WO2024031306A1 - 一种双荧光素酶报告基因检测系统及其应用 - Google Patents

一种双荧光素酶报告基因检测系统及其应用 Download PDF

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WO2024031306A1
WO2024031306A1 PCT/CN2022/111058 CN2022111058W WO2024031306A1 WO 2024031306 A1 WO2024031306 A1 WO 2024031306A1 CN 2022111058 W CN2022111058 W CN 2022111058W WO 2024031306 A1 WO2024031306 A1 WO 2024031306A1
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luciferase
mutated
gaussia
dual
nucleotide
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PCT/CN2022/111058
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English (en)
French (fr)
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卓世添
滕波
章文蔚
陈奥
徐讯
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深圳华大生命科学研究院
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/66—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving luciferase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence

Definitions

  • the invention relates to the technical field of molecular biology, and in particular to a dual-luciferase reporter gene detection system and its application.
  • Luciferase is the collective name for enzymes that can produce bioluminescence in nature. Luciferase reporter assay refers to a detection system that uses luciferin as a substrate to detect luciferase activity. It is used in cell imaging. , amino acid labeling, protein labeling and positioning, specific recognition of antibodies, nucleic acid labeling, construction of dual reporter genes and gene sequencing, etc., are widely used.
  • Coelenterazine is the most abundant natural fluorescein in nature. Coelenterazine can serve 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), is more convenient for in vivo bioluminescence research, and has higher luminescence brightness. Therefore, coelenterazine is often used as a luminescent substrate for reporter gene assays based on fluorescence analysis as well as for live animal assays.
  • ATP adenosine triphosphate
  • Dual luciferase luminescence systems are widely used in gene sequencing, construction of dual reporter genes, and protein localization. This method has the advantages of high sensitivity, low interference, convenient detection, and wide detection range. Compared with single luciferase detection, multiplex detection is more suitable for the following aspects: studying the expression regulation of multiple genes at the same time; reducing off-target effects; identifying the interaction between two or more signaling pathways; and reducing "artifacts" produced by the experimental system. "Normalized.
  • the existing dual-luciferase luminescence system is mainly a dual-color detection composed of firefly luciferase and Renilla luciferase.
  • Firefly luciferase requires the simultaneous presence of luciferin, oxygen, ATP and magnesium ions to emit light; while Renilla luciferase only requires coelenterazine and oxygen to emit light.
  • the difference in reaction conditions between the two luciferase systems limits their application in the same scenario.
  • the present invention provides a dual-luciferase reporter gene detection system and its application.
  • the invention provides a dual-luciferase reporter gene detection system, including Gaussia luciferase, Pleuromamma xiphias luciferase and substrate,
  • the substrate is a compound represented by formula (I), or a stereoisomer, geometric isomer, tautomer, salt, nitrogen oxide, or hydrate of a compound represented by formula (I) or solvate:
  • R 1 and R 2 are independently H, D, F, Cl, Br, I, OH, NH 2 , NO 2 , CN, N 3 , C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1-6 haloalkyl, C 1-6 alkoxy, C 1-6 hydroxyalkyl or C 1-6 alkylamino;
  • R 3 is optionally substituted aryl, heterocyclyl or heteroaryl
  • a 1 is 0 or any integer from 1 to 6,
  • a 2 is 0 or any integer from 1 to 6,
  • b 0 or 1
  • c 0, 1 or 2.
  • the Gaussia luciferase is any one of the following I) to III):
  • luciferase having at least 85% homology with the amino acid sequence of the Gaussia luciferase described in I) and having the same or similar functions as the Gaussia luciferase described in I);
  • a luciferase in which the amino acid sequence of Gaussia luciferase as described in I) is modified, substituted, deleted or added with one or more amino acid residues and still has Gaussia luciferase activity.
  • the Gaussia luciferase is not optimized, it 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).
  • it has at least 85% homology with the amino acid sequence of Gaussia luciferase in i), such as at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93% , 94%, 95%, 96%, 97%, 98% or 99% homology, and a luciferase having the same or similar functions as the Gaussia luciferase in i).
  • the Gaussia luciferase is an optimized mutant, that is, a Gaussia luciferase mutant.
  • the Gaussia luciferase mutant has the following mutations. At least one of the 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 bits.
