Ratio-type probes for measuring extracellular ATP

Abstract

This invention relates to a DNA construct and a vector containing the construct, a cell line transfected with the vector or the construct, isolated mRNA transcribed from the construct, and their use in measuring extracellular ATP levels, the DNA construct comprising the following elements operatively linked from 5' to 3': i) a DNA sequence encoding an extracellular ATP-dependent luciferase; ii) an IRES sequence; and iii) a DNA sequence encoding a cytoplasmic ATP-independent luciferase.

Description

Technical Field

[0001] This invention relates to a DNA construct and a vector containing the construct, a cell line transfected with the vector or the construct, isolated mRNA transcribed from the construct, and their use for measuring extracellular ATP levels. The DNA construct comprises the following elements operatively linked from 5' to 3': i) a DNA sequence encoding an extracellular ATP-dependent luciferase; ii) an IRES sequence; and iii) a DNA sequence encoding a cytoplasmic ATP-independent luciferase. Background Technology

[0002] ATP is widely recognized as a ubiquitous extracellular messenger. This nucleotide-induced response depends on concentration and the P2R isoform expressed by the target cell, encompassing a wide range of responses from chemotaxis (Oshimi et al., 1999) to cell adhesion (Freyer et al., 1998), from cytokine release (Perregaux & Gabel, 1994) to neurotransmitter secretion (Illles & Norenberg, 1993), and from apoptosis activation (Zanovello et al., 1990) to cell proliferation stimulation (Neary et al., 2003). Furthermore, multiple mediators (neurotransmitters, cytokines, hormones) work synergistically to increase or decrease ATP release in the microenvironment immediately adjacent to the cell membrane, where these factors exert paracrine or autocrine effects, or act as endogenous ligands. While many pathological conditions (trauma, inflammation, ischemia) are generally considered to result in elevated extracellular ATP concentrations as a simple consequence of cellular damage, the pathway supporting non-lytic release of ATP remains unclear. There is growing interest in signals that can alert the immune system in the early stages of tissue damage or pathogen invasion (Matzinger, 2002; La SaIa et al., 2003; Skobemeet et al., 2004). Intracellular nucleotides are considered potential candidates for this role due to their widespread distribution, high intracellular concentration, extremely low extracellular levels at rest, specific receptors, and ability to regulate dendritic cell differentiation. The additional features described in this paper (revealing a non-lytic and self-sustaining release mechanism) make ATP an even more important warning signal.

[0003] ATP's extremely low extracellular levels at rest, rapid increases induced by various stimuli, rapid degradation in the extracellular space, and the presence of specific receptors make it an ideal extracellular messenger (Burnstock, 2004; Zimmermann, 2000). However, the lack of sufficient probes to accurately measure extracellular concentration has hindered our understanding of ATP's role as an extracellular signal.

[0004] The main methods for measuring extracellular ATP are the soluble firefly luciferase method, the IgG-coupled firefly luciferase method, the patch-clamp method, and the pmeLUC method. Therefore, the firefly luciferase method (commonly known as Fireflyluciferase) is currently the most widely used method due to its various derivative forms.

[0005] The advantage of soluble firefly luciferase is its simplicity and ease of operation. However, it involves sample processing, which may interfere with experimental results. Furthermore, while this system can measure ATP present in the extracellular space, it cannot detect ATP changes occurring in the pericellular space. Moreover, this system is not convenient for in vivo use.

[0006] Firefly luciferase can also couple with IgG immunoglobulins, thereby binding to specific epitopes present on the cell membrane. However, a drawback of this system is the need for specific antibodies, which can also alter the physiological properties of the cell. Furthermore, the possibility of antibody endocytosis and redistribution must also be considered.

[0007] pmeLUC consists of a chimeric firefly luciferase fused inframe with the following sequences: a GPI sequence fused to the C-terminus for anchoring to the plasma membrane, and an endoplasmic reticulum targeting sequence fused to the N-terminus. This system offers numerous advantages, such as the ability to measure rapid changes in ATP in the cell periphery. It is well-suited for in vitro use, but even more so for in vivo use. The main drawback of pmeLUC relates to its expression in host cells. In fact, the process is highly dependent on multiple factors, including transfection efficiency and cellular metabolism. The expression level of pmeLUC clearly affects the measurement results.

[0008] Another system currently in use consists of a patch-clamp micropipette. However, this system is very complex and often produces human-caused results.

[0009] Given the above, there is clearly a need for a reliable, simple, and reproducible method for measuring ATP levels in the pericellular space that can provide more reliable measurements than existing methods. Summary of the Invention

[0010] This invention relates to a DNA construct for measuring extracellular ATP levels.

[0011] The present invention also relates to a bicistronic plasmid vector having two recombinant genes separated by an IRES (Internal Ribosome Entry site) sequence.

[0012] Currently, there are two methods for expressing heterologous genes in a single vector: the first method uses two separate promoters, and the second method uses an IRES sequence. In the plasmid described above, IRES technology was used, thus transcribed two genes into a single mRNA. Subsequently, the first gene was translated in a cap-dependent manner, while the second gene was translated in a cap-independent manner. In fact, it is the IRES sequence that enables translation by recruiting the 40S subunit of the ribosome near the AUG codon.

[0013] Using bicistronic vectors containing the IRES sequence offers significant advantages over using two separate promoters: both genes are transcribed into a single mRNA at the same level. When using two promoters, the transcriptional levels of the two genes are more unpredictable and more susceptible to interference.

[0014] The DNA construct of this invention expresses two luciferases. The first, an ATP-dependent luciferase (anchored to the plasma membrane and facing the extracellular space), detects extracellular ATP. This localization allows us to measure abrupt changes in ATP near the cell membrane both in vivo and in vitro. Using a cytoplasmic ATP-independent luciferase allows us to overcome a major limitation of using pmeLUC in vivo, namely that the intensity of the luminescent signal depends not only on the effective concentration of extracellular ATP but also on the expression level of the probe (which may vary in different tissues). Therefore, using a cytoplasmic ATP-independent luciferase, particularly Renilla luciferase, in the construct of this invention allows for the normalization of the extracellular ATP-dependent luciferase signal, resulting in a value that depends solely on ATP concentration.

