Fusion protein for screening mitophagy inducers, and use thereof

Abstract

A fusion protein for screening mitophagy inducers, and the use thereof. Mitophagy can be conveniently indicated via the establishment of a high-throughput screening model for mitophagy and by virtue of the characteristics of an mtFirefly-EGFP protein.

Description

Fusion proteins for screening mitochondrial autophagy inducers and their applications

[0001] Cross-reference declaration

[0002] This invention claims priority to Chinese Patent Application No. 2024116302382, filed on November 14, 2024, entitled "Fusion Protein for Screening Mitochondrial Autophagy Inducers and Its Application", the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to the field of biomedicine, and in particular to a fusion protein for screening mitochondrial autophagy inducers and its applications. Background Technology

[0004] Mitochondria are organelles found in most eukaryotic cells, enclosed by two membranes. They are the cells' energy-producing structures and the primary site of cellular energy metabolism. Mitochondrial damage, dysfunction, or abnormality refers to energy metabolism disorders caused by factors such as mitochondrial membrane disruption, respiratory chain inhibition, reduced enzyme activity, and mtDNA damage, leading to a series of interacting damaging processes. The accumulation of mitochondrial dysfunction and damage can trigger many diseases, including diabetes and its complications, certain neurodegenerative diseases such as Alzheimer's and Parkinson's diseases, and cardiovascular diseases.

[0005] Mitophagy is a highly conserved cellular process in eukaryotic cells that selectively removes dysfunctional or excess mitochondria through autophagy. This process effectively regulates mitochondrial number and maintains cellular energy metabolism stability. Therefore, inducing mitophagy may be an effective therapeutic approach for diseases related to mitochondrial dysfunction and damage. However, existing mitophagy inducers are limited, making the screening of new, safe, and effective mitophagy inducers an urgent need.

[0006] Currently, there is a lack of models and methods for effectively screening mitophagy inducers. Therefore, there is an urgent need in this field to establish a safe and reliable screening system and develop corresponding screening methods to screen mitophagy inducers. Summary of the Invention

[0007] The purpose of this invention is to provide a fusion protein.

[0008] Another object of the present invention is to provide a polynucleotide sequence encoding the above-mentioned fusion protein.

[0009] Another object of the present invention is to provide a vector containing the above-mentioned polynucleotide sequence.

[0010] Another object of the present invention is to provide cells containing the above-described carrier.

[0011] Another object of the present invention is to provide the use of the above-mentioned fusion protein.

[0012] Another object of the present invention is to provide a method for screening mitochondrial autophagy inducers.

[0013] To address the aforementioned technical problems, a first aspect of the present invention provides a fusion protein, the fusion protein comprising: a luciferase containing a mitochondrial localization sequence; and

[0014] Fluorescent proteins.

[0015] In some preferred embodiments, the fusion protein comprises the structure shown in formula (I):

[0016] FLE, Formula (I)

[0017] Wherein, F represents luciferase containing mitochondrial localization sequences;

[0018] L represents any cuttable connector;

[0019] E represents fluorescent protein.

[0020] In some preferred embodiments, the luciferase is firefly luciferase.

[0021] In some preferred embodiments, L represents 2A peptide, more preferably T2A.

[0022] In some preferred embodiments, the fluorescent protein is green fluorescent protein.

[0023] In some preferred embodiments, the structure of the fusion protein is as follows:

[0024] Luciferase-T2A-fluorescent protein containing mitochondrial localization sequences.

[0025] In some preferred embodiments, the structure of the fusion protein is as follows:

[0026] Firefly luciferase-T2A-green fluorescent protein containing mitochondrial localization sequences.

[0027] In some preferred embodiments, the amino acid sequence of the fusion protein is shown in SEQ ID NO.2.

[0028] In a second aspect, the present invention provides a polynucleotide encoding the aforementioned fusion protein.

[0029] In some preferred embodiments, the sequence of the polynucleotide is as shown in SEQ ID NO.1.

[0030] In a third aspect, the present invention provides a vector containing the above-described polynucleotide sequence.

[0031] In some preferred embodiments, the vector is a lentiviral vector, more preferably a lentiviral plasmid pLV.

[0032] In a fourth aspect, the present invention provides a host cell containing the aforementioned carrier.

