Dual luciferase reporter gene detection system and its applications

Abstract

A dual-luciferase reporter gene detection system and its applications are provided. The detection system comprises Gaussia luciferase, Pleuromamma xiphias luciferase, and a substrate, the substrate being coelenterazine or a derivative thereof. Compared with other dual-luciferase reporter gene detection systems, the dual-luciferase reporter gene detection system of the present invention has a stronger signal and simpler reaction conditions.

Description

[Technical Field]

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

[0002] Luciferase is a general term for enzymes that can naturally produce bioluminescence. A luciferase reporter assay is a detection system that detects luciferase activity using luciferin as a substrate. It has a wide range of applications, including cell imaging, amino acid labeling, protein labeling and localization, specific antibody identification, nucleic acid labeling, construction of dual reporter genes, and gene sequencing.

[0003] Coelenterazine is the most abundant natural luciferin in nature. Coelenterazine can be used as a substrate for many luciferases, including Renilla luciferase, Gaussia luciferase-secreted luciferase, and jellyfish photoprotein. Unlike the beetle luciferin / luciferase system, the coelenterazine / luciferase system does not require adenosine triphosphate (ATP), making it easier to study biofluorescence in vivo and producing higher luminescence brightness. For this reason, coelenterazine is often used as a luminescent substrate for fluorescence-based reporter gene detection and live animal detection.

[0004] Different coelenterazine derivatives have different emission wavelengths, cell membrane permeability, and photon efficiencies, resulting in different experimental results for the same application. Because different luciferases have different emission 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 have been widely applied in fields such as gene sequencing, dual reporter gene construction, and protein localization. This method has advantages such as high sensitivity, low interference, easy detection, and a wide detection range. Compared to single luciferase detection, multiplex detection is more widely used for simultaneously studying the expression regulation of multiple genes, reducing off-target effects, interplay between two or more signal pathways, and normalizing "phantoms" in experimental systems.

[0006] Currently, conventional dual-luciferase luminescence systems are primarily two-color detection systems consisting of firefly luciferase and Renilla luciferase. Firefly luciferase can only emit light in the simultaneous presence of luciferin, oxygen, ATP, and magnesium ions, while Renilla luciferase can only emit light in the presence of coelenterazine and oxygen. The difference in reaction conditions between the two luciferase systems limits their application in the same setting. Summary of the Invention [Problem to be solved by the invention]

[0007] In view of this, the present invention provides a dual-luciferase reporter gene detection system and its applications. [Means for solving the problem]

[0008] The present invention provides a dual luciferase reporter gene detection system, comprising Gaussia luciferase, Pleuromamma xiphias luciferase and a substrate, and the substrate is a compound of formula (I), or a stereoisomer, geometric isomer, tautomer, salt, nitroxide, hydrate, or solvate of a compound of formula (I); [ka] where R1 and R2 are independently H, D, F, Cl, Br, I, OH, NH2, NO2, CN, N3, or C 1-6Alkyl group, C 2-6 Alkenyl group, C 2-6 Alkynyl group, C 1-6 Halogenated alkyl groups, C 1-6 Alkoxy group, C 1-6 Hydroxyalkyl group or C 1-6 is an alkylamino group, R3 is an optionally substituted aryl, heterocyclic, or heteroaryl group; 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.

[0009] In the present invention, the Gaussia luciferase is I) the Gaussia luciferase whose amino acid sequence is shown in SEQ ID NO:1; II) A luciferase having at least 85% homology with 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) or I), any one of luciferases in which one or more amino acid residues have been modified, substituted, deleted or added to the amino acid sequence of Gaussia luciferase and still have Gaussia luciferase activity.

[0010] In some embodiments, the Gaussia luciferase is not optimized and is derived from Gaussia luciferase, and the amino acid sequence is the sequence of GenBank accession number AY015993.1 (shown in SEQ ID NO:1).

[0011] In some embodiments, the luciferase has at least 85%, for example 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).

[0012] In some embodiments, the Gaussia luciferase is an optimized variant, i.e., a Gaussia luciferase variant, which, compared to the amino acid sequence of SEQ ID NO:1, has at least one mutation at 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.

[0013] In some embodiments, the Gaussia luciferase mutant comprises at least one of the following mutations: 1) The E mutation at position 24 is K; 2) the F mutation at position 26 is R or L; 3) the N mutation at position 27 is D; 4) the V mutation at position 29 is F or L; 5) the A mutation at position 30 is G or D; 6) the V mutation at position 31 is I; 7) The A mutation at position 32 is V; 8) the S mutation at position 33 is E, R, or K; 9) The A mutation at position 36 is V or I; 10) The T mutation at position 37 is N or E; 11) The L mutation at position 40 is I or T; 12) the K mutation at position 66 is P, S, I, R, or N; 13) The H mutation at position 79 is K; 14) The P mutation at position 84 is A, L, K, or V; 15) The K mutation at position 88 is R; 16) The E mutation at position 102 is D, A, S, K, or N; 17) The S mutation at position 103 is T; 18) The A mutation at position 104 is G; 19) The E mutation at position 110 is P, G, or A; 20) The D mutation at position 123 is N; 21) The L mutation at position 124 is M, G, or I; 22) The V mutation at position 138 is E or D; 23) The Q mutation at position 152 is R or H; 24) The Q mutation at position 163 is D; 25) The S mutation at position 170 is N or T; 26) The G mutation at position 174 is K; 27) The Q mutation at position 175 is E; 28) The K mutation at position 178 is T; 29) The A mutation at position 182 is M; 30) The G mutation at position 183 is N or A.

[0014] Compared with wild-type Gaussia luciferase, the mutant has a broader substrate spectrum, stronger specificity, and significantly enhanced luminescence brightness, resulting in significantly improved practical detection accuracy.

[0015] In some embodiments, the Gaussia luciferase may or may not include a signal peptide amino acid sequence.

[0016] In the present invention, the Pleuromamma xiphias luciferase is i) Pleuromamma xiphias luciferase, the amino acid sequence of which is shown in SEQ ID NO:3; ii) A luciferase having at least 85% homology with 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); iii) Any one of luciferases in which one or more amino acid residues have been modified, substituted, deleted or added to the amino acid sequence of Pleuromamma xiphias luciferase described in i) and still have Pleuromamma xiphias luciferase activity.

[0017] In some embodiments, the Pleuromamma xiphias luciferase is not optimized, is derived from Pleuromamma xiphias, and has the amino acid sequence PMID number 23886588 (shown in SEQ ID NO:3).

[0018] In some embodiments, the luciferase has at least 85%, for example 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 Pleuromamma xiphias luciferase described in i) and has the same or similar function as the Pleuromamma xiphias luciferase described in i).

[0019] In some embodiments, the Pleuromamma xiphias luciferase is an optimized mutant, i.e., a Pleuromamma xiphias luciferase mutant, and compared to the amino acid sequence shown in SEQ ID NO:3, the Pleuromamma xiphias luciferase mutant has at least one of mutation sites at positions 81, 82, 83, and 84.

[0020] In some embodiments, the Pleuromamma xiphias luciferase mutant comprises at least one of the following mutations: (1) the G mutation at position 81 is L or P or Q or S or T; (2) the Q mutation at position 82 is R or W or I or Y or A or L or F or V or P or E or M; (3) the G mutation at position 83 is S or Q or R or W or T or A or L; (4) The G mutation at position 84 is F or R or S or C or Y or L or I or K or V or P.

[0021] Compared with wild-type Pleuromamma xiphias luciferase, the mutant has a broader substrate spectrum, stronger specificity, and significantly enhanced luminescence brightness, resulting in significantly improved practical detection accuracy.

[0022] In some embodiments, the Pleuromamma xiphias luciferase may or may not include a signal peptide amino acid sequence.

[0023] In some embodiments, the Pleuromamma xiphias luciferase comprises a signal peptide amino acid sequence, and preferably, the Pleuromamma xiphias luciferase amino acid sequence including the signal peptide is set forth in SEQ ID NO:4.

[0024] In embodiments of the present invention, the compound of formula (I) may include at least one of the following additional technical features:

[0025] In some specific embodiments, R1 is H.

[0026] In some specific embodiments, R2 is H, NH2, OH, or C 1-6 It is an alkylamino group, preferably H, NH2, 3-OH, 4-OH or dimethylamino.

[0027] 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.

[0028] In some specific embodiments, a1 is 0 or 1.

[0029] In some specific embodiments, a2 is 0 or 1.

[0030] In some specific embodiments, a1 is 0 and a2 is 1; a1 is 1 and a2 is 0; or a1 is 0 and a2 is 0.

[0031] In some specific embodiments, b is 1.

[0032] In some specific embodiments, c is 1.

[0033] In some embodiments, the compound of formula (I) is [ka] Any two of the following are included.

[0034] In some embodiments, the Gaussia luciferase substrate is [ka] is.

[0035] In some embodiments, the substrate for Pleuromamma xiphias luciferase is [ka] is.

[0036] In some embodiments, the molar ratio of Gaussia luciferase to Pleuromamma xiphias luciferase is 1:(0.01-100); In some embodiments, the molar ratio of Gaussia luciferase to substrate is 1:(1-1000); In some embodiments, the molar ratio of the Pleuromamma xiphias luciferase to the substrate is 1:(1-1000).