[0015] The IRES sequence linking the two genes in the construct of this invention provides two additional advantages. The first advantage lies in the simultaneous transcription of extracellular ATP-dependent luciferase and cytoplasmic ATP-independent luciferase (which are essentially controlled by the same promoter). Therefore, extracellular ATP-dependent luciferase and cytoplasmic ATP-independent luciferase are transcribed into a single mRNA. This characteristic is advantageous because transcription occurs simultaneously, and cytoplasmic ATP-independent luciferase itself serves as an excellent normalization system. If the two genes are controlled by different promoters, transcription will proceed independently, and each gene will be influenced by the surrounding genomic environment. Therefore, even using the same cell line, different relative expression levels may exist.

[0016] A second advantage of using the IRES sequence is that the second gene is expressed at a lower rate compared to the first, because IRES-mediated translation is less efficient at initiating than cap-dependent translation. However, this effect itself is favorable for normalization. In fact, cytoplasmic ATP-independent luciferases expressed in the cytoplasm will emit a signal that depends solely on their expression and tends to be very strong; conversely, extracellular ATP-dependent luciferases will only signal in the presence of extracellular ATP (the signal is variable and definitely lower than the normalization reference signal). Without the IRES sequence, the signal of cytoplasmic ATP-independent luciferase would be too high compared to that of extracellular ATP-dependent luciferase, impairing its normalization function and potentially rendering it unmeasurable because it exceeds the sensitivity range of many commercially available chemiluminescence analyzers.

[0017] The system for measuring extracellular ATP uses extracellular ATP-dependent luciferase as a sensor of ATP concentration near the plasma membrane and cytoplasmic ATP-independent luciferase as an internal control to normalize the signal. Therefore, this invention enables accurate measurement of extracellular ATP.

[0018] Therefore, the objective of this invention is: A DNA construct for measuring extracellular ATP levels, the DNA construct comprising or consisting of the following elements operably linked from 5' to 3': i) The DNA sequence encoding extracellular ATP-dependent luciferase; ii) IRES sequence; iii) The DNA sequence encoding cytoplasmic ATP-independent luciferase; A vector comprising a DNA construct according to the present invention; Cell lines transfected with the vector of the present invention or the DNA construct of the present invention; An isolated mRNA transcribed from a DNA construct according to the invention; The DNA constructs of the present invention, the cell lines of the present invention, or the mRNA of the present invention are used in vitro for measuring extracellular ATP levels. A method for screening target compounds that can regulate extracellular ATP levels, the method comprising the following steps: a) Contact the cell line defined according to any embodiment described herein with a solution containing the substrate and the target compound; b) Detect the percentage of emitted photons or light emission; c) Normalize the value of emitted photons or percentage of luminescence from ATP-dependent luciferase by dividing the value of emitted photons or percentage of luminescence from ATP-independent luciferase by the value of emitted photons or percentage of luminescence from ATP-independent luciferase to obtain a value that depends only on extracellular ATP. A biosensor comprising a cell line characterized in that the cell line is transfected with a vector according to the invention or a DNA construct according to the invention; The biosensor according to the invention is used for measuring extracellular ATP levels in an in vitro system.

[0019] Other advantages and / or embodiments of the invention will become apparent from the following detailed description. Attached Figure Description

[0020] Figure 1 : Schematic diagram of the pmeLUC / nilla probe.

[0021] Figure 2 Transcription and translation results of bicistronic mRNA of pmeLUC / nilla.

[0022] Figure 3 Schematic diagram of the reaction catalyzed by pmeLUC and Renilla luciferase.

[0023] Figure 4 Schematic diagram of the pmeLUC / Renilla probe in operation. The concentration of ATP in the extracellular space changes rapidly with the activity of various factors. pmeLUC is used to measure ATP concentration, while Renilla, expressed in the cytoplasm, allows for signal normalization.

[0024] Figure 5Calibration curves were obtained using HEK293 cells transfected with pmeLUC / renilla. The pmeLUC signal was dependent on ATP concentration. However, the Renilla signal was independent of extracellular ATP concentration. Therefore, the ratio of pmeLUC luminescence to Renilla luminescence increased with increasing ATP concentration.

[0025] Figure 6 (a) Schematic diagram of the pmeLUC / nilla probe structure compared with (b) a conventional dual-promoter expression system. (c) Difference in bioluminescence between the pmeLUC / nilla probe equipped with the IRES sequence and vectors expressing the two proteins independently. In the second case, Renilla's luminescence was significantly higher than that of pmeLUC, which could cause several problems from an experimental perspective. Data are presented as mean ± SD, N=4. Detailed Implementation

[0026] Different embodiments of the present invention will be described below. It should be understood that, where compatible, the features of the various embodiments can be combined.

[0027] Generally, subsequent implementation schemes will describe the differences from the previously described implementation schemes.

[0028] 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 this invention pertains.

[0029] DNA construct This invention relates to a DNA construct comprising or consisting of, or composed of, elements operably linked from 5' to 3': i) The DNA sequence encoding extracellular ATP-dependent luciferase; iii) IRES sequence; iv) DNA sequence encoding cytoplasmic ATP-independent luciferase.

[0030] For the purposes of this invention, the term "operably linked from 5' to 3'" means that the elements of a DNA construct are linked in such a way that the direction of the DNA strand can be read from one 5' end to the other 3' end.

[0031] For the purposes of this invention, the term "leader sequence" refers to a DNA region that regulates the initiation of gene transcription associated with it, providing a binding site for RNA polymerase and other proteins necessary to initiate the transcription process.

[0032] For the purposes of this invention, the term "IRES sequence" refers to a sequence capable of translating mRNA (messenger RNA) into protein in a cap-independent manner, thereby enabling efficient translation under specific conditions (e.g., conditions necessary for gene expression in a DNA construct).

[0033] For the purposes of this invention, the term "extracellular ATP-dependent luciferase" refers to a luciferase anchored to the cell membrane, particularly to the outer surface of the cell membrane, whose luminescence intensity depends on the concentration of extracellular ATP.

[0034] For the purposes of this invention, the term "cytoplasmic ATP-independent luciferase" refers to a luciferase whose luminescence intensity is independent of ATP concentration and which can perform its enzymatic function in the cytoplasm.

[0035] In the presence of oxygen and ATP in the extracellular environment, extracellular ATP-dependent luciferase breaks down ATP into ADP and phosphate, and oxidizes luciferin. Each molecule of ATP consumed emits a photon (light). Therefore, the emission of light is directly proportional to the concentration of extracellular ATP.