[0033] In some preferred embodiments, the host cell is a human embryonic kidney transformed cell (HEK293T).

[0034] A fifth aspect of the invention provides the use of (i) the fusion protein described above, (ii) the polynucleotide described above, (iii) the vector described above, and (iv) the cell described above for screening mitochondrial autophagy inducers.

[0035] A sixth aspect of the present invention provides a method for screening mitophagy inducers, the method comprising the steps of:

[0036] S1, obtaining the RLU value of the experimental group; including the following steps: treating human embryonic kidney transformed cells infected with lentivirus with the drug to be screened, culturing the cells under suitable conditions for a period of time, measuring the fluorescence intensity GFP of the system, then lysing the cells, adding luciferase substrate to detect the chemiluminescence intensity FLU of the system, and calculating the RLU value of the experimental group, wherein the RLU value of the experimental group is experimental group FLU / experimental group GFP.

[0037] S2, obtain the RLU value of the control group; treat human embryonic kidney transformed cells infected with lentivirus with blank reagent, and then calculate the RLU value of the control group in the same way as the experimental group;

[0038] S3. Compare the RLU values ​​of the experimental group and the control group to determine whether the drug can induce mitophagy.

[0039] In some preferred embodiments, if the RLU value of the experimental group is lower than the RLU value of the control group, the drug is determined to induce mitophagy.

[0040] In some preferred embodiments, if the RLU value of the experimental group is lower than that of the control group and the difference is significant, then the drug is determined to induce mitophagy.

[0041] In some preferred embodiments, the method is a high-throughput screening method.

[0042] In some preferred embodiments, the method includes the steps of: placing the lentivirus-infected human embryonic kidney transformed cells into several wells, adding the same or different screening drugs to each well, and culturing the cells under suitable conditions.

[0043] In some preferred embodiments, the number of holes is 384 or 1536.

[0044] In some preferred embodiments, the suitable conditions are 37°C and 5% CO2.

[0045] In some preferred embodiments, the cell culture time is 18-30 hours, for example 24 hours.

[0046] In some preferred embodiments, the detection system for chemiluminescence intensity (FLU) includes the following steps: adding 100 μL of lysis buffer and pipetting the cells to obtain cell lysates; adding 10 μL of the cell lysates to 80 μL of luciferase substrate; and then detecting the chemiluminescence intensity (FLU) of the system.

[0047] Compared with the prior art, the present invention has at least the following advantages:

[0048] (1) This invention establishes a high-throughput screening model for mitophagy, and through the characteristics of the mtFirefly-EGFP protein, it can conveniently indicate mitophagy.

[0049] (2) Compared with fluorescence-based methods, the present invention can detect mitochondrial autophagy in cells within minutes without relying on fluorescence photography and complex photo analysis.

[0050] (3) Compared with fluorescence analysis, the present invention can be used for detection in 384 wells or 1536 wells, which is suitable for high-throughput detection.

[0051] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0052] One or more embodiments are illustrated by way of example with reference to the accompanying drawings, and these illustrative descriptions do not constitute a limitation on the embodiments.

[0053] Figure 1 is a schematic diagram illustrating the results of verifying whether the HEK293TmtFirefly-EGFP system, a human embryonic kidney transformed cell, can indicate mitochondrial autophagy, according to an embodiment of the present invention. Detailed Implementation

[0054] Through extensive and in-depth research, the inventors have developed a fusion protein system for screening mitophagy inducers. The system incorporates a mitochondrial localization sequence (MTS) onto firefly luciferase and uses a linker sequence to connect firefly luciferase and green fluorescent protein (EGFP). Utilizing the properties of both, when mitophagy occurs, the firefly protein localized to the mitochondria is phagocytosed by lysosomes. The addition of firefly luciferase substrate significantly reduces its chemiluminescence intensity, while EGFP protein is expressed in the cytoplasm with unchanged fluorescence intensity. Using the fluorescence intensity of EGFP protein as an internal control, the ratio of its fluorescence intensity to the chemiluminescence intensity produced by firefly binding to the substrate is used to evaluate the level of mitophagy, improving accuracy, reducing interference, and enabling rapid, high-throughput screening of mitophagy inducers.