[0037] 10 is a view showing the use of the dual luciferase reporter gene detection system according to the present invention in the preparation of a detection reagent.

[0038] In the present invention, the detection reagent includes a cell imaging reagent, an amino acid labeling reagent, a protein labeling and localization reagent, an antibody specific recognition reagent, a nucleic acid labeling reagent or a gene sequencing reagent.

[0039] The present invention further provides a fluorescence detection kit comprising the dual luciferase reporter gene detection system described in the present invention.

[0040] In the present invention, the fluorescence detection reagent further comprises a reaction buffer, which comprises water, Tris-HCl, NaCl and Tween-20.

[0041] The present invention further provides a method for detecting luciferase luminescent signals, which comprises the steps of contacting a sample with a substrate in the dual-luciferase reporter gene detection system described in the present invention, and detecting luminescent signals generated by the Gaussia luciferase and Pleuromamma xiphias luciferase, wherein the sample contains Gaussia luciferase and Pleuromamma xiphias luciferase.

[0042] In the present invention, the sample is a nucleic acid, a nucleic acid analog, a cell, a culture containing a cell, or a product produced from a culture containing a cell. The cell may be a bacterial cell, a fungal cell, an animal cell, a plant cell, or a human cell. The nucleic acid may be DNA or RNA, but the present invention is not limited thereto.

[0043] In some embodiments, the sample is a cell, a culture containing cells, or a cell lysate that expresses Gaussia luciferase and Pleuromamma xiphias luciferase in the dual-luciferase reporter gene detection system described in the present invention.

[0044] In some other embodiments, the sample is a nucleotide analog that specifically binds to Gaussia luciferase and Pleuromamma xiphias luciferase in the dual-luciferase reporter gene detection system described in the present invention, and the nucleotide analog specifically binds to the luciferase via a nucleophilic reagent.

[0045] The nucleophile is an antigen-antibody complex, biotin-streptavidin or digoxin-digoxin antibody.

[0046] The nucleotide analogues are composed of nucleotide molecules containing four different base types, wherein the first nucleotide molecule is capable of specifically binding to Gaussia luciferase, the second nucleotide molecule is capable of specifically binding to Pleuromamma xiphias luciferase, the third nucleotide molecule is capable of specifically binding to Gaussia luciferase and Pleuromamma xiphias luciferase, and the fourth nucleotide molecule does not bind to either Gaussia luciferase or Pleuromamma xiphias luciferase.

[0047] In the present invention, the method further comprises the step of determining the type of the nucleotide molecule according to the luminescence signal.

[0048] Additionally, the present invention further provides a method for sequencing a nucleic acid molecule, comprising: (1) providing a nucleic acid molecule to be sequenced that is bound to a support, or binding the nucleic acid molecule to be sequenced to a support; (2) forming a reaction system including a solution phase and a solid phase by adding a primer for initiating a nucleotide polymerization reaction, a polymerase for carrying out a nucleotide polymerization reaction, and four kinds of compounds, wherein the four kinds of compounds are derivatives of nucleotides A, (T / U), C, and G, respectively, and have base-complementary pairing ability, and the hydroxyl group (-OH) at the 3' position of ribose or deoxyribose of the four kinds of compounds is protected by a protecting group, and a first molecular beacon attached to the first compound; a second molecular beacon attached to the second compound; The first molecular label and the second molecular label are bound to the third compound, or the first molecular label is bound to some of the third compounds and the second molecular label is bound to other parts of the third compounds; a fourth compound to which no molecular beacon is attached; (3) annealing a primer to the nucleic acid molecule to be sequenced, the primer forming a duplex that is bound to the support together with the nucleic acid molecule to be sequenced as an initial growing nucleic acid strand; (4) incorporating one of the four compounds onto the 3' end of the growing nucleic acid strand by performing a nucleotide polymerization reaction using a polymerase under conditions that allow the polymerase to perform a nucleotide polymerization reaction; (5) contacting the duplex from the previous step with Gaussia luciferase and Pleuromamma xiphias luciferase in the dual-luciferase reporter gene detection system of the present invention to carry out a binding reaction, wherein the Gaussia luciferase and Pleuromamma xiphias luciferase can specifically bind to the first molecular label and the second molecular label, respectively; causing the luciferase to undergo a fluorescent reaction in the presence of a substrate; and detecting the emitted fluorescent signal; (6) removing the protecting groups and molecular labels of the incorporated nucleotides; (7) optionally repeating steps (4)-(6) or (3)-(6) one or more times, thereby obtaining sequence information of the nucleic acid molecule.

[0049] In the implementation of the present invention, the binding of luciferase to a nucleotide derivative enables spontaneous luminescence detection of the nucleotide to be sequenced, eliminating the need for an additional excitation light source. In a specific implementation, the binding of luciferase to a nucleotide is achieved by specific binding between a label on the luciferase and a corresponding label on the nucleotide derivative. In a specific implementation, a first luciferase is bound to a first nucleotide, a second luciferase is bound to a second nucleotide, the first luciferase and the second luciferase are bound to a third nucleotide, and no luciferase is bound to a fourth nucleotide. Next, the corresponding substrates of the two luciferases are introduced, and the luminescence signals of the four bases are detected. When the substrate of the first luciferase is introduced, the first and third nucleotides emit light. When the substrate of the second luciferase is introduced, the second and third nucleotides emit light. When the substrate of the first luciferase and the substrate of the second luciferase are introduced, neither the fourth nucleotide emits light. Therefore, the bases can be identified based on the luminescence of the four nucleotides.

[0050] Furthermore, the method for sequencing a nucleic acid molecule according to the present invention comprises: (1) providing a nucleic acid molecule to be sequenced that is bound to a support, or binding the nucleic acid molecule to be sequenced to a support; (2) forming a reaction system including a solution phase and a solid phase by adding a primer for initiating a nucleotide polymerization reaction, a polymerase for carrying out a nucleotide polymerization reaction, and four kinds of compounds, wherein the four kinds of compounds are derivatives of nucleotides A, (T / U), C, and G, respectively, and have base-complementary pairing ability, and the hydroxyl group (-OH) at the 3' position of ribose or deoxyribose of the four kinds of compounds is protected by a protecting group, and a first molecular beacon attached to the first compound; a second molecular beacon attached to the second compound; a step of binding a first molecular label and a second molecular label to a third compound, or binding a first molecular label to some of the third compounds and a second molecular label to other some of the third compounds, and no molecular label to a fourth compound; (3) annealing a primer to the nucleic acid molecule to be sequenced, the primer forming a duplex that is bound to the support together with the nucleic acid molecule to be sequenced as an initial growing nucleic acid strand; (4) incorporating one of the four compounds onto the 3' end of the growing nucleic acid strand by performing a nucleotide polymerization reaction using a polymerase under conditions that allow the polymerase to perform a nucleotide polymerization reaction; (5) removing the solution phase of the reaction system from the previous step, leaving the double strand bound 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 label and the second molecular label, respectively; (6) removing unbound luciferase with an elution buffer; (7) adding a substrate for the first luciferase and detecting a first fluorescent signal; (8) removing the solution from the reaction of the previous step; (9) adding a substrate for a second luciferase and detecting a second fluorescent signal; (10) removing the solution from the reaction of the previous step; (11) removing the protecting group and molecular tag of the incorporated nucleotide; (12) optionally removing the solution from the reaction of the previous step; (13) optionally repeating steps (3)-(12) or (4)-(11) one or more times, thereby obtaining sequence information of the nucleic acid molecule.

[0051] The present invention combines wild-type / mutant Gaussia luciferase (Gaussia luciferase) with wild-type / mutant Pleuromamma xiphias luciferase (Pleuromamma xiphias, AB716975). By screening the luminescence interactions between different coelenterazine derivatives and Gaussia luciferase and Pleuromamma xiphias luciferase, a dual-luciferase reporter gene detection system with low cross-interference was obtained. Compared with other dual-luciferase combinations, the reaction conditions for this dual-luciferase combination are simpler, requiring only coelenterazine and oxygen, making the operation easier. DETAILED DESCRIPTION OF THE INVENTION

[0052] The present invention discloses a dual-luciferase reporter gene detection system and its applications, and those skilled in the art can appropriately improve the process parameters and realize the system by referring to the contents of this document. It should be particularly noted that all similar substitutions and variations are obvious to those skilled in the art and are considered to be included in the present invention. Although the method and application of the present invention have been described through preferred embodiments, it goes without saying that those skilled in the art can change or appropriately modify and combine the methods and applications described herein to realize and apply the technology of the present invention without departing from the content, spirit, and scope of the present invention.

[0053] Definitions and General Terms

[0054] In describing the present invention, unless otherwise specified, "plurality" means two or more than two.

[0055] The present invention details the literature corresponding to the content of the determined embodiment, and the examples are accompanied by structural and chemical formula illustrations. The present invention is intended to anticipate and cover all options, variations, and equivalents that may fall within the prior art of the invention as defined in the claims. Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which may be applied to the practice of the present invention. The present invention is in no way limited to the description of the methods and materials. There are many literature and similar materials that are distinct from or inconsistent with the application of the present invention, including, but not limited to, definitions of terms, usage of terms, descriptions of techniques, or scope governed by the application of the present invention.