[0036] Unlike extracellular ATP-dependent luciferases, cytoplasmic ATP-independent luciferases are confined to the cytoplasm. They interact with cofactors (such as coelentrin) to generate a light signal, but this light signal is independent of the presence of ATP. Therefore, the light emitted by cytoplasmic ATP-independent luciferases is directly proportional to their intracellular concentration, thus indicating protein mass very accurately. The light signal can be easily normalized by the ratio between the light emitted by extracellular ATP-dependent luciferases and that emitted by cytoplasmic ATP-independent luciferases, allowing for reliable quantitative measurement of extracellular ATP concentration.

[0037] According to a preferred embodiment of the present invention, the DNA sequence encoding extracellular ATP-dependent luciferase comprises or consists of the following sequences: a) A sequence that encodes a leading sequence.

[0038] b) The sequence encoding a protein with ATP-dependent luciferase activity.

[0039] c) Sequences encoding cell membrane anchoring sequences.

[0040] According to a preferred embodiment, sequences a), b) and c) are operatively linked from 5' to 3', the sequence encoding the leader sequence having SEQ ID NO:4 and / or the sequence encoding the cell membrane anchoring sequence having SEQ ID NO:6.

[0041] According to any of the embodiments described herein, the construct may also include, for example, a sequence encoding a tag between sequence b) and sequence c), particularly a sequence encoding a myc tag, such as SEQ ID NO:5.

[0042] In one embodiment, the protein having ATP-dependent luciferase activity is selected from Photinus Pyralis luciferase (Uniprot Registry No. P08659), Luciola cruciate luciferase (Japanese firefly or Genji-botaru luciferase, Uniprot Registry No. (entry) P13129); Luciferase Luciola italic (Italian firefly luciferase, Uniprot Registry No. Q1AG35), Luciferase Luciola lateralis (Japanese firefly (Heike) luciferase, Uniprot Registry No. Q2ABY2), Luciferase Luciola mingrelica (Eastern European firefly luciferase, Uniprot Registry No. Q26304), Luciferase Photuris pennsylvanica (Pennsylvanian firefly luciferase, Uniprot Registry No. Q27757); Luciferase Pyrophorus plagiophthalamus (Clickbeetle luciferase, Uniprot accession number Q718A5), Luciferase Phrixothrix hirtus (Railroad worm luciferase, Uniprot accession number Q9U4U7) or their variants, especially variants with the same or improved enzyme activity.

[0043] In this invention, when referring to the Uniprot database, it means "version 2023_04".

[0044] In this invention, when referring to the Genbank database, it refers to the version updated as of October 24, 2023.

[0045] In this invention, when referring to the PDB database, it refers to the version updated as of October 24, 2023.

[0046] For the purposes of this invention, the term "variant" refers to a group of very similar proteins that are derived from a single gene or gene family and are the result of genetic differences.

[0047] For the purposes of this invention, “variants” retain the same function, for example, variants of which are considered suitable for the DNA sequences of this invention, these sequences encode the same protein, or these sequences have sequence identity greater than 90%, preferably greater than 95% or 99%, and maintain equivalent or improved enzymatic functionality compared to wild-type proteins. The term “enhanced activity” or similar terms refer to a detectable increase in the activity of a protein or enzyme. As used herein, “enhanced activity” can refer to a modified protein or enzyme exhibiting higher activity than similar proteins or enzymes of the same type (e.g., proteins or enzymes without specific genetic modification (e.g., original or wild-type proteins or enzymes, or the activity level of host cell proteins or enzymes that serve as the starting point for genetic modification)).

[0048] For example, the activity of a modified or engineered protein or enzyme can be about 5%, about 10%, about 15%, about 20%, about 30%, about 50%, about 60%, about 70%, or about 100% higher than that of an unengineered protein or enzyme of the same type (e.g., wild-type protein or enzyme, or the activity exhibited by a protein or enzyme in a host cell). The host cell of the protein or enzyme exhibiting improved enzymatic activity can be verified by any method known in the art.

[0049] Wild-type (WT) expression refers to the typical form of a species' phenotype in nature.

[0050] In a preferred embodiment, the extracellular ATP-dependent luciferase is firefly (Photinus pyralis) luciferase or a variant thereof.

[0051] pmeLuc In a preferred embodiment, the extracellular ATP-dependent luciferase is a luciferase defined as pmeLUC, namely the firefly (Photinus pyralis) luciferase expressed on the cell membrane and referred to as "pmeLUC" (plasma membrane luciferase) as described in European Patent No. RM2005A000252-SG / IC.

[0052] The membrane luciferase pmeLUC is expressed with its catalytic site facing the outer side of the plasma membrane, and can measure extracellular ATP by adding its substrate luciferin.

[0053] In a specific preferred embodiment, the DNA sequence encodes an extracellular ATP-dependent luciferase pmeLUC having the sequence shown in SEQ ID NO:2.

[0054] In one embodiment, the cytoplasmic ATP-independent luciferase is selected from Renilla reniformis Luciferase (Renilla luciferase, Unipol accession number P27652), Cypridina noctiluca Luciferase (Cypridina luciferase, Unipol accession number A0A387LB08), Cypridinahilgendorfii Luciferase (Cypridina (Vargula) luciferase, Unipol accession number P17554), Luciferase Metridia longa (Metridia luciferase), also known as MLuc7, Unipol accession number A0A0B4UFT5, Luciferase Oplophorus gracilorostris (OLuc, Unipol accession number Q9GV45), Nanoluc (SEQ ID NO. 8), LumiLuc (SEQ ID NO. 9), or variants thereof. It should be noted that Nanoluc and Lumiluc are engineered mutants of Oplophorus gracilorostris Luciferase.

[0055] Renilla Renilla luciferase (Uniprot accession number P27652) is a cytoplasmic luciferase isolated from the coelenterate *Renillareniformis*. Cytoplasmic Renilla luciferase is ATP-independent and emits photons by oxidizing its substrate, coelentrin. Renilla allows for the tracking of probe expression independently of the presence of ATP, thus providing the possibility of normalizing pmeLUC signals.

[0056] In a specific preferred embodiment, the DNA sequence encodes a cytoplasmic ATP-independent Renilla luciferase having the sequence shown in SEQ ID NO:3.

[0057] In one embodiment, the sequence encoding the leader sequence has the sequence shown in SEQ ID NO:4 or a variant thereof.

