspend in 1 mL of pre-cooled PBS, centrifuge at 4°C, 650 g for 4 min, discard the supernatant, and keep the precipitate.

[0110] (6) Prepare magnetic beads: Take 50 μL of Tiandirenhe Streptavidin Magpoly Beads, wash three times with KPBS, and resuspend in 50 μL KPBS.

[0111] (7) Cell membrane lysis: Add 1 mL of KBPS to the cell pellet after centrifugation in step (5) to resuspend the pellet, then add 1 μL of 100 mg / mL digitonin, gently pipette and lyse at 4°C for 5 min, and centrifuge (4°C, 1000 g, 2 min).

[0112] (8) Extract the cytoplasm: Aspirate the supernatant after centrifugation, being careful not to aspirate the suspended matter.

[0113] (9) Extraction of mitochondria: Add 50 μL of washed beads to the cytoplasmic supernatant of step (8), incubate on a shaker at room temperature for 15 min, separate the beads and supernatant using a magnetic stand, and remove the supernatant. Wash the beads three times with KPBS, and finally resuspend in 100 μL KPBS and boil the protein to obtain the purified cytoplasmic supernatant mitochondrial protein.

[0114] Among them, steps (7) to (9) are all carried out on ice, and the reagents need to be pre-cooled on ice in advance.

[0115] KPBS composition: 136mM KCl, 10mM KH2PO4, 1mM Dithiothreitol (DTT), 1×ProteaseInhibitor Cocktail, pH=7.25. (KOH adjusts the pH to 7.25 to avoid the introduction of Na + , filtered with a 0.22 μm filter head and stored at 4°C. KPBS is stable for several months).

[0116] Example 3

[0117] This example provides another method for extracting mammalian mitochondria, which is performed using the recombinant plasmid PLV3-CMV-3×FLAG-TurboID-MAO-Puro-HA. The other steps and process conditions are the same as those in Example 1.

[0118] Example 4

[0119] This example provides another method for extracting mammalian mitochondria, which is performed using the recombinant plasmid PLV3-CMV-3×FLAG-TurboID-MAVS-Puro-HA. The other steps and process conditions are the same as those in Example 1.

[0120] Example 5

[0121] This example provides another method for extracting mammalian mitochondria, using the recombinant plasmid PLV3-CMV-3×FLAG-TOM20-TurboID-Puro-HA in Example 1, with the only difference being the lentiviral infection step. The details are as follows:

[0122] 1. Lentivirus infection: The recombinant plasmid is infected with lentivirus through cell transfection and screening for subsequent mitochondrial extraction. The specific cell transfection and screening steps are as follows:

[0123] (1) 293T cells (CL-0005, Pronocell) were transferred to a cell culture dish in advance and cultured to a cell density of 60% to 80%. Fresh DMEM complete medium was replaced before transfection. The DMEM complete medium was composed of DMEM basal medium (gibco, Lot: 6124064) containing 10% fetal bovine serum and 1% penicillin-streptomycin.

[0124] (2) Select a stable transfection system (as shown in Table 1) and prepare the transfection system according to the corresponding target plasmid transfection method.

[0125] (3) Plasmid transfection: Liposome transfection was used to mix psPAX2, pMD2.G and the target plasmid with Opti-MEM according to the system in Table 1. The mixture was added to a sterile EP tube, gently blown to mix, and placed in a 37°C incubator for 5 min.

[0126] (4) Add Lipo Fiter reagent and the remaining Opti-MEM medium into a sterile EP tube, blow gently to mix, and place in a 37°C incubator for 5 min.

[0127] (5) Add the plasmid-Opti-MEM mixture to the Lipo Fiter-Opti-MEM mixture, pipette to mix, and let stand at room temperature for 20 min.

[0128] (6) Add the prepared transfection mixture dropwise into the cell culture dish and place in a 37°C, 5% CO2 incubator for cell transfection.

[0129] (7) 8 to 12 hours after transfection, replace the culture medium with fresh one and continue culturing for 24 to 36 hours; collect the culture supernatant, filter it with a 0.22 μm filter membrane to remove the suspended 293T cells, divide it into sterile EP tubes, and freeze it at -80°C.