[0056] The present invention applies the following definitions unless otherwise indicated. For purposes of the present invention, chemical elements are defined in accordance with the Periodic Table of the Elements, CAS Edition, and the Chemicals Handbook, Vol. 75, thEd, 1994. General principles of organic chemistry are also described in "Organic Chemistry," 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, the entire contents of which are incorporated by reference.

[0057] The term "comprises" is an open-ended expression, i.e. includes the subject matter specified in the present invention, but does not exclude the subject matter of other embodiments.

[0058] The compounds described herein, such as compounds of the general formula in the present invention, or specific examples, subclasses, and compounds included in the examples, may be optionally substituted with one or more substituents. It should be understood that the term "optionally substituted" can be 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 a specified substituent. Unless otherwise indicated, one optional substituent may have one substituent substituted at each substitutable position of the group. When more than one position in a given structural formula can be substituted with one or more substituents selected from a specified group, the substituents may be the same or different and may be substituted at each position. The substituents include hydrogen, F, Cl, Br, I, a nitro group, a cyano group, an oxo group (=O), a hydroxyl group, an alkyl group, a hydroxyalkyl group, an alkylamino group, an aminoalkyl group, a haloalkoxy group, a cycloalkyl group, an amino group, an aryl group, a heterocyclic group, a heteroaryl group, an alkenyl group, an alkynyl group, a cycloalkoxy group, an alkoxy group, an alkoxyalkyl group, a halogenated alkyl group, -COOH, an -alkylene-C(=O)O-alkyl group, an -alkylene-S(=O)2-alkyl group, an -alkylene-S(=O)2-amino group, an -S(=O)2-alkyl group, an -S(=O)2-amino group, an -S(=O)2OH, an -O-alkylene-C(=O)O-alkyl group, an -O-alkylene-S(=O)2-a alkyl group, -O-alkylene-S(=O)2-amino group, -O-alkylene-S(=O)2OH, -C(=O)NH2, -C(=O)NH-alkyl group, -C(=O)N(alkyl)-alkyl group, -C(=O)NHS(=O)2-alkyl group, -C(=O)NHS(=O)2-amino group, -C(=O)NHS(=O)2OH, -N(halogenated alkyl group)-alkyl group, -N(alkyl)-S(=O)2-alkyl group, -NHS(=O)2-alkyl group, -NHS(=O)2-halogenated alkyl group, -N(alkyl)S(=O)2-halogenated alkyl group, -N(alkyl)S(=O)2-alkylamino group, -NHC(=O)-alkyl group, -NHC(=O)-halogenated alkyl group-N(alkyl group)C(=O)-halogenated alkyl group, -N(alkyl group)C(=O)-alkylamino group, -N(alkyl group)C(=O)O-alkyl group, -NHC(=O)O-alkyl group, -NHC(=O)O-halogenated alkyl group, -N(alkyl group)C(=O)O-halogenated alkyl group, -N(alkyl group)C(=O)O-aminoalkyl group, -NHC(=O)-NH2, -NHC(=O)NH-(alkyl group), -NHC(=O)NH(halogenated alkyl group), -NHC(=O)N(alkyl group)-alkyl group, -OC(=O)-alkyl group, -OC(=O)-amino group, -OC(=O)-alkylamino group, -OC(=O)-aminoalkyl group, -OC(=O )-alkoxy group, -C(=O)N(alkyl group)S(=O)2-alkyl group, -C(=O)N(alkyl group)S(=O)2-amino group, -C(=O)NH-S(=O)2OH, -C(=NH)NH2, -C(=NH)NH-alkyl group, -C(=NH)N(alkyl group)-alkyl group, -C(=N-alkyl group)-NH2, -C(=O)NH-alkylene-S(=O)2OH, -C(=O)NHC(=O)OH, -C(=O)NHC(=O)O-alkyl group, -C(=O)N(alkyl group)C(=O)O-alkyl group, -C(=O)NH-alkylene-C(=O)OH and -C(=O)NH-alkylene-C(=O)O-alkyl group.

[0059] The term "alkyl group" as used herein includes saturated, straight- or branched-chain monovalent hydrocarbon groups of 1 to 20 carbon atoms, or 1 to 10 carbon atoms, or 1 to 6 carbon atoms, or 1 to 4 carbon atoms, or 1 to 3 carbon atoms, or 1 to 2 carbon atoms, which alkyl groups may independently and optionally be substituted with one or more substituents described herein. Further examples of alkyl groups include 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 (-CH2CH2CH2CH2CH2CH2CH3), 2-hexyl (-CH(CH3)CH2CH2CH2CH2CH3), 3-hexyl (-CH(CH2CH3)(CH2CH2CH3)), 2-methyl-2-pentyl (-C(CH3)2CH2CH2CH3), 3-methyl-2-pentyl (-CH(CH3)CH(CH3)CH2CH3), 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, Examples include, but are not limited to, 3-dimethyl-2-butyl (-C(CH3)2CH(CH3)2), 3,3-dimethyl-2-butyl (-CH(CH3)C(CH3)3), n-heptyl, and n-octyl. As used herein, the term "alkyl group" and its prefix "alkyl" are inclusive of both straight and branched saturated carbon chains.

[0060] The term "alkenyl group" refers to a straight- or branched-chain 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, in which at least one position is unsaturated, i.e., one carbon-carbon bond is sp 2 The double bond, alkenyl group radical may be independently and optionally substituted with one or more substituents described herein, including "reverse," "forward," or "E," "Z" orientations of the radical, and specific examples include, but are not limited to, vinyl (-CH=CH), allyl (-CHCH=CH), and alkenylbutyl (-CHCHCH=CH).

[0061] The term "alkynyl group" refers to a straight or branched monovalent hydrocarbon radical of 2-12 carbon atoms, or 2-8 carbon atoms, or 2-6 carbon atoms, or 2-4 carbon atoms, in which at least one position is unsaturated, i.e., one carbon-carbon is an sp triple bond, and in which 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 (-CHC≡CH).

[0062] The term "halogen" refers to F, Cl, Br or I.

[0063] As used herein, the term "unsaturated" means partially containing one or more unsaturations.

[0064] The term "alkoxy group" or "alkyloxy group" as used herein refers to an alkyl group, as defined herein, that is connected to the rest of the compound molecule via an oxygen atom, and in some embodiments, an alkoxy group is C 1-4 Alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, and butoxy, and the alkoxy groups may independently be unsubstituted or substituted with one or more of the substituents described herein.

[0065] The terms "halogenated alkyl group," "halogenated alkenyl group," and "halogenated alkyloxy group" refer to alkyl, alkenyl, or alkyloxy groups that can be substituted with one or more halogen atoms, and in some embodiments, the halogenated alkyl group is a halogenated C 1-6 In some other embodiments, the halogenated alkyl group is a halogenated C 1-3 In some embodiments, the halogenated alkyloxy or halogenated alkoxy group is a halogenated C 1-6 Alkyloxy group or halogenated C 1-6 In some other embodiments, the halogenated alkyloxy group or halogenated alkoxy group is a halogenated C 1-3 Alkyloxy group or halogenated C 1-3 Alkoxy groups include, but are not limited to, trifluoromethyl, 2-chlorovinyl, 2,2-difluoroethyl, and trifluoromethoxy. The "halogenated alkyl group," "halogenated alkenyl group," and "halogenated alkyloxy group" groups may be independently and optionally substituted with one or more substituents described herein.

[0066] The term "cycloalkyl group" or "cycloalkane" refers to a monovalent or polyvalent saturated monocyclic, bicyclic, or tricyclic carbocyclic ring system containing 3-12 carbon atoms, but does not include any 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 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 be independently unsubstituted or substituted with one or more of the substituents described herein.

[0067] The terms "heterocyclic group" and "heterocycle" are used interchangeably herein and refer to a saturated or partially unsaturated monocyclic, bicyclic, or tricyclic ring containing 3-12 ring atoms, in any case not including aromatic rings, of which at least one ring atom is a heteroatom. Unless otherwise specified, a heterocyclic group may be a carbon or nitrogen group, and the heteroatom has the meaning described herein. Examples of heterocyclic groups include, but are not limited to, ethylene oxide, azetacyclobutyl, oxacyclobutyl, thiozerocyclobutyl, pyrrolidinyl, 2-pyrroline, 3-pyrroline, pyrazolino, pyrazolidine, imidazolinyl, imidazolidine, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, 1,3-dioxocyclopentyl, dithiocyclopentyl, tetrahydropyranyl, dihydropyranyl, 2H-pyranyl, 4H-pyranyl, tetrahydrothiayl, piperidinyl, morpholinyl, thiomorpholinyl, piperazinyl, dioxanyl, dithianyl, thioxanyl, homopiperazinyl, homopiperidinyl, oxoheptyl, thioheptyl, oxazepinyl, diazepinyl, thiazepinyl, and 2-oxa-5-azabicyclo[2.2.1]hept-5-yl. Examples of heterocyclic groups in which the -CH2- group is replaced with -C(=O)- include, but are not limited to, 2-oxopyrrolidinyl, oxo-1,3-thiazolidinyl, 2-piperidinone, 3,5-dioxopiperidinyl, and pyrimidinedione. Examples of heterocyclic groups in which the sulfur atom is oxidized include, but are not limited to, cyclobutylsulfone and 1,1-dioxothiolinomolinyl. The heterocyclic groups may be optionally substituted with one or more of the substituents described herein.