[0058] In one embodiment, the sequence encoding the cell membrane anchoring sequence has the sequence shown in SEQ ID NO:6 or a variant thereof.

[0059] In another embodiment, the IRES sequence has the sequence shown in SEQ ID NO:7 or a variant thereof.

[0060] Therefore, in a preferred embodiment, the DNA construct of the present invention comprises the following elements operably linked from 5' to 3': i) The DNA sequence encoding pmeLUC luciferase or a variant thereof; ii) IRES sequence; iii) The DNA sequence encoding Renilla luciferase or a variant thereof.

[0061] In other words, in a preferred embodiment, the DNA construct of the present invention encodes two genes, such as membrane luciferase pmeLUC and cytoplasmic luciferase Renilla. Given the structure of this preferred embodiment, the probe is named pmeLUC / nilla.

[0062] In a specific preferred embodiment, the construct of the present invention has the sequence shown in SEQ ID NO:1, or has greater than 90% identity with the sequence shown in SEQ ID NO:1, preferably greater than 95% identity, more preferably greater than 99% identity.

[0063] In the context of nucleotide or amino acid sequences, the term "sequence identity percentage" refers to the number of identical residues between two sequences after a maximum match alignment. The length of a sequence identity comparison can be a segment of at least about 9 nucleotides, typically at least about 18 nucleotides, more typically at least about 24 nucleotides, typically at least about 28 nucleotides, more typically at least about 32 nucleotides, and preferably at least about 36 or more 48 nucleotides. Numerous different algorithms known in the art can be used to measure nucleotide sequence identity. For example, FASTA, Gap, or Bestfit (all available programs that provide alignment results and sequence identity percentages of the best overlapping regions between the query and search sequences) can be used to compare polynucleotide sequences (Pearson, Methods Enzymol. 183:63-98 (1990); Pearson, MoI Biol. 276:71-84 (1998); which are incorporated herein by reference).

[0064] When referring to nucleic acids or their fragments or amino acids, the term "substantial similarity" or "substantial sequence similarity" means that, when optimally aligned with another nucleic acid (or its complementary strand) by appropriate nucleotide insertions or deletions, at least about 85% of the nucleotide bases, preferably at least about 90% of the nucleotide bases, more preferably at least about 95%, 96%, 97%, 98%, or 99% of the nucleotide bases, have nucleotide sequence identity, which can be measured by any known sequence identity algorithm (e.g., FASTA, BLAST, or Gap) discussed above. When applied to peptides, the term "substantial identity" means that, when two peptide sequences are optimally aligned by, for example, the GAP or BESTFIT program using the default vacancy weights provided by the program, they have at least 70%, 75%, or 80% sequence identity, preferably at least 90% or 95% of the sequence identity, more preferably at least 97%, 98%, or 99% of the sequence identity.

[0065] In one embodiment, the positions of dissimilar residues differ due to conserved amino acid substitutions. A “conserved amino acid substitution” refers to replacing one amino acid residue with another amino acid residue whose side chain R group has similar chemical properties (e.g., charge or hydrophobicity). Typically, conserved amino acid substitutions do not substantially alter the functional properties of a protein. When two or more amino acid sequences differ from each other due to conserved substitutions, the percentage of sequence identity can be adjusted upwards to correct for the conservation of the substitution. Methods for making such adjustments are well known to those skilled in the art.

[0066] For example, see Pearson, Methods MoI. Biol. 243:307-31 (1994). Examples of amino acid groupings with side chains of similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic-hydroxy side chains: serine and threonine; 3) starch-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid; and 7) sulfur-containing side chains: cysteine ​​and methionine. Conserved substitution groupings of amino acids include: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conserved substitution refers to any change that has a positive value in the PAM250 likelihood matrix as described in Gonnet et al., Science 256:1443-45 (1992) (which is incorporated herein by reference). A “moderately conserved” substitution refers to any change that has a non-negative value in the PAM250 likelihood matrix. Sequence identity of peptides is typically measured using sequence analysis software. Protein analysis software uses similarity measures assigned to various substitutions, deletions, and other changes, including conserved amino acid substitutions, to match sequences. For example, GCG includes programs such as “Gap” and “Bestfit”, which can determine sequence homology or sequence identity between closely related peptides (e.g., homologous peptides from different species) or between a wild-type protein and its mutant (mutein) using default parameters specified by the program.

[0067] In one embodiment, the construct further includes a promoter operably connected upstream of the construct in a 5' to 3' direction.

[0068] Another objective of this invention is to isolate mRNA transcribed from the DNA construct of this invention.

[0069] In this patent application, the phrase "isolated mRNA transcribed from the DNA construct of the present invention" refers to messenger RNA (mRNA) synthesized or generated using the DNA construct described in the present invention. In other words, it is mRNA generated from a specific DNA construct identified in the present invention.

[0070] The pmeLUC / nilla ratio probe was obtained as follows: pmeLUC luciferase cDNA was amplified from the pcDNA3-pmeLUC plasmid available in our laboratory using the following primers: 5'-ATG TGC TAG CAT GGC TCA GCG GATGAC AAC-3' and 5'-GCA TTG AAT TCC TCG AGG TCG ACG GTA TCA AG-3'. The PCR product was transferred to the pIRES vector (BD Biotech) digested with NheI and EcoRI, cloning pmeLUC upstream of the IRES sequence. Renilla cDNA was amplified from the pMirGLO plasmid (Promega) using the following primers: 5'-GCT TTC TAG AAT GGC TTC CAA GGT GTA CGA C-3' and 5'-TAA CCG CCG GCG TTACTG CTC GTT CTT CAG CAC G. The PCR product was ligated into the pIRES-pmeLUC vector digested with XbaI and NotI, cloning Renilla downstream of the IRES sequence. The clone was validated by sequence analysis performed by BMR Genomics (Padua). A schematic diagram of the final construct is shown below. Figure 1 As shown.

[0071] Carrier containing the construct of the present invention Another object of the present invention is to provide a vector comprising a DNA construct according to any embodiment of the present invention.

[0072] The vectors described herein contain nucleic acid regions with sequences capable of being transcribed. Therefore, sequences encoding mRNA, tRNA, and rRNA are all included in this definition. All vectors suitable for expressing the DNA constructs contained therein using appropriate cellular systems are included in the definition of vectors of this invention.