  • the Gaussia fluorescein mutant has at least one of the following mutations:
  • V at position 29 is mutated to F or L;
  • a at position 30 mutates to G or D;
  • a at position 32 is mutated to V
  • a at position 36 is mutated to V or I;
  • T at position 37 is mutated to N or E;
  • the L at position 40 is mutated to I or T;
  • the K at position 66 is mutated to P, S, I, R or N;
  • P at position 84 is mutated to A, L, K or V;
  • the E at position 102 is mutated to D, A, S, K or N;
  • a at position 104 is mutated to G;
  • the E at position 110 is mutated to P, G or A;
  • the L at position 124 is mutated to M, G or I;
  • V at position 138 is mutated to E or D;
  • the Q at position 152 is mutated to R or H;
  • the S at position 170 is mutated to N or T;
  • a at position 182 is mutated to M
  • the G at position 183 is mutated to N or A.
  • the mutant Compared with wild-type Gaussia luciferase, the mutant has a broader substrate spectrum, stronger specificity, significantly enhanced luminescence brightness, and significantly improved detection accuracy in practical applications.
  • the Gaussia luciferase may or may not include a signal peptide amino acid sequence.
  • the Pleuromamma xiphias luciferase is any one of the following i) to iii):
  • a luciferase that has at least 85% homology with the amino acid sequence of the Pleuromamma xiphias luciferase described in i), and has the same or similar functions as the Pleuromamma xiphias luciferase described in i);
  • the Pleuromamma xiphias luciferase is not optimized, it is from Pleuromamma xiphias, and the amino acid sequence is the sequence with PMID number 23886588 (as shown in SEQ ID NO: 3).
  • it has at least 85% homology with the amino acid sequence of i) Pleuromamma xiphias luciferase, such as at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98% or 99% homology, and a luciferase having the same or similar functions as the Pleuromamma xiphias luciferase in i).
  • the Pleuromamma xiphias luciferase is an optimized mutant, that is, the Pleuromamma xiphias luciferase mutant. Compared with the amino acid sequence shown in SEQ ID NO: 3, the Pleuromamma xiphias luciferase mutant It has at least one of the following mutation sites: position 81, position 82, position 83 and position 84.
  • the Pleuromamma xiphias fluorescein mutant has at least one of the following mutations:
  • the G at position 81 is mutated to L or P or Q or S or T;
  • Q at position 82 is mutated to R or W or I or Y or A or L or F or V or P or E or M;
  • the G at position 83 is mutated to S or Q or R or W or T or A or L;
  • the G at position 84 is mutated to F or R or S or C or Y or L or I or K or V or P.
  • the mutant Compared with wild-type Pleuromamma xiphias luciferase, the mutant has a wider substrate spectrum, stronger specificity, significantly enhanced luminescence brightness, and the detection accuracy is significantly improved in practical applications.
  • the Pleuromamma xiphias luciferase may or may not include a signal peptide amino acid sequence.
  • the Pleuromamma xiphias luciferase includes a signal peptide amino acid sequence; preferably, the Pleuromamma xiphias luciferase amino acid sequence including a signal peptide is as shown in SEQ ID NO: 4.
  • the compound represented by formula (I) may further include at least one of the following additional technical features:
  • R 1 is H.
  • the R 2 is H, NH 2 , OH or C 1-6 alkylamino, 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 FC 6 H 5 , more preferably 4-OH-C 6 H 5 or 4-FC 6 H 5 .
  • a 1 is 0 or 1.
  • a 2 is 0 or 1.
  • a 2 when a 1 is 0, a 2 is 1; when a 1 is 1, a 2 is 0; or when a 1 is 0, a 2 is 0.
  • b is 1.
  • c is 1.
  • the compound represented by formula (I) includes any two of the following:
  • the substrate of Gaussia luciferase is
  • the substrate of Pleuromamma xiphias luciferase is
  • the molar ratio of Gaussia luciferase and Pleuromamma xiphias luciferase is 1: (0.01-100);
  • the molar ratio of Gaussia luciferase to substrate is 1:(1-1000);
  • the molar ratio of Pleuromamma xiphias luciferase to substrate is 1:(1-1000).
  • 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.
  • the present invention also provides a fluorescence detection kit, which includes the dual-luciferase reporter gene detection system of the present invention.
  • the fluorescence detection reagent further includes a reaction buffer, and the reaction buffer includes water, Tris-HCl, NaCl and Tween-20.
  • the invention also provides a method for detecting luciferase luminescence signals, which includes contacting the substrate in the dual-luciferase reporter gene detection system of the invention with a sample, and detecting Luminescent signal generated by luciferase; the sample contains Gaussia luciferase and Pleuromamma xiphias luciferase.
