Fusion protein for screening mitochondrial autophagy inducer and application thereof

Abstract

The invention discloses a fusion protein for screening a mitochondrial autophagy inducer and application of the fusion protein. In the application, the mitochondrial autophagy can be conveniently indicated through the characteristics of the mtFirefly-EGFP protein by establishing the high-throughput screening model of the mitochondrial autophagy.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a fusion protein for screening a mitochondrial autophagy inducer and an application thereof. Background Art

[0002] Mitochondria are a type of organelles that exist in most eukaryotic cells and are surrounded by two membranes. They are structures that produce energy in cells and are the main site of cellular energy metabolism. Mitochondrial damage, mitochondrial dysfunction, or mitochondrial dysfunction refer to energy metabolism disorders caused by mitochondrial membrane damage, respiratory chain inhibition, reduced enzyme activity, mtDNA damage, etc., which in turn lead to a series of interacting damage processes. Mitochondrial dysfunction and the accumulation of damage can cause many diseases, including diabetes and its complications, certain neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, and cardiovascular diseases.

[0003] Mitophagy is a highly conserved cellular process in which eukaryotic cells selectively remove dysfunctional or redundant mitochondria through the autophagy mechanism. This process can well adjust the number of mitochondria and maintain the stability of cellular energy metabolism. Therefore, inducing mitophagy may be an effective treatment for diseases related to mitochondrial dysfunction and damage. However, there are few existing mitophagy inducers, and the need to screen new safe and effective mitophagy inducers is urgent.

[0004] Currently, there is still a lack of models and methods that can effectively screen mitophagy inducers. Therefore, there is an urgent need to establish a safe and reliable screening system and develop corresponding screening methods to screen mitophagy inducers. Summary of the invention

[0005] The object of the present invention is to provide a fusion protein.

[0006] Another object of the present invention is to provide a polynucleotide sequence encoding the above fusion protein.

[0007] Another object of the present invention is to provide a vector containing the above polynucleotide sequence.

[0008] Another object of the present invention is to provide cells containing the above vector.

[0009] Another object of the present invention is to provide uses of the above fusion protein.

[0010] Another object of the present invention is to provide a method for screening a mitochondrial autophagy inducer.

[0011] In order to solve the above technical problems, the first aspect of the present invention provides a fusion protein, wherein the fusion protein comprises:

[0012] luciferase containing a mitochondrial localization sequence; and

[0013] Fluorescent protein.

[0014] In some preferred embodiments, the fusion protein comprises a structure represented by the following formula (I):

[0015] FLE, formula (I)

[0016] Among them, F represents luciferase containing a mitochondrial localization sequence;

[0017] L represents any cleavable linker;

[0018] E indicates fluorescent protein.

[0019] In some preferred embodiments, the luciferase is firefly luciferase.

[0020] In some preferred embodiments, L is represented by a 2A peptide, more preferably T2A.

[0021] In some preferred embodiments, the fluorescent protein is green fluorescent protein.

[0022] In some preferred embodiments, the structure of the fusion protein is as follows:

[0023] Luciferase-T2A-fluorescent protein containing a mitochondrial localization sequence.

[0024] In some preferred embodiments, the structure of the fusion protein is as follows:

[0025] Firefly luciferase-T2A-green fluorescent protein containing a mitochondrial localization sequence.

[0026] In some preferred embodiments, the amino acid sequence of the fusion protein is shown in SEQ ID NO.2.

[0027] The second aspect of the present invention provides a polynucleotide encoding the above fusion protein.

[0028] In some preferred embodiments, the sequence of the polynucleotide is as shown in SEQ ID NO.1.

[0029] The third aspect of the present invention provides a vector, wherein the vector contains the above-mentioned polynucleotide sequence.

[0030] In some preferred embodiments, the vector is a lentiviral vector, more preferably a lentiviral plasmid pLV.

[0031] The fourth aspect of the present invention provides a host cell, wherein the cell contains the above-mentioned vector.

