Dual-luciferase reporter gene detection system and application thereof

Abstract

The invention discloses a dual-luciferase reporter gene detection system and application thereof. The detection system comprises Gaussian luciferase, Pleuroma xiphias luciferase and a substrate, and the substrate is coelenteran or a derivative of coelenteran. Compared with other dual-luciferase reporter gene detection systems, the dual-luciferase reporter gene detection system has stronger signals and simple reaction conditions.

Description

A dual-luciferase reporter gene detection system and its application Technical Field

[0001] The present invention relates to the technical field of molecular biology, and in particular to a dual-luciferase reporter gene detection system and applications thereof. Background Art

[0002] Luciferase is a general term for enzymes that can produce biological fluorescence in nature. The luciferase reporter assay refers to a detection system that uses luciferin as a substrate to detect luciferase activity. It has a wide range of applications in cell imaging, amino acid labeling, protein labeling and localization, antibody-specific recognition, nucleic acid labeling, construction of dual reporter genes, and gene sequencing.

[0003] Coelenterazine is the most abundant natural luciferin. It serves as a substrate for many luciferases, such as Renilla luciferase, Gaussia secreted luciferase, and aequorin. Unlike the beetle luciferin / luciferase system, the coelenterazine / luciferase system does not require adenosine triphosphate (ATP), making it more convenient for in vivo bioluminescence studies and producing higher luminescence brightness. Therefore, coelenterazine is often used as a luminescent substrate in fluorescence-based reporter gene assays and in vivo animal assays.

[0004] Different coelenterazine derivatives vary in their luminescence wavelength, cell membrane permeability, and quantum efficiency, resulting in varying experimental results for the same application. Because different luciferases have different luminescence spectral characteristics and substrates, this provides a theoretical basis for constructing a dual-luciferase luminescence system with low cross-interference and high specificity.

[0005] Dual-luciferase luminescence systems are widely used in gene sequencing, dual-reporter gene construction, and protein localization. This method offers advantages such as high sensitivity, minimal interference, ease of detection, and a wide detection range. Compared to single-luciferase assays, multiplexed assays are more suitable for simultaneously studying the expression regulation of multiple genes; reducing off-target effects; identifying interactions between two or more signaling pathways; and normalizing artifacts generated by experimental systems.

[0006] Currently available dual-luciferase luminescence systems primarily utilize two-color detection systems combining firefly luciferase and Renilla luciferase. Firefly luciferase requires the simultaneous presence of luciferin, oxygen, ATP, and magnesium ions to emit light, while Renilla luciferase requires only coelenterazine and oxygen. The differences in reaction conditions between the two luciferase systems limit their application in the same scenario.

[0007] Summary of the Invention

[0008] In view of this, the present invention provides a dual-luciferase reporter gene detection system and applications thereof.

[0009] The present invention provides a dual luciferase reporter gene detection system, comprising Gaussia luciferase, Pleuromammamma xiphias luciferase and a substrate.

[0010] And the substrate is a compound represented by formula (I), or a stereoisomer, geometric isomer, tautomer, salt, nitrogen oxide, hydrate or solvate of the compound represented by formula (I):

[0011]

[0012] Wherein, R1 and R2 are independently H, D, F, Cl, Br, I, OH, NH2, NO2, CN, N3, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy, C 1-6 Hydroxyalkyl or C 1-6 Alkylamino;

[0013] R3 is optionally substituted aryl, heterocyclyl or heteroaryl;

[0014] a1 is 0 or any integer from 1 to 6,

[0015] a2 is 0 or any integer from 1 to 6,

[0016] b is 0 or 1,

[0017] c is 0, 1 or 2.

[0018] In the present invention, the Gaussia luciferase is any one of the following I) to III):

[0019] 1), the amino acid sequence of the Gaussia luciferase is shown in SEQ ID NO: 1;

[0020] II), a luciferase having at least 85% homology to the amino acid sequence of the Gaussia luciferase described in I) and having the same or similar function as the Gaussia luciferase described in I);

[0021] III) A luciferase having the Gaussia luciferase activity as described in I) with its amino acid sequence modified, substituted, deleted or added with one or more amino acid residues.

[0022] In some embodiments, the Gaussia luciferase is not optimized, and is derived from Gaussia Luciferase, and the amino acid sequence is the sequence with GenBank accession number AY015993.1 (as shown in SEQ ID NO: 1).

[0023] In some embodiments, the luciferase has at least 85% homology, such as at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology to the amino acid sequence of the Gaussia luciferase described in i), and has the same or similar function as the Gaussia luciferase described in i).

[0024] In some embodiments, the Gaussia luciferase is an optimized mutant, i.e., a Gaussia luciferase mutant, which has at least one of the following mutation sites compared to the amino acid sequence shown in SEQ ID NO: 1: positions 24, 26, 27, 29, 30, 31, 32, 33, 36, 37, 40, 66, 79, 84, 88, 102, 103, 104, 110, 123, 124, 138, 152, 163, 167, 170, 174, 175, 178, 182, and 183.

[0025] In some embodiments, the Gaussia luciferin mutant has at least one of the following mutations:

[0026] 1) E at position 24 was mutated to K;

[0027] 2) F at position 26 mutated to R or L;

[0028] 3) N at position 27 was mutated to D;

[0029] 4) V at position 29 mutated to F or L;

[0030] 5) A at position 30 mutated to G or D;

[0031] 6) V at position 31 was mutated to I;

[0032] 7) A at position 32 was mutated to V;

[0033] 8) S at position 33 is mutated to E, R, or K;

[0034] 9) A at position 36 is mutated to V or I;

[0035] 10) T at position 37 mutated to N or E;

[0036] 11) L at position 40 is mutated to I or T;

[0037] 12) K at position 66 is mutated to P, S, I, R, or N;

[0038] 13) H at position 79 mutated to K;

[0039] 14) P at position 84 is mutated to A, L, K or V;

[0040] 15) K at position 88 mutated to R;

[0041] 16) E at position 102 is mutated to D, A, S, K or N;

[0042] 17) S at position 103 mutated to T;

[0043] 18) A at position 104 was mutated to G;

[0044] 19) E at position 110 is mutated to P, G, or A;

[0045] 20) D at position 123 was mutated to N;

[0046] 21) L at position 124 is mutated to M, G, or I;

[0047] 22) V at position 138 mutated to E or D;

[0048] 23) Q at position 152 is mutated to R or H;

[0049] 24) Q at position 163 was mutated to D;

[0050] 25) S at position 170 mutated to N or T;

[0051] 26) G at position 174 was mutated to K;

[0052] 27) Q at position 175 was mutated to E;

[0053] 28) K at position 178 mutated to T;

[0054] 29) A at position 182 was mutated to M;

[0055] 30) The G at position 183 was mutated to N or A.

[0056] Compared with wild-type Gaussia luciferase, the mutant has a wider substrate spectrum, stronger specificity, and significantly enhanced luminescence brightness, and the detection accuracy is significantly improved in practical applications.

[0057] In some embodiments, the Gaussia luciferase may or may not include a signal peptide amino acid sequence.

[0058] In the present invention, the Pleuromamma xiphias luciferase is any one of the following i) to iii):

[0059] i) The amino acid sequence of the Pleuromammamma xiphias luciferase is shown in SEQ ID NO: 3;

[0060] ii) a luciferase having at least 85% homology to the amino acid sequence of the Pleuromamma xiphias luciferase described in i) and having the same or similar function as the Pleuromamma xiphias luciferase described in i);

[0061] iii) A luciferase having the Pleuromammamma xiphias luciferase activity, wherein the amino acid sequence of the Pleuromammamma xiphias luciferase described in i) is modified, substituted, deleted or added with one or more amino acid residues.

[0062] In some embodiments, the Pleuromammamma xiphias luciferase is not optimized, is derived from Pleuromammamma xiphias, and has an amino acid sequence of PMID 23886588 (as shown in SEQ ID NO: 3).

[0063] In some embodiments, the luciferase has at least 85% homology to the amino acid sequence of the Pleuromammamma xiphias luciferase described in i), such as at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology, and has the same or similar function as the Pleuromammamma xiphias luciferase described in i).

[0064] In some embodiments, the Pleuromammamma xiphias luciferase is an optimized mutant, i.e., a Pleuromammamma xiphias luciferase mutant. Compared with the amino acid sequence shown in SEQ ID NO: 3, the Pleuromammamma xiphias luciferase mutant has at least one of the following mutation sites: position 81, position 82, position 83, and position 84.

[0065] In some embodiments, the Pleuromamma xiphias luciferin mutant has at least one of the following mutations:

[0066] (1) G at position 81 is mutated to L, P, Q, S, or T;

[0067] (2) Q at position 82 is mutated to R, W, I, Y, A, L, F, V, P, E, or M;

[0068] (3) G at position 83 is mutated to S, Q, R, W, T, A, or L;

[0069] (4) The G at position 84 is mutated to F, R, S, C, Y, L, I, K, V, or P.

[0070] Compared with the wild-type Pleuromammamma xiphias luciferase, the mutant has a wider substrate spectrum, stronger specificity, and significantly enhanced luminescence brightness, and the detection accuracy is significantly improved in practical applications.

[0071] In some embodiments, the Pleuromammamma xiphias luciferase may or may not include a signal peptide amino acid sequence.

[0072] In some embodiments, the Pleuromammamma xiphias luciferase includes a signal peptide amino acid sequence; preferably, the Pleuromammamma xiphias luciferase amino acid sequence including the signal peptide is as shown in SEQ ID NO: 4.

[0073] In the embodiment of the present invention, the compound represented by formula (I) may further include at least one of the following additional technical features:

[0074] In some specific embodiments, R1 is H.