[0055] Fusion protein

[0056] As used herein, the term "fusion protein" refers to a novel protein molecule formed by fusing two or more distinct protein sequences together using genetic engineering techniques. Such proteins typically consist of at least two domains encoded by separate genes and expressed in host cells through genetic engineering. In some embodiments, the fusion protein comprises two domains linked by any bond or linker. In embodiments of the invention, the fusion protein has the structure shown in FLE formula (I), where F represents a luciferase containing a mitochondrial localization sequence, L represents any cleavable linker, and E represents a fluorescent protein. The term "luciferase containing a mitochondrial localization sequence" refers to a luciferase linked with a mitochondrial localization sequence, the connection between the mitochondrial localization sequence and the luciferin sequence being a bond or any non-cleavable connection, preferably a bond. In some embodiments, the mitochondrial localization sequence is linked to the N-terminus of the luciferase. In some embodiments, the mitochondrial localization sequence is linked to the C-terminus of the luciferase. In a preferred embodiment of the invention, the sequence of the luciferase containing the mitochondrial localization sequence is as shown in SEQ ID NO. 7.

[0057] As used herein, the term "mitochondrial targeting sequence" refers to a protein sequence, typically located at the N-terminus of a mitochondrial protein, that guides these proteins into the mitochondria and ultimately to their specific subcellular regions. This sequence is cleaved after the protein has been transported into the mitochondria and is therefore also referred to as a presequence. Examples of mitochondrial targeting sequences include, but are not limited to: COX IV (cytochrome c oxidase subunit IV) targeting sequence (SEQ ID NO. 8: MKQKVEALLVLFALGYL), Cytochrome c (cytochrome c) targeting sequence (SEQ ID NO. 9: MKLVLLFLGAAGSLQKL), Tom20 (mitochondrial outer membrane protein 20) targeting sequence (SEQ ID NO. 10: MKLLVALLLAFVAGASAK), or TP synthase subunit 6 (ATP synthase subunit 6) targeting sequence (SEQ ID NO. 11: MKLILLALLLVLLGLL). In a preferred embodiment of the invention, the mitochondrial targeting sequence is MLSLRQSIRFFKPATRTLCSSR (SEQ ID NO. 3).

[0058] The term "linker" refers to a short amino acid chain that serves as a linker between two proteins in a fusion protein, allowing the proteins on either side to perform their respective independent functions. The terms "cleavable linker" or "shearable linker" refer to linkers that can be cleaved intracellularly by enzymes, such as 2A peptides. In a preferred embodiment of the invention, the shearable linker is a 2A peptide, more preferably T2A or P2A. In the most preferred embodiment, the amino acid is T2A, with the amino acid sequence shown in SEQ ID NO. 5. The T2A sequence exhibits higher shearing efficiency in the fusion protein system of the present invention.

[0059] As used in this invention, the term "firefly luciferase" is a general term for enzymes in nature that can produce bioluminescence, specifically referring to enzymes that catalyze the oxidation of luciferin to produce light. Firefly luciferase, in the presence of ATP, magnesium ions, and oxygen, can catalyze the oxidation of luciferin (D-luciferin) to oxyluciferin, emitting bioluminescence in the process. In a preferred embodiment of this invention, the firefly luciferase is firefly luciferase, whose amino acid sequence is shown in SEQ ID NO. 4.

[0060] As used herein, the term "green fluorescent protein (GFP)" refers to a protein composed of approximately 238 amino acids, originally isolated from the jellyfish *Aequorea victoria* in the northwestern Pacific Ocean, which emits green fluorescence when excited by ultraviolet or blue light. In embodiments of the present invention, the amino acid sequence of green fluorescent protein is shown in SEQ ID NO. 6.

[0061] Target gene and its expression

[0062] As used herein, the term "polynucleotide" refers to a nucleobase polymer or oligomer wherein nucleobases are linked by glycophosphate bonds (glycophosphate backbone). Exemplary polynucleotides and oligonucleotides include polymers of 2'-deoxyribonucleotides (DNA) and polymers of ribonucleotides (RNA). Polynucleotides may consist entirely of ribonucleotides, entirely of 2'-deoxyribonucleotides, or combinations thereof. In embodiments of the invention, the polynucleotide is a separate polynucleotide. In embodiments of the invention, the polynucleotide is capable of encoding the fusion protein of the invention. In embodiments of the invention, the sequence of the polynucleotide is shown in SEQ ID NO. 1.