[0032] In some preferred embodiments, the host cell is a human embryonic kidney transformed cell (HEK293T).

[0033] The fifth aspect of the present invention provides the use of any one of (i) the above-mentioned fusion protein, (ii) the above-mentioned polynucleotide, (iii) the above-mentioned vector, and (iv) the above-mentioned cell for screening mitochondrial autophagy inducers.

[0034] A sixth aspect of the present invention provides a method for screening a mitochondrial autophagy inducer, the method comprising the steps of:

[0035] S1, obtaining the value of RLU of the experimental group; comprising the steps of: 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 and adding luciferase substrate to detect the chemiluminescence intensity FLU of the system, calculating the value of RLU of the experimental group, and the value of RLU of the experimental group is the experimental group FLU / experimental group GFP;

[0036] S2, obtaining the RLU value of the control group; using a blank reagent to treat the human embryonic kidney transformed cells infected with the lentivirus, and then calculating the RLU value of the control group in the same manner as the experimental group;

[0037] S3, comparing the RLU value of the experimental group with the RLU value of the control group to determine whether the drug can induce mitochondrial autophagy.

[0038] In some preferred embodiments, if the RLU value of the experimental group is lower than the RLU value of the control group, it is determined that the drug is able to induce mitochondrial autophagy.

[0039] In some preferred embodiments, if the RLU value of the experimental group is lower than the RLU value of the control group and shows a significant difference, it is determined that the drug can induce mitochondrial autophagy.

[0040] In some preferred embodiments, the method is a high-throughput screening method.

[0041] In some preferred embodiments, the method comprises the steps of placing the human embryonic kidney transformed cells infected with the lentivirus in a plurality of wells, adding the same or different drugs to be screened to each well for treatment, and culturing the cells under appropriate conditions.

[0042] In some preferred embodiments, the number of holes is 384 holes or 1536 holes.

[0043] In some preferred embodiments, the suitable conditions are 37°C and 5% CO2.

[0044] In some preferred embodiments, the cell culture time is 18-30 hours, such as 24 hours.

[0045] In some preferred embodiments, the detection system chemiluminescence intensity FLU includes the steps of adding 100ul lysis solution to blow the cells to obtain the cell lysate, adding 10ul of the cell lysate to 80ul of luciferase substrate, and then detecting the system chemiluminescence intensity FLU.

[0046] Compared with the prior art, the present invention has at least the following advantages:

[0047] (1) The present invention establishes a high-throughput screening model for mitochondrial autophagy and can conveniently indicate mitochondrial autophagy through the characteristics of the mtFirefly-EGFP protein.

[0048] (2) Compared with fluorescence-based methods, the present invention can detect mitochondrial autophagy of cells within a few minutes without relying on fluorescence photography and complex photo analysis.

[0049] (3) Compared with fluorescence analysis, the present invention can be used for detection in 384 wells or 1536 wells, and is suitable for high-throughput detection.

[0050] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] One or more embodiments are exemplarily described by the pictures in the corresponding drawings, and these exemplary descriptions do not constitute limitations on the embodiments.

[0052] Figure 1 This is a schematic diagram of the results of verifying whether the human embryonic kidney transformed cell HEK293TmtFirefly-EGFP system can indicate mitochondrial autophagy according to an embodiment of the present invention. DETAILED DESCRIPTION

[0053] After extensive and in-depth research, the inventors have developed a fusion protein system for screening mitochondrial autophagy inducers, in which the mitochondrial localization sequence (MTS) is combined with firefly luciferase, and the firefly luciferase and green fluorescent protein (EGFP) are connected using a linker sequence. By utilizing the characteristics of the two, when mitochondrial autophagy occurs, the Firefly protein located on the mitochondria will be engulfed by the lysosomes, and its chemiluminescence intensity will significantly decrease when the firefly luciferase substrate is added, while the EGFP protein is expressed in the cytoplasm with unchanged fluorescence intensity. The fluorescence intensity of the EGFP protein is used as an internal reference, and the ratio of the fluorescence intensity of the EGFP protein to the chemiluminescence intensity generated by the combination of Firefly and the substrate is used to evaluate the level of mitochondrial autophagy, thereby improving accuracy, reducing interference, and realizing rapid high-throughput screening of mitochondrial autophagy inducers.