[0075] In some specific embodiments, R2 is H, NH2, OH or C 1-6 Alkylamino is preferably H, NH2, 3-OH, 4-OH or dimethylamino.

[0076] In some specific embodiments, R3 is a substituted aryl group, preferably OH-C6H5 or F-C6H5, more preferably 4-OH-C6H5 or 4-F-C6H5.

[0077] In some specific embodiments, a1 is 0 or 1.

[0078] In some specific embodiments, a2 is 0 or 1.

[0079] In some specific embodiments, when a1 is 0, a2 is 1; when a1 is 1, a2 is 0; or when a1 is 0, a2 is 0.

[0080] In some specific embodiments, b is 1.

[0081] In some specific embodiments, c is 1.

[0082] In some embodiments, the compound represented by formula (I) includes any two of the following:

[0083]

[0084]

[0085] In some embodiments, the substrate of Gaussia luciferase is

[0086] In some embodiments, the substrate of the Pleuromammamma xiphias luciferase is

[0087] In some embodiments, the molar ratio of Gaussia luciferase to Pleuromammamma xiphias luciferase is 1:(0.01-100);

[0088] In some embodiments, the molar ratio of Gaussia luciferase to substrate is 1:(1-1000);

[0089] In some embodiments, the molar ratio of Pleuromammamma xiphias luciferase to substrate is 1:(1-1000).

[0090] The dual-luciferase reporter gene detection system of the present invention is used in the preparation of detection reagents.

[0091] In the present invention, the detection reagents include: cell imaging reagents, amino acid labeling reagents, protein labeling and positioning reagents, antibody specific recognition reagents, nucleic acid labeling reagents or gene sequencing reagents.

[0092] The present invention also provides a fluorescence detection kit, which includes the dual-luciferase reporter gene detection system of the present invention.

[0093] In the present invention, the fluorescence detection reagent further comprises a reaction buffer, and the reaction buffer comprises water, Tris-HCl, NaCl and Tween-20.

[0094] The present invention also provides a method for detecting a luciferase luminescent signal, which comprises contacting the substrate in the dual-luciferase reporter gene detection system of the present invention with a sample, and detecting the luminescent signal generated by the Gaussia luciferase and the Pleuromammamma xiphias luciferase; the sample contains the Gaussia luciferase and the Pleuromammamma xiphias luciferase.

[0095] In the present invention, the sample is a nucleic acid, a nucleic acid analog, a cell, a culture containing a cell, or a product obtained from a culture containing a cell. The cell is a bacterial cell, a fungal cell, an animal cell, a plant cell, or a human cell. The nucleic acid is DNA or RNA, which is not limited in the present invention.

[0096] In some embodiments, the sample is a cell, a culture containing the cell, or a cell lysate expressing Gaussia luciferase and Pleuroma xiphias luciferase described in the dual-luciferase reporter gene detection system of the present invention.

[0097] In other embodiments, the sample is a nucleotide analog that specifically binds to Gaussia luciferase and Pleuromammamma xiphias luciferase in the dual-luciferase reporter gene detection system of the present invention, and the nucleotide analog specifically binds to the luciferase through a nucleophilic reagent.

[0098] The nucleophilic reagent is an antigen-antibody complex, biotin-streptavidin or digoxin-digoxin antibody.

[0099] The nucleotide analogues are composed of nucleotide molecules containing four different base types, wherein the first nucleotide molecule can specifically bind to Gaussia luciferase, the second nucleotide molecule can specifically bind to Pleuromammamma xiphias luciferase, the third nucleotide molecule can specifically bind to Gaussia luciferase and Pleuromammamma xiphias luciferase, and the fourth nucleotide molecule can neither bind to Gaussia luciferase nor to Pleuromammamma xiphias luciferase.

[0100] In the present invention, the method further comprises: determining the type of the nucleotide molecule by the luminescent signal.

[0101] Furthermore, the present invention also provides a method for sequencing a nucleic acid molecule, comprising the following steps:

[0102] (1) providing a nucleic acid molecule to be sequenced attached to a support, or attaching the nucleic acid molecule to be sequenced to a support;

[0103] (2) adding a primer for initiating nucleotide polymerization reaction, a polymerase for carrying out nucleotide polymerization reaction, and four compounds to form a reaction system containing a solution phase and a solid phase; wherein the four compounds are derivatives of nucleotides A, (T / U), C, and G, respectively, and have base complementary pairing capabilities; and the hydroxyl group (-OH) at the 3' position of the ribose or deoxyribose of the four compounds is protected by a protecting group; and,

[0104] The first compound is linked to a first molecular label,

[0105] The second compound is linked to a second molecular label,

[0106] The third compound is connected to the first molecular marker and the second molecular marker, or part of the third compound is connected to the first molecular marker and another part of the third compound is connected to the second molecular marker, and the fourth compound is not connected to the molecular marker;

[0107] (3) annealing the primer to the nucleic acid molecule to be sequenced, wherein the primer serves as the initial growing nucleic acid chain and forms a duplex connected to the support together with the nucleic acid molecule to be sequenced;

[0108] (4) performing a nucleotide polymerization reaction using a polymerase under conditions that allow the polymerase to perform a nucleotide polymerization reaction, thereby incorporating one of the four compounds into the 3′ end of the growing nucleic acid chain;

[0109] (5) contacting the duplex in the previous step with Gaussia luciferase and Pleuromammamma xiphias luciferase in the dual luciferase reporter gene detection system of the present invention and performing a binding reaction, wherein the Gaussia luciferase and Pleuromammamma xiphias luciferase are capable of specifically binding to the first molecular marker and the second molecular marker, respectively, and then causing the luciferase to undergo a fluorescence reaction in the presence of a substrate, and detecting the emitted fluorescence signal;

[0110] (6) removing the protective groups of the incorporated nucleotides and the molecular labels;

[0111] (7) Optionally repeat steps (4)-(6) or (3)-(6) one or more times to obtain sequence information of the nucleic acid molecule.

[0112] In an embodiment of the present invention, the self-luminescence detection of the nucleotides to be sequenced is achieved by connecting luciferase to nucleotide derivatives, thereby eliminating the need for an additional excitation light source. In a specific embodiment, the connection between luciferase and nucleotides is achieved by specific binding of a label on the luciferase to a corresponding label on the nucleotide derivative. In a specific embodiment, the first nucleotide is connected to the first luciferase, the second nucleotide is connected to the second luciferase, the third nucleotide is connected to the first luciferase and the second luciferase, and the fourth nucleotide is not connected to any luciferase. The corresponding substrates of the two luciferases are then introduced to detect the luminescence signals of the four bases; when the substrate of the first luciferase is introduced, the first nucleotide and the third nucleotide emit light, when the substrate of the second luciferase is introduced, the second nucleotide and the third nucleotide emit light; and when the substrate of the first luciferase and the substrate of the second luciferase are introduced, the fourth nucleotide does not emit light. Therefore, base identification can be performed based on the luminescence of the four nucleotides.

[0113] Furthermore, the method for sequencing a nucleic acid molecule according to the present invention comprises:

[0114] (1) providing a nucleic acid molecule to be sequenced attached to a support, or attaching the nucleic acid molecule to be sequenced to a support;

[0115] (2) adding a primer for initiating nucleotide polymerization reaction, a polymerase for carrying out nucleotide polymerization reaction, and four compounds to form a reaction system containing a solution phase and a solid phase; wherein the four compounds are derivatives of nucleotides A, (T / U), C, and G, respectively, and have base complementary pairing capabilities; and the hydroxyl group (-OH) at the 3' position of the ribose or deoxyribose of the four compounds is protected by a protecting group; and,

[0116] The first compound is linked to a first molecular label,

[0117] The second compound is linked to a second molecular label,

[0118] The third compound is connected to the first molecular marker and the second molecular marker, or part of the third compound is connected to the first molecular marker and another part of the third compound is connected to the second molecular marker, and the fourth compound is not connected to the molecular marker;

[0119] (3) annealing the primer to the nucleic acid molecule to be sequenced, wherein the primer serves as the initial growing nucleic acid chain and forms a duplex connected to the support together with the nucleic acid molecule to be sequenced;

[0120] (4) performing a nucleotide polymerization reaction using a polymerase under conditions that allow the polymerase to perform a nucleotide polymerization reaction, thereby incorporating one of the four compounds into the 3′ end of the growing nucleic acid chain;

[0121] (5) removing the solution phase of the reaction system of the previous step, retaining the duplex connected to the support, and adding two different luciferases to carry out a binding reaction, wherein the two luciferases can specifically bind to the first molecular marker and the second molecular marker respectively;

[0122] (6) Remove unbound luciferase with elution buffer;

[0123] (7) adding a first luciferase substrate and detecting a first fluorescent signal;

[0124] (8) removing the solution from the previous reaction;

[0125] (9) adding a second luciferase substrate and detecting a second fluorescent signal;

[0126] (10) removing the solution from the previous reaction;

[0127] (11) removing the protective groups of the incorporated nucleotides and the molecular labels;

[0128] (12) optionally removing the solution from the previous reaction;

[0129] (13) Optionally repeat steps (3)-(12) or (4)-(11) one or more times to obtain sequence information of the nucleic acid molecule.

