Recombinant expression vector and application thereof in preparation of mitochondria

Abstract

The invention discloses a recombinant expression vector and application thereof in preparation of mitochondria. The recombinant expression vector comprises a skeleton vector and the following items: 1) a gene 1 for coding a peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC1 alpha); and 2) a gene 2 encoding a human basic fibroblast growth factor gene (hbFGF). By using the cell containing the recombinant expression vector to prepare mitochondria, the preparation time of the mitochondria is greatly shortened, and the production cost is reduced.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a recombinant expression vector and its use in preparing mitochondria. BACKGROUND

[0002] Mitochondria is a semi-autonomous organelle in eukaryotic cells, known as the "energy factory" of cells, mainly responsible for generating ATP (adenosine triphosphate) through oxidative phosphorylation (OXPHOS). Mitochondria plays a core role in cell energy metabolism, signal transduction and maintaining cell function stability. In pathological conditions such as aging, metabolic diseases, and neurodegenerative diseases, mitochondrial dysfunction and reduction in number are common phenomena, which have a serious impact on the normal physiological function of cells. Therefore, mitochondria has a wide application prospect in medical research and clinical treatment, mainly including: disease mechanism research and therapeutic targets, gene therapy and base editing, and drug development, etc.

[0003] Therefore, it is of great scientific and clinical significance to develop a new technology that can effectively increase the number of mitochondria and improve their function, and there is an urgent need in the art to develop a method for efficiently preparing mitochondria. SUMMARY

[0004] Therefore, at least to provide a recombinant expression vector and its use in preparing mitochondria.

[0005] In the first aspect of the present application, a recombinant expression vector for preparing mitochondria is provided, comprising a backbone vector and the following items:

[0006] 1) a gene 1 encoding peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC1a); and,

[0007] 2) a gene 2 encoding human basic fibroblast growth factor gene (hbFGF).

[0008] In the second aspect of the present application, a genetically engineered bacterium is provided, comprising the recombinant expression vector for preparing mitochondria as defined in the first aspect.

[0009] In the third aspect of the present application, a recombinant cell is provided, expressing the gene 1 and the gene 2 as defined in the first aspect.

[0010] In the fourth aspect of the present application, a lipid nanoparticle is provided, comprising the recombinant expression vector for preparing mitochondria as defined in the first aspect.

[0011] In the fifth aspect of the present application, the use of the recombinant expression vector for preparing mitochondria as defined above, the genetically engineered bacterium as defined above, the recombinant cell as defined above, or the lipid nanoparticle as defined above in preparing mitochondria is provided.

[0012] One aspect of the present application provides a method for efficient production of mitochondria by transfecting plasmid DNA containing humanized optimized PGC1a and hbFGF genes, significantly improving the mitochondrial production and proliferation capacity of human fibroblasts (BJ cells). Overexpression of PGC1a promotes the biosynthesis of mitochondria, while overexpression of hbFGF significantly enhances the efficiency of cell proliferation. Combined with the optimized three-dimensional culture system (spheroid culture and shaking culture), the yield of mitochondria can reach 10 times that of wild-type BJ cells within 36 hours.

[0013] This method greatly shortens the preparation time of mitochondria and reduces the production cost. Through cell lysis and ultrafiltration technology, high-quality mitochondria are successfully extracted and purified. The prepared mitochondria can be widely used in the development of oral supplements to improve energy metabolism, delay aging and support cell function recovery. At the same time, mitochondria have important potential application value in drug research and development and treatment of mitochondrial-related diseases (such as neurodegenerative diseases and metabolic diseases). The present application provides an innovative solution for the large-scale production of mitochondria and their application in the field of nutritional supplements and regenerative medicine. DETAILED DESCRIPTION

[0014] In order to facilitate the understanding of the present application, the present application will be described more fully below by examples. However, the present application can be implemented in many different forms, and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present application more thorough and comprehensive.