[0068] The term "heteroaryl group" refers to monocyclic, bicyclic, and tricyclic ring systems containing 5-12 ring atoms, or 5-10 ring atoms, or 5-6 ring atoms, where at least one ring is aromatic and contains one or more heteroatoms, and each ring contains 5-7 atoms and has one or more points of attachment to other parts of the molecule. The term "heteroaryl group" can be used interchangeably with the terms "heteroaryl ring" or "heteroaryl compound." The heteroaryl group may optionally be substituted with one or more of the substituents described herein. In one embodiment, the 5-10 atom heteroaryl group contains 1, 2, 3, or 4 heteroatoms independently selected from O, S, and N, and the nitrogen atom can be further oxidized.

[0069] Examples of heteroaryl groups include furanyl, imidazole (e.g., N-imidazole, 2-imidazole, 4-imidazole, 5-imidazole), isoxazolyl, oxazolyl (e.g., 2-oxazolyl, 4-oxazolyl, 5-oxazolyl), pyrrole (e.g., N-pyrrole, 2-pyrrole, 3-pyrrole), pyridine, pyrimidinyl (e.g., 2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl), pyridazinyl, thiazole (e.g., 2-thiazole, 4-thiazole, 5-thiazole), and tetrazolyl. (e.g., 5-tetrazolyl), triazole, thiophenyl (e.g., 2-thiophenyl, 3-thiophenyl), pyrazolyl, isothiazolyl, 1,2,3-oxadiazolyl, 1,2,5-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,3-triazole, 1,2,3-oxadiazolyl, 1,3,4-oxadiazolyl, 1,2,5-oxadiazolyl, pyrazinyl, 1,3,5-triazinyl, but are not limited to benzozolyl, benzofuranyl, benzothienyl, indole (e.g., Bicycles include, but are in no way limited to, bicycles such as [1,2,4]triazolo[4,3-b]pyridazinyl, [1,2,4]triazolo[1,5-a]pyridinyl, [1,2,4]triazolo[1,5-a]pyrimidinyl, and [1,2,4]triazolo[1,5-a]pyridine.

[0070] The term "aminoalkyl group" refers to a C substituted with one or more amino groups. 1-10 In some embodiments, the aminoalkyl group is a C substituted with one or more amino groups. 1-6Examples of alkyl groups include, but are not limited to, aminomethyl, aminoethyl, aminopropyl, aminobutyl, and aminohexyl, which may be optionally substituted with one or more of the substituents described herein.

[0071] The term "hydroxyalkyl group" refers to an alkyl group substituted with one or more hydroxyl groups, wherein the alkyl group has the meaning described herein. Examples of such groups include, but are not limited to, hydroxymethyl, hydroxyethyl, and 1,2-dihydroxyethyl.

[0072] As described herein, a ring system formed by a substituent drawn with a bond attached to the ring at the center indicates that the substituent can be substituted at any substitutable position on that ring. For example, formula (a) shows that the substituent R o can be mono- or polysubstituted at any substitutable position on the E ring. [ka]

[0073] It should also be explained that unless otherwise specified, the descriptive methods used throughout this specification "each ... and ... independently," "... and ... independently of each other," and "... and ... independently of each other" are interchangeable and should be understood in a broad sense, and may mean that specific options represented by the same symbol in different groups do not affect each other, or may mean that specific options represented by the same symbol in the same group do not affect each other.

[0074] Unless otherwise indicated, the structural formulae depicted in the present invention encompass all homo isomers (e.g., enantiomerism, diastereomerism, geometric isomerism, or conformational isomerism), such as the R,S configurations containing asymmetric centers, (Z), (E) isomers of double bonds, and (Z), (E) conformational isomers. Thus, any single stereochemical isomer or mixture of enantiomers, diastereomers, geometric isomers, or conformational isomers of the compounds of the present invention are within the scope of the present invention.

[0075] Unless otherwise indicated, the structural formulas and compounds described herein include all homoisomers (e.g., enantiomers, diastereomers, geometric isomers, or conformational isomers), salts, nitroxides, hydrates, or solvates. Thus, single stereochemical isomers, enantiomers, diastereomers, geometric isomers, conformational isomers, salts, nitroxides, hydrates, and solvates of the compounds of the present invention are also included within the scope of the present invention. Additionally, unless otherwise indicated, the structural formulas of the compounds described herein include enriched isotopes of one or more of the different atoms.

[0076] The definitions and conventions of stereochemistry used herein generally refer to 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 or chiral centers, and therefore exist in 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, are part of the present invention. Many organic compounds exist in optically active forms, i.e., have the ability to rotate the plane of plane-polarized light. In describing an optically active compound, the prefixes D, L, or R, S are used to indicate the absolute configuration of the molecule's chiral centers. The prefixes d, l, (+), and (-) are used to designate the sign of a compound's rotation of plane-polarized light; (-) or l means the compound is left-handed, and (+) or d means the compound is right-handed. These stereoisomers have the same chemical structure but differ in their configuration. A specific stereoisomer may be an enantiomer, and a mixture of isomers is usually called an enantiomeric mixture. A 50:50 mixture of enantiomers is called a racemic mixture or racemate, and there may be no stereoselectivity or stereoorientation during a chemical reaction. The terms "racemic mixture" and "racemate" refer to a mixture of two enantiomers in equimolar amounts, lacking optical activity.

[0077] The term "tautomer" or "tautomeric form" refers to structural isomers of different energetic structures that can be converted into each other via a low energy barrier. For example, proton tautomers (i.e., proton shift tautomers) involve tautomerization via proton migration, e.g., ketone-enol and imine-enamine structural isomerizations. Valence tautomers involve tautomerization of rearranged bond electrons.

[0078] The term "hydrate" as used herein refers to an association formed between solvent molecules and water.

[0079] The term "solvate" as used herein refers to an association formed by one or more solvent molecules and a compound of the present invention. Solvents that form solvates include, but are not limited to, water, isopropyl alcohol, ethanol, methanol, dimethyl sulfoxide, ethyl acetate, acetic acid, and aminoethanol.

[0080] The term "ester" as used herein refers to a compound of Formula (I) containing a hydroxyl group that forms an ester that can be hydrolyzed in vivo. Such an ester is, for example, a pharmaceutically acceptable ester that can be hydrolyzed in the human or animal body to produce the parent alcohol. Examples of ester groups that can be hydrolyzed in the human or animal body include, but are not limited to, phosphate, acetoxymethoxy, 2,2-dimethylpropionyloxymethoxy, alkanoyl, benzoyl, phenylacetyl, alkoxycarbonyl, dialkylcarbamoyl, and N-(dialkylaminoethyl)-N-alkylcarbamoyl.

[0081] The term "nitrogen oxide" as used herein means that, when a compound contains several amine functional groups, one or more nitrogen atoms can be oxidized to form an N-oxide. Specific examples of N-oxides include the N-oxides of tertiary amines or nitrogen atoms of nitrogen-containing heterocycles. N-oxides can be formed using an oxidizing agent such as hydrogen peroxide or by treating the corresponding amine with a peracid (e.g., peroxycarboxylic acid) (see Advanced Organic Chemistry, Wiley Interscience, Version 4, Jerry March, pages 1977-1987). In particular, N-oxides can be prepared by the method of L.W. Ready (Syn. Comm. 1977, 7, 509-514), for example, by reacting an amine compound with m-chloroperbenzoic acid (MCPBA) in an inert solvent (e.g., dichloromethane).

[0082] In the description herein, a statement referring to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples" means that a particular feature, structure, material, or characteristic described with reference to that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, general references to such terms do not necessarily refer to the same embodiment or example. In addition, a particular feature, structure, material, or characteristic described may be incorporated in any suitable manner in any one or more embodiments or examples.

[0083] Unless otherwise defined, all technical terms used herein have the same meaning as understood by those skilled in the art. For definitions and terms in the field, experts can specifically refer to Current Protocols in Molecular Biology (Ausubel). Abbreviations for amino acid residues are standard three-letter and / or one-letter codes used in the field to refer to one of the 20 common L-amino acids.

[0084] The luciferase combination provided by the present invention contains two luciferases: one 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 20 kDa and is derived from a bioluminescent enzyme found in the marine copepod Gaussia princeps. The Gaussia luciferase receptor molecule is very small, and it does not require ATP supplementation. It catalyzes the oxidative luminescence of coelenterazine in the presence of molecular oxygen. Pleuromamma xiphias luciferase, also known as myridine luciferase, is a copepod luciferase that can catalyze the oxidative luminescence of coelenterazine in the presence of molecular oxygen. Screening of a fluorescent enzyme combination in the present invention has demonstrated that it generates stronger fluorescence than other fluorescent enzyme combinations and is free of cross-interference.

[0085] In the fluorescent enzyme reporter gene detection system according to the present invention, the luciferase may be present in the form of an encoding nucleic acid, an expression module, an expression vector, a recombinant strain, an expression product of a recombinant strain, or a purified expression product.

[0086] Taking the encoding nucleic acid as an example, the detection system described in the present invention is a mixture of a nucleic acid encoding Gaussia luciferase and a nucleic acid encoding Pleuromamma xiphias luciferase, and may be two independently existing nucleic acids encoding Gaussia luciferase and Pleuromamma xiphias luciferase, or may be a fusion fragment formed by a nucleic acid encoding Gaussia luciferase and a nucleic acid encoding Pleuromamma xiphias luciferase, and the present invention is not limited thereto.