[0130] The present invention combines wild-type / mutant Gaussia luciferase (Gaussia Luciferase) and wild-type / mutant Pleuromammamma xiphias luciferase (Pleuromamma xiphias, AB716975).By screening the luminescence of different coelenterazine derivatives and Gaussia luciferase and Pleuromammamma xiphias luciferase, a dual-luciferase reporter gene detection system with low cross-interference is obtained.Relative to the combination of other dual-luciferases, the dual-luciferase combination reaction conditions are simple, only coelenterazine and oxygen are required, and operation is simpler. DETAILED DESCRIPTION

[0131] The present invention discloses a dual-luciferase reporter gene detection system and its application. Those skilled in the art can refer to the contents of this article and appropriately improve the process parameters for implementation. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0132] Definitions and General Terms

[0133] In the description of the present invention, unless otherwise specified, "plurality" means two or more.

[0134] The present invention will list the literature corresponding to the specific content of the invention in detail, and the examples are accompanied by diagrams of structural formulas and chemical formulas. The present invention is intended to cover all options, variations and equivalents that may be included in the existing invention field as defined by the claims. Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which can be applied to the practice of the present invention. The present invention is in no way limited to the description of methods and materials. There are many documents and similar materials that differ or conflict with the present application, including but not limited to the definition of terms, the usage of terms, the technology described, or the scope controlled by the present application.

[0135] The following definitions apply to the present invention unless otherwise indicated. For purposes of the present invention, the chemical elements are defined according to the Periodic Table of the Elements, CAS version, and the Chemical Handbook, 75th Ed, 1994. In addition, general principles of organic chemistry are described in "Organic Chemistry," by Thomas Sorrell, University Science Books, Sausalito: 1999, and "March's Advanced Organic Chemistry," by Michael B. Smith and Jerry March, John Wiley & Sons, New York: 2007, all of which are incorporated herein by reference.

[0136] The term "comprising" is an open expression, that is, including the contents specified in the present invention, but not excluding other contents.

[0137] Compounds as described herein may optionally be substituted with one or more substituents, as described in the general formulae of the present invention, or as described in the specific examples, subclasses, and classes of compounds encompassed by the present invention. It should be understood that the term "optionally substituted" is used interchangeably with the term "substituted or unsubstituted." In general, the term "optionally," whether preceded by the term "substituted," indicates that one or more hydrogen atoms in a given structure are replaced with the specified substituent. Unless otherwise indicated, an optionally substituted group may have a substituent at each substitutable position of the group. When more than one position in a given formula can be substituted with one or more substituents selected from the specified group, the substituents may be the same or different at each position.The substituents may be, but are not limited to, hydrogen, F, Cl, Br, I, nitro, cyano, oxo (=O), hydroxy, alkyl, hydroxyalkyl, alkylamino, aminoalkyl, haloalkoxy, cycloalkyl, amino, aryl, heterocyclyl, heteroaryl, alkenyl, alkynyl, cycloalkyloxy, alkoxy, alkoxyalkyl, haloalkyl, -COOH, -alkylene-C (=O) O-alkyl, -alkylene-S (=O) 2 -alkyl, -alkylene-S (=O) 2 -amino, -S (=O) 2 -alkyl, -S (=O) 2 -amino, -S (=O) 2 OH, -O-alkylene-C (=O) O-alkyl, -O-alkylene-S (=O) 2 -alkyl -alkyl, -O-alkylene-S(=O)2-amino, -O-alkylene-S(=O)2OH, -C(=O)NH2, -C(=O)NH-alkyl, -C(=O)N(alkyl)-alkyl, -C(=O)NHS(=O)2-alkyl, -C(=O)NHS(=O)2-amino, -C(=O)NHS(=O)2OH, -N(haloalkyl)-alkyl, -N(alkyl)-S(=O)2-alkyl, -NHS(=O)2-alkyl, -NHS(=O)2-haloalkyl, -N(alkyl)S(=O)2-haloalkyl, -N(alkyl)S(=O)2-alkylamino, -NHC(=O)-alkyl, -NHC(=O)-haloalkyl, -N(alkyl)C(=O)-haloalkyl, -N(alkyl)C(=O)-alkylamino, -N(alkyl)C(=O)O-alkyl, -NHC(=O)O-alkyl, -NHC(=O)O-haloalkyl, -N(alkyl)C(=O)O-haloalkyl, -N(alkyl)C(=O)O-aminoalkyl, -NHC(=O)-NH2, -NHC(=O)NH-(alkyl), -NHC(=O)NH(haloalkyl), -NHC(=O)N(alkyl)-alkyl, -OC(=O)-alkyl, -OC(=O)-amino, -OC(=O)-alkylamino, -OC(=O)-aminoalkyl, -OC(=O) )-alkoxy, -C(=O)N(alkyl)S(=O)2-alkyl, -C(=O)N(alkyl)S(=O)2-amino, -C(=O)NH-S(=O)2OH, -C(=NH)NH2, -C(=NH)NH-alkyl, -C(=NH)N(alkyl)-alkyl, -C(=N-alkyl)-NH2, -C(=O)NH-alkylene-S(=O)2OH, -C(=O)NHC(=O)OH, -C(=O)NHC(=O)O-alkyl, -C(=O)N(alkyl)C(=O)O-alkyl, -C(=O)NH-alkylene-C(=O)OH and -C(=O)NH-alkylene-C(=O)O-alkyl, and the like.

[0138] As used herein, the term "alkyl" includes saturated linear or branched monovalent hydrocarbon groups of 1-20 carbon atoms, or 1-10 carbon atoms, or 1-6 carbon atoms, or 1-4 carbon atoms, or 1-3 carbon atoms, or 1-2 carbon atoms, wherein the alkyl group may be independently optionally substituted with one or more substituents described herein. Further examples of alkyl groups include, but are not limited to, methyl (Me, -CH3), ethyl (Et, -CH2CH3), n-propyl (n-Pr, -CH2CH2CH3), isopropyl (i-Pr, -CH(CH3)2), n-butyl (n-Bu, -CH2CH2CH2CH3), isobutyl (i-Bu, -CH2CH(CH3)2), sec-butyl (s-Bu, -CH(CH3)CH2CH3), tert-butyl (t- -Bu, -C(CH3)3), n-pentyl (-CH2CH2CH2CH2CH3), 2-pentyl (-CH(CH3)CH2CH2CH3), 3-pentyl (-CH(CH2CH3)2), 2-methyl-2-butyl (-C(CH3)2CH2CH3), 3-methyl-2-butyl (-CH(CH3)CH(CH3)2), 3-methyl-1-butyl (-CH2CH2CH(CH3)2), 2-methyl -1-butyl (-CH2CH(CH3)CH2CH3), n-hexyl (-CH2CH2CH2CH2CH2CH3), 2-hexyl (-CH(CH3)CH2CH2CH2CH3), 3-hexyl (-CH(CH2CH3)(CH2CH2CH3)), 2-methyl-2-pentyl (-C(CH3)2CH2CH2CH3), 3-methyl-2-pentyl (-CH(CH3)CH(CH3)CH2C H3), 4-methyl-2-pentyl (-CH(CH3)CH2CH(CH3)2), 3-methyl-3-pentyl (-C(CH3)(CH2CH3)2), 2-methyl-3-pentyl (-CH(CH2CH3)CH(CH3)2), 2,3-dimethyl-2-butyl (-C(CH3)2CH(CH3)2), 3,3-dimethyl-2-butyl (-CH(CH3)C(CH3)3), n-heptyl and n-octyl, etc. The term "alkyl" and its prefix "alkane" as used herein include both straight and branched saturated carbon chains.

[0139] The term "alkenyl" refers to a linear or branched monovalent hydrocarbon radical of 2 to 12 carbon atoms, or 2 to 8 carbon atoms, or 2 to 6 carbon atoms, or 2 to 4 carbon atoms, wherein at least one position is unsaturated, i.e., one carbon-carbon is sp 2Double bond, wherein the alkenyl group can be independently optionally substituted with one or more substituents described in the present invention, including groups with "trans", "normal" or "E" or "Z" orientations, specific examples of which include, but are not limited to, vinyl (-CH=CH2), allyl (-CH2CH=CH2) and butyl (-CH2CH2CH=CH2) and the like.

[0140] The term "alkynyl" refers to a linear or branched monovalent hydrocarbon group of 2-12 carbon atoms, or 2-8 carbon atoms, or 2-6 carbon atoms, or 2-4 carbon atoms, wherein at least one position is unsaturated, i.e., one carbon-carbon triple bond is sp, wherein the alkynyl group may be independently and optionally substituted with one or more substituents described herein, specific examples of which include, but are not limited to, ethynyl (-C≡CH) and propargyl (-CH2C≡CH).

[0141] The term "halogen" refers to F, Cl, Br or I.

[0142] As used herein, the term "unsaturated" means that the moiety contains one or more degrees of unsaturation.

[0143] The term "alkoxy" or "alkyloxy" as used herein refers to an alkyl group, as defined herein, attached to the rest of the compound molecule via an oxygen atom. In some embodiments, the alkoxy group is C 1-4 Alkoxy groups; examples thereof include, but are not limited to, methoxy, ethoxy, propoxy, and butoxy, etc., and the alkoxy groups may be independently unsubstituted or substituted with one or more substituents described herein.

[0144] The terms "haloalkyl", "haloalkenyl" and "haloalkyloxy" refer to alkyl, alkenyl or alkyloxy groups that may be substituted with one or more halogen atoms. In some embodiments, the haloalkyl group is a haloC 1-6 In other embodiments, the haloalkyl group is a haloalkyl group. 1-3 In some embodiments, the haloalkyloxy or haloalkoxy group is a haloalkyl group. 1-6 Alkyloxy or halogenated C 1-6 In other embodiments, the haloalkyloxy or haloalkoxy group is a haloalkyloxy group. 1-3 Alkyloxy or halogenated C 1-3 Alkoxy. Examples include, but are not limited to, trifluoromethyl, 2-chloro-vinyl, 2,2-difluoroethyl, and trifluoromethoxy. The "haloalkyl," "haloalkenyl," and "haloalkyloxy" groups may be independently and optionally substituted with one or more substituents described herein.