[0015] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application belongs. The terminology used in the specification of the present application is only for the purpose of describing specific embodiments and is not intended to limit the present application. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0016] In the present application, "one or more" means any one of the listed items or any combination of the listed items, unless otherwise specified. Similarly, "one or more" and the like otherwise indicate "one or more" are also understood in the same way, unless otherwise specified.

[0017] As used in the present application, "combinations thereof", "any combination thereof", "any combination thereof" and the like include all suitable combinations of any two or more of the listed items.

[0018] In the present application, "suitable", in "suitable combination", "suitable manner", "any suitable manner" and the like, is subject to the ability to implement the technical solutions of the present application, solve the technical problems of the present application, and achieve the intended technical effects of the present application.

[0019] In the present application, "further", "still further", "in particular", "for example", "such as", "example", "for instance" and the like are used for description purposes, indicating that the different technical solutions before and after are related in terms of coverage, but should not be understood as limiting the previous technical solution, nor as limiting the protection scope of the present application. In the present application, A (such as B) means that B is one non-limiting example of A, and it can be understood that A is not limited to B.

[0020] In the present application, "optionally", "optional" and "optional" mean optional, i.e. selected from either of the two parallel schemes "with" or "without". If there are multiple "optional" in a technical solution, and there is no contradictory or mutually restrictive relationship, each "optional" is independent. In the present application, "optionally includes", "optionally contains" and the like are described, for example, "optionally includes" means "may include or not include".

[0021] The terms "contain", "include" and "comprise" used in the present application are synonymous terms, which are inclusive or open-ended, and do not exclude additional, unmentioned members or features. Members or features, such as materials or components, structures, elements, instruments, etc.; non-limiting examples of members or features also include actions, conditions, timing, states, etc.

[0022] In the present application, the technical features or technical solutions described in open-ended language include both closed technical features or technical solutions consisting of the listed contents and open technical features or technical solutions containing the listed contents.

[0023] In the present application, the exemplary description involving "in some embodiments (or examples)", "in one embodiment (or example)" and the like can cover but is not limited to the following meanings: these schemes can be combined with other schemes in a suitable manner to form new technical solutions.

[0024] In the present application, the terms "first", "second", "third", "fourth" and the like in "first aspect", "second aspect", "third aspect", "fourth aspect" and the like are only for description purposes, and cannot be understood as indicating or implying relative importance or quantity, nor can it be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth" and the like only serve the purpose of non-exhaustive enumeration description, and it should be understood that they do not constitute a closed limitation on the quantity.

[0025] In this application, when a numerical interval (i.e., a numerical range) is involved, the distribution of the selectable numbers in the numerical interval is considered to be continuous and includes both numerical endpoints (i.e., the minimum and maximum values) of the numerical interval and every number between the two numerical endpoints, unless otherwise specified. When a numerical interval refers only to integers within the numerical interval, including both endpoints and every integer between the two endpoints, it is equivalent to directly listing each integer, unless otherwise specified. When multiple numerical ranges are provided to describe a feature or a characteristic, the numerical ranges can be combined. In other words, unless otherwise indicated, numerical ranges disclosed herein are to be understood to include any and all sub-ranges of the numbers subsumed therein. A "number" in a numerical interval can be any quantitative value, such as a number, a percentage, a ratio, etc. A "numerical interval" is intended to broadly include numerical interval types such as percentage intervals, ratio intervals, and the like.

[0026] In this application, when a method flow involves multiple steps, unless otherwise specified, the execution of the steps does not have strict order restrictions and can be executed in other orders than described. Moreover, any step can include multiple sub-steps or multiple stages, which do not necessarily have to be executed at the same time, but can be executed at different times, and the execution order does not necessarily have to be sequential, but can be executed alternately or simultaneously with other steps or sub-steps or stages of other steps.