  • the sample is a nucleic acid, a nucleic acid analog, a cell, a cell-containing culture, or a product prepared from a cell-containing culture.
  • the cells are bacterial cells, fungal cells, animal cells, plant cells or human cells.
  • the nucleic acid is DNA or RNA, which is not limited in the present invention.
  • the sample is a cell expressing Gaussia luciferase and Pleuromamma xiphias luciferase described in the dual-luciferase reporter gene detection system of the present invention, a culture containing cells, or a cell lysate.
  • the sample is a nucleotide analog that specifically binds Gaussia luciferase and Pleuromamma xiphias luciferase in the dual-luciferase reporter gene detection system of the present invention, and the nucleotide is similar to The substance binds specifically to luciferase via a nucleophile.
  • the nucleophile is an antigen-antibody complex, biotin-streptavidin or digoxin-digoxin antibody.
  • the nucleotide analog consists of nucleotide molecules containing four different base types, of which the first nucleotide molecule can specifically bind to Gaussia luciferase, and the second nucleotide molecule can bind to Pleuromamma
  • the third nucleotide molecule specifically binds to Gaussia luciferase and Pleuromamma xiphias luciferase.
  • the fourth nucleotide molecule neither binds to Gaussia luciferase nor to Pleuromamma xiphias luciferase. Pleuromamma xiphias luciferase binding.
  • the method further includes: determining the type of the nucleotide molecule through the luminescent signal.
  • the present invention also provides a method for sequencing nucleic acid molecules, which includes the following steps:
  • nucleic acid molecules to be sequenced connected to the support, or connect nucleic acid molecules to be sequenced to the support;
  • the first compound has a first molecular label attached to it
  • the second compound has a second molecular label attached to it
  • the third compound is connected to a first molecular label and a second molecular label, or part of the third compound is connected to a first molecular label and another part of the third compound is connected to a second molecular label, and the fourth compound is not connected to a molecular label;
  • Gaussia luciferase and Pleuromamma xiphias luciferase in the dual-luciferase reporter gene detection system of the present invention and perform a binding reaction.
  • the Gaussia luciferase and Pleuromamma xiphias luciferase xiphias luciferase can specifically bind to the first molecular marker and the second molecular marker respectively, and then cause the luciferase to undergo a fluorescent reaction in the presence of a substrate to detect the emitted fluorescent signal;
  • connection of luciferase and nucleotide derivatives through the connection of luciferase and nucleotide derivatives, self-luminescence detection of nucleotides to be sequenced is achieved, thereby eliminating the need for additional excitation light sources.
  • the connection of luciferase to nucleotides is achieved through specific binding of a label on luciferase to a corresponding label on the nucleotide derivative.
  • the first nucleotide is connected to a first luciferase
  • the second nucleotide is connected to a second luciferase
  • the third nucleotide is connected to the first luciferase and the second luciferase.
  • the fourth nucleotide does not have any luciferase attached to it. Then the corresponding substrates of the two luciferase enzymes are passed in to detect the luminescence signals of the four bases; when the substrate of the first luciferase is passed in, the first nucleotide and the third nucleotide emit light.
  • the substrate of the second luciferase is passed in, the second nucleotide and the third nucleotide emit light; when the substrate of the first luciferase and the substrate of the second luciferase are passed in, the fourth core None of the glucosides emit light. Therefore, the bases can be identified based on the luminescence of the four nucleotides.
  • the method for sequencing nucleic acid molecules according to the present invention includes:
  • nucleic acid molecules to be sequenced connected to the support, or connect nucleic acid molecules to be sequenced to the support;
  • the first compound has a first molecular label attached to it
  • the second compound has a second molecular label attached to it
  • the third compound is connected to a first molecular label and a second molecular label, or part of the third compound is connected to a first molecular label and another part of the third compound is connected to a second molecular label, and the fourth compound is not connected to a molecular label;
  • the present invention combines wild-type/mutant Gaussia Luciferase (Gaussia Luciferase) and wild-type/mutant Pleuromamma xiphias luciferase (Pleuromamma xiphias, AB716975).
  • Gaussia Luciferase Gaussia Luciferase
  • Pleuromamma xiphias luciferas AB716975.
  • the invention discloses a dual-luciferase reporter gene detection system and its application. Those skilled in the art can learn from the content of this article and appropriately improve the process parameters for implementation. It should be noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are deemed to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments. Relevant persons can obviously make modifications or appropriate changes and combinations to the methods and applications described herein without departing from the content, spirit and scope of the present invention to achieve and Apply the technology of this invention.