[0145] The term "cycloalkyl" or "cycloalkane" refers to a monovalent or multivalent saturated monocyclic, bicyclic, or tricyclic carbon ring system containing 3-12 carbon atoms, but never containing aromatic rings. In one embodiment, the cycloalkyl group contains 3-12 carbon atoms; in another embodiment, the cycloalkyl group contains 3-8 carbon atoms; and in yet another embodiment, the cycloalkyl group contains 3-6 carbon atoms. Examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. The cycloalkyl groups may independently be unsubstituted or substituted with one or more substituents described herein.

[0146] The terms "heterocyclyl" and "heterocycle" are used interchangeably herein and refer to a saturated or partially unsaturated monocyclic, bicyclic, or tricyclic ring containing 3 to 12 ring atoms, never aromatic, wherein at least one ring atom is a heteroatom. Unless otherwise specified, a heterocyclyl group may be a carbon group or a nitrogen group, and heteroatoms have the meanings set forth herein. Examples of heterocyclic groups include, but are not limited to, oxiranyl, azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, 2-pyrrolinyl, 3-pyrrolinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, 1,3-dioxolane, dithiolanyl, tetrahydropyranyl, dihydropyranyl, 2H-pyranyl, 4H-pyranyl, tetrahydrothiopyranyl, piperidinyl, morpholinyl, thiomorpholinyl, piperazinyl, dioxanyl, dithianyl, thioxanyl, homopiperazinyl, homopiperidinyl, oxepanyl, thiepanyl, oxazepine Base, diazepine thiazolinone Examples of heterocyclic groups in which the -CH2- group is replaced by -C(=O)- include, but are not limited to, 2-oxopyrrolidinyl, oxo-1,3-thiazolidinyl, 2-piperidonyl, 3,5-dioxopiperidinyl, and pyrimidinedione. Examples of heterocyclic groups in which the sulfur atom is oxidized include, but are not limited to, sulfolane and 1,1-dioxothiomorpholinyl. The heterocyclic groups may be optionally substituted with one or more substituents described herein.

[0147] The term "heteroaryl" refers to monocyclic, bicyclic, and tricyclic ring systems containing 5-12 ring atoms, or 5-10 ring atoms, or 5-6 ring atoms, wherein at least one ring system is aromatic and at least one ring system contains one or more heteroatoms, wherein each ring contains 5-7 ring atoms and has one or more points of attachment to the rest of the molecule. The term "heteroaryl" can be used interchangeably with the terms "heteroaromatic ring" or "heteroaromatic compound". The heteroaryl group is optionally substituted with one or more substituents described herein. In one embodiment, the 5-10 heteroaryl group contains 1, 2, 3, or 4 heteroatoms independently selected from O, S, and N, wherein the nitrogen atom can be further oxidized.

[0148] Examples of heteroaryl groups include, but are not limited to, furanyl, imidazolyl (e.g., N-imidazolyl, 2-imidazolyl, 4-imidazolyl, 5-imidazolyl), isoxazolyl, oxazolyl (e.g., 2-oxazolyl, 4-oxazolyl, 5-oxazolyl), pyrrolyl (e.g., N-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl), pyridinyl, pyrimidinyl (e.g., 2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl), pyridazinyl, thiazole 1,2,3-oxadiazolyl, 1,2,5-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,3-triazolyl, 1,2,3-thiodiazolyl, 1,3,4-thiodiazolyl, 1,2,5-thiodiazolyl, 1,2,3 ... oxadiazole, pyrazinyl, 1,3,5-triazinyl; also include the following bicyclic rings, but are in no way limited to these bicyclic rings: benzimidazolyl, benzofuranyl, benzothiophenyl, indolyl (such as 2-indolyl), purinyl, quinolyl (such as 2-quinolyl, 3-quinolyl, 4-quinolyl), 1,2,3,4-tetrahydroisoquinolyl, 1,3-benzodioxolyl, indolinyl, isoquinolyl (such as 1-isoquinolyl), [1,2,4]triazolo[4,3-b]pyridazinyl, [1,2,4]triazolo[1,5-a]pyrimidinyl and [1,2,4]triazolo[1,5-a]pyridinyl, and the like.

[0149] The term "aminoalkyl" includes C 1-10 In some embodiments, aminoalkyl is a C substituted with one or more amino groups. 1-6Alkyl groups, such as aminomethyl, aminoethyl, aminopropyl, aminobutyl and aminohexyl, can be optionally substituted with one or more substituents described herein.

[0150] The term "hydroxyalkyl" refers to an alkyl group substituted with one or more hydroxy groups, wherein the alkyl group has the meaning described herein. Examples include, but are not limited to, hydroxymethyl, hydroxyethyl, and 1,2-dihydroxyethyl.

[0151] As described in the present invention, a substituent is connected to the central ring by a bond to form a ring system, which means that the substituent can be substituted at any substitutable position on the ring. For example, formula (a) represents a substituent R o Any possible substitution position on the E ring can be monosubstituted or polysubstituted.

[0152]

[0153] In addition, it should be noted that, unless otherwise explicitly stated, the descriptions used throughout this document, “each ... and ... are independently,” “... and ... are each independently,” and “... and ... are respectively independently,” are interchangeable and should be understood in a broad sense. They may mean that in different groups, the specific options expressed by the same symbols do not affect each other, or that in the same group, the specific options expressed by the same symbols do not affect each other.

[0154] Unless otherwise indicated, the structural formulas described herein include all isomeric forms (e.g., enantiomers, diastereomers, geometric isomers, or conformational isomers): for example, R and S configurations containing asymmetric centers, (Z) and (E) isomers of double bonds, and (Z) and (E) conformational isomers. Therefore, individual stereochemical isomers of the compounds of the present invention, or mixtures of such enantiomers, diastereomers, geometric isomers, or conformational isomers thereof, are within the scope of the present invention.

[0155] Unless otherwise indicated, the structural formulas and compounds described herein include all isomeric forms (e.g., enantiomers, diastereomers, geometric isomers, or conformers), salts, N-oxides, hydrates, or solvates thereof. Therefore, individual stereochemical isomers, enantiomers, diastereomers, geometric isomers, conformers, salts, N-oxides, hydrates, and solvates of the compounds of the present invention are also within the scope of the present invention. Furthermore, unless otherwise indicated, the structural formulas of the compounds described herein include isotopically enriched forms of one or more different atoms.

[0156] The definitions and conventions of stereochemistry used herein are generally those of S.P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., "Stereochemistry of Organic Compounds", John Wiley & Sons, Inc., New York, 1994. The compounds of the present invention may contain asymmetric centers or chiral centers and therefore exist as different stereoisomers. All stereoisomeric forms of the compounds of the present invention, including but not limited to diastereomers, enantiomers, atropisomers, and mixtures thereof, such as racemic mixtures, form part of the present invention. Many organic compounds exist in optically active forms, i.e., they have the ability to rotate the plane of plane-polarized light. When describing an optically active compound, the prefix D, L or R, S is used to indicate the absolute configuration of the molecule about its chiral center. The prefixes d, l, or (+), (-) are used to designate the sign of rotation of plane-polarized light in a compound. (-) or l means the compound is levorotatory, and the prefix (+) or d means the compound is dextrorotatory. These stereoisomers have the same chemical structure, but their stereostructures are different. Specific stereoisomers can be enantiomers, and a mixture of isomers is often called an enantiomeric mixture. A 50:50 mixture of enantiomers is called a racemic mixture or racemate, which may result in a lack of stereoselectivity or stereospecificity during chemical reactions. The terms "racemic mixture" and "racemate" refer to an equimolar mixture of two enantiomers that lacks optical activity.

[0157] The term "tautomer" or "tautomeric form" refers to structural isomers of different energies that are interconvertible via a low energy barrier. For example, proton tautomers (i.e., prototropic tautomers) include interconversions via proton migration, such as keto-enol and imine-enamine isomerizations. Valence tautomers include interconversions by reorganization of bonding electrons.

[0158] The "hydrate" of the present invention refers to an association compound formed when the solvent molecule is water.

[0159] The "solvate" of the present invention refers to an association formed between one or more solvent molecules and the compound of the present invention. Solvents that form solvates include, but are not limited to, water, isopropanol, ethanol, methanol, dimethyl sulfoxide, ethyl acetate, acetic acid, and aminoethanol.

[0160] "Esters" herein refer to esters of compounds of formula (I) containing hydroxy groups that are hydrolyzable in vivo. Such esters are, for example, pharmaceutically acceptable esters that hydrolyze in the human or animal body to produce the parent alcohol. Examples of in vivo hydrolyzable esters of compounds of formula (I) containing hydroxy groups include, but are not limited to, phosphate, acetoxymethoxy, 2,2-dimethylpropionyloxymethoxy, alkanoyl, benzoyl, phenylacetyl, alkoxycarbonyl, dialkylcarbamoyl, and N-(dialkylaminoethyl)-N-alkylcarbamoyl groups.

[0161] The "nitrogen oxide" of the present invention refers to when a compound contains several amine functional groups, one or more nitrogen atoms can be oxidized to form an N-oxide. Special examples of N-oxides are N-oxides of tertiary amines or N-oxides of nitrogen atoms in nitrogen-containing heterocyclic rings. The corresponding amine can be treated with an oxidizing agent such as hydrogen peroxide or a peracid (e.g., peroxycarboxylic acid) to form an N-oxide (see Advanced Organic Chemistry, Wiley Interscience, 4th edition, Jerry March, pages). In particular, N-oxides can be prepared by the method of LW Deady (Syn. Comm. 1977, 7, 509-514), for example, in an inert solvent (e.g., dichloromethane), by reacting the amine compound with m-chloroperoxybenzoic acid (MCPBA).