[0027] One aspect of the present application proposes a new technology for improving the proliferation ability of human fibroblasts (BJ cells) and increasing the number of mitochondria by co-transfecting plasmid DNA containing PGC1α gene (peroxisome proliferator-activated receptor γ coactivator 1α) and human basic fibroblast growth factor gene (hbFGF). PGC1α is a key regulator of mitochondrial biogenesis, and its overexpression can significantly enhance the ability of mitochondria to generate; at the same time, hbFGF is an important cell growth factor that can significantly promote the proliferation of BJ cells, thereby providing an adequate source of cells for the preparation of mitochondria.

[0028] Through this technology, the optimized double gene transfection significantly improves the viability and proliferation efficiency of BJ cells, and successfully purifies a high number and high quality of mitochondria through cell lysis and ultrafiltration. This technology has the following several significant features:

[0029] 1) Co-transfection of genes: Co-transfection of PGC1α and hbFGF genes enhances mitochondrial production and cell proliferation, respectively, and synergistically improves the yield of mitochondria.

[0030] 2) Efficiency: By optimizing the cell lysis and ultrafiltration process, efficient extraction and purification of mitochondria are achieved.

[0031] 3) Wide applicability: This technology not only applies to mitochondrial research, but also serves as a potential tool for treating diseases related to mitochondrial dysfunction, providing new solutions for regenerative medicine and cell therapy.

[0032] In the first aspect of the present application, a recombinant expression vector for preparing mitochondria is provided, which comprises a backbone vector and the following items:

[0033] 1) a gene 1 encoding peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC1a); and,

[0034] 2) a gene 2 encoding human basic fibroblast growth factor gene (hbFGF).

[0035] Wherein, the gene 1 and the gene 2 in the recombinant expression vector can be independently at least one copy. It should be understood that the two genes in the recombinant expression vector can be arranged in any order.

[0036] In some embodiments, the gene 1 comprises a nucleotide fragment with a sequence as shown in SEQ ID NO: 1.

[0037] In some embodiments, the sequence of the gene 1 is as shown in SEQ ID NO: 1.

[0038] In some embodiments, the gene 2 comprises a nucleotide fragment with a sequence as shown in SEQ ID NO: 2.

[0039] In some embodiments, the sequence of the gene 2 is as shown in SEQ ID NO: 2.

[0040] In some embodiments, the backbone vector is selected from the group consisting of pcDNA3.1, pcDNA3.0, pcDNA4 / 5, pEF1a, pSV2-neo and pCI-neo. Illustratively, the backbone vector is pcDNA3.1.

[0041] In some embodiments, the recombinant expression vector further comprises a human CMV promoter.

[0042] In other embodiments, the recombinant expression vector further comprises 5' and 3' untranslated regions (UTRs) of a- globin. Illustratively, the sequence of the 5' untranslated region of a- globin is as shown in SEQ ID NO: 3, and the sequence of the 3' untranslated region of a- globin is as shown in SEQ ID NO: 4.

[0043] In one embodiment, the recombinant expression vector further comprises a human CMV promoter and 5' and 3' untranslated regions of a- globin.

[0044] In some embodiments, the recombinant expression vector is a combination product comprising a recombinant expression vector 1 and a recombinant expression vector 2; wherein the recombinant expression vector 1 comprises the gene 1 and the recombinant expression vector 2 comprises the gene 2.

[0045] In the second aspect of the present application, a genetically engineered bacterium comprising the recombinant expression vector for preparing mitochondria as defined in the first aspect is provided.

[0046] In some embodiments, the host cell of the genetically engineered bacterium comprises a prokaryotic cell and a eukaryotic cell.

[0047] Illustratively, the prokaryotic cell can comprise or be Escherichia coli; and the eukaryotic cell can comprise or be yeast.

[0048] It should be understood that the genetically engineered bacterium aims to amplify the plasmid comprising the recombinant expression vector for subsequent transfection into a mammal to prepare a recombinant cell.

[0049] In the third aspect of the present application, a recombinant cell expressing the gene 1 and the gene 2 as defined above is provided.

[0050] In some embodiments, the recombinant cell comprises the recombinant expression vector for preparing mitochondria as defined in the first aspect.