[0073] A vector is a tool that enables or facilitates the transfer of an entity from one environment to another. For example, some vectors used in recombinant DNA technologies can transfer entities such as DNA fragments (e.g., heterologous DNA fragments, such as heterologous cDNA fragments) into target cells. Optionally, once inside the target cell, the vector can maintain the heterologous DNA within the cell or serve as a unit for DNA replication. Examples of vectors used in recombinant DNA technologies include plasmids, chromosomes, artificial chromosomes, and viruses.

[0074] Specifically, as used herein, the term "plasmid" refers to an extrachromosomal circular double-stranded DNA molecule to which other DNA fragments can be linked. A plasmid is a vector, that is, a nucleic acid molecule capable of carrying another nucleic acid to which it is linked. Some plasmids can replicate autonomously after being introduced into a host cell (e.g., bacterial plasmids with bacterial origins of replication and mammalian free plasmids). Other vectors (e.g., mammalian non-free vectors (episomal plasmids)) can integrate into the host cell's genome after being introduced into the host cell and replicate along with the host genome. Some plasmids can direct the expression of genes to which they are operatively linked.

[0075] Non-viral delivery systems include, but are not limited to, DNA transfection methods. Here, transfection includes the process of delivering genes into target mammalian cells using non-viral vectors.

[0076] In one embodiment, the vector is selected from expression vectors, viral vectors, pCMV, CMV, pcDNA3, and VR1012.

[0077] In a preferred embodiment, the viral vector is adeno-associated virus (AAV), adenovirus, or lentivirus.

[0078] In one embodiment, the carrier suitable for the construct of the present invention is a carrier containing a promoter. Specifically, the carrier is a carrier with a strong promoter, such as CMV, SV40, or β-hCG.

[0079] Transfected cell lines Another objective of this invention is to transfect cell lines using the expression vector of this invention and the DNA construct according to this invention.

[0080] In this invention, the term "transfected cell line" refers to any cell line that has been in contact with any of the vectors of this invention. Specifically, the term "transfection" also includes the terms "conversion" or "infection".

[0081] Transfection can be achieved by any known method for introducing polynucleotides into host cells. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art, including dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of polynucleotides in liposomes, and direct microinjection of DNA into the cell nucleus. Furthermore, nucleic acid molecules can also be introduced into mammalian cells via viral vectors.

[0082] Methods for transfecting cells are well known in the art (see, for example, U.S. Patents 4,399,216, 4,912,040, 4,740,461, and 4,959,455, which are incorporated herein by reference). Methods for transfecting plant cells are well known in the industry, including, for example, Agrobacterium-mediated transformation, gene gun transformation, direct injection, electroporation, and viral transformation. Methods for transfecting bacterial and yeast cells are also well known in the art. Mammalian cell lines that can serve as expression hosts are well known in the industry, including many immortalized cell lines available from the American Type Culture Collection (ATCC). These cell lines include, but are not limited to, HEK293, HeLa, ACN, N9, N13, PC12, J774, and A549 cells. Particularly preferred cell lines are selected by identifying which cell lines have high expression levels.

[0083] In one embodiment, the cell line of the present invention is characterized by expressing a target receptor, the activation of which leads to an increase in extracellular ATP levels.

[0084] In a preferred embodiment, the target receptor is selected from members of the P2Y and P2X purinergic receptor subfamilies.

[0085] ATP can be released into the extracellular space through various pathways, including cell damage, P2X7 receptor activation, pannexin-1 activation, or secretory exocytosis. Subsequently, ATP binds to P2X and P2Y receptors or is degraded by extracellular ATPases (ecto-ATPases) such as CD39 and CD73.

[0086] In a preferred embodiment, the cell line of the present invention is capable of expressing both the extracellular ATP-dependent luciferase and the cytoplasmic ATP-independent luciferase. In a preferred embodiment, the cell line of the present invention is capable of expressing pmeLUC luciferase and Renilla luciferase.

[0087] Uses and methods A further object of the present invention is the use of the DNA construct, the cell line, or the mRNA of the present invention for in vitro determination of extracellular ATP levels. Preferably, the construct or cell line can be used as an experimental model in a screening method to screen target compounds capable of regulating extracellular ATP levels.

[0088] Specifically, the present invention also aims to provide a method for screening target compounds capable of regulating extracellular ATP levels, the method comprising the following steps: a) Contact the cell line defined according to any embodiment described herein with a solution containing the substrate and the target compound; b) Detect the percentage of emitted photons or light emission; c) Normalize the value of emitted photons or luminescence percentage from ATP-dependent luciferase by dividing the value of emitted photons or luminescence percentage from ATP-independent luciferase by the value of emitted photons or luminescence percentage from ATP-independent luciferase to obtain a value that depends only on extracellular ATP.

[0089] In a preferred embodiment, step b) includes the following sub-steps: i) First detection, detect the percentage of photons or light emitted by the product obtained in step b) to obtain the value of the percentage of photons or light emitted from ATP-dependent luciferase; ii) The cell line was kept at 37°C for 1 hour to ensure complete consumption of the substrate and to prevent luciferase signal overlap. iii) Add a prosthetic group; iv) Second detection, detecting the percentage of photons or light emitted by the product obtained in steps b-iii) to obtain a value of the percentage of photons or light emitted from ATP-independent luciferase.

[0090] In one embodiment, in step a), when the extracellular ATP-dependent luciferase is pmeLUC, the substrate is D-luciferin, and it is added before, after, or simultaneously with the addition of the target compound.

[0091] In one embodiment, the cell line is suspended in a culture medium in a well plate. The number of wells can be determined according to requirements known to those skilled in the art, such as 24 or 48 wells.

[0092] In one embodiment, a calibration step is provided, which includes adding a known amount of ATP in increasing concentrations (e.g., from 10 µM to 1.0 mM) to successive wells.

[0093] In one embodiment, step b-ii) includes the steps of aspirating the culture medium and adding fresh culture medium before incubating the cell line.

[0094] In one embodiment, in step a), the substrate solution is added to obtain a final concentration of 1.5 mg / mL.

[0095] In one embodiment, in step b), an imaging device (preferably IVIS Perkin-Elmer) is used to detect the emitted photons or the percentage of light emitted.

[0096] The prosthetic group is a prosthetic group that is compatible with ATP-dependent luciferase.

[0097] In one embodiment, when the cytoplasmic ATP-independent luciferase is Renilla luciferase, the cofactor is coelenterin, and the cofactor is added to obtain a final concentration of 50 μM.