[0162] Throughout this specification, reference to terms such as "one embodiment," "an embodiment of the present invention," "some embodiments," "illustrative embodiments," "examples," "specific examples," or "some examples" means that a specific feature, structure, material, or characteristic described in conjunction with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

[0163] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as understood by those of ordinary skill in the art. For definitions and terminology in this field, professionals are specifically referred to Current Protocols in Molecular Biology (Ausubel). The abbreviations for amino acid residues are the standard three-letter and / or one-letter codes used in the art to designate one of the 20 commonly used L-amino acids.

[0164] In the luciferase combination provided by the present invention, two luciferases are included, one of which is Gaussia luciferase and the other is Pleuromamma xiphias luciferase. Gaussia luciferase, also known as " Gaussia luciferase", is a protein with a molecular weight of 20kDa, which is derived from the bioluminescent enzyme of the animal Gaussia matrix (Gaussia princeps) of marine copepods. The Gaussia luciferase receptor molecule is very small and does not require the assistance of ATP. In the presence of oxygen molecules, it catalyzes the oxidation of coelenterazine to produce luminescence. Pleuromamma xiphias luciferase is a copepod luciferase, also known as sword-milk-spotted water flea luciferase, which can catalyze the oxidation of coelenterazine to produce luminescence in the presence of oxygen molecules. The present invention has screened the combination of luciferases, and the results show that, relative to other luciferase combinations, stronger fluorescence can be produced without cross-interference.

[0165] In the luciferase reporter gene detection system of the present invention, the luciferase can exist in the form of an encoding nucleic acid, an expression module, an expression vector, a recombinant strain, an expression product of the recombinant strain, or a purified expression product.

[0166] Taking the encoding nucleic acid as an example, the detection system described in the present invention includes a mixture of a nucleic acid encoding Gaussia luciferase and a nucleic acid encoding Pleuromammamma xiphias luciferase, or two independently existing nucleic acids encoding Gaussia luciferase and nucleic acids encoding Pleuromammamma xiphias luciferase, or a fusion fragment formed by a nucleic acid encoding Gaussia luciferase and a nucleic acid encoding Pleuromammamma xiphias luciferase. The present invention is not limited to this.

[0167] Taking expression module as example, described expression module comprises promoter, encoding nucleic acid and terminator.Described nucleic acid encoding Gaussia luciferase and nucleic acid encoding Pleuromammamma xiphias luciferase are in the same expression module, also can be in two different expression modules.The promoter of two expression modules at the place of two encoding nucleic acids can be identical or different, and the present invention does not limit this.

[0168] By way of example, the expression vector includes a backbone vector and an encoding nucleic acid. The expression vector includes, but is not limited to, a plasmid vector or a viral vector. The nucleic acid encoding Gaussia luciferase and the nucleic acid encoding Pleuroma xiphias luciferase can be expressed in the same expression vector or in different expression vectors, which is not limited by the present invention. When the nucleic acids are expressed in different vectors, the backbone vectors can be the same or different.

[0169] The expression strain of the present invention is also referred to as a recombinant host, which can express Gaussia luciferase and / or Pleuromammamma xiphias luciferase, i.e., Gaussia luciferase and Pleuromammamma xiphias luciferase are expressed with the same host, or they can be expressed in different hosts, which is not limited by the present invention. The recombinant host includes a prokaryotic host or a eukaryotic host, which can be constructed by electroporation or by viral infection. It can be Escherichia coli, yeast, animal cell, or human cell, which is not limited by the present invention. Expressing two luciferases in two different hosts can adopt the same host cell or different host cells.

[0170] The expression product or purified expression product of the recombinant strain of the present invention is derived from the aforementioned expression strain or recombinant host, and may be a mixture of two luciferase-expressing strains, or a product of two luciferase-expressing strains.

[0171] In the present invention, "reporter gene" is a molecular biology concept, referring to a type of gene that is expressed in cells, tissues / organs, or individuals under specific conditions and causes them to produce easily detectable traits that would not otherwise be produced by the experimental material. In other words, it is a gene that encodes a detectable protein or enzyme. As a reporter gene, it must meet the following conditions in genetic selection and screening detection: 1. It must have been cloned and its entire sequence determined; 2. The expression product must not be native to the recipient cell, i.e., there is no background, and no similar endogenous expression products are present in the transfected cells; 3. Its expression product can be quantitatively measured. When used, it includes but is not limited to the following methods: fusing the reporter gene with a gene expression regulatory sequence to form a chimeric gene, or fusing it with other target genes to express the nucleic acid under the control of the regulatory sequence, thereby using its expression product to detect the expression regulation of the target gene and study the nucleic acid.

[0172] The luciferase reporter gene detection system described herein, also known as a dual-luciferase luminescence system, comprises the luciferase composition described herein and a substrate. The luciferase composition and substrate exist independently and, when mixed in the detection system, emit light. The present invention screens the substrates to obtain a luciferase reporter gene detection system that exhibits higher fluorescence intensity, thereby improving detection sensitivity.

[0173] The detection reagent includes a substance capable of causing the analyte to overexpress Gaussia luciferase and Pleuromammamma xiphias luciferase. The substance capable of causing the analyte to overexpress Gaussia luciferase and Pleuromammamma xiphias luciferase includes, but is not limited to, a plasmid vector or viral vector containing a nucleic acid encoding Gaussia luciferase and / or a nucleic acid encoding Pleuromammamma xiphias luciferase.

[0174] As used herein, the term "polynucleotide" refers to deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or an analog thereof. A polynucleotide can be single-stranded, double-stranded, or contain both single-stranded and double-stranded sequences. A polynucleotide molecule can be derived from a double-stranded DNA (dsDNA) form (e.g., genomic DNA, PCR and amplification products, etc.), or can be derived from a single-stranded form of DNA (ssDNA) or RNA and converted to a dsDNA form, and vice versa. The exact sequence of a polynucleotide molecule can be known or unknown. The following are illustrative examples of polynucleotides: a gene or gene fragment (e.g., a probe, primer, EST or SAGE tag), genomic DNA, a genomic DNA fragment, an exon, an intron, a messenger RNA (mRNA), a transfer RNA, a ribosomal RNA, a ribozyme, a cDNA, a recombinant polynucleotide, a synthetic polynucleotide, a branched polynucleotide, a plasmid, a vector, an isolated DNA of any sequence, an isolated RNA of any sequence, a nucleic acid probe, a primer, or an amplified copy of any of the above sequences.

[0175] A polynucleotide may comprise nucleotides or nucleotide analogs. Nucleotides generally contain a sugar (such as ribose or deoxyribose), a base, and at least one phosphate group. Nucleotides include deoxyribonucleotides, modified deoxyribonucleotides, ribonucleotides, modified ribonucleotides, modified phosphate sugar backbone nucleotides, and mixtures thereof. Examples of nucleotides include, for example, adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), thymidine monophosphate (TMP), thymidine diphosphate (TDP), thymidine triphosphate (TTP), cytidine monophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate (CTP), guanosine monophosphate (GMP), guanosine diphosphate (GDP), Guanosine triphosphate (GTP), uridine monophosphate (UMP), uridine diphosphate (UDP), uridine triphosphate (UTP), deoxyadenosine monophosphate (dAMP), deoxyadenosine diphosphate (dADP), deoxyadenosine triphosphate (dATP), deoxythymidine monophosphate (dTMP), deoxythymidine diphosphate (dTDP), deoxythymidine triphosphate (dTTP), deoxycytidine monophosphate (dCMP), deoxycytidine diphosphate (dCDP), deoxycytidine triphosphate (dCTP), deoxyguanosine monophosphate (dGMP), deoxyguanosine diphosphate (dGDP), deoxyguanosine triphosphate (dGTP), deoxyuridine monophosphate (BUMP), deoxyuridine diphosphate (dUDP), and deoxyuridine triphosphate (dUTP).

[0176] Nucleotide analogs comprising modified bases can also be used in the methods described herein. Exemplary modified bases that can be included in polynucleotides, whether having a native backbone or an analogous structure, include, for example, inosine, xanthine (xathanine), hypoxathanine (hypoxathanine), isocytosine, isoguanine, 2-aminopurine, 5-methylcytosine, 5-hydroxymethylcytosine, 2-aminoadenine, 6-methyladenine, 6-methylguanine, 2-propylguanine, 2-propyladenine, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 15-halouracil, 15-halocytosine, 5 ...aminopurine, 5-hydroxymethylcytosine, 2-aminopurine, 6-methyladenine, 6-methylguanine, 2-propylguanine, 2-propyladenine, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 15-halouracil, 15-halocytosine, 5-aminopurine, 5-aminopurine, 5-methylcytosine, 5-aminopurine, 5-aminopurine, 5-aminopurine, 5-aminopurine, 5-aminopurine, 5-aminopurine, 5-aminopurine, 5-aminopurine, 5-aminopurine, 5 -propynyluracil, 5-propynylcytosine, 6-azouracil, 6-azocytosine, 6-azothymine, 5-uracil, 4-thiouracil, 8-haloadenine or guanine, 8-aminoadenine or guanine, 8-thioadenine or guanine, 8-sulfanyladenine or guanine, 8-hydroxyadenine or guanine, 5-halogen-substituted uracil or cytosine, 7-methylguanine, 7-methyladenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, and the like.