[0054] Fusion Protein

[0055] As used in the present invention, the term "fusion protein" refers to a novel protein molecule formed by fusing two or more different protein sequences together through genetic engineering technology. This protein is usually composed of at least two domains, which are encoded by separate genes and expressed in host cells through genetic engineering means. In some embodiments, the fusion protein includes two domains, which are connected by and through any bond or linker. In an embodiment of the present invention, the fusion protein has a structure shown in FLE formula (I), wherein 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 connected to a mitochondrial localization sequence, and the connection between the mitochondrial localization sequence and the luciferin sequence is a bond or any non-cleavable mode, preferably a bond. In some embodiments, the mitochondrial localization sequence is connected to the N-terminus of the luciferase. In some embodiments, the mitochondrial localization sequence is connected to the C-terminus of the luciferase. In a preferred embodiment of the present invention, the sequence of the luciferase containing the mitochondrial localization sequence is shown in SEQ ID NO.7.

[0056] As used in the present invention, the term "mitochondrial targeting sequence" refers to a protein sequence (mitochondrial targeting sequence, MTS), which is usually located at the N-terminus of mitochondrial proteins and is used to guide these proteins to enter mitochondria and ultimately localize in their specific subcellular regions. This sequence will be removed after the protein is transported to the mitochondria, so it is also called a leader sequence. 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 present invention, the mitochondrial targeting sequence is MLSLRQSIRFFKPATRTLCSSR (SEQ ID NO.3).

[0057] The term "linker" refers to a short amino acid chain that acts as a linker between two proteins in a fusion protein as an element of a fusion protein, allowing the proteins on both sides to perform their own independent functions. The term "cleavable linker" or "cleavable linker" refers to a linker that can be cleaved by the action of an enzyme inside a cell, such as a 2A peptide. In a preferred embodiment of the present invention, the cleavable linker is a 2A peptide, more preferably T2A or P2A. In a most preferred embodiment, the amino acid is T2A, and its amino acid sequence is shown in SEQ ID NO.5. The T2A sequence has a higher cleavage efficiency in the fusion protein system of the present invention.

[0058] As used in the present invention, the term "firefly luciferase" is a general term for enzymes that can produce bioluminescence in nature, and refers to an enzyme that can catalyze the oxidation of luciferin to produce light. Firefly luciferase can catalyze the oxidation of luciferin (D-luciferin) to oxidized luciferin (Oxyluciferin) in the presence of ATP, magnesium ions and oxygen, and emit bioluminescence in the process. In a preferred embodiment of the present invention, the firefly luciferase is firefly firefly luciferase, and its amino acid sequence is shown in SEQ ID NO.4.

[0059] As used herein, the term "green fluorescent protein (GFP)" refers to a protein consisting of about 238 amino acids originally isolated from the Pacific Northwest jellyfish Aequorea victoria, which emits green fluorescence when excited by ultraviolet light or blue light. In an embodiment of the present invention, the amino acid sequence of the green fluorescent protein is shown in SEQ ID NO.6.

[0060] Target gene and its expression

[0061] As used in the present invention, the term "polynucleotide" refers to a nucleobase polymer or oligomer in which the nucleobases are connected by sugar-phosphate bonds (sugar-phosphate backbone). Exemplary polynucleotides and oligonucleotides include polymers of 2' deoxyribonucleotides (DNA) and polymers of ribonucleotides (RNA). The polynucleotide can be composed entirely of ribonucleotides, entirely of 2' deoxyribonucleotides, or a combination thereof. In an embodiment of the present invention, the polynucleotide is an isolated polynucleotide. In an embodiment of the present invention, the polynucleotide can encode a fusion protein of the present invention. In an embodiment of the present invention, the sequence of the polynucleotide is as shown in SEQ ID NO.1.