[0098] Biosensors Another object of the present invention is a biosensor comprising a cell line characterized in that the cell line is transfected with a vector according to the invention or a DNA construct according to the invention.

[0099] A biosensor is a device that uses a living organism, part of an organism, or biomolecule to detect specific compounds or environmental changes and transmits that information in a measurable form. Therefore, in the context of this invention, a biosensor consists of a cell line that has been transfected by introducing a vector according to the invention or a DNA construct as described herein.

[0100] Another object of the present invention is the use of the biosensor of the present invention for measuring extracellular ATP levels in an in vitro system.

[0101] Another object of the present invention is the use of the biosensor of the present invention for measuring extracellular ATP levels in an in vivo system.

[0102] In any part of this specification and claims, the term "comprising" may be replaced by the term "consisting of".

[0103] The following examples are intended to better illustrate the methods disclosed in this specification, and these examples should in no way be considered as limiting the foregoing description and claims.

[0104] Example In a preferred embodiment, the DNA construct encodes two genes: a firefly luciferase (Photinus pyralis) expressed on the cell membrane, referred to as "pmeLUC" (plasma membrane luciferase), which is the subject of European Patent Office Patent No. RM2005A000252-SG / IC (see point 5), and a cytoplasmic luciferase called Renilla luciferase isolated from Renilla reniformis. The membrane luciferase pmeLUC is expressed with its catalytic site facing outward from the plasma membrane and measures extracellular ATP by adding its substrate luciferin. The cytoplasmic Renilla luciferase is ATP-independent and emits photons by oxidizing its substrate coelenterate. Renilla allows the probe expression to be tracked independently of the presence of ATP, thus providing the possibility of normalizing the pmeLUC signal. Given its structure, the probe is named pmeLUC / nilla.

[0105] In a preferred embodiment, the nucleotide sequence encoding the membrane luciferase pmeLUC is followed by the IRES sequence, then the Renilla and derived sequences.

[0106] The proteins pmeLUC and Renilla are derived from the transcription and translation of the aforementioned nucleotide sequence.

[0107] This invention provides the possibility of obtaining normalized measurements of extracellular ATP both in vivo and in vitro. Therefore, it can be used to study ATP as a signaling molecule in physiological and pathological processes. Furthermore, it facilitates the research and development of drugs that act on purinergic receptors or more broadly regulate inflammatory processes. To avoid mutual interference, the detection of pmeLUC and Renilla signals must be appropriately spaced out (approximately 1 hour).

[0108] It is recommended to use fluorescein and coelenterate formulations suitable for specific experimental needs. For in vivo use, it is recommended to use highly luminescent fluorescein and coelenterate with appropriate biodistribution capabilities.

[0109] pmeLUC / nilla must be expressed in a host organism. Both cell lines and animal models can be used. In the presence of oxygen and ATP in the extracellular environment, pmeLUC breaks down ATP into ADP and phosphate, and oxidizes luciferin, emitting one photon (light) for every molecule of ATP consumed. Therefore, light emission is proportional to the concentration of extracellular ATP. Unlike pmeLUC, Renilla luciferase remains confined to the cytoplasm; it generates a light signal through interaction with the cofactor coenzyme, but this light signal is independent of the presence of ATP.

[0110] Therefore, the light emitted by Renilla luciferase is directly proportional to its intracellular concentration, thus indicating the amount of protein very accurately. The relationship between the light emitted by pmeLUC and that emitted by Renilla luciferase allows for easy normalization of the light signal to reliably quantify the concentration of extracellular ATP.

[0111] The system for measuring extracellular ATP uses pmeLUC as a sensor for ATP concentration near the plasma membrane and Renilla as an internal control to normalize the signal. ATP is released into the extracellular space through various processes, including cell damage and P2X7 receptor activation. Subsequently, ATP binds to P2X and P2Y receptors or is degraded by extracellular ATPases (ecto-ATPases) such as CD39 and CD73. Therefore, this invention enables the precise measurement of extracellular ATP.

[0112] To evaluate the effectiveness of our invention, several cell lines were transfected using the previously described ATP measurement system. Subsequently, calibration curves were generated in which the bioluminescence of the signal correlated with ATP concentration.

[0113] Example: In vitro experimental protocol for calibration curves and compound testing HEK293-pmeLUC / nilla cells were detached by continuous treatment with trypsin-EDTA at 37°C for 5 minutes and resuspended in complete DMEM-F12 medium. Cells were counted and then centrifuged at 200g for 5 minutes at room temperature. The medium was removed, and the cells were centrifuged at 7 × 10⁻⁶ cells / min. 4 Resuspend the cells at a concentration of 10 cells / ml in DMEM-F12 medium supplemented with 10% fetal bovine serum. Gently shake the cell suspension to avoid cell aggregation. Then, seed 1 ml of the cell suspension into 24-well cell culture plates and incubate the cells in a humidified incubator at 37°C and 5% CO2 for 24 hours.

[0114] Various target compounds can be used to treat cell monolayers. To construct ATP calibration curves, incremental concentrations of ATP (from 1 µM to 1 mM) are added to, for example, HEK293-pmeLUC cells or other target cell types seeded in 24-well plates (at least three replicates for each ATP concentration). After each ATP addition, gentle stirring is performed to ensure uniform reagent distribution. Then, a solution of d-luciferin sodium salt is added to each well to a final concentration of 1.5 mg / mL.

[0115] Data can be collected using a chemiluminescence microplate reader, an IVIS Perkin-Elmer system, or a similar system.

[0116] Place the plate in the IVIS Perkin-Elmer and perform acquisition as follows: Select the "Luminescent" option with the following parameters: "Exposure time" is 3 minutes, "Binning" is 4, and "F / STOP" is 1. Enable the "Photography, Auto Exposure" and "Overlay" options, and select option "D" from the "Field of View" list.

[0117] Remove the plate from the IVIS Perkin-Elmer, aspirate the culture medium, and replace it with fresh medium. Incubate the cells at 37°C and 5% CO2 for 1 hour to allow complete clearance of luciferin, thereby shutting down the luciferase signal from *Photinus pyralis*. At this point, add coelentrin dissolved in methanol to a final concentration of 50 µM. Repeat the sampling process as described above to detect the luminescence of *Renilla reniformis*.