[0051] In some embodiments, the host cell of the recombinant cell can be, for example, a BJ human fibroblast, 293T, H9c2, or 3T3, etc. Illustratively, the BJ human fibroblast can be ATCC CRL-2522.

[0052] In some embodiments, the method for preparing the recombinant cell comprises:

[0053] The recombinant cell is prepared after transfecting the above-mentioned host cell using a lipid nanoparticle comprising the recombinant expression vector for preparing mitochondria as defined in the first aspect.

[0054] In the fourth aspect of the present application, a lipid nanoparticle comprising the recombinant expression vector for preparing mitochondria as defined in the first aspect is provided.

[0055] In the fifth aspect of the present application, the recombinant expression vector for preparing mitochondria as defined in the first aspect, the genetically engineered bacterium as defined in the second aspect, the recombinant cell as defined in the third aspect, or the lipid nanoparticle as defined in the fourth aspect is used for preparing mitochondria.

[0056] In some embodiments, the use comprises:

[0057] S100. Cultivating the recombinant cell according to the third aspect;

[0058] S200. Lysing the cultured recombinant cells and collecting the supernatant; and,

[0059] S300. Isolation and purification of mitochondria using ultrafiltration technology.

[0060] In some embodiments, in step S100, a three-dimensional culture system is used for culture.

[0061] The three-dimensional culture system comprises:

[0062] culturing the recombinant cells upside down to form spheroids; and,

[0063] The spheroids were transferred to liquid culture medium and cultured in a shaking incubator.

[0064] In some embodiments, each spheroid comprises at least 5,000 cells.

[0065] In some embodiments, the liquid culture medium is a high-glucose DMEM medium containing 15% to 25% (eg, 20%) fetal bovine serum by volume.

[0066] The shaking culture conditions may be: 37°C, 5% CO2.

[0067] In some embodiments, in step S200, the recombinant cells are resuspended in ice-cold mitochondrial isolation buffer, mechanically lysed, and the supernatant collected. Exemplarily, the mitochondrial isolation buffer comprises 200 mM to 300 mM sucrose, 8 mM to 12 mM HEPES (pH 7.4), and 0.8 mM to 1.2 mM EDTA.

[0068] In some embodiments, the mitochondrial isolation buffer comprises 250 mM sucrose, 10 mM HEPES pH 7.4, and 1 mM EDTA.

[0069] In some embodiments, in step S300, the supernatant is centrifuged at 8000 g to 12,000 g (e.g., 10,000 g) for 15 minutes to 25 minutes (e.g., 20 minutes), the precipitate is collected, the precipitate is resuspended in mitochondrial isolation buffer, and then filtered through an ultrafiltration membrane to prepare mitochondria.

[0070] Some examples are provided below.

[0071] The embodiments of the present application will be described in detail below with examples. It should be understood that these examples are only used to illustrate the present application and not to limit the scope of the present application. The experimental methods in the following examples without specified conditions should be understood as the priority of the instructions given in the present application, and can also be according to the experimental manual or conventional conditions in the art, or according to the conditions suggested by the manufacturer, or according to the experimental methods known in the art.

[0072] Example 1 Preparation of mitochondria

[0073] 1. Plasmid construction and preparation

[0074] pcDNA3.1 vector was used as an expression vector for expressing human PGC1a and hbFGF genes. Human PGC1a and hbFGF gene sequences were human codon optimized, and the sequences are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively; the expression frame contains a human CMV promoter to achieve high-level expression and 5' and 3' untranslated regions (UTRs) of a globin protein (sequences are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively) to enhance mRNA stability and translation efficiency.

[0075] The synthesized plasmid DNA was transformed into E. coli DH5a by heat shock method. The transformed strain was inoculated in Luria-Bertani (LB) medium containing ampicillin and cultured at 37°C for 16 hours with shaking. Then, large-scale plasmid DNA was extracted by alkaline lysis method and further purified using a plasmid extraction kit (such as Qiagen Plasmid Maxi Kit). The plasmid concentration was determined by NanoDrop spectrophotometer, and the purity (A260 / A280) should reach 1.8-2.0.