[0062] As used in the present invention, the term "vector" refers to an extra chromosomal element that usually 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 integration sequence, a phage or a nucleotide sequence, linear or circular, in which many nucleotide sequences have been connected or recombined. Converted into a unique structure that can introduce the promoter fragment and DNA sequence of the selected gene product and the appropriate 3' non-translated sequence into the cell. In an embodiment of the present invention, the vector is a lentiviral vector, preferably a lentiviral plasmid pLV.

[0063] As used in the present invention, the term "host cell" refers to a cell used to replicate a vector. In a preferred embodiment of the present invention, the host cell is a cell used to produce a lentivirus, such as a human embryonic kidney transformed cell (HEK293T). In some embodiments of the present invention, a vector packaging plasmid containing a gene encoding a fusion protein of the present invention is transfected into a human embryonic kidney transformed cell (HEK293T) to produce a lentivirus.

[0064] Lentivirus production

[0065] The method of producing lentivirus is a technique well known to those skilled in the art. It generally includes the steps of 1) constructing a lentiviral vector, 2) transfecting a host cell with the lentiviral vector, and 3) culturing cells to produce lentivirus. The construction of a lentiviral vector includes the step of constructing a plasmid system for packaging lentivirus. Commonly used plasmid systems include three-plasmid or four-plasmid systems. The three-plasmid system consists of a plasmid containing an exogenous gene, a psPAX2 plasmid (carrying gag, pol, rev genes) and a pMD2G plasmid (providing an envelope protein vsvg gene). The four-plasmid system places the REV gene on a separate expression plasmid and removes the Tat gene, which reduces the risk of accidentally producing active viruses compared to the three-plasmid system. In an embodiment of the present invention, a plasmid pLV and pMD2.0G containing an exogenous gene and a psPAX2 packaging plasmid are used to form a plasmid system for packaging lentivirus, and HEK293T cells are transfected, and cells are cultured to obtain lentiviral particles, and the resulting lentiviral particles carry the exogenous target gene.

[0066] Method for screening mitophagy inducers

[0067] The present invention also relates to realizing high-throughput screening of mitochondrial autophagy inducers by using a lentivirus containing a target gene in an embodiment of the present invention, specifically comprising the steps of: S1, obtaining the RLU value of an experimental group; comprising the steps of: using a drug to be screened to treat human embryonic kidney transformed cells infected with a lentivirus, culturing the cells under suitable conditions for a period of time, measuring the system fluorescence intensity GFP, then lysing the cells and adding a luciferase substrate to detect the chemiluminescence intensity FLU of the system, calculating the RLU value of the experimental group, and the RLU value of the experimental group is the experimental group FLU / experimental group GFP; S2, obtaining the RLU value of a control group; using a blank reagent to treat human embryonic kidney transformed cells infected with a lentivirus, and then calculating the RLU value of the control group according to the same method 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 mitochondrial autophagy.

[0068] The above method uses a lentivirus containing a target gene in an embodiment of the present invention to infect human embryonic kidney transformed cells (HEK293T) cells, and then determines the chemiluminescence intensity and bioluminescence intensity of the system, and uses the bioluminescence emitted by green fluorescent protein as an internal reference, and uses the value of chemiluminescence intensity / bioluminescence intensity as an indicator for screening drugs. Compared with the chemiluminescence intensity FLU of the substrate detection system for luciferase alone, this method effectively corrects the error caused by the internal matrix of the cell, the screened drugs are not affected by the cells themselves, and the accuracy of evaluating the level of mitochondrial autophagy is high.

[0069] To make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and weight parts. The experimental materials and reagents used in the following examples can be obtained from commercial channels unless otherwise specified.