  • substituents When more than one position in a given structural formula can be substituted by one or more substituents selected from a specific group, the substituents may be identically or differently substituted at each position.
  • alkyl as used herein includes 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 A saturated straight-chain or branched-chain monovalent hydrocarbon group with 1-2 carbon atoms, in which the alkyl group can be independently optionally substituted by one or more substituents described in the present invention.
  • alkyl groups include, but are not limited to, methyl (Me, -CH 3 ), ethyl (Et, -CH 2 CH 3 ), n-propyl (n-Pr, -CH 2 CH 2 CH 3 ), isopropyl (i-Pr, -CH(CH 3 ) 2 ), n-butyl (n-Bu, -CH 2 CH 2 CH 2 CH 3 ), isobutyl (i-Bu, -CH 2 CH(CH 3 ) 2 ), sec-butyl (s-Bu, -CH(CH 3 )CH 2 CH 3 ), tert-butyl (t-Bu, -C(CH 3 ) 3 ), n-pentyl (- CH 2 CH 2 CH 2 CH 3 ), 2-pentyl (-CH(CH 3 )CH 2 CH 2 CH 3 ), 3-pentyl (-CH(CH 2 CH 3 ) 2 ), 2-methyl Base-2-butyl (-C(CH 3 ), methyl
  • alkynyl means a linear or branched monovalent hydrocarbon group 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 It is in an unsaturated state, that is, one carbon-carbon is an sp triple bond, in which the alkynyl group can be independently optionally substituted by one or more substituents described in the present invention.
  • substituents described in the present invention include, but are not limited to, acetylene. radical (-C ⁇ CH) and propargyl (-CH 2 C ⁇ CH).
  • halogen refers to F, Cl, Br or I.
  • unsaturated as used herein means a moiety containing one or more degrees of unsaturation.
  • alkoxy or "alkyloxy” as used in the present invention refers to an alkyl group, as defined in the present invention, connected to other parts of the compound molecule through an oxygen atom.
  • alkyl The oxygen group is a C 1-4 alkoxy group; such examples include, but are not limited to, methoxy, ethoxy, propoxy, butoxy, and the like.
  • the alkoxy group may be independently unsubstituted or substituted by one or more substituents described herein.
  • haloalkyl refers to the case where an alkyl, alkenyl or alkyloxy group may be substituted by one or more halogen atoms.
  • the haloalkyl group is Halogenated C 1-6 alkyl.
  • the haloalkyl group is haloC 1-3 alkyl.
  • haloalkyloxy or haloalkoxy is haloC 1-6 alkyloxy or haloC 1-6 alkoxy.
  • haloalkyloxy or haloalkoxy is haloC 1-3 alkyloxy or haloC 1-3 alkoxy.
  • Such examples include, but are not limited to, trifluoromethyl, 2-chloro-vinyl, 2,2-difluoroethyl, trifluoromethoxy, and the like.
  • the "haloalkyl”, “haloalkenyl” and “haloalkyloxy” groups can be independently optionally substituted by one or more substituents described in the present invention.
  • cycloalkyl or "cycloalkane” means a monovalent or multivalent saturated monocyclic, bicyclic or tricyclic carbocyclic ring system containing 3 to 12 carbon atoms, but never containing aromatic rings.
  • the cycloalkyl group contains 3-12 carbon atoms; in another embodiment, the cycloalkyl group contains 3-8 carbon atoms; in yet another embodiment, the cycloalkyl group contains 3-6 carbon atoms carbon atom.
  • Such examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like.
  • the cycloalkyl groups may independently be unsubstituted or substituted with one or more substituents described herein.
  • heterocyclyl and “heterocycle” are used interchangeably herein and both refer to a saturated or partially unsaturated monocyclic, bicyclic or tricyclic ring containing 3 to 12 ring atoms, and never include aromatic rings, where At least one ring atom is a heteroatom.
  • the heterocyclyl group may be a carbon group or a nitrogen group, and the heteroatoms have the meanings described herein.
  • heterocyclyl 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-dioxopentyl, disulfide ring Pentyl, tetrahydropyranyl, dihydropyranyl, 2H-pyranyl, 4H-pyranyl, tetrahydrothiopyranyl, piperidyl, morpholinyl, thiomorpholinyl, piperazinyl , dioxanyl, dithianyl, thioxanyl, homopiperazinyl,
  • Examples of oxidized sulfur atoms in heterocyclyl groups include, but are not limited to, sulfolane groups and 1,1-dioxothiomorpholinyl groups.