[0063] As used herein, the term "vector" refers to an additional chromosomal element that typically carries a gene and is usually in the form of a circular double-stranded DNA molecule. Such an element can be an autonomously replicating sequence of single-stranded or double-stranded DNA or RNA derived from any source, a genome-integrated sequence, a bacteriophage, or a nucleotide sequence, linear or circular, wherein many nucleotide sequences have been ligated or recombined. This is transformed into a unique structure capable of introducing a promoter fragment and DNA sequence of a selected gene product, along with a suitable 3' untranslated sequence, into a cell. In embodiments of the invention, the vector is a lentiviral vector, preferably a lentiviral plasmid pLV.

[0064] As used herein, the term "host cell" refers to the cell used for the replication vector. In a preferred embodiment of the invention, the host cell is a cell used for producing lentivirus, such as a human embryonic kidney transformed cell (HEK293T). In some embodiments of the invention, a vector packaging plasmid containing a gene encoding the fusion protein of the invention is transfected into human embryonic kidney transformed cells (HEK293T) to produce lentivirus.

[0065] Lentiviral production

[0066] The production of lentiviruses is a technique well-known to those skilled in the art. It typically involves 1) constructing a lentiviral vector, 2) transfecting the lentiviral vector into host cells, and 3) culturing the cells to produce lentiviruses. Constructing a lentiviral vector includes the step of constructing a plasmid system for packaging lentiviruses. Commonly used plasmid systems include three-plasmid or four-plasmid systems. A three-plasmid system consists of a plasmid containing a foreign gene, a psPAX2 plasmid (carrying the gag, pol, and rev genes), and a pMD2G plasmid (providing the envelope protein vsvg gene). A four-plasmid system places the REV gene on a separate expression plasmid and removes the Tat gene, reducing the risk of accidental generation of live viruses compared to a three-plasmid system. In an embodiment of this invention, a plasmid system for packaging lentiviruses is constructed using plasmids pLV and pMD2.0G containing foreign genes and the psPAX2 packaging plasmid. This system is then transfected into HEK293T cells, and the cells are cultured to obtain lentiviral particles carrying the foreign target gene.

[0067] Methods for screening mitophagy inducers

[0068] This invention also relates to using lentiviruses containing the target gene in the embodiments of this invention to achieve high-throughput screening of mitophagy inducers, specifically including the following steps: S1, obtaining the RLU value of the experimental group; including the steps of: treating human embryonic kidney transformed cells infected with the lentivirus with the drug to be screened, culturing the cells under suitable conditions for a period of time, measuring the fluorescence intensity GFP of the system, then lysing the cells, adding luciferase substrate to detect the chemiluminescence intensity FLU of the system, and calculating the RLU value of the experimental group, wherein the RLU value of the experimental group is experimental group FLU / experimental group GFP; S2, obtaining the RLU value of the control group; treating human embryonic kidney transformed cells infected with the lentivirus with a blank reagent, and then calculating the RLU value of the control group in the same way as the experimental group; and S3, comparing the RLU value of the experimental group with the RLU value of the control group to determine whether the drug can induce mitophagy.

[0069] The above method involves infecting human embryonic kidney transformed cells (HEK293T) with a lentivirus containing the target gene, as described in this invention. The chemiluminescence and biofluorescence intensities of the system are then measured, with the biofluorescence emitted by green fluorescent protein serving as an internal control. The ratio of chemiluminescence intensity to biofluorescence intensity is used as an indicator for drug screening. Compared to measuring the chemiluminescence intensity (FLU) of a substrate detection system using luciferase alone, this method effectively corrects for errors caused by the intracellular matrix. The screened drugs are not affected by the cell itself, and the accuracy of evaluating mitophagy levels is high.

[0070] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight. Unless otherwise specified, the experimental materials and reagents used in the following embodiments are commercially available.

[0071] Unless otherwise specified, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. It should be noted that the terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the exemplary embodiments of this application.

[0072] Unless otherwise specified, the term “or” means the term “and / or” and is used interchangeably with the term “and / or”.