[0130] (8) The M3 liver cancer cells to be infected (C6303, Bio-Tech) were subcultured to a density of 15% to 20%, and an appropriate amount of virus (the supernatant obtained in step (7)) was mixed with fresh DMEM medium (containing 10% fetal bovine serum + 1% streptomycin) at a ratio of 1:1 (v / v), and added to the cells to be infected. At the same time, polybrene was added at a final concentration of 8 μg / mL to improve the efficiency of virus infection; (In addition, a cell that was not infected with the virus was retained as a control group for subsequent screening).

[0131] (9) 12 to 24 hours after virus infection, replace the culture medium with fresh one and continue culturing for another 12 to 24 hours.

[0132] (10) Add appropriate amount of Puromycin (Puro) to the experimental and control cells respectively (the final concentration depends on the sensitivity of the cell line, and a concentration gradient pre-experiment can be performed in advance) for resistance screening. When all the cells in the control group die, remove the screening medium, wash once with PBS, and replace with fresh medium for continued culture.

[0133] The subsequent steps and process conditions such as mitochondrial extraction are the same as those in Example 1.

[0134] Example 6

[0135] This example provides another method for extracting mammalian mitochondria, which is performed using the recombinant plasmid PLV3-CMV-3×FLAG-TurboID-MAO-Puro-HA. The other steps and process conditions are the same as those in Example 5.

[0136] Example 7

[0137] This example provides another method for extracting mammalian mitochondria, which is performed using the recombinant plasmid PLV3-CMV-3×FLAG-TurboID-MAVS-Puro-HA. The other steps and process conditions are the same as those in Example 5.

[0138] Testing and result analysis

[0139] 1. Western Blot

[0140] The experimental results in Examples 2-4 were characterized and analyzed by Western Blot. Figure 5 ,in, Figure 5 A is the expression verification of TOM20, MAO and MAVS cells; Figure 5 B is the verification of each protein in the mitochondrial products extracted from TOM20 cells treated with and without biotin; Figure 5 C is the verification of each protein in the mitochondrial products extracted from MAO cells treated with and without biotin; Figure 5 D is the verification of each protein in the mitochondrial products extracted from MAVS cells treated with and without biotin.

[0141] Note: The TOM20, MAO, and MAVS cells are the abbreviations of the cell systems transfected with the recombinant plasmids PLV3-CMV-3×FLAG-TOM20-TurboID-Puro-HA, PLV3-CMV-3×FLAG-TurboID-MAO-Puro-HA, and PLV3-CMV-3×FLAG-TurboID-MAVS-Puro-HA in Examples 2-4, respectively;

[0142] Biotin (-) means no biotin treatment was used before extraction;

[0143] Biotin (+) indicates treatment with biotin before extraction;

[0144] C represents the supernatant obtained by centrifugation after lysing the cell membrane, i.e., the soluble cytoplasmic component;

[0145] AC represents the supernatant protein after incubation with magnetic beads and separation of beads using a magnetic stand;

[0146] CM indicates mitochondrial proteins adsorbed by magnetic beads, i.e., the final extracted components;

[0147] C=AC+CM.

[0148] Total represents the total protein of untreated cells.

[0149] pass Figure 5 A It can be concluded that since the recombinant plasmids all carry a 3×Flag tag, by detecting the tag, it can be seen that TOM20, MAO or MAVS cells are all overexpressed successfully.

[0150] Figure 5 B~ Figure 5 D shows that cells infected with PLV3-CMV-3×FLAG-TOM20-TurboID-Puro-HA, PLV3-CMV-3×FLAG-TurboID-MAO-Puro-HA, and PLV3-CMV-3×FLAG-TurboID-MAVS-Puro-HA achieved efficient extraction of mitochondria after treatment with biotin, and the extracted mitochondrial proteins contained almost no other cytoplasmic impurities and no nuclear protein contamination.

[0151] 2. Immunofluorescence analysis

[0152] The HCCLM3 cells transfected with the recombinant plasmid in Example 5-7 were subjected to immunofluorescence staining analysis. The results are as follows: Figure 6-Figure 8 As shown, Figure 5-8 M3-TOM20-TurboID, M3-MAO-TurboID, and M3-MAVS-TurboID are HCCLM3 cells transfected with PLV3-CMV-3×FLAG-TOM20-TurboID-Puro-HA, PLV3-CMV-3×FLAG-TurboID-MAO-Puro-HA, and PLV3-CMV-3×FLAG-TurboID-MAVS-Puro-HA, respectively. Mito tracker marks mitochondria, and DAPI stains the cell nucleus (indicating the cell location).