[0070] Unless otherwise specified, the technical and scientific terms used herein have the same meaning as commonly understood by ordinary technicians in the technical field to which the application belongs. It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments of the present application.

[0071] Unless otherwise indicated, the term "or" means and is used interchangeably with the term "and / or".

[0072] As used herein, including the appended claims, singular forms of words such as "a," "an," and "the" include their corresponding plural referents unless the context clearly dictates otherwise.

[0073] The present invention will be further described below in conjunction with specific implementation modes, and the advantages and features of the present invention will become clearer as the description proceeds. However, the present invention is not limited to the following implementation modes.

[0074] Example 1. Establishment of dual fluorescence system model

[0075] 1. Design principles

[0076] In the present embodiment, firefly luciferase (Firefly luciferase) protein and mitochondrial localization sequence (MTS) and green fluorescent protein (EGFP) are fused into mtFirefly-T2A-EGFP protein. When the protein is expressed in the cell, Firefly and EGFP proteins will be cut, and the Firefly protein with mitochondrial localization sequence (SEQ ID NO.3) will be located on the mitochondria, while the EGFP protein is expressed in the cytoplasm. When mitochondria undergo autophagy, the EGFP protein level expressed in the cytoplasm remains unchanged, and the Firefly protein will be degraded by lysosomes. At this time, after lysing cells, the chemiluminescence intensity of the addition of firefly luciferase substrate will significantly decrease. Therefore, using EGFP fluorescence intensity as an internal reference, the constant level of protein expression in the cell is represented, and the chemiluminescence intensity produced by Firefly in combination with the substrate can evaluate the mitochondrial autophagy level.

[0077] 2. Construction of Stable Cell Lines

[0078] A human embryonic kidney transformed cell HEK293TmtFirefly-T2A-EGFP stable cell line was constructed based on the mtFirefly-T2A-EGFP fusion protein; construction steps:

[0079] 1) Preparation of lentiviral plasmid: The mtFirefly-T2A-EGFP sequence was synthesized according to the sequence (sequence source NCBI: MK484108, MN517551) and then linked into the lentiviral plasmid pLV to form PLV-mtFirefly-T2A-EGFP by restriction digestion and ligation, and the endotoxin-free plasmid PLV-mtFirefly-EGFP, lentiviral packaging plasmid pMD2.0G and psPAX2 were extracted.

[0080] 2) Lentivirus preparation: HEK293T cells were cultured at 1*10 6 The cells were seeded in a T75 culture flask at a density of 100 / ml. After 24 hours, three plasmids, PLV-mtFirefly-T2A-EGFP, pMD2.0G and psPAX2, were transfected, each plasmid was 4μg. The supernatants were collected 48 hours and 72 hours after transfection, combined and filtered with a 0.45μm filter membrane, and an appropriate amount of concentrated solution (25% PEG8000, 0.75M NaCl) was added at a ratio of 7.5ml of virus concentrated solution per 30ml of filtered supernatant. After mixing, the mixture was inverted 3-5 times every 30 minutes, for a total of 4 times. It was placed at 4℃ overnight. The next day, 3000g was centrifuged at 4℃ for 20 minutes, the supernatant was discarded, and the virus precipitate was resuspended with 1ml PBS (phosphate buffer). It was stored at -80℃.

[0081] 3) Lentivirus infection: HEK293T cells were inoculated with 5*10 5 / ml in a T75 culture flask. After 24 hours, the prepared lentivirus was taken out of the minus 80 degree refrigerator, fully dissolved and dripped into the cell culture. After 48 hours, the infection rate was observed, and the infection rate was greater than 90% and was evaluated as qualified. 1μg / ml of puromycin was added to the qualified infected cell culture to eliminate uninfected cells. After 2-3 days, the medium was changed to pass the cells, the culture was expanded, and the cells were frozen to obtain the human embryonic kidney transformed cell HEK293TmtFirefly-T2A-EGFP stable cell line. After that, single clones were obtained by flow cytometry sorting.