[0118] To obtain normalized extracellular ATP concentration values, the luminescence of luciferase from *Photinus pyralis* (the first obtained luminescence) was divided by the luminescence of luciferase from *Renilla reniformis* (the second obtained luminescence). The data were then calibrated to ATP concentration using a calibration curve plotted by adding known amounts of ATP to parallel-prepared samples.

[0119] In vivo experimental protocol example: HEK-293-pmeLUC / nilla cells were used as a reporter system to test compounds. The effect of substances on the release of ATP in the body.

[0120] HEK293-pmeLUC / nilla cells were detached by continuous treatment with trypsin-EDTA at 37°C for 5 minutes and resuspended in DMEM-F12 medium supplemented with 10% fetal bovine serum. Cells were counted and then centrifuged at 200g for 5 minutes at room temperature. The medium was removed, and the cells were centrifuged at 7.5 × 10⁻⁶ cells / min. 6 The cells were resuspended in phosphate-buffered saline (PBS) at a concentration of 1.5 × 10⁻⁶ cells / ml and injected intraperitoneally into mice in a volume of 200 µL, resulting in a final concentration of 1.5 × 10⁻⁶ cells / ml. 6 Cells / mouse. Mice were then subjected to the target treatment. Then, 200 µL of fluorescein solution was injected intraperitoneally at a concentration of 15 mg / ml.

[0121] Mice were anesthetized with isoflurane and placed in an IVIS Perkin-Elmer or similar small animal luminometer. The acquisition configuration was as follows: the “Luminescent” option was selected, with the following parameters: “Exposure time” of 5 minutes, “Binning” of 4, and “F / STOP” of 1. The “Photography, AutoExposure” and “Overlay” options were selected, and option “D” was chosen from the “Field of View” list.

[0122] One hour after injection of D-luciferin, coelenterate (200 µL per mouse, 50 μg / mL) was injected intraperitoneally, and the mice were then placed in an IVIS Perkin-Elmer or similar luminometer. The luminescence was collected using the same method as for luciferase from *Photinus pyralis*. Finally, the luminescence of pmeLUC was normalized using the luminescence of Renilla.

[0123] The following outlines an experimental protocol for screening target compounds that can regulate extracellular ATP levels.

[0124] Material: • Cells expressing pmeLUC / nilla • Cell culture medium • Target compound to be tested •D-fluorescein solution • ATP solution •Coelenterazine solution • Perkin-Elmere IVIS luminometer or chemiluminescent microplate reader or similar instrument.

[0125] Experimental plan: 1. Seed pmeLUC / nilla expression cells suspended in culture medium into 48-well plates.

[0126] 2. Treat cells with the target compound for an appropriate time.

[0127] 3. Add D-fluorescein solution to each well to achieve a final concentration of 1.5 mg / mL.

[0128] 4. Add known amounts of ATP in increasing concentrations (from 10 µM to 1.0 mM) to a series of wells for calibration.

[0129] 5. Place the plate in a chemiluminescent microplate reader and begin data acquisition. Three replicates are recommended.

[0130] 7. Remove the soil and replace it with fresh soil. Place the cells in a 37°C incubator for 1 hour to allow the luciferin to be completely consumed. This step is crucial to prevent overlap between the luminescence from Photinus pyralis luciferase and Renilla luciferase.

[0131] 8. Add coelenterate to a final concentration of 50 μM.

[0132] 9. Place the plate in the chemiluminescent microplate reader and begin data acquisition.

[0133] 10. The luminescence of each well was normalized by dividing the luminescence of pmeLUC by that of Renilla. The normalized values ​​depended only on extracellular ATP and were independent of probe expression.

[0134] 11. Check whether the target compound alters ATP levels.

[0135] sequence list SEQ ID NO. 1: pmeLUC / nilla SEQ ID NO.2: pmeLUC MAQRMTTQLLLLLVWVAVVGEAQTRIAEQKLISEEDLLQMEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDAHIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAPANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQSMYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHARDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYKIQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGYGLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMSGYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESILLQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVVFVDEVPKGLTGKLDARKIREILIKAKKGGKIAVAAAMSGAGPWAAWPFLLSLALMLLWLLS SEQ ID NO.3: Renilla MASKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAENAVIFLHGNAASSYLWRHVVPHIEPVARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAWFELLNLPKKIIFVGHDWGACLAFHYSYEHQDKIKAIVHAESVVDVIESWDEWPDIEEDIALIKSEEGEKMVLENNFFVETMLPSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWPREIPLVKGGKPDVVQIVRNYNAYLRASDDLPKMFIESDPGFFSNAIVEGAKKFPNTEFVKVKGLHFSQEDAPDEMGKYIKSFVERVLKNEQ SEQ ID NO. 4: Leader sequence ATGGCTCAGCGGATGACAACACAGCTGCTGCTCCTTCTAGTGTGGGTGGCTGTAGTAGGGGAGGCTCAGACAAGGATTGCA SEQ ID NO. 5: Myc tag GAACAAAAACTAATAAGCGAGGAGGACCTG SEQ ID NO. 6: GPI sequence CTGCAGCCATGAGTGGGGCTGGGCCCTGGGCAGCCTGGCCTTTCCTGCTTAGCCTGGCCCTAATGCTGCTGTGGCTGCTCAGCTGATGA SEQ ID NO.7: IRES sequence GCCCCTCTCCCTCCCCCCCCCCTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTGATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTTAGTCGAGGTTAAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAA SEQ ID NO.8: Lumiluc protein sequence KVFTLGDFVGDWRQTAGYNQAQVLEQGGLTSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSCDQMAQIEKIFKVVYPVDDHHFKAILHYGTLVIDGVTPNMIDYFGQPYEGIAKFDGKKITVTGTLWNGNTIIDERLINPDGSLLFRVTINGVTGWRLHERILA SEQ ID NO. 9: Nanoluc protein sequence MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPV DDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILA SEQ ID NO. 10: pmeLuc cDNA construct ATG TGC TAG CAT GGC TCA GCG GAT GAC AAC SEQ ID NO. 11: cDNA pme Luc construct GCA TTG AAT TCC TCG AGG TCG ACG GTA TCA AG SEQ ID NO. 12: Renilla cDNA synthesis GCT TTC TAG AAT GGC TTC CAA GGT GTA CGA C SEQ ID NO. 13: Renilla cDNA synthesis TAA CCG CCG GCG TTA CTG CTC GTT CTT CAG CAC G

Claims

1. A DNA construct for measuring extracellular ATP levels, said DNA construct comprising the following elements operatively linked from 5' to 3': i) The DNA sequence encoding extracellular ATP-dependent luciferase; ii) IRES sequence; iii) DNA sequence encoding cytoplasmic ATP-independent luciferase.