  • the heterocyclyl group may be optionally substituted with one or more substituents described herein.
  • heteroaryl means monocyclic, bicyclic and tricyclic ring systems containing 5 to 12 ring atoms, or 5 to 10 ring atoms, or 5 to 6 ring atoms, at least one of which is an aromatic ring, and At least one ring system contains one or more heteroatoms, each of which contains a ring of 5-7 atoms and has one or more points of attachment to the rest of the molecule.
  • heteroaryl may be used interchangeably with the term “heteroaryl ring” or “heteroaromatic compound.”
  • the heteroaryl group is optionally substituted with one or more substituents described herein.
  • the heteroaryl group consisting of 5 to 10 atoms contains 1, 2, 3 or 4 heteroatoms independently selected from O, S and N, wherein the nitrogen atom can be further oxidized.
  • heteroaryl groups include, but are not limited to: furyl, imidazolyl (such as N-imidazolyl, 2-imidazolyl, 4-imidazolyl, 5-imidazolyl), isoxazolyl, oxazolyl (such as 2-oxazolyl, 4-oxazolyl, 5-oxazolyl), pyrrolyl (such as N-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl), pyridyl, pyrimidinyl (such as 2- Pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl), pyridazinyl, thiazolyl (such as 2-thiazolyl, 4-thiazolyl, 5-thiazolyl), tetrazolyl (such as 5-tetrazolyl), Triazolyl, thienyl (such as 2-thienyl, 3-thienyl), pyrazolyl, isothiazolyl, 1,2,3-oxadiazol
  • aminoalkyl includes C 1-10 straight or branched alkyl groups substituted with one or more amino groups. Some examples are that the aminoalkyl group is a C 1-6 alkyl group substituted by one or more amino groups. Such examples include, but are not limited to: aminomethyl, aminoethyl, aminopropyl, Aminobutyl and aminohexyl. The aminoalkyl group may be optionally substituted with one or more substituents described herein.
  • hydroxyalkyl means an alkyl group substituted with one or more hydroxyl groups, wherein the alkyl group has the meaning described herein. Such examples include, but are not limited to, hydroxymethyl, hydroxyethyl, 1,2-dihydroxyethyl, and the like.
  • a ring system formed by a substituent bonded to a central ring means that the substituent may be substituted at any substitutable position on the ring.
  • formula (a) represents that the substituent R o can be mono- or poly-substituted at any position on the E ring that may be substituted.
  • the structural formulas described in the present invention include all isomeric forms (such as enantiomers, diastereomers, geometric isomers or conformational isomers): for example, R, S containing an asymmetric center Configuration, double bond (Z), (E) isomers, and (Z), (E) conformational isomers. Therefore, individual stereochemical isomers of the compounds of the invention or mixtures of enantiomers, diastereomers, geometric isomers or conformational isomers thereof are within the scope of the invention.
  • the structural formulas and compounds described in the present invention include all isomeric forms (such as enantiomers, diastereomers, geometric isomers or conformational isomers), salts, nitrogen oxides, etc. substance, hydrate or solvate. Therefore, the compounds of the present invention are individual stereochemical isomers, enantiomers, diastereomers, geometric isomers, conformational isomers, salts, nitrogen oxides, hydrates, solvates of the compounds also belong to 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 different atoms.
  • stereochemistry used in this invention are generally referred to the following documents: 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, constitute the present invention. part.
  • racemic mixture A 50:50 mixture of enantiomers is called a racemic mixture or racemate, which may result in a chemical reaction without stereoselectivity or stereospecificity.
  • racemic mixture and racemate refer to an equimolar mixture of two enantiomers that lacks optical activity.
  • tautomer or “tautomeric form” refers to isomers of structures of different energies that can be converted into each other through a low energy barrier.
  • proton tautomers i.e., proton-shifting tautomers
  • tautomers by proton migration such as keto-enol and imine-enamine isomerizations.
  • Valence (valency) tautomers include recombination of bonding electrons.
  • Hydrophilrate in the present invention refers to an association in which solvent molecules are water.
  • Solvents that form solvates include, but are not limited to: water, isopropyl alcohol, ethanol, methanol, dimethyl sulfoxide, ethyl acetate, acetic acid, and aminoethanol.
  • esters in the present invention refers to a compound of formula (I) containing a hydroxyl group forming an ester that is hydrolyzable in vivo.
  • esters are, for example, pharmaceutically acceptable esters that hydrolyze in humans or animals to yield the parent alcohol.