[0177] Generally speaking, nucleotides include nucleotides A, C, G, T or U. As used herein, the term "nucleotide A" refers to a nucleotide containing adenine (A) or a modification or analog thereof, such as ATP, dATP. "Nucleotide G" refers to a nucleotide containing guanine (G) or a modification or analog thereof, such as GTP, dGTP. "Nucleotide C" refers to a nucleotide containing cytosine (C) or a modification or analog thereof, such as CTP, dCTP. "Nucleotide T" refers to a nucleotide containing thymine (T) or a modification or analog thereof, such as TTP, dTTP. "Nucleotide U" refers to a nucleotide containing uracil (U) or a modification or analog thereof, such as UTP, dUTP.

[0178] Nucleotide labeling

[0179] The present invention relates to labeling nucleotides with different labels, alone or in combination, so that different luciferases can be connected to the nucleotides. As used herein, the molecular marker for labeling nucleotides and the label specifically bound thereto can be any molecular pairing that can specifically bind to each other. The specific binding between the pairing members realizes the connection between the nucleotide and the luciferase. Exemplary pairing members include, but are not limited to: (a) haptens or antigenic compounds combined with corresponding antibodies or their binding portions or fragments, such as digoxin-digoxin antibodies, N3G-N3G antibodies, FITC-FITC antibodies; (b) nucleic acid aptamers and proteins; (c) non-immune binding pairs (e.g., biotin-avidin, biotin-streptavidin, biotin-neutravidin); (d) hormone-hormone binding protein; (e) receptor-receptor agonist or antagonist; (f) homolectin-carbohydrate; (g) enzyme-enzyme cofactor and (h) enzyme-enzyme inhibitor;.

[0180] In a specific embodiment, the first molecular marker and the second molecular marker are small molecule markers selected from biotin, digoxigenin, N3G or FITC. The two luciferases can specifically bind to the first molecular marker and the second molecular marker, respectively. For example, in a specific embodiment, the first molecular marker is biotin, then the first luciferase can be a luciferase labeled with streptavidin; the second molecular marker is digoxigenin, then the second luciferase can be a luciferase different from the first luciferase labeled with a digoxigenin antibody. Preferably, the first luciferase is the Gaussia luciferase or a mutant thereof described in the present invention, and the second luciferase is the Pleuromammamma xiphias luciferase or a mutant thereof described in the present invention.

[0181] As used herein, the expression "a first compound is linked to a first molecular marker" means that all of the first compounds are linked to the first molecular marker, or that some of the first compounds are linked to the first molecular marker but the rest of the first compounds are not linked to the molecular marker. Similarly, the expression "a second compound is linked to a second molecular marker" means that all of the second compounds are linked to the second molecular marker, or that some of the second compounds are linked to the second molecular marker but the rest of the second compounds are not linked to the molecular marker. The expression "a third compound is linked to a first molecular marker and a second molecular marker" means that all of the third compounds are linked to the first molecular marker and the second molecular marker, or that some of the third compounds are linked to the first molecular marker and the second molecular marker but the rest of the third compounds are not linked to the molecular marker.

[0182] Polynucleotide sequencing

[0183] Preferably, the dual luciferase reporter gene detection system of the present invention is suitable for synthesis sequencing. As used herein, synthesis sequencing is a variety of synthesis sequencing methods well known in the art. Basically, synthesis sequencing involves first hybridizing the nucleic acid molecule to be sequenced with a sequencing primer, and then, in the presence of a polymerase, polymerizing nucleotides or nucleotide analogs as described herein at the 3' end of the sequencing primer using the nucleic acid molecule to be sequenced as a template. After polymerization, the type of nucleotide molecule is identified by detecting the fluorescent signal emitted by the luciferase. After removing the luciferase from the labeled nucleotide, the next polymerization sequencing cycle is performed.

[0184] Methods for determining the sequence of a target polynucleotide can be performed by denaturing the target polynucleotide sequence, contacting the target polynucleotide with different nucleotides to form complements of the target nucleotides, and detecting incorporation of the nucleotides. The method utilizes polymerization, allowing a polymerase to extend the complementary chain by incorporating the correct nucleotides complementary to the target. The polymerization reaction also requires specific primers to initiate polymerization.

[0185] For each round of reaction, the incorporation of the nucleotide is carried out by a polymerase, and the incorporation event is subsequently determined. There are many different polymerases, and it is easy for a person of ordinary skill in the art to determine the most suitable polymerase. Preferred enzymes include DNA polymerase I, Klenow fragment, DNA polymerase III, T4 or T7 DNA polymerase, Taq polymerase, or Vent polymerase. Polymerases engineered to have specific properties can also be used.

[0186] The sequencing method is preferably performed on target polynucleotides arranged on a solid support. A plurality of target polynucleotides can be immobilized on the solid support via a linker molecule, or can be attached to particles such as microspheres, which can also be attached to a solid support material.

[0187] The polynucleotides can be attached to the solid support by a variety of methods, including the use of biotin-streptavidin interactions. Methods for immobilizing polynucleotides on solid supports are well known in the art and include lithographic techniques and spotting each polynucleotide at a specific location on a solid support. Suitable solid supports are well known in the art and include glass slides and beads, ceramic and silicon surfaces, and plastic materials.

[0188] The support is typically a plane, although microbeads (microspheres) may also be used and the latter may also be attached to other solid supports by known methods. The microspheres may be of any suitable size, typically 10-100 nanometers in diameter. In a preferred embodiment, the polynucleotides are directly attached to a plane, preferably a flat glass surface. Connection is preferably carried out in the form of a covalent bond. The array used is preferably a single molecule array, which includes polynucleotides located in a unique optically resolvable region, such as described in International Application No. WO 00 / 06770.

[0189] The necessary conditions for polymerization are well known to those skilled in the art. In order to carry out the polymerase reaction, it is usually necessary to first anneal a primer sequence to the target polynucleotide. The primer sequence is recognized by the polymerase and serves as a starting point for subsequent extension of the complementary chain. The primer sequence can be added as an independent component relative to the target polynucleotide. In addition, the primer and target polynucleotide can each be part of a single-stranded molecule, with the primer portion and a portion of the target forming an intramolecular duplex, i.e., a hairpin loop structure. The structure can be fixed to the solid support at any site on the molecule. Other conditions necessary for carrying out the polymerase reaction are well known to those skilled in the art, and include temperature, pH, and buffer composition.

[0190] Subsequently, the labeled nucleotides or nucleotide analogs of the present invention are contacted with the target polynucleotide to enable polymerization. The nucleotides or nucleotide analogs can be added sequentially, i.e., each type of nucleotide (A, C, G or T / U) is added separately, or multiple types of nucleotides (A, C, G or T / U) are added simultaneously.

[0191] The polymerization step is allowed to proceed for a time sufficient to incorporate one nucleotide.

[0192] Unincorporated nucleotides are then removed, for example, by removing the solution phase of the reaction from the previous step, leaving the duplex attached to the support.

[0193] Subsequently, two luciferases containing different luciferases can be added to carry out a binding reaction. The two luciferases can each specifically bind to the molecular label on the nucleotide, thereby achieving the connection between the luciferase and the incorporated nucleotide. The incorporated nucleotide is then identified by adding the corresponding luciferase substrate and detecting the fluorescent signal.

[0194] In one specific embodiment, four deoxyribonucleotide analogs are labeled with different small molecule markers, biotin (abbreviated as B) and digoxigenin (abbreviated as D), respectively. For example, nucleotide A is labeled with B, nucleotide C is labeled with B and D, nucleotide T is labeled with D, and nucleotide G is unlabeled. The 3'-terminal hydroxyl groups of the four deoxyribonucleotide analogs labeled with different small molecules are blocked to ensure that only one deoxyribonucleotide is bound in each sequencing reaction. During the sequencing reaction, the four labeled deoxyribonucleotide analogs and sequencing polymerase mixture are first introduced. Under the action of the polymerase, according to the principle of base complementary pairing, one deoxyribonucleotide analog is incorporated into the 3' end of the growing nucleic acid chain. By removing the solution phase of the reaction system from the previous step and retaining the duplex attached to the support, unbound deoxyribonucleotide analogs can be removed. Then, two luciferases containing different luciferases are added. The first luciferase is labeled with streptavidin and binds to nucleotide A or nucleotide C labeled with small molecule B. The second luciferase is labeled with digoxigenin antibody and binds to nucleotide C or nucleotide T labeled with small molecule D. After removing unbound luciferase using elution buffer, the substrate of the first luciferase is added, the nucleotide linked to the first luciferase emits light, and the signal is detected using a detector. The substrate of the second luciferase is added, the nucleotide linked to the second luciferase emits light, and the signal is detected using a detector. The luminescence conditions shown in the table below are obtained, and base recognition can be performed (1 indicates that a fluorescent signal is detected, and 0 indicates that no fluorescent signal is detected).

[0195] First test Second test A10C11G00T01

[0196] In a specific embodiment, the connection of two luciferases (Gaussia luciferase or its mutant, and Pleuroma xiphias luciferase or its mutant) containing different luciferases to labeled nucleotides and signal detection can be carried out separately. First, a first luciferase is added, which is labeled with streptavidin and combined with nucleotide A or nucleotide C labeled with small molecule B. After removing the unbound first luciferase with elution buffer, the substrate of the first luciferase is added, the nucleotide connected with the first luciferase emits light, and a detector is used to detect the signal. After removing the reaction solution, a second luciferase labeled with a digoxigenin antibody is added, which is combined with nucleotide C or nucleotide T labeled with small molecule D, and then the unbound second luciferase is removed with elution buffer, the substrate of the second luciferase is added, the base connected with the second luciferase emits light, and a detector is used to detect the signal, thereby deriving the luminescence shown in the above table, and the base can be identified.