[0076] 2. Preparation of lipid nanoparticles and plasmid transfection

[0077] The extracted plasmid DNA was wrapped by lipid nanoparticles (LNP) for transfection. The lipid nanoparticles were prepared using a commercial kit (such as Invitrogen Lipofectamine 3000) according to the manufacturer's instructions. The wrapped plasmid was transfected into BJ cells by LNP.

[0078] 3. Culture and three-dimensional culture system of BJ cells

[0079] BJ human fibroblasts (ATCC CRL-2522) were cultured in high glucose Dulbecco's Modified Eagle Medium (DMEM) containing 20% Fetal Bovine Serum (FBS) at 37°C in a 5% CO2 incubator.

[0080] In the three-dimensional culture system, BJ cells were first cultured upside-down in culture dishes. After the cells formed spheroids (each spheroid contained at least 5000 cells), they were transferred to a shaker flask and cultured in a shaker incubator at 37°C in 5% CO2 to maintain the growth of the three-dimensional spheroids.

[0081] 4. Mitochondria purification

[0082] BJ cells were collected 48 hours after transfection and mitochondria were isolated and purified by cell lysis and ultrafiltration techniques. The specific steps are as follows:

[0083] (1) Cell collection and lysis:

[0084] The transfected BJ cells were collected, washed twice with PBS, and resuspended with ice-cold mitochondria isolation buffer (e.g., 250 mM sucrose, 10 mM HEPES pH 7.4, 1 mM EDTA, with protease inhibitors).

[0085] The cells were mechanically lysed using a Dounce homogenizer for 20 strokes on ice.

[0086] Centrifuged at 600 g for 10 minutes to remove nuclei and unlysed cells, and the supernatant was collected.

[0087] (2) Mitochondria isolation and purification:

[0088] The supernatant was centrifuged at 10,000 g for 20 minutes, and the precipitate (crude mitochondrial fraction) was collected.

[0089] The crude mitochondrial precipitate was resuspended with mitochondria isolation buffer and filtered through a 0.22 μm ultrafiltration membrane to remove cell debris and impurities, obtaining purified mitochondria.

[0090] The purified mitochondrial suspension can be directly used for downstream experiments or stored at -80°C for future use.

[0091] (3) Mitochondria quantification analysis

[0092] Mitochondrial number was quantitatively analyzed by MitoTracker fluorescent probe staining kit (e.g., Thermo Fisher Scientific MitoTracker Green FM). The specific steps are as follows:

[0093] a. The BJ cells were gently washed twice with PBS.

[0094] b. Prepare MitoTracker working solution (final concentration 200 nM) and add it to the cell culture medium, incubate at 37°C for 30 minutes.

[0095] c. After incubation, wash the cells with PBS three times to remove unbound dye.

[0096] d. Use a fluorescence microscope to detect the fluorescence signal (Ex / Em = 490 / 516 nm).

[0097] e. After normalizing the mitochondrial fluorescence intensity to the number of cells, it is used for quantitative comparison of mitochondria.

[0098] (4) Western Blot analysis of PGC1α and hbFGF expression

[0099] Western Blot is used to detect the expression of PGC1α and hbFGF in BJ cells after transfection. The specific steps are as follows:

[0100] a. Protein extraction:

[0101] Collect the transfected BJ cells and lyse the cells with ice-cold RIPA lysis buffer (containing 1% PMSF and protease inhibitors), incubate on ice for 30 minutes.

[0102] Centrifuge at 12,000 g for 10 minutes, collect the supernatant, and determine the protein concentration by BCA protein concentration determination method.

[0103] b. Protein separation and membrane transfer:

[0104] Load 20 μg of total protein per well and use SDS-PAGE (10% separating gel) for electrophoretic separation.

[0105] Transfer the separated proteins to a PVDF membrane (0.22 μm pore size) by wet transfer, and after membrane transfer, block with 5% skim milk at room temperature for 1 hour.