[0087] Taking an expression module as an example, the expression module includes a promoter, a coding nucleic acid, and a terminator. The nucleic acid encoding Gaussia luciferase and the nucleic acid encoding Pleuromamma xiphias luciferase may be located in the same expression module or in two different expression modules. The promoters of the two expression modules in which the two coding nucleic acids are located may be the same or different, and the present invention is not limited thereto.

[0088] For example, the expression vector may include a skeleton vector and an encoding nucleic acid. The expression vector may include a plasmid vector or a viral vector. The nucleic acid encoding Gaussia luciferase and the nucleic acid encoding Pleuromamma xiphias luciferase may be expressed in the same expression vector or in different expression vectors, but the present invention is not limited thereto. When the nucleic acids are expressed in different vectors, the skeleton vectors may be the same or different.

[0089] The expression strain described in the present invention is also called a recombinant host and can express Gaussia luciferase and / or Pleuromamma xiphias luciferase. That is, Gaussia luciferase and Pleuromamma xiphias luciferase can be expressed in the same host or in different hosts, but the present invention is not limited thereto. The recombinant host can be a prokaryotic or eukaryotic host and can be constructed by electrotransformation or viral infection. It can also be E. coli, yeast, or animal or human cells, but the present invention is not limited thereto. To express two luciferases in two different hosts, the same host cell or different host cells can be used.

[0090] The expression product or purified expression product of the recombinant strain of the present invention is derived from the above-mentioned expression strain or recombinant host, and may be a mixture of the two fluorescent enzyme-expressing strains after expression, or may be a product expressed by the two fluorescent enzyme-expressing strains.

[0091] In the present invention, the term "reporter gene" refers to a molecular biology concept: a gene that is expressed in a cell, tissue / organ, or individual under specific conditions, produces a detectable characteristic that is not naturally produced in experimental materials, i.e., a gene encoding a detectable protein or enzyme. A reporter gene must satisfy the following conditions for genetic selection and screening detection: 1) be cloned and its entire sequence can be measured; 2) its expression product is not naturally present in recipient cells, i.e., there is no background and no endogenous expression product similar to that of transfected cells; and 3) its expression product can be quantitatively measured. When used, a reporter gene can be fused with a gene expression regulatory sequence to form a hybrid gene, or fused with another gene to express nucleic acid under the control of a regulatory sequence, and the expression product can be used to detect the expression regulation of the target gene and study nucleic acid. This includes, but is not limited to, these methods.

[0092] In the present invention, the luciferase reporter gene detection system, also known as a dual luciferase luminescence system, comprises the fluorescent enzyme composition and substrate described in the present invention. The fluorescent enzyme composition and substrate exist independently and emit light after mixing in the detection system during detection. The present invention selects a substrate, and the resulting luciferase reporter gene detection system can have higher fluorescence intensity, thereby improving detection sensitivity.

[0093] The detection reagent contains a substance that induces overexpression of Gaussia luciferase and Pleuromamma xiphias luciferase in the analyte, including, but not limited to, a nucleic acid encoding Gaussia luciferase and / or a plasmid vector or viral vector of a nucleic acid encoding Pleuromamma xiphias luciferase.

[0094] As used herein, the term "polynucleotide" refers to deoxyribose nucleic acid (DNA), ribose nucleic acid (RNA), or analogs thereof. Polynucleotides may be single-stranded, double-stranded, or contain both single- and double-stranded sequences. Polynucleotide molecules may be derived from double-stranded DNA (dsDNA) forms (e.g., genomic DNA, PCR and amplification products, etc.), or may be derived from single-stranded forms of DNA (ssDNA) or RNA and converted to dsDNA forms, or vice versa. The exact sequence of the nucleotide molecule may or may not be known. The following are illustrative examples of polynucleotides: a gene or gene fragment (e.g., a probe, primer, EST or SAGE label), genomic DNA, a genomic DNA fragment, an exon, an intron, messenger RNA (mRNA), transporter RNA, 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.

[0095] A polynucleotide can comprise nucleotides or nucleotide analogs. Nucleotides typically comprise a sugar (e.g., ribose or deoxyribose), a base, and at least one phosphate group. Nucleotides include deoxyribose nucleotides, modified deoxyribose nucleotides, ribose nucleotides, modified ribose nucleotides, 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 (U TP}, 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).

[0096] Nucleotide analogs containing modified bases are used in the methods described herein. Exemplary modified bases that can be included in polynucleotides, whether with a natural backbone or a similar structure, include (for example) inosine, xanthine, hypoxanthine, 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-haloureacil, 15-halocytosine, 15-haloureacil ... Examples of uracils include 5-aminoadenine, 5-propyluracil, 5-propanylcytosine, 6-azouracil, 6-azocytosine, 6-azothymine, 5-uracil, 4-thiouracil, 8-haloadenine or guanine, 8-aminoadenine or guanine, 8-thioadenine or guanine, 8-hydroxyadenine or guanine, 5-halogen substituted uracil or cytosine, 7-methylguanine, 7-methyladenine, 8-azaguanine, 8-azadenine, 7-deazaguanine, 7-deazadenine, 3-deazaguanine, 3-deazadenine, and the like.

[0097] Typically, a nucleotide includes the nucleotides A, C, G, T, or U. As used herein, the term "nucleotide A" refers to a nucleotide containing denine (A) or a modified or analogous thereof, e.g., ATP, dATP. "nucleotide G" refers to a nucleotide containing guanine (G) or a modified or analogous thereof, e.g., GTP, dGTP. "nucleotide C" refers to a nucleotide containing cytosine (C) or a modified or analogous thereof, e.g., CTP, dCTP. "nucleotide T" refers to a nucleotide containing thymine (T) or a modified or analogous thereof, e.g., TTP, dTTP. "nucleotide U" refers to a nucleotide containing uracil (U) or a modified or analogous thereof, e.g., UTP, dUTP.

[0098] Nucleotide labeling

[0099] The present invention relates to labeling nucleotides with different labels, either singly or in combination, to allow the binding of different luciferases to the nucleotides. As used herein, the molecular label that labels a nucleotide and the label that specifically binds to it may be any molecular pair that specifically binds to each other. The binding of the nucleotide to the luciferase is achieved by specific binding between the pair members. Exemplary pair members include, but are not limited to, (a) a half-antigen or antigenic compound combined with a corresponding antibody or its binding portion or fragment, e.g., digoxin-digoxin antibody, N3G-N3G antibody, FITC-FITC antibody; (b) a nucleic acid aptamer and a protein; (c) a non-immune binding pair (e.g., biotin-antibiotin protein, biotin-streptavidin, biotin-neutral antibiotic protein); (d) a hormone-hormone binding protein; (e) a receptor-receptor agonist or antagonist; (f) a homolectin-carbohydrate; (g) an enzyme-enzyme cofactor; and (h) an enzyme-enzyme inhibitor.

[0100] In a specific embodiment, the first and second molecular labels are small molecule labels selected from biotin, digoxin, N3G, and FITC. Two luciferases can specifically bind to the first and second molecular labels, respectively. For example, in one specific embodiment, when the first molecular label is biotin, the first luciferase can be a luciferase labeled with streptavidin; when the second molecular label is digoxin, the second luciferase can be a luciferase different from the first luciferase labeled with a digoxin antibody. Preferably, the first luciferase is a Gaussia luciferase or a variant thereof described in the present invention, and the second luciferase is a Pleuromamma xiphias luciferase or a variant thereof described in the present invention.

[0101] As used herein, the phrase "a first molecular label is bound to a first compound" means that the first molecular label is bound to all of the first compounds, or that the first molecular label is bound to some of the first compounds, but that the remaining first compounds do not have a molecular label bound to them. Similarly, the phrase "a second molecular label is bound to a second compound" means that the second molecular label is bound to all of the second compounds, or that the second molecular label is bound to some of the second compounds, but that the remaining second compounds do not have a molecular label bound to them. The phrase "a first molecular label and a second molecular label are bound to a third compound" means that the first molecular label and a second molecular label are bound to all of the third compounds, or that the first molecular label and a second molecular label are bound to some of the third compounds, but that the remaining third compounds do not have a molecular label bound to them.

[0102] Polynucleotide sequencing

[0103] Preferably, the dual luciferase reporter gene detection system of the present invention is applied to synthetic sequencing. As used herein, synthetic sequencing refers to various synthetic sequencing methods known in the art. Essentially, synthetic sequencing involves first hybridizing a sequencing primer with the nucleic acid molecule to be sequenced, and then polymerizing a nucleotide or nucleotide analog described herein at the 3' end of the sequencing primer in the presence of a polymerase, 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 transmitted by the luciferase. After removing the luciferase from the labeled nucleotide, the next cycle of polymerized sequencing is carried out.

[0104] A method for determining a target polynucleotide sequence 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, such that a polymerase extends the complementary strand by incorporating the correct nucleotide complementary to the target. The polymerization reaction also requires a special primer to initiate the polymerization process.

[0105] For each reaction, the incorporation of the nucleotide is performed by a polymerase, and the incorporation event is then measured. Many different polymerases exist, and the optimal polymerase can be easily determined by one of skill in the art. 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 may also be used.