[0153] It can be seen that the co-localization of Flag and mito tracker markers indicates that the plasmid introduced in the present invention is accurately positioned in the mitochondria; the co-localization of Biotin and mito tracker markers indicates that the Biotin marker in the present invention is well marked before the mitochondrial extraction process.

[0154] It can be seen from the above examples and characterization results that the method for extracting mammalian cell mitochondria provided in the present invention can quickly and efficiently extract mitochondria, and the extracted mitochondria have complete structure and function, contain almost no other cytoplasmic miscellaneous proteins, and have no nuclear protein contamination.

[0155] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0156] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A recombinant plasmid, characterized in that: The recombinant plasmid is obtained by introducing a mitochondrial localization sequence into a backbone vector, wherein the backbone vector contains a TurboID sequence as shown in SEQ ID NO.

2.

2. The recombinant plasmid according to claim 1, characterized in that The recombinant plasmid has a backbone vector with a sequence as shown in SEQ ID NO.1, and is obtained by recombining in any one of the following ways (1) to (3): (1) replacing the multiple cloning site fragment of SEQ ID NO.3 in the backbone vector with the TOM20 sequence of SEQ ID NO.5, and inserting a linker of SEQ ID NO.8 after the TOM20 sequence; (2) deleting the multi-cloning site fragment of the backbone vector sequence such as SEQ ID NO.3, and replacing the HA tag of the backbone vector sequence such as SEQ ID NO.4 with a linker sequence such as SEQ ID NO.8 and a MAO sequence such as SEQ ID NO.6; (3) The multiple cloning site fragment of the backbone vector sequence such as SEQ ID NO.3 is deleted, and the HA tag of the backbone vector sequence such as SEQ ID NO.4 is replaced with a linker sequence such as SEQ ID NO.8 and a MAVS sequence such as SEQ ID NO.

7.

3. A recombinant lentivirus, characterized in that: It is a packaging body obtained after the recombinant plasmid according to claim 1 or 2 and the lentiviral auxiliary packaging plasmid are co-transfected into a host cell.

4. Use of the recombinant plasmid according to claim 1 or 2 or the recombinant lentivirus according to claim 3 in extracting mitochondria from mammalian cells.

5. A method for extracting mitochondria from mammalian cells, characterized in that: The following steps are involved: Constructing and screening a recombinant lentivirus containing the recombinant plasmid according to claim 1 or 2, and culturing the screened cells to a density of 80% to 90%; After the cultured cells were subjected to biotin labeling, cell digestion, and cell counting, the cells were resuspended and centrifuged; The cell pellet after centrifugation is treated to lyse the cell membrane and centrifuged; The supernatant after centrifugation was aspirated to obtain the cytoplasmic fraction; The mitochondrial proteins in the cytoplasm are enriched by coupling streptavidin with biotin, and then purified to obtain the extracted mitochondrial proteins.

6. The method for extracting mitochondria from mammalian cells according to claim 5, characterized in that: The biotin labeling is achieved by replacing the original culture medium with a complete culture medium containing 0.05 to 500 μM biotin and then culturing; Preferably, the culturing is carried out at 37°C for 10 min to 18 h.

7. The method for extracting mitochondria from mammalian cells according to claim 5, characterized in that: The cell digestion comprises the following steps: After adding cell digestion solution into the cell culture container, digestion is performed; After observing that all cells have become round and completely detached from the bottom of the cell culture container, add a culture medium equal to the volume of the cell digestion solution to terminate the digestion; Preferably, the cell digestion solution is trypsin.

8. The method for extracting mitochondria from mammalian cells according to claim 5, characterized in that: The cell membrane lysis is achieved by adding Digitonin lysis solution; Preferably, 100 mg / mL of Digitonin lysis solution is added for every 2 to 5 million cells.

9. The method for extracting mitochondria from mammalian cells according to claim 5, characterized in that: The method of coupling biotin with streptavidin is to use streptavidin magnetic beads.

10. A kit for extracting mitochondria from mammalian cells, characterized in that: Containing the recombinant plasmid according to claim 1 or 2 or the recombinant lentivirus according to claim 3; Preferably, the kit further contains a culture solution containing 0.05 to 500 μM biotin; Preferably, the kit further contains 100 mg / mL Digitonin lysis solution.

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