[0106] c. Antibody incubation:

[0107] Primary antibody: Incubate with anti-PGC1α antibody (e.g., Abeam, 1:1000 dilution) and anti-hbFGF antibody (e.g., Cell Signaling Technology, 1:1000 dilution) at 4°C overnight, respectively.

[0108] Secondary antibody: Incubate with HRP-labeled secondary antibody (1:5000 dilution) matching the primary antibody at room temperature for 1 hour.

[0109] d. Signal detection:

[0110] Develop with ECL chemiluminescence kit (e.g., Thermo Fisher Scientific SuperSignal West Pico) and detect protein signal using gel imaging system.

[0111] Internal reference: Use β-actin or GAPDH, and analyze band gray value by ImageJ software to quantify the expression level of target protein.

[0112] Results show:

[0113] 1. PGC1α transfection promotes the increase of mitochondrial production

[0114] Detect mitochondrial production by MitoTracker reagent, the results show that compared with wild-type BJ cells, BJ cells transfected with PGC1α plasmid have significantly increased mitochondrial production, reaching 2.2 times that of wild-type cells. This indicates that overexpression of PGC1α significantly promotes the biosynthesis of mitochondria.

[0115] 2. hbFGF transfection promotes cell proliferation

[0116] BJ cells transfected with hbFGF plasmid have significantly enhanced proliferation ability. Experimental results show that compared with wild-type BJ cells, BJ cells transfected with hbFGF have increased proliferation rate by 1.8 times, and have obtained more cell number in the same culture time. This indicates that overexpression of hbFGF can significantly promote the proliferation of BJ cells, providing more cell sources for further preparation of mitochondria.

[0117] 3. Synergistic effect of PGC1α and hbFGF combined transfection

[0118] In BJ cells with combined transfection of PGC1α and hbFGF, the production of mitochondria is further greatly increased. After 36 hours of culture, the mitochondrial production of combined transfection cells is 5 times that of wild-type BJ cells. This indicates that the synergistic effect of PGC1α and hbFGF can significantly improve the efficiency of mitochondrial generation.

[0119] 4. Further optimization of three-dimensional culture system

[0120] In the three-dimensional culture system (cell spheroid culture combined with shaking culture), the mitochondrial production of BJ cells transfected with PGC1a and hbFGF was further improved. Within 36 hours of culture, the mitochondrial production reached 10 times that of wild-type BJ cells. This result shows that the three-dimensional culture system can significantly optimize the growth environment of cells and further improve the efficiency of mitochondrial production.

[0121] Conclusion

[0122] This study developed a new technology for efficient preparation of mitochondria. Through the combined transfection of PGC1a and hbFGF and the optimized three-dimensional culture system, the production of mitochondria was significantly improved. Within 36 hours of culture, the mitochondrial production of the combined transfection cells in the three-dimensional culture system reached 10 times that of wild-type BJ cells. Compared with traditional methods, this technology not only significantly shortens the time required for mitochondrial preparation, but also greatly reduces the production cost by improving the cell proliferation efficiency and mitochondrial production efficiency.

[0123] This efficient preparation technology of mitochondria provides the possibility for the practical application of mitochondria in multiple fields. For example, high-purity mitochondria can be used to develop oral supplements to supplement cell function and improve energy metabolism, which has potential benefits against aging and metabolic diseases. In addition, mitochondria can also be used as an important tool for drug development to treat diseases related to mitochondrial dysfunction (such as neurodegenerative diseases and cardiovascular diseases), and has important application prospects in the fields of regenerative medicine and cell therapy.

[0124] The technical features of the above-described embodiments can be combined in any manner. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described, but as long as the combinations of the technical features do not exist, they should be considered as within the scope of the present disclosure.

[0125] The above-described embodiments only express several implementation manners of the present application, and the description is more specific and detailed, but it should not be understood as a limitation on the scope of the patent. It should be noted that for ordinary skilled persons in the art, without departing from the concept of the present application, several modifications and improvements can be made, which are within the scope of the present application. Therefore, the scope of protection of the present patent should be subject to the appended claims, and the description can be used to explain the content of the claims.