[0106] The sequencing method is preferably performed on target polynucleotides arrayed on a solid support, to which multiple target polynucleotides can be immobilized by linker molecules or attached to particles such as microspheres, which can be bound to the solid support material.

[0107] The polynucleotides may be attached to the solid support by several methods, including using biotin-streptavidin interactions. Methods for immobilizing polynucleotides on solid supports are well known in the art and include lithographic printing techniques and spotting various polynucleotides onto specific locations on the solid support. Suitable solid supports are well known in the art and include glass slides and beads, ceramic and silicon surfaces, and plastic materials.

[0108] The support is typically flat, and microbeads (microspheres) can be used, although the latter can be attached to other solid supports by known methods. The microspheres can be of any suitable size, typically 10-100 nanometers in diameter. In a preferred implementation, the polynucleotides are directly attached to the flat surface, preferably to a flat glass surface. Attachment is preferably by covalent bonding. The array used is preferably a single-molecule array containing polynucleotides in unique, optically distinguishable regions, as described in International Application No. WO 00 / 06770.

[0109] The essential conditions for polymerization are well known to those skilled in the art. To carry out the polymerase reaction, a primer sequence must first be annealed to the target polynucleotide. The primer sequence is recognized by the polymerase and serves as the initiation region from which the complementary strand subsequently extends. The primer sequence can be added to the target polynucleotide as an independent component. Alternatively, the primer and target polynucleotide may each be part of a single-stranded molecule, with a portion of the primer and a portion of the target forming an intramolecular double-stranded, i.e., a hairpin ring structure. This structure can be immobilized to the solid support at any site on the molecule. Other conditions required for the polymerase reaction are known to those skilled in the art, including temperature, pH, and buffer composition.

[0110] The labeled nucleotides or nucleotide analogs of the present invention are then contacted with the target polynucleotide so that they can be polymerized. 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.

[0111] The polymerization step is carried out for a time sufficient to incorporate one nucleotide.

[0112] Unincorporated nucleotides are then removed, eg, by removing the solution phase of the reaction system from the previous step, leaving the duplex bound to the support.

[0113] Then, two luciferases, including different luciferases, can be added to carry out a binding reaction, and the two luciferases can specifically bind to the molecular labels on the nucleotides, thereby realizing the binding of the luciferases with the incorporated nucleotides.Then, the substrates of the corresponding luciferases are added to detect the fluorescent signals, thereby realizing the identification of the incorporated nucleotides.

[0114] In one specific implementation, four deoxyribose nucleotide analogs are labeled with different small molecule labels, biotin (abbreviated as B) and digoxin (abbreviated as D). 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 deoxyribose nucleotide analogs labeled with different small molecules are all blocked to ensure that only one deoxyribose nucleotide is attached per sequencing reaction. The sequencing reaction begins by introducing the four labeled deoxyribose nucleotide analogs and a sequencing polymerase mixture. Under the action of the polymerase, one deoxyribose nucleotide analog is incorporated into the 3' end of the growing nucleic acid strand according to the principle of base-complementary pairing. The solution phase of the reaction system from the previous step is then removed, leaving the double strand attached to the support, and any unattached deoxyribose nucleotide analogs are removed. Next, two different luciferases are added, where the first luciferase is labeled with streptavidin, and small molecule B binds to labeled nucleotide A or nucleotide C. The second luciferase is labeled with digoxin antibody, and small molecule D binds to labeled nucleotide C or nucleotide T. After removing unbound luciferase using an elution buffer, a substrate for the first luciferase is added, causing the nucleotide bound to the first luciferase to emit light and the signal to be detected by a detector. A substrate for the second luciferase is added, causing the nucleotide bound to the second luciferase to emit light and the signal to be detected by a detector. This results in the light emission patterns shown in the table below, allowing base discrimination (1 indicates that a fluorescent signal is detected, 0 indicates that a fluorescent signal is not detected). TIFF2025528138000006.tif32131

[0115] In one specific implementation, two different luciferases (Gaussia luciferase or its variant and Pleuromamma xiphias luciferase or its variant) can be bound to labeled nucleotides and their signals detected separately. First, a first luciferase is added, labeled with streptavidin, and small molecule B binds to nucleotide A or nucleotide C. After removing unbound first luciferase using elution buffer, a substrate for the first luciferase is added, causing the nucleotide bound to the first luciferase to emit light and a signal is detected by a detector. After removing the reaction solution, a second luciferase labeled with a digoxin antibody binds to nucleotide C or nucleotide T labeled with small molecule D. Next, unbound second luciferase is removed using elution buffer, and a substrate for the second luciferase is added, causing the nucleotide bound to the second luciferase to emit light and a signal is detected by a detector. This results in the light-emitting patterns shown in the table above, allowing base discrimination.

[0116] Fluorescence signal detection

[0117] Methods for detecting fluorescent signals are well known in the art. For example, they can be realized by devices that detect the wavelength of fluorescence. Such devices are well known in the art. For example, such devices may be a confocal scanning microscope, which scans the surface of a solid support with a laser to image the fluorophores directly bound to the sequenced nucleic acid molecules. Alternatively, each generated signal can be observed 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.

[0118] All reagents and instruments used in the present invention can be purchased commercially. Gaussia luciferase (GenBank: AY015993.1) used in the examples was prepared using an E. coli expression system, and the expression vector was pET28a. Pleuromamma xiphias luciferase (PMID: 23886588) was also prepared using an E. coli expression system, and the expression vector was pET28a.

[0119] The present invention will be further explained below with reference to a combination of examples.

[0120] Example 1 Paired luminescence detection of dual luciferase system (wild type)

[0121] Experimental objective: To test the luminescence intensity of wild-type Gaussia luciferase and wild-type Pleuromamma xiphias luciferase on different substrates and to select a luminescence combination with high brightness and low cross-interference.

[0122] Experimental steps: (1) Wild-type Gaussia luciferase (WT-Gluc) has the amino acid sequence shown below: KPTENNEDFNIVAVASNFATTDLDADRGKLPGKKLPLEVLKEMEANARKAGCTRGCLICLSHIKCTPKMKKFIPGRCHTYEGDKESAQGGIGEAIVDIPEIPGFKDLEPMEQFIAQVDLCVDCTTGCLKGLANVQCSDLLKKWLPQRCATFASKIQGQVDKIKGAGGD(SEQ ID NO:1). The wild-type Gaussia luciferase (WT-Gluc) gene has the nucleotide sequence shown below: AAACCAACTGAAAACAATGAAGATTTCAACATTGTAGCTGTAGCTAGCAACTTTGCTACAACGGATCTCGATGCTGACCGTGGTAAATTGCCCGGAAAAAAATTACCACTTGAGGTACTCAAAGAAATGGAAGCCAATGCTGGAAAGCTGGCTGCACTAGGGGATGTCTGATATGCCTGTCACACATCAAGTGTACACCCAAAATGAAGAAGTTTATCCCAGGAAGATGCCACACCTATGAAGGAGACAAAGA AAGTGCACAGGGAGGAATAGGAGAGGCTATTGTTGACATTCCTGAAATTCCTGGGTTTAAGGATTTGGAACCCATGGAACAATTCATTGCACAAGTTGACCTATGTGTAGACTGCACAACTGGATGC CTCAAAGGTCTTGCCAATGTGCAATGTTCTGATTTACTCAAGAAATGGCTGCCACAAAGATGTGCAACTTTTGCTAGCAAAATTCAAGGCCAAGTGGACAAAATAAAGGGTGCCGGTGGTGAT(SEQ ID NO:2). Wild-type Pleuromamma xiphias luciferase (WT-Pxluc) has the amino acid sequence shown below: QPTENKQESHIVDSDLDGDRGRKLPGKKLPIEVLKIMEANARRAGCTRGCLICLSKIKCTAKMKRYIPGRCHTYEGDKSIGQGGIGGPIVDIPEIIGFKNMEPMDQFIAQVDLCADCTTGCLKGLANVRCNDLLKKWLPDRCAGFADKIQNEVDSIKGMAGDR(SEQ ID NO:3) After adding a signal peptide to wild-type Pleuromamma xiphias luciferase (WT-Pxluc), it has the amino acid sequence shown below: MYIKVWFGLACLSLVLAOPTENKOESHIVDSDLDGDRGRKLPGKKLPIEVLKIMEANARRAGCTRGCLICLSKIKCTAKMKRYIPGRCHTYEGDKSIGOGGIGGPIVDIPEIIGFKNMEPMDOFIAOVDLCADCTTGCLKGLANVRCNDLLKKWLPDRCAGFADKIQNEVDSIKGMAGDR(SEQ ID NO:4) The wild-type Pleuromamma xiphias luciferase (WT-Pxluc) gene has the nucleotide sequence shown below: CAACCAACTGAAAACAAGCAGGAGTCTCATATTGTAGATTCAGATCTTGATGGCGACCGTGGTAGGAAGTTGCCCGGAAAAAAGCTTCCTATAGAAGTACTCAAAATCATGGAAGCCAATGCCAGGAGAGCTGGTTGCACTAGAGGATGTCTCATATGTCTTTCAAAAATCAAGTGTACAGCCAAAATGAAGCGATACATTCCAGGGAGATGTCATACTTATGAAGGAGATAAATCTATTGGACAG GGAGGCATAGGTGGCCCTATTGTTGATATTCCTGAAATTATTGGATTCAAGAACATGGAACCCATGGATCAGTTCATCGCACAAGTTGATCTGTGCGCCGACTGTACAACTGGGTGCCTGAAAGGCCTTGCTAATGTTAGGTGCAATGACTTGCTGAAGAAATGGCTGCCTGACAGATGTGCTGGTTTTGCCGACAAAATTCAAAATGAAGTGGATAGTATCAAGGGCATGGCTGGGGATCGC(SEQ ID NO:5) (2) Coelenterazine derivatives as substrates: Substrates f-CTZ, N0, N1, N3, N4, N5, N6, N7 and N8 are synthesized by referring to the method disclosed in patent application PCT / CN2018 / 107646. [ka] (3) Expression vectors (plasmids) containing the wild-type Gaussia luciferase gene and the wild-type Pleuromamma xiphias luciferase gene were constructed according to conventional methods, such as those disclosed in PCT / CN2017 / 082180. The plasmids were transformed into E. coli OrigamiB (DE3), plates were smeared, and single colonies were picked from the plates and cultured overnight at 37°C. The following day, the colonies were diluted 1:100 and transferred to 3 mL of fresh LB medium containing ampicin resistance (100 μg / mL). The colonies were cultured at 37°C with shaking at 200 rpm for 4 hours, followed by cooling on ice for 1 hour. The inducer IPTG was added to a final concentration of 1 mM, and the resulting mixture was induced overnight at 16°C.