[0082] mtFirefly-T2A-EGFP sequence:

[0083] ATGCTGAGCCTGCGCCAGAGTATCCGCTTCTTCAAGCCCGCCACCAGGACTCTGTGCAG

[0084] TTCCAGGATGGAAGATGCCAAAAACATTAAGAAGGGCCCAGCGCCATTCTACCCACTCG

[0085] AAGACGGGACCGCCGGCGAGCAGCTGCACAAAGCCATGAAGCGCTACGCCCTGGTGCC

[0086] CGGCACCATCGCCTTTACCGACGCACATATCGAGGTGGACATTACCTACGCCGAGTACTT

[0087] CGAGATGAGCGTTCGGCTGGCAGAAGCTATGAAGCGCTATGGGCTGAATACAAACCATC

[0088] GGATCGTGGTGTGCAGCGAGAATAGCTTGCAGTTCTTCATGCCCGTGTTGGGTGCCCTG

[0089] TTCATCGGTGTGGCTGTGGCCCCAGCTAACGACATCTACAACGAGCGCGAGCTGCTGAA

[0090] CAGCATGGGCATCAGCCAGCCCACCGTCGTATTCGTGAGCAAGAAAGGGCTGCAAAAG

[0091] ATCCTCAACGTGCAAAAGAAGCTACCGATCATACAAAAGATCATCATCATGGATAGCAA

[0092] GACCGACTACCAGGGCTTCCAAAGCATGTACACCTTCGTGACTTCCCATTTGCCACCCG

[0093] GCTTCAACGAGTACGACTTCGTGCCCGAGAGCTTCGACCGGGACAAAACCATCGCCCT

[0094] GATCATGAACAGTAGTGGCAGTACCGGATTGCCCAAGGGCGTAGCCCTACCGCACCGCA

[0095] CCGCTTGTGTCCGATTCAGTCATGCCCGCGACCCCATCTTCGGCAACCAGATCATCCCCG

[0096] ACACCGCTATCCTCAGCGTGGTGCCATTTCACCACGGCTTCGGCATGTTCACCACGCTGG

[0097] GCTACTTGATCTGCGGCTTTCGGGTCGTGCTCATGTACCGCTTCGAGGAGGAGCTATTCT

[0098] TGCGCAGCTTGCAAGACTATAAGATTCAATCTGCCCTGCTGGTGCCCACACTATTTAGCT

[0099] TCTTCGCTAAGAGCACTCTCATCGACAAGTACGACCTAAGCAACTTGCACGAGATCGCC

[0100] AGCGGCGGGGCGCCGCTCAGCAAGGAGGTAGGTGAGGCCGTGGCCAAACGCTTCCACC

[0101] TACCAGGCATCCGCCAGGGCTACGGCCTGACAGAAACAACCAGCGCCATTCTGATCACC

[0102] CCCGAAGGGGACGACAAGCCTGGCGCAGTAGGCAAGGTGGTGCCCTTCTTCGAGGCTA

[0103] AGGTGGTGGACTTGGACACCGGTAAGACACTGGGTGTGAACCAGCGCGGCGAGCTGTG

[0104] CGTCCGTGGCCCCATGATCATGAGCGGCTACGTTAACAACCCCGAGGCTACAAACGCTC

[0105] TCATCGACAAGGACGGCTGGCTGCACAGCGGCGACATCGCCTACTGGGACGAGGACGA

[0106] GCACTTCTTCATCGTGGACCGGCTGAAGAGCCTGATCAAATACAAGGGCTACCAGGTAG

[0107] CCCCAGCCGAACTGGAGAGCATCCTGCTGCAACACCCCAACATCTTCGACGCCGGGGT

[0108] CGCCGGCCTGCCCGACGACGATGCCGGCGAGCTGCCCGCCGCAGTCGTCGTGCTGGAA

[0109] CACGGTAAAACCATGACCGAGAAGGAGATCGTGGACTATGTGGCCAGCCAGGTTACAA

[0110] CCGCCAAGAAGCTGCGCGGTGGTGTTGTGTTCGTGGACGAGGTGCCTAAAGGACTGAC

[0111] CGGCAAGTTGGACGCCCGCAAGATCCGCGAGATTCTCATTAAGGCCAAGAAGGGCGGC

[0112] AAGATCGCCGTGGAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATC

[0113] CCGGCCCTGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGA

[0114] GCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGAT

[0115] GCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCC

[0116] CTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCG

[0117] ACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGA

[0118] GCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTC

[0119] GAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACG

[0120] GCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATG

[0121] GCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGG

[0122] ACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCC

[0123] CGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCC

[0124] AACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAG(SEQ ID NO.1)