  • the hydrolyzable ester groups of the compound of formula (I) containing hydroxyl groups include, but are not limited to: phosphate group, acetoxymethoxy group, 2,2-dimethylpropionyloxymethoxy group, alkanoyl group, Benzoyl, phenylacetyl, alkoxycarbonyl, dialkylcarbamoyl and N-(dialkylaminoethyl)-N-alkylcarbamoyl, etc.
  • the "nitrogen oxide” of the present invention means that when the compound contains several amine functional groups, one or more nitrogen atoms can be oxidized to form N-oxide.
  • 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 such as peroxycarboxylic acid to form an N-oxide (see Advanced Organic Chemistry, Wiley Interscience, 4th edition, Jerry March, pages).
  • the N-oxides can be prepared by the method of L.W. Deady (Syn. Comm. 1977, 7, 509-514), for example by reacting an amine compound with m-chloroperoxybenzoic acid (MCPBA) in an inert solvent such as methylene chloride. )reaction.
  • MCPBA m-chloroperoxybenzoic acid
  • the luciferase combination provided by the invention includes two luciferases, one of which is Gaussia luciferase and the other is Pleuromamma xiphias luciferase.
  • Gaussia luciferase also known as "Gaussian luciferase"
  • Gaussia luciferase is a protein with a molecular weight of 20kDa. It is derived from the bioluminescent enzyme of the marine copepod animal Gaussian matrix (Gaussia princeps).
  • the Gaussian luciferase receptor molecule is very It is small and does not require the assistance of ATP. In the presence of oxygen molecules, it catalyzes the oxidation of coelenterazine and emits light.
  • Pleuromamma xiphias luciferase is a copepod luciferase, also known as Daphnia luciferase. It can catalyze the oxidation of coelenterazine and emit light in the presence of oxygen molecules.
  • the present invention screens a combination of luciferases, and the results show that compared with other luciferase combinations, it can produce stronger fluorescence without causing cross-interference.
  • the luciferase in the luciferase reporter gene detection system of the present invention, can exist in the form of encoded nucleic acid, expression module, expression vector, recombinant strain, expression product of the recombinant strain or purified expression product.
  • the detection system of the present invention includes a mixture of a nucleic acid encoding Gaussia luciferase and a nucleic acid encoding Pleuromamma xiphias luciferase, or it can also be two independently existing nucleic acids encoding Gaussia luciferase. It can also be a fusion fragment formed by a nucleic acid encoding Pleuromamma xiphias luciferase or a nucleic acid encoding Gaussia luciferase and a nucleic acid encoding Pleuromamma xiphias luciferase, which is not limited by the present invention.
  • the expression module includes a promoter, coding nucleic acid and terminator.
  • the nucleic acid encoding Gaussia luciferase and the nucleic acid encoding Pleuromamma xiphias luciferase are in the same expression module, or they can be in two different expression modules.
  • the promoters of the two expression modules where the two coding nucleic acids are located may be the same or different, and the invention is not limited thereto.
  • the expression vector includes a backbone vector and coding nucleic acid.
  • the expression vector includes, but is not limited to, plasmid vectors or viral vectors.
  • the nucleic acid encoding Gaussia luciferase and the nucleic acid encoding Pleuromamma xiphias luciferase can be expressed in the same expression vector, or they can be expressed in different expression vectors. The present invention does not limit this.
  • the backbone carriers can be the same or different.
  • the expression strain of the present invention is also called a recombinant host, which can express Gaussia luciferase and/or Pleuromamma xiphias luciferase, that is, Gaussia luciferase and Pleuromamma xiphias luciferase are expressed in the same host, or they can be expressed in different Expression in the host 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 virus infection. It can be Escherichia coli, yeast, animal cells or human cells, which is not limited in the present invention. To express two luciferases in two different hosts, the same host cell or different host cells can be used.
  • 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. It can be a mixture of two strains expressing luciferase expressed separately, or it can be a mixture of strains expressing two luciferases. product.
  • reporter gene is a molecular biology concept, which refers to a type of gene that is expressed in cells, tissues/organs or individuals under specific circumstances and causes them to produce traits that are easy to detect and that the experimental materials would not otherwise produce.
  • a gene is a gene that codes for a protein or enzyme that can be detected. As a reporter gene, it must meet the following conditions in terms of genetic selection and screening detection: 1. It has been cloned and the full sequence has been determined; 2. The expression product does not exist in the recipient cells, that is, there is