[0123] The next day, the bacteria were collected by centrifugation and suspended in 500 μL of cracker solution (50 mM Tris-HCl pH 8.0, 500 mM NaCl, 0.2% lysozyme, PMSF). The suspension was then digested at room temperature for 20 minutes. The suspension was then frozen and thawed three times in liquid nitrogen, centrifuged at 18,000 g for 30 minutes, and the supernatant was collected and purified using a centrifugal Ni column.

[0124] The protein eluted from the Ni column was dialyzed overnight at 4°C against dialysis buffer (25 mM Tris, pH 8.0, 250 mM NaCl). TM Pierce TM Accurately measure the luciferase concentration using the BCA Protein Assay Kit.

[0125] Dilute both luciferases to 1 μg / mL in diluent (50 mM Tris-HCl pH 8.0, 100 mM NaCl, 0.1% (v / v) Tween-20) and add 10 μL of each to a black 96-well plate. Dilute the substrate to 100 μM in the same diluent and add 90 μL to each well of the 96-well plate containing the luciferase. Read the luminescence intensity using the enzyme marker's autoluminescence module.

[0126] The test data for Gaussia luciferase and Pleuromamma xiphias luciferase with different substrates are shown in Table 1. The relative intensities are the ratios of the activities of wild-type Pleuromamma xiphias luciferase and compound N0. The results show that the fluorescence intensity is highest when Gaussia luciferase is combined with the substrate f-CTZ, and the fluorescence intensity is highest when Pleuromamma xiphias luciferase is combined with the substrate N0. In other words, in this dual-luciferase reporter gene detection system, Gaussia luciferase is detected with the substrate f-CTZ, and Pleuromamma xiphias luciferase is detected with the substrate N0. After selection, an optimal luminescence combination with high fluorescence brightness and low cross-interference was obtained.

[0127] [Table 1]

[0128] Example 2: Paired luminescence detection of dual luciferase system (mutant type)

[0129] Experimental objective: To screen for a high-intensity, low-cross-interference luminescence combination by testing the luminescence intensity of mutant Gaussia luciferase and mutant Pleuromamma xiphias luciferase with different substrates.

[0130] Experimental steps: (1) Mutant Gaussia luciferases E1-A3, G2-F11, and G2-F8: a) E1-A3, based on SEQ ID NO: 1, mutated at the following sites: F26R, V29F, A32V, S33E, A36V, L40I, K66P, H79K, P84L, E102S, S103T, A104G, E110P, L124M, V138E; The gene for the E1-A3 mutant has the nucleotide sequence shown below: ATGAAACCAACTGAAAACAATGAAGATCGCAACATTTTCGCTGTAGTTGAGAACTTTGTTACGACGGATATTGATGCTGACCGTGGTAAATTGCCCGGAAAAAAATTACCACTTGAGGTACTCAAAG AAATGGAAGCCAATGCTAGGCCTGCTGGCTGCACTAGGGGATGTCTGATATGCCTGTCAAAAATCAAGTGTACACTCAAAAATGAAGAAGTTTATCCCAGGAAGATGCCACACCTATGAAGGAGACAAA TCAACTGGACAGGGAGGAATAGGACCGGCTATTGTTGACATTCCTGAAATTCCTGGGTTTAAGGATATGGAACCCATGGAACAATTCATTGCACAAGTTGACCTATGTGAAGACTGCACAACTGGATGCCTCAAAGGTCTTGCCAATGTGCAATGTTCTGATTTACTCAAGAAATGGCTGCCACAAAGATGTGCAACTTTTGCTAGCAAAATTCAAGGCCAAGTGGACAAAATAAAGGGTGCCGGTGGTGAT(SEQ ID NO:6) b) G2-F11, based on SEQ ID NO: 1, mutated at the following sites: 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); The gene for the G2-F11 mutant has the nucleotide sequence shown below: ATGAAACCAACTGAAAACAATAAAGATTTCAACATTGTAGCTGTAGCTAGCAACTTTGCTACAACGGATCTCGATGCTGACCGTGGTAAATTGCCCGGAAAAAAATTACCACTTGAGGTACTCAAAG AAATGGAAGCCAATGCTAGGAAAGCTGGCTGCACTAGGGGATGTCTGATATGCCTGTCAAAAATCAAGTGTACACTCAAAAATGAAGAAGTTTATCCCAGGAAGATGCCACACCTATGAAGGAGACAAA TCAACTGGACAGGGAGGAATAGGACCGGCTATTGTTGACATTCCTGAAATTCCTGGGTTTAAGGATGGGGAACCCATGGAACAATTCATTGCACAAGTTGACCTATGTGTAGACTGCACAACTGGATGCCTCAAAGGTCTTGCCAATGTGCGATGTTCTGATTTACTCAAGAAATGGCTGCCAGACAGATGTGCAACTTTTGCTAACAAAATTCAAGGCGAAGTGGACACAATAAAGGGTATGAACGGTGAT(SEQ ID NO:7) c) G2-F8, based on SEQ ID NO:1, with the following mutations: H79K, P84L, E102S, S103T, A104G, E110P, L124M, Q152H, Q163D, T167S, S170T, G174K, A182M, G183A. The gene for the G2-F8 mutant has the nucleotide sequence shown below: ATGAAACCAACTGAAAACAATGAAGATTTCAACATTGTAGCTGTAGCTAGCAACTTTGCTACAACGGATCTCGATGCTGACCGTGGTAAATTGCCCGGAAAAAAATTACCACTTGAGGTACTCAAAG AAATGGAAGCCAATGCTAGGAAAGCTGGCTGCACTAGGGGATGTCTGATATGCCTGTCAAAAATCAAGTGTACACTCAAAAATGAAGAAGTTTATCCCAGGAAGATGCCACACCTATGAAGGAGACAAA TCAACTGGACAGGGAGGAATAGGACCGGCTATTGTTGACATTCCTGAAATTCCTGGGTTTAAGGATATGGAACCCATGGAACAATTCATTGCACAAGTTGACCTATGTGTAGACTGCACAACTGGATGCCTCAAAGGTCTTGCCAATGTGCATTGTTCTGATTTACTCAAGAAATGGCTGCCAGACAGATGTGCAAGTTTTGCTACCAAAATTCAAAAGCAAGTGGACAAAATAAAGGGTATGGCTGGTGAT(SEQ ID NO:8) Mutant Pleuromamma xiphias luciferase P26-95: d) P26-95, based on SEQ ID NO:3, mutating the following sites: G83A, G84P. The gene for the P26-95 mutant has the nucleotide sequence shown below: CAACCAACTGAAAACAAGCAGGAGTCTCATATTGTAGATTCAGATCTTGATGGCGACCGTGGTAGGAAGTTGCCCGGAAAAAAGCTTCCTATAGAAGTACTCAAAATCATGGAAGCCAATGCCAGGAGAGCTGGTTGCACTAGAGGATGTCTCATATGTCTTTCAAAAATCAAGTGTACAGCCAAAATGAAGCGATACATTCCAGGGAGATGTCATACTTATGAAGGAGATAAATCTATTGGACAG GCACCGATAGGTGGCCCTATTGTTGATATTCCTGAAATTATTGGATTCAAGAACATGGAACCCATGGATCAGTTCATCGCACAAGTTGATCTGTGCGCCGACTGTACAACTGGGTGCCTGAAAGGCCTTGCTAATGTTAGGTGCAATGACTTGCTGAAGAAATGGCTGCCTGACAGATGTGCTGGTTTTGCCGACAAAATTCAAAATGAAGTGGATAGTATCAAGGGCATGGCTGGGGATCGC(SEQ ID NO:9)

[0131] According to the method described in Example 1, an expression vector containing the Gaussia luciferase mutant gene and the Pleuromamma xiphias luciferase mutant gene is constructed, a recombinant bacterium is constructed, the mutant luciferase is expressed, and the substrate and luciferase luminescence intensity are detected.