[0197] Detection of fluorescence signals

[0198] Methods for detecting fluorescent signals are well known in the art. For example, this can be achieved by a device that detects the wavelength of fluorescence. Such devices are well known in the art. For example, such a device can be a confocal scanning microscope that uses a laser to scan the surface of a solid support so as to image the fluorophores directly bound to the nucleic acid molecules being sequenced. In addition, each signal generated can be observed, for example, using a sensitive 2-D detector, such as a charge coupled device (CCD). Other techniques such as scanning near-field optical microscopy (SNOM) can also be used.

[0199] The reagents and instruments used in the present invention can be purchased commercially. The Gaussia luciferase (GenBank: AY015993.1) used in the examples was produced using an E. coli expression system in the pET28a vector. Pleuromamma xiphias luciferase (PMID: 23886588) was also produced using an E. coli expression system in the pET28a vector.

[0200] The present invention will be further described below with reference to the embodiments.

[0201] Example 1 Paired Luminescence Detection of Dual-Luciferase System (Wild Type)

[0202] Experimental purpose: By testing the luminescence intensity of wild-type Gaussia luciferase and wild-type Pleuromammamma xiphias luciferase with different substrates, a luminescence combination with high brightness and low cross-interference was screened.

[0203] Experimental steps:

[0204] (1) Wild-type Gaussia luciferase (WT-Gluc) has the following amino acid sequence:

[0205]

[0206] The wild-type Gaussia luciferase (WT-Gluc) gene has the nucleotide sequence shown below:

[0207]

[0208] Wild-type Pleuromamma xiphias luciferase (WT-Pxluc) has the amino acid sequence shown below:

[0209]

[0210] The wild-type Pleuromammamma xiphias luciferase (WT-Pxluc) has the following amino acid sequence after adding the signal peptide:

[0211]

[0212] The wild-type Pleuroma xiphias luciferase (WT-Pxluc) gene has the nucleotide sequence shown below:

[0213]

[0214] (2) Coelenterazine derivatives as substrates: The substrates f-CTZ, N0, N1, N3, N4, N5, N6, N7 and N8 were synthesized with reference to the method disclosed in patent application PCT / CN2018 / 107646.

[0215]

[0216] (3) According to conventional methods, such as the method disclosed in PCT / CN2017 / 082180, an expression vector (plasmid) containing the above-mentioned wild-type Gaussia luciferase gene and wild-type Pleuroma xiphias luciferase gene was constructed. The plasmid was transformed into Escherichia coli OrigamiB (DE3), plated, and a single colony was picked from the plate and cultured overnight at 37°C. The next day, it was diluted at a ratio of 1:100 and transferred to a fresh 3mL LB medium containing ampicillin resistance (100μg / mL), cultured at 37°C and 200rpm for 4 hours, and cooled on ice for 1 hour. The inducer IPTG was added at a final concentration of 1mM and induced overnight at 16°C.

[0217] The next day, the cells were harvested by centrifugation and resuspended in 500 μL of lysis buffer (50 mM Tris-HCl pH 8.0, 500 mM NaCl, 0.2% lysozyme, PMSF). Lysed at room temperature for 20 minutes, the cells were frozen and thawed three times in liquid nitrogen, and centrifuged at 18,000 g for 30 minutes. The supernatant was collected and purified using a centrifugal Ni column.

[0218] The protein eluted from the Ni column was dialyzed with dialysis buffer (25 mM Tris, pH 8.0, 250 mM NaCl) at 4°C overnight. TM Pierce TM The concentration of luciferase was accurately determined using the BCA Protein Assay Kit.

[0219] Dilute both luciferases to 1 μg / mL using a diluent (50 mM Tris-HCl pH 8.0, 100 mM NaCl, 0.1% (v / v) Tween-20). Add 10 μL of each to a black 96-well plate. Dilute the substrate to 100 μM using the same diluent, add 90 μL to the 96-well plate containing the luciferase (90 μL / well), and read the luminescence intensity using a microplate reader with a self-luminescence module.

[0220] Table 1 shows the test data for Gaussia luciferase and Pleuromamma xiphias luciferase against different substrates. Relative intensities are percentages relative to the activity of wild-type Pleuromamma xiphias luciferase and compound N0. The results show that the fluorescence intensity of Gaussia luciferase combined with the substrate f-CTZ reached the highest level, while that of Pleuromamma xiphias luciferase combined with the substrate N0 reached the highest level. This means that in this dual-luciferase reporter gene assay, detecting Gaussia luciferase with the substrate f-CTZ and detecting Pleuromamma xiphias luciferase with the substrate N0 represents the optimal luminescence combination, identified after screening, for high fluorescence brightness and low cross-interference.

[0221] Table 1 Luminescence intensity of various substrates and luciferase

[0222]

[0223]

[0224] Example 2 Paired Luminescence Detection of Dual-Luciferase System (Mutant Type)

[0225] Experimental purpose: By testing the luminescence intensity of different substrates on mutant Gaussia luciferase and mutant Pleuromammamma xiphias luciferase, a luminescence combination with high brightness and low cross-interference was screened.

[0226] Experimental steps:

[0227] (1) Mutant Gaussia luciferase E1-A3, G2-F11 and G2-F8:

[0228] a) E1-A3, based on SEQ ID NO: 1, with the following mutations: F26R, V29F, A32V, S33E, A36V, L40I, K66P, H79K, P84L, E102S, S103T, A104G, E110P, L124M, V138E;

[0229] The gene of the E1-A3 mutant has the nucleotide sequence shown below:

[0230]

[0231] b) G2-F11, based on SEQ ID NO: 1, with the following mutations: E24K, H79K, P84L, E102S, S103T, A104G, E110P, L124G, Q152R, Q163D, S170N, Q175E, K178T, A182M, G183N); G2-E1 (H79K, P84L, E102S, S103T, A104G, E110P, L124I, Q152H, Q163D, T167S, S170T, G174K, A182M, G183A);

[0232] The gene of the G2-F11 mutant has the nucleotide sequence shown below:

[0233]

[0234]

[0235] c) G2-F8, based on SEQ ID NO: 1, mutate the following sites: H79K, P84L, E102S, S103T, A104G, E110P, L124M, Q152H, Q163D, T167S, S170T, G174K, A182M, G183A.

[0236] The gene of the G2-F8 mutant has the nucleotide sequence shown below:

[0237]

[0238] Mutant Pleuromamma xiphias luciferase P26-95:

[0239] d) P26-95, based on SEQ ID NO: 3, mutate the following sites: G83A, G84P.

[0240] The gene of the P26-95 mutant has the nucleotide sequence shown below:

[0241]

[0242] According to the method shown in Example 1, expression vectors containing the above-mentioned Gaussia luciferase mutant gene and Pleuromammamma xiphias luciferase mutant gene were constructed, recombinant bacteria were constructed, the mutant luciferase was expressed, and the substrate and luciferase luminescence intensity were detected.

[0243] The results showed that the fluorescence intensity of Gaussia luciferase mutants E1-A3, G2-F11, and G2-F8 was the highest when combined with the substrate f-CTZ, while the fluorescence intensity of Pleuromamma xiphias luciferase mutant P26-95 was the highest when combined with the substrate N0. In other words, in this dual-luciferase reporter gene assay system, detecting Gaussia luciferase mutants E1-A3, G2-F11, or G2-F8 with the substrate f-CTZ and detecting Pleuromamma xiphias luciferase mutant P26-95 with the substrate N0 was the optimal luminescence combination obtained after screening, with high fluorescence brightness and low cross-interference.

[0244] Example 3 Use of the Dual Luciferase Reporter Gene Detection System

[0245] The expression plasmids with wild-type Gaussia luciferase (WT-Gluc) gene and wild-type Pleuromamma xiphias luciferase (WT-Pxluc) gene as reporter genes were constructed and transfected into 0.5x10 4 HEK293 cells were cultured in a 24-well plate in a cell culture incubator at 5% CO2 and 37°C overnight.

[0246] The next day, remove the culture medium from the plate / dish and gently wash the cells with ample PBS. Completely remove the PBS wash solution. Add 50 μL of 1x Passive Lysis Buffer (Promega) to lyse the cells. Transfer the cell suspension to a 1.5 mL centrifuge tube and thoroughly vortex for 30 seconds. The lysed cell suspension can be used for luminescence analysis. Alternatively, centrifuge at 100 g for 30 seconds and collect the supernatant for luminescence analysis.

[0247] 50 μL of cell suspension or supernatant was added to a black 96-well plate. Coelenterazine f-CTZ was diluted to 100 μM with diluent (50 mM Tris-HCl pH 8.0, 100 mM NaCl, 0.1% (v / v) Tween-20), and 50 μL was added to the 96-well plate containing the cell suspension or supernatant (50 μL / well). The luminescence intensity was read using a microplate reader with a self-luminescence module. After the measurement was completed, 1x stop & glo reagent (Promega) was added to stop the luminescence of f-CTZ, and the NO substrate diluted to 100 μM with the same diluent was added to the same system, and the luminescence intensity was read using a microplate reader with a self-luminescence module.

[0248] The results showed that the dual-luciferase reporter gene detection system can be used to detect luciferase expressed in cells.

[0249] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A dual luciferase reporter gene detection system, characterized in that: It comprises Gaussia luciferase, Pleuromammamma xiphias luciferase and a substrate, and the substrate is a compound represented by formula (I), or a stereoisomer, geometric isomer, tautomer, salt, nitrogen oxide, hydrate or solvate of the compound represented by formula (I): Wherein, R1 and R2 are independently H, D, F, Cl, Br, I, OH, NH2, NO2, CN, N3, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy, C 1-6 Hydroxyalkyl or C 1-6 Alkylamino; R3 is optionally substituted aryl, heterocyclyl or heteroaryl; a1 is 0 or any integer from 1 to 6, a2 is 0 or any integer from 1 to 6, b is 0 or 1, c is 0, 1 or 2.