Claims

1. A recombinant expression vector for preparing mitochondria, characterized in that: It contains a backbone vector and the following items: 1) gene encoding peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC1α); and, 2) Gene 2 encoding human basic fibroblast growth factor gene (hbFGF).

2. The recombinant expression vector for preparing mitochondria according to claim 1, wherein The recombinant expression vector satisfies one or more of the following conditions (1) to (3): (1) The gene 1 comprises a nucleotide fragment having a sequence as shown in SEQ ID NO: 1; (2) the gene 2 comprises a nucleotide fragment having a sequence as shown in SEQ ID NO: 2; and (3) The backbone vector is selected from the group consisting of pcDNA3.1, pcDNA3.0, pcDNA4 / 5, pEF1α, pSV2-neo and pCI-neo.

3. The recombinant expression vector for preparing mitochondria according to claim 2, wherein: The recombinant expression vector further comprises a human CMV promoter and one or more of the 5' and 3' untranslated regions (UTRs) of α-globin.

4. The recombinant expression vector for preparing mitochondria according to any one of claims 1 to 3, wherein The recombinant expression vector is a combination product, which includes recombinant expression vector 1 and recombinant expression vector 2; wherein, the recombinant expression vector 1 includes the gene 1, and the recombinant expression vector 2 includes the gene 2.

5. A genetically engineered bacterium, characterized in that: It comprises the recombinant expression vector for preparing mitochondria according to any one of claims 1 to 4; Optionally, the host cells of the genetically engineered bacteria include prokaryotic cells and eukaryotic cells; The prokaryotic cells may optionally include Escherichia coli; The eukaryotic cell optionally includes yeast.

6. A recombinant cell, characterized in that It expresses gene 1 and gene 2 as defined in claim 1; Optionally, the recombinant cell comprises the recombinant expression vector for preparing mitochondria according to any one of claims 1 to 4; Optionally, the recombinant cells comprise one or more of BJ human fibroblasts, 293T, H9c2 and 3T3; wherein the BJ human fibroblasts are optionally ATCC CRL-2522.

7. Lipid nanoparticles, characterized in that It comprises the recombinant expression vector for preparing mitochondria according to any one of claims 1 to 4.

8. Use of the recombinant expression vector for preparing mitochondria according to any one of claims 1 to 4, the genetically engineered bacteria according to claim 5, the recombinant cell according to claim 6, or the lipid nanoparticles according to claim 7 in preparing mitochondria.

9. The use according to claim 8, characterized in that The uses include: Cultivating the recombinant cell according to claim 6; Lysing the cultured recombinant cells and collecting the supernatant; and, Mitochondria were isolated and purified by ultrafiltration technology.

10. The use according to claim 8 or 9, characterized in that It satisfies one or more of the following conditions (1) to (3): (1) In the step of culturing the recombinant cells, a three-dimensional culture system is used for culturing; The three-dimensional culture system comprises: culturing the recombinant cells upside down to form spheroids; and, transferring the spheroids into liquid culture medium for shaking culture; Wherein, the liquid culture medium may optionally be a high-glucose DMEM culture medium containing 15% to 25% fetal bovine serum by volume, and the shaking culture conditions may optionally include: 37° C., 5% CO 2 ; (2) In the step of lysing the recombinant cells obtained by culture, the recombinant cells are resuspended in ice-cold mitochondrial isolation buffer, mechanically lysed and the supernatant is collected; optionally, the mitochondrial isolation buffer contains 200 mM to 300 mM sucrose, 8 mM to 12 mM HEPES with a pH of 7.4 and 0.8 mM to 1.2 mM EDTA; and, (3) In the step of separating and purifying mitochondria using ultrafiltration technology, the supernatant is centrifuged at 8000 g~10,000 g for 15 minutes to 25 minutes, the precipitate is collected, the precipitate is resuspended in mitochondrial separation buffer and then filtered through an ultrafiltration membrane to prepare mitochondria.