[0073] As used herein, including the appended claims, unless the context clearly indicates otherwise, the singular forms of words such as “an,” “a,” and “the” include their respective plural referents.

[0074] The present invention will be further described below with reference to specific embodiments. The advantages and features of the present invention will become clearer as the description proceeds, but the present invention is not limited to the following embodiments.

[0075] Example 1. Establishment of a dual-fluorescence system model

[0076] 1. Design Principles

[0077] In this embodiment, firefly luciferase protein, mitochondrial localization sequence (MTS), and green fluorescent protein (EGFP) are fused to form mtFirefly-T2A-EGFP protein. When this protein is expressed intracellularly, Firefly and EGFP proteins are cleaved. The Firefly protein with the mitochondrial localization sequence (SEQ ID NO.3) localizes to the mitochondria, while the EGFP protein is expressed in the cytoplasm. During mitophagy, the level of EGFP protein expressed in the cytoplasm remains unchanged, while Firefly protein is degraded by lysosomes. Therefore, after cell lysis, the chemiluminescence intensity of the added firefly luciferase substrate significantly decreases. Thus, using EGFP fluorescence intensity as an internal control to represent a constant level of intracellular protein expression, the chemiluminescence intensity produced by Firefly binding to the substrate can be used to evaluate the level of mitophagy.

[0078] 2. Construction of stable cell lines

[0079] Based on the mtFirefly-T2A-EGFP fusion protein, a stable cell line of human embryonic kidney transformed cells HEK293TmtFirefly-T2A-EGFP was constructed; the construction steps are as follows:

[0080] 1) Lentiviral plasmid preparation: The mtFirefly-T2A-EGFP sequence was synthesized by full gene synthesis according to the sequences (sequence sources NCBI: MK484108, MN517551), and then ligated into the lentiviral plasmid pLV by restriction enzyme digestion and ligation methods to form PLV-mtFirefly-T2A-EGFP. The endotoxin-free plasmid PLV-mtFirefly-EGFP, the lentiviral packaging plasmids pMD2.0G and psPAX2 were extracted.

[0081] 2) Lentivirus preparation: HEK293T cells were seeded in a T75 culture flask at a density of 1×10 6 cells / ml. After 24 hours, three plasmids, PLV-mtFirefly-T2A-EGFP, pMD2.0G and psPAX2, were transfected, with 4 μg of each plasmid. At 48 hours and 72 hours after transfection, the supernatants were collected, combined, and filtered through a 0.45 μm filter membrane. An appropriate amount of the concentration solution (25% PEG8000, 0.75 M NaCl) was added at a ratio of 7.5 ml of the concentration solution per 30 ml of the filtered supernatant. After mixing, it was inverted and mixed 3 - 5 times every 30 minutes for a total of 4 times. It was placed at 4°C overnight. The next day, it was centrifuged at 3000g for 20 minutes at 4°C, the supernatant was discarded, and the virus pellet was resuspended with 1 ml of PBS (phosphate buffer). It was stored at -80°C.

[0082] 3) Lentivirus infection: HEK293T cells were seeded in a T75 culture flask at a density of 5×10 5 cells / ml. After 24 hours, the prepared lentivirus was taken out from the -80°C refrigerator, fully dissolved, and then dropped into the cell culture. The infection rate was observed 48 hours later. An infection rate greater than 90% was evaluated as qualified. Puromycin at a concentration of 1 μg / ml was added to the qualified infected cell culture to eliminate the uninfected cells. After 2 - 3 days, the cells were changed to a new medium and passaged, expanded in culture, and cryopreserved to obtain a stable cell line of human embryonic kidney transformed cells HEK293TmtFirefly-T2A-EGFP. Then, monoclonal cells were sorted by flow cytometry.