2. The dual luciferase reporter gene detection system according to claim 1, characterized in that The Gaussia luciferase is any one of the following I) to III): 1), the amino acid sequence of the Gaussia luciferase is shown in SEQ ID NO: 1; II), a luciferase having at least 85% homology to the amino acid sequence of the Gaussia luciferase described in I) and having the same or similar function as the Gaussia luciferase described in I); III) A luciferase having the Gaussia luciferase activity as described in I) with its amino acid sequence modified, substituted, deleted or added with one or more amino acid residues.

3. The dual luciferase reporter gene detection system according to claim 2, characterized in that: The Gaussia luciferase is an optimized mutant. Compared with the amino acid sequence shown in SEQ ID NO: 1, the Gaussia luciferase mutant has at least one of the following mutation sites: positions 24, 26, 27, 29, 30, 31, 32, 33, 36, 37, 40, 66, 79, 84, 88, 102, 103, 104, 110, 123, 124, 138, 152, 163, 167, 170, 174, 175, 178, 182 and 183.

4. The dual luciferase reporter gene detection system according to claim 3, characterized in that The Gaussia luciferin mutant has at least one of the following mutations: 1) E at position 24 was mutated to K; 2) F at position 26 mutated to R or L; 3) N at position 27 was mutated to D; 4) V at position 29 mutated to F or L; 5) A at position 30 mutated to G or D; 6) V at position 31 was mutated to I; 7) A at position 32 was mutated to V; 8) S at position 33 is mutated to E, R, or K; 9) A at position 36 is mutated to V or I; 10) T at position 37 mutated to N or E; 11) L at position 40 is mutated to I or T; 12) K at position 66 is mutated to P, S, I, R, or N; 13) H at position 79 mutated to K; 14) P at position 84 is mutated to A, L, K or V; 15) K at position 88 mutated to R; 16) E at position 102 is mutated to D, A, S, K or N; 17) S at position 103 mutated to T; 18) A at position 104 was mutated to G; 19) E at position 110 is mutated to P, G, or A; 20) D at position 123 was mutated to N; 21) L at position 124 is mutated to M, G, or I; 22) V at position 138 mutated to E or D; 23) Q at position 152 is mutated to R or H; 24) Q at position 163 was mutated to D; 25) S at position 170 mutated to N or T; 26) G at position 174 was mutated to K; 27) Q at position 175 was mutated to E; 28) K at position 178 mutated to T; 29) A at position 182 was mutated to M; 30) The G at position 183 was mutated to N or A.

5. The dual luciferase reporter gene detection system according to claim 1, characterized in that The Pleuromamma xiphias luciferase is any one of the following i) to iii): i) The amino acid sequence of the Pleuromammamma xiphias luciferase is shown in SEQ ID NO: 3; ii) a luciferase having at least 85% homology to the amino acid sequence of the Pleuromammamma xiphias luciferase described in i) and having the same or similar function as the Pleuromammamma xiphias luciferase described in i); iii) A luciferase having the Pleuromammamma xiphias luciferase activity, wherein the amino acid sequence of the Pleuromammamma xiphias luciferase described in i) is modified, substituted, deleted or added with one or more amino acid residues.

6. The dual-luciferase reporter gene detection system according to claim 5, characterized in that: The Pleuromamma xiphias luciferase is an optimized mutant. Compared with the amino acid sequence shown in SEQ ID NO: 3, the Pleuromamma xiphias luciferase mutant has at least one of the following mutation sites: position 81, position 82, position 83 and position 84.

7. The dual-luciferase reporter gene detection system according to claim 6, characterized in that: The Pleuromamma xiphias luciferin mutant has at least one of the following mutations: (1) G at position 81 is mutated to L, P, Q, S, or T; (2) Q at position 82 is mutated to R, W, I, Y, A, L, F, V, P, E, or M; (3) G at position 83 is mutated to S, Q, R, W, T, A, or L; (4) The G at position 84 is mutated to F, R, S, C, Y, L, I, K, V, or P.

8. The dual luciferase reporter gene detection system according to claim 1, characterized in that: The compound represented by formula (I) may further include at least one of the following additional technical features: Said R1 is H; R2 is H, NH2, OH or C 1-6 Alkylamino; preferably H, NH2, 3-OH, 4-OH or dimethylamino; R3 is a substituted aryl group, preferably OH-C6H5 or F-C6H5, more preferably 4-OH-C6H5 or 4-F-C6H5; a1 is 0 or 1; a2 is 0 or 1; Preferably, when a1 is 0, a2 is 1; when a1 is 1, a2 is 0; or when a1 is 0, a2 is 0; b is 1; The c is 1.

9. The dual-luciferase reporter gene detection system according to any one of claim 8, characterized in that: The compound represented by formula (I) includes any two of the following:

10. The dual-luciferase reporter gene detection system according to claim 9, characterized in that: The substrate of the Gaussia luciferase is The substrate of the Pleuromamma xiphias luciferase is 11. The dual-luciferase reporter gene detection system according to claim 1, characterized in that: The molar ratio of the Gaussia luciferase to the Pleuromammamma xiphias luciferase is 1:(0.01-100); The molar ratio of the Gaussia luciferase to the substrate is 1:(1-1000); The molar ratio of the Pleuromamma xiphias luciferase to the substrate is 1:(1-1000).

12. Use of the dual-luciferase reporter gene detection system according to any one of claims 1 to 11 in the preparation of a detection reagent.

13. The use according to claim 12, characterized in that The detection reagents include: cell imaging reagents, amino acid labeling reagents, protein labeling and positioning reagents, antibody specific recognition reagents, nucleic acid labeling reagents or gene sequencing reagents.

14. A fluorescence detection kit comprising the dual-luciferase reporter gene detection system according to any one of claims 1 to 11.

15. The fluorescence detection kit according to claim 14, characterized in that Also included is a reaction buffer comprising water, Tris-HCl, NaCl, and Tween-20.

16. A method for detecting a luciferase luminescent signal, comprising contacting the substrate according to any one of claims 1 to 11 with a sample, and detecting the luminescent signal generated by the Gaussia luciferase and Pleuromammamma xiphias luciferase; the sample contains the Gaussia luciferase and Pleuromammamma xiphias luciferase according to any one of claims 1 to 11.

17. The method according to claim 16, characterized in that The sample is a cell expressing the Gaussia luciferase and Pleuromammamma xiphias luciferase according to any one of claims 1 to 11, a culture containing the cell, or a cell lysate.

18. The method according to claim 16, characterized in that The sample is a nucleotide analogue that specifically binds to the Gaussia luciferase and Pleuromammamma xiphias luciferase according to any one of claims 1 to 11, and the nucleotide analogue specifically binds to the luciferase through a nucleophilic reagent.

19. The method according to claim 18, characterized in that The nucleophilic reagent is an antigen-antibody complex, biotin-streptavidin or digoxin-digoxin antibody.

20. The method according to claim 18 or 19, characterized in that The nucleotide analogues are composed of nucleotide molecules containing four different base types, wherein the first nucleotide molecule can specifically bind to Gaussia luciferase, the second nucleotide molecule can specifically bind to Pleuromammamma xiphias luciferase, the third nucleotide molecule can specifically bind to Gaussia luciferase and Pleuromammamma xiphias luciferase, and the fourth nucleotide molecule can neither bind to Gaussia luciferase nor to Pleuromammamma xiphias luciferase.

21. The method according to any one of claims 16 to 20, characterized in that The method further includes: determining the type of the nucleotide molecule according to the luminescent signal.

22. A method for sequencing a nucleic acid molecule, comprising the steps of: (1) providing a nucleic acid molecule to be sequenced attached to a support, or attaching the nucleic acid molecule to be sequenced to a support; (2) adding a primer for initiating nucleotide polymerization reaction, a polymerase for carrying out nucleotide polymerization reaction, and four compounds to form a reaction system containing a solution phase and a solid phase; wherein the four compounds are derivatives of nucleotides A, (T / U), C, and G, respectively, and have base complementary pairing capabilities; and the hydroxyl group (-OH) at the 3' position of the ribose or deoxyribose of the four compounds is protected by a protecting group; and, The first compound is linked to a first molecular label, The second compound is linked to a second molecular label, The third compound is connected to the first molecular marker and the second molecular marker, or part of the third compound is connected to the first molecular marker and another part of the third compound is connected to the second molecular marker, and the fourth compound is not connected to the molecular marker; (3) annealing the primer to the nucleic acid molecule to be sequenced, wherein the primer serves as the initial growing nucleic acid chain and forms a duplex connected to the support together with the nucleic acid molecule to be sequenced; (4) performing a nucleotide polymerization reaction using a polymerase under conditions that allow the polymerase to perform a nucleotide polymerization reaction, thereby incorporating one of the four compounds into the 3′ end of the growing nucleic acid chain; (5) contacting the duplex from the previous step with the Gaussia luciferase and Pleuromammamma xiphias luciferase according to any one of claims 1 to 11 and performing a binding reaction, wherein the Gaussia luciferase and Pleuromammamma xiphias luciferase are capable of specifically binding to the first molecular marker and the second molecular marker, respectively, and then causing the luciferase to undergo a fluorescence reaction in the presence of a substrate, and detecting the emitted fluorescence signal; (6) removing the protective groups of the incorporated nucleotides and the molecular labels; (7) Optionally repeat steps (4)-(6) or (3)-(6) one or more times to obtain sequence information of the nucleic acid molecule.