[0132] The results showed that Gaussia luciferase variants E1-A3, G2-F11, and G2-F8 exhibited the highest fluorescence intensity when combined with the f-CTZ substrate, while Pleuromamma xiphias luciferase variant P26-95 exhibited the highest fluorescence intensity when combined with the N0 substrate. This dual-luciferase reporter gene detection system detects Gaussia luciferase variants E1-A3, G2-F11, or G2-F8 with the f-CTZ substrate, and Pleuromamma xiphias luciferase variant P26-95 with the N0 substrate. This combination of high fluorescence intensity and low cross-interference was selected as the optimal combination.

[0133] Example 3 Use of a Dual-Luciferase Reporter Gene Detection System

[0134] Expression plasmids containing the wild-type Gaussia luciferase (WT-Gluc) gene and the wild-type Pleuromamma xiphias luciferase (WT-Pxluc) gene as reporter genes were constructed and used in 0.5x10 4 HEK293 cells were pre-transfected into a 24-well plate, which was then placed in a cell culture tank and cultured overnight under conditions of 5% CO 2 and 37°C.

[0135] The next day, discard the culture medium from the culture plate / dish, add a sufficient amount of PBS, and gently wash the cells. Completely discard the PBS washing 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 vortex thoroughly for 30 seconds. The lysed cell suspension is used for luminescence measurement. Centrifuge at 100 g for 30 seconds, and then remove the supernatant for luminescence measurement.

[0136] Place 50 μL of cell suspension or supernatant into a black 96-well plate. Dilute coelenterazine f-CTZ to 100 μM in diluent (50 mM Tris-HCl pH 8.0, 100 mM NaCl, 0.1% (v / v) Tween-20). Add 50 μL of this solution to each well of the 96-well plate containing the cell suspension or supernatant. Read the luminescence intensity using the enzyme marker's autoluminescence module. After the measurement is complete, add 1x stop&glo reagent (Promega) to quench the f-CTZ luminescence. Add 100 μM N0 substrate in the same diluent to the same well. Read the luminescence intensity using the enzyme marker's autoluminescence module.

[0137] The results show that the dual-luciferase reporter gene detection system can detect luciferase expressed in cells.

[0138] It should be pointed out that the above description is only a preferred embodiment of the present invention, and those skilled in the art can make some improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered as the protection scope of the present invention.

Claims

1. A dual-luciferase reporter gene detection system, comprising Gaussia luciferase, Pleuromamma xiphias luciferase and a substrate, wherein the substrate is a compound of formula (I) or a stereoisomer, geometric isomer, tautomer, salt, nitroxide, hydrate or solvate of a compound of formula (I); 【Chemical 1】 Here, R １ , R ２ are each independently H, D, F, Cl, Br, I, OH, or NH ２ , NO ２ , C.N., N. ３ , C １－６ Alkyl group, C ２－６ Alkenyl group, C ２－６ Alkynyl group, C １－６ Halogenated alkyl group, C １－６ Alkoxy group, C １－６ Hydroxyalkyl group or C １－６ is an alkylamino group, R ３ is an optionally substituted aryl, heterocyclic, or heteroaryl group; a １ is 0 or any integer from 1 to 6, a ２ is 0 or any integer from 1 to 6, b is 0 or 1, A dual luciferase reporter gene detection system, wherein c is 0, 1 or 2.

2. The Gaussia luciferase is I) Gaussia luciferase, the amino acid sequence of which is shown in SEQ ID NO: 1; II) A luciferase having at least 85% homology with 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 Gaussia luciferase activity even when one or more amino acid residues are modified, substituted, deleted or added to the amino acid sequence of the Gaussia luciferase described in I); 2. The dual-luciferase reporter gene detection system according to claim 1, wherein the dual-luciferase reporter gene detection system is any one of the following:

3. 3. The dual-luciferase reporter gene detection system of claim 2, wherein the Gaussia luciferase is an optimized variant, and the Gaussia luciferase variant has at least one of mutation sites at 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 compared to the amino acid sequence of SEQ ID NO:

1.

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

5. The Pleuromamma xiphias luciferase is i) the Pleuromamma xiphias luciferase whose amino acid sequence is shown in SEQ ID NO: 3; ii) a luciferase having at least 85% homology with 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); iii) a luciferase that still has Pleuromamma xiphias luciferase activity even when one or more amino acid residues are modified, substituted, deleted, or added to the amino acid sequence of the Pleuromamma xiphias luciferase described in i). The dual-luciferase reporter gene detection system of claim 1, wherein the luciferase is any one of the following:

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

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

8. The compound of formula (I) comprises at least one of the following additional technical features: The R １ is H, The R ２ is H, NH ２ , OH or C １－６ Alkylamino group, preferably H, NH ２ , 3-OH, 4-OH or dimethylamino; The R ３ is a substituted aryl group, preferably OH—C ６ H ５ or F.C. ６ H ５ and more preferably 4-OH-C ６ H ５ or 4-F-C ６ H ５ and a １ is 0 or 1, a ２ is 0 or 1, Preferably a １ is 0, then a ２ is 1, and a １ is 1, then a ２ is 0, or a １ is 0, then a ２ is 0, The b is 1, The dual-luciferase reporter gene detection system according to claim 1 , wherein c is 1.

9. The compound of formula (I) is 【Chemistry 2】 The dual-luciferase reporter gene detection system according to claim 8, comprising any two of the following:

10. The substrate for the Gaussia luciferase is 【Chemistry 3】 and The substrate for the Pleuromamma xiphias luciferase is 【Chemistry 4】 10. The dual luciferase reporter gene detection system according to claim 9, wherein:

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

12. 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 reagent comprises a cell imaging reagent, an amino acid labeling reagent, a protein labeling and localization reagent, a specific recognition reagent for an antibody, a nucleic acid labeling reagent or a gene sequencing reagent.

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

15. 15. The fluorescence detection kit according to claim 14, further comprising a reaction buffer, the reaction buffer comprising water, Tris-HCl, NaCl and Tween-20.

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

17. The detection method according to claim 16, wherein the sample is a cell, a culture containing the cell, or a cell lysate that expresses Gaussia luciferase and Pleuromamma xiphias luciferase according to any one of claims 1 to 11.

18. The detection method according to claim 16, wherein the sample is a nucleotide analog that specifically binds to Gaussia luciferase and Pleuromamma xiphias luciferase described in any one of claims 1 to 11, and the nucleotide analog specifically binds to the luciferase via a nucleophilic reagent.

19. 19. The method of claim 18, wherein the nucleophilic reagent is an antigen-antibody complex, biotin-streptavidin, or digoxin-digoxin antibody.

20. 20. The detection method of claim 18 or 19, wherein the nucleotide analogs are composed of nucleotide molecules containing four different base types, wherein a first nucleotide molecule is capable of specifically binding to Gaussia luciferase, a second nucleotide molecule is capable of specifically binding to Pleuromamma xiphias luciferase, a third nucleotide molecule is capable of specifically binding to Gaussia luciferase and Pleuromamma xiphias luciferase, and a fourth nucleotide molecule is capable of not binding to either Gaussia luciferase or Pleuromamma xiphias luciferase.

21. The detection method according to any one of claims 16 to 20, further comprising the step of determining the type of the nucleotide molecule based on the luminescence signal.

22. 1. A method for sequencing a nucleic acid molecule, comprising: (1) providing a nucleic acid molecule to be sequenced that is bound to a support, or binding the nucleic acid molecule to be sequenced to a support; (2) forming a reaction system including a solution phase and a solid phase by adding a primer for initiating a nucleotide polymerization reaction, a polymerase for carrying out a nucleotide polymerization reaction, and four kinds of compounds, wherein the four kinds of compounds are derivatives of nucleotides A, (T / U), C, and G, respectively, and have base-complementary pairing ability, and the hydroxyl group (—OH) at the 3′ position of ribose or deoxyribose in the four kinds of compounds is protected by a protecting group, and a first molecular beacon attached to the first compound; a second molecular beacon attached to the second compound; a first molecular label and a second molecular label are bound to a third compound, or a first molecular label is bound to some of the third compounds and a second molecular label is bound to other parts of the third compounds; a fourth compound having no molecular beacon attached thereto; (3) annealing a primer to the nucleic acid molecule to be sequenced, the primer forming a duplex that is bound to the support together with the nucleic acid molecule to be sequenced as an initial growing nucleic acid strand; (4) incorporating one of the four compounds onto the 3'-end of the growing nucleic acid strand by performing a nucleotide polymerization reaction using a polymerase under conditions that allow the polymerase to perform a nucleotide polymerization reaction; (5) contacting the double strand from the previous step with the Gaussia luciferase and Pleuromamma xiphias luciferase according to any one of claims 1 to 11 to carry out a binding reaction, wherein the Gaussia luciferase and Pleuromamma xiphias luciferase can specifically bind to the first molecular label and the second molecular label, respectively; causing the luciferase to undergo a fluorescent reaction in the presence of a substrate; and detecting the emitted fluorescent signal; (6) removing the protecting groups and molecular tags of the incorporated nucleotides; (7) optionally repeating steps (4)-(6) or (3)-(6) one or more times, thereby obtaining sequence information of the nucleic acid molecule.

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