[0125] Amino acid sequence of mtFirefly-T2A-EGFP::

[0126] MLSLRQSIRFFKPATRTLCSSRMEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTI

[0127] AFTDAHIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAV

[0128] APANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQSMY

[0129] TFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHARDPIFG

[0130] NQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYKIQSALLVPTLF

[0131] SFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGYGLTETTSAILITPEGD

[0132] DKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMSGYVNNPEATNALIDKD

[0133] GWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESILLQHPNIFDAGVAGLPDDDA

[0134] GELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVVFVDEVPKGLTGKLDARKIREI

[0135] LIKAKKGGKIAVEGRGSLLTCGDVEENPGPVSKGEELFTGVVPILVELDGDVNGHKFSVSGE

[0136] GEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYV

[0137] QERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMA

[0138] DKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK(SEQ ID NO.2)

[0139] MTS mitochondrial localization sequence:

[0140] MLSLRQSIRFFKPATRTLCSSR(SEQ ID NO.3)

[0141] Firefly luciferase sequence:

[0142] MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDAHIEVDITYAEYFEMSV

[0143] RLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAPANDIYNERELLNSMGISQP

[0144] TVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQSMYTFVTSHLPPGFNEYDFVPESF

[0145] DRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHARDPIFGNQIIPDTAILSVVPFHHGFGM

[0146] FTTLGYLICGFRVVLMYRFEEELFLRSLQDYKIQSALLVPTLFSFFAKSTLIDKYDLSNLHEIA

[0147] SGGAPLSKEVGEAVAKRFHLPGIRQGYGLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVD

[0148] LDTGKTLGVNQRGELCVRGPMIMSGYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIV

[0149] DRLKSLIKYKGYQVAPAELESILLQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVVFVDEVPKGLTGKLDARKIREILIKAKKGGKIAV(SEQ ID NO.4)

[0150] Firefly luciferase sequence containing a mitochondrial localization sequence

[0151] MLSLRQSIRFFKPATRTLCSSR

[0152] MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDAHIEVDITYAEYFEMSV

[0153] RLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAPANDIYNERELLNSMGISQP

[0154] TVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQSMYTFVTSHLPPGFNEYDFVPESF

[0155] DRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHARDPIFGNQIIPDDAILSVVPHFHHGFGM

[0156] FTTLGYLICGFRVVLMYRFEEELFLRSLQDYKIQSALLVPTLFSFFAKSTLIDKYDLSNLHEIA

[0157] SGGAPLSKEVGEAVAKRFHLPGIRQGYGLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVD

[0158] LDTGKTLGVNQRGELCVRGPMIMSGYVNNPEATNALIDKDGWLHSGDIAYWDDEDEHFFIV

[0159] DRLKSLIKYKGYQVAPAELESILLQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVVFVDEVPKGLTGKLDARKIREILIKAKKGGKIAV(SEQ ID NO.7)

[0160] T2A connection sequence:

[0161] EGRGSLLTCGDVEENPGP(SEQ ID NO.5)

[0162] EGFP sequence:

[0163] VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVT

[0164] TLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRI

[0165] ELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK(SEQ ID NO.6)

[0166] Example 2. Dual fluorescence system model for detecting mitochondrial autophagy

[0167] 1. Experimental Methods

[0168] Human embryonic kidney transformed cells HEK293TmtFirefly-T2A-EGFP cells were seeded in a 96-well black ELISA plate at 5000 cells / well, 100μl per well, and drugs were added after 24 hours: DMSO was added to the control group, and carbonyl cyanide chloride p-chlorophenylhydrazone (CCCP) (3μM, 0.5μM) was added to the experimental group. Each treatment was repeated 3 times, and the green fluorescence intensity (GFP) was directly detected in the ELISA instrument after 24 hours of treatment at 37°C and 5% CO2. Then the cells were lysed and the substrate was added to detect the chemiluminescence intensity (FLU). Specific steps:

[0169] 1) Lyse cells: aspirate the culture medium, add 100ul lysis solution to pipette cells, and centrifuge at 220r for 15min;

[0170] 2) Place 10ul of lysate in a new 96-well plate;

[0171] 3) Add 80ul of firefly luciferase substrate, mix well and detect chemiluminescence (FLU) using a microplate reader.

[0172] 2. Data processing

[0173] The green fluorescence intensity emitted by EGFP was used as an internal reference to represent the constant expression level of cellular protein, and the FLU intensity was used to measure the level of mitochondrial autophagy. Therefore, this study used RLU (FLU / GFP) as the standard for evaluating the level of mitochondrial autophagy. Figure 1 .

[0174] like Figure 1 As shown, * represents a significant difference between the experimental group and the DMSO group. CCCP is known to be a drug that can induce mitochondrial autophagy. The DMSO group is the experimental control group. When the CCCP concentration is greater than 0.5 μM, the RLU is significantly reduced compared with the DMSO group. Through the analysis of the positive control results, this system can be used to evaluate the level of mitochondrial autophagy.

[0175] Those skilled in the art will appreciate that the above-mentioned embodiments are specific examples for implementing the present invention, and in actual applications, various changes may be made thereto in form and detail without departing from the spirit and scope of the present invention.

Claims

1. A fusion protein, characterized in that The fusion protein comprises: luciferase containing a mitochondrial localization sequence; and Fluorescent protein.

2. The fusion protein according to claim 1, characterized in that The fusion protein comprises the structure shown in the following formula (I): FLE, formula (I) Among them, F represents luciferase containing a mitochondrial localization sequence; L represents a cleavable linker; E indicates fluorescent protein.

3. The fusion protein according to claim 2, characterized in that The luciferase is firefly luciferase; and / or, L is represented by a 2A sequence, more preferably T2A; And / or, the fluorescent protein is green fluorescent protein.

4. The fusion protein according to claim 3, characterized in that 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 according to any one of claims 1 to 4.

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

1.

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

8. A host cell, characterized in that The cell contains the vector according to claim 7. Preferably, it is a human embryonic kidney transformed cell (HEK293T).

9. Use of the fusion protein according to any one of claims 1 to 4; the polynucleotide according to claim 5 or 6; the vector according to claim 7; and / or the cell according to claim 8 for screening a mitochondrial autophagy inducer.

10. A method for screening a mitochondrial autophagy inducer, characterized in that: The method comprises the steps of: S1, obtaining the value of RLU of the experimental group; comprising the steps of: 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 and adding luciferase substrate to detect the chemiluminescence intensity FLU of the system, calculating the value of RLU of the experimental group, and the value of RLU of the experimental group is the experimental group FLU / experimental group GFP; S2, obtaining the RLU value of the control group; comprising the steps of: using a blank reagent to treat the human embryonic kidney transformed cells infected with the lentivirus, and then calculating the RLU value of the control group in the same manner as the experimental group; S3, comparing the RLU value of the experimental group with the RLU value of the control group to determine whether the drug can induce mitochondrial autophagy.

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