[0083] The mtFirefly-T2A-EGFP sequence:

[0084] The amino acid sequence of mtFirefly-T2A-EGFP::

[0085] MTS mitochondrial localization sequence:

[0086] Firefly luciferase sequence:

[0087] Firefly luciferase sequence containing mitochondrial localization sequence

[0088] T2A connection subsequence:

[0089] EGFP sequence:

[0090] Example 2. Detection of mitophagy using a dual-fluorescence system model

[0091] 1. Experimental Methods

[0092] Human embryonic kidney transformed cells HEK293TmtFirefly-T2A-EGFP were seeded at 5000 cells / well in 96-well black ELISA plates, with 100 μl per well. After 24 hours, the following drugs were added: DMSO was added to the control group, and 3 μM and 0.5 μM chlorinated p-chlorophenylhydrazone (CCCP) were added to the experimental group. Each treatment was performed in triplicate. Cells were cultured at 37°C and 5% CO2 for 24 hours. The green fluorescence intensity (GFP) was then directly measured using an ELISA reader. Cells were then lysed, substrate was added, and chemiluminescence intensity (FLU) was measured. Specific steps:

[0093] 1) Cell lysis: Aspirate the culture medium, add 100 μL of lysis buffer, pipette the cells, and centrifuge at 220 rpm for 15 min;

[0094] 2) Take 10 μL of lysis buffer into a new 96-well plate;

[0095] 3) Add 80 μL of firefly luciferase substrate, mix well, and then detect chemiluminescence (FLU) using an ELISA reader.

[0096] 2. Data Processing

[0097] The intensity of green fluorescence emitted by EGFP was used as an internal control to represent a constant level of cellular protein expression, and the intensity of FLU was used to measure the level of mitophagy. Therefore, this study used RLU (FLU / GFP) as the standard for evaluating the level of mitophagy. The results are shown in Figure 1.

[0098] As shown in Figure 1, * represents a significant difference between the experimental group and the DMSO group. CCCP is known to induce mitophagy. The DMSO group served as the experimental control group. Treatment of cells with CCCP concentrations greater than 0.5 μM resulted in a significant decrease in RLU compared to the DMSO group. Analysis of the positive control results demonstrates that this system can be used to evaluate mitophagy levels.

[0099] Those skilled in the art will understand that the above embodiments are specific examples of implementing the present invention, and in practical applications, various changes in form and detail may be made without departing from the spirit and scope of the present invention.

Claims

1. A fusion protein, characterized in that, The fusion protein includes: Luciferase containing mitochondrial localization sequences; and Fluorescent proteins.

2. The fusion protein of claim 1, wherein, The fusion protein comprises the structure shown in formula (I): FLE, Formula (I) Wherein, F represents luciferase containing mitochondrial localization sequences; L indicates a shearable connector; E represents fluorescent protein.

3. The fusion protein of claim 2, wherein, The luciferase is firefly luciferase. And / or, L represents a 2A sequence, more preferably T2A; And / or, the fluorescent protein is green fluorescent protein.

4. The fusion protein of claim 3, wherein, The amino acid sequence of the fusion protein is shown in SEQ ID NO.

2.

5. A polynucleotide, characterized in that, The polynucleotide is used to encode the fusion protein as described in any one of claims 1-4.

6. The polynucleotide according to claim 5, characterized in that, The sequence of the polynucleotide is shown in SEQ ID NO.

1.

7. A carrier, characterized in that, The vector contains the polynucleotide as described in claim 5 or 6. Preferably, it is a lentiviral vector.

8. A host cell, characterized in that, The cells contain the carrier as described in claim 7. Preferably, they are human embryonic kidney transformed cells (HEK293T).

9. The fusion protein as described in any one of claims 1-4; the polynucleotide as described in claim 5 or 6; the vector as described in claim 7; and / or the use in cells as described in claim 8, for screening mitophagy inducers.

10. A method for screening mitophagy inducers, characterized in that, The method includes the following steps: S1, obtaining the RLU value of the experimental group; including the following steps: treating human embryonic kidney transformed cells infected with lentivirus with the drug to be screened, culturing the cells under suitable conditions for a period of time, measuring the fluorescence intensity GFP of the system, then lysing the cells, adding luciferase substrate to detect the chemiluminescence intensity FLU of the system, and calculating the RLU value of the experimental group, wherein the RLU value of the experimental group is experimental group FLU / experimental group GFP. S2, Obtain the RLU value of the control group; including the following steps: treat human embryonic kidney transformed cells infected with lentivirus with blank reagent, and then calculate the RLU value of the control group in the same way as the experimental group; S3. Compare the RLU values ​​of the experimental group and the control group to determine whether the drug can induce mitophagy.

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