2. The DNA construct according to claim 1, wherein the DNA sequence encoding extracellular ATP-dependent luciferase comprises or consists of the following sequences: a) A sequence that encodes a leader sequence; b) The sequence encoding a protein with ATP-dependent luciferase activity; c) Sequences encoding cell membrane anchoring sequences.

3. The DNA construct according to claim 2, wherein the protein having extracellular ATP-dependent luciferase activity is selected from Luciferase Photinus Pyralis, Luciferase Luciola cruciate, Luciferase Luciola italic, Luciferase Luciola lateralis, Luciferase Luciola mingrelica, Luciferase Photuris pennsylvanica, Luciferase Pyrophorus plagiophthalamus, Luciferase Phrixothrix hirtus, or variants thereof.

4. The DNA construct according to any one of claims 1 to 3, wherein the extracellular ATP-dependent luciferase is firefly luciferase (Photinus pyralis), plasma membrane luciferase (pmeLUC), or a variant thereof.

5. The DNA construct according to any one of claims 1 to 4, wherein the DNA sequence encodes an extracellular ATP-dependent luciferase having SEQ ID NO:

2.

6. The DNA construct according to any one of claims 1 to 5, wherein the cytoplasmic ATP-independent luciferase is selected from Luciferase Renilla reniformis, Luciferase Cypridina noctiluca, Luciferase Cypridina hilgendorfii, Luciferase Metridia longa, Luciferase Oplophorus gracilorostris, or variants thereof.

7. The DNA construct according to any one of claims 1 to 6, wherein the cytoplasmic ATP-independent luciferase is luciferase Renilla or a variant thereof.

8. The DNA construct according to any one of claims 1 to 7, wherein the DNA sequence encodes a cytoplasmic ATP-independent luciferase or a variant thereof having SEQ ID NO:

3.

9. The DNA construct according to any one of claims 2 to 8, wherein the leader sequence has SEQ ID NO:4 and / or the sequence encoding the cell membrane anchoring sequence has SEQ ID NO:

6.

10. The DNA construct according to any one of claims 1 to 9, wherein the DNA construct further comprises a sequence encoding a tag, preferably a sequence encoding a myc tag.

11. The DNA construct according to any one of claims 1 to 10, wherein the IRES sequence has SEQ ID NO:7 or a variant thereof.

12. The DNA construct according to any one of claims 1 to 11, having SEQ ID NO:1, or having greater than 90% identity with SEQ ID NO:1, preferably greater than 95% identity, more preferably greater than 99% identity.

13. A vector comprising a DNA construct according to any one of claims 1 to 12.

14. The vector according to claim 13, wherein the vector is selected from expression vectors, viral vectors, pCMV, CMV, pcDNA3, and VR1012.

15. The vector according to any one of claims 13 or 14, wherein the viral vector is adeno-associated virus (AAV), adenovirus, or lentivirus.

16. The vector according to any one of claims 13 to 15, wherein the vector comprises a promoter operatively linked upstream of the DNA construct in a 5' to 3' orientation.

17. A cell line transfected with a vector according to any one of claims 13 to 15, or a DNA construct according to any one of claims 1 to 12.

18. The cell line according to claim 17, characterized in that... The target receptor is expressed, and activation of the target receptor leads to an increase in extracellular ATP levels.

19. The cell line according to any one of claims 17 or 18, wherein the target receptor is selected from P2Y and P2X.

20. The cell line according to any one of claims 17 to 19, wherein the P2X receptor is P2X7.

21. The cell line according to any one of claims 17 to 20, wherein the cell line is selected from the group consisting of HEK293, HeLa, ACN, N9, N13, PC12, J774, and A549.

22. The cell line according to any one of claims 17 to 21, wherein the cell line is capable of expressing the extracellular ATP-dependent luciferase and the cytoplasmic ATP-independent luciferase.

23. An isolated mRNA, said isolated mRNA being transcribed from a DNA construct according to any one of claims 1 to 12.

24. The DNA construct according to any one of claims 1 to 12, or the cell line according to any one of claims 17 to 22, or the mRNA according to claim 23 for in vitro use in measuring extracellular ATP levels.

25. A method for screening target compounds capable of regulating extracellular ATP levels, the screening method comprising the following steps: a) Contacting the cell line as defined in any one of claims 17 to 22 with a solution containing the substrate and the target compound; b) Detect the percentage of emitted photons or light emission; c) Normalize the number of photons emitted or the percentage of light emitted from ATP-dependent luciferase by dividing the value of the number of photons emitted or the percentage of light emitted from ATP-independent luciferase by the value of the number of photons emitted or the percentage of light emitted from ATP-independent luciferase to obtain a value that depends only on extracellular ATP.

26. The screening method according to claim 25, wherein step b) comprises the following sub-steps: i) First detection, detect the percentage of photons or light emitted by the product obtained in step b) to obtain the value of the percentage of photons or light emitted from ATP-dependent luciferase; ii) The cell line was kept at 37°C for 1 hour to ensure complete consumption of the substrate and to prevent luciferase signal overlap. iii) Add a prosthetic group; iv) Second detection, detecting the percentage of photons or light emitted by the product obtained in steps b-iii) to obtain a value of the percentage of photons or light emitted from ATP-independent luciferase.

27. The screening method according to any one of claims 25 or 26, wherein in step a), when the extracellular ATP-dependent luciferase is pmeLUC, the substrate is D-luciferin, and the substrate is added to obtain a final concentration of 1.5 mg / mL.

28. The screening method according to any one of claims 25 to 27, wherein the detection of emitted photons or percentage of emission in step b) is performed by an imaging device, preferably IVIS Perkin-Elmer.

29. The screening method according to any one of claims 25 to 28, wherein when the cytoplasmic ATP-independent luciferase is luciferase Renilla, the cofactor is coelenterin, and the cofactor is added to obtain a final concentration of 50 μM.

30. A biosensor comprising a cell line characterized in that it is transfected with a vector according to any one of claims 13 to 16, or a DNA construct according to any one of claims 1 to 12.

31. Use of the biosensor according to claim 30 for measuring extracellular ATP levels in an in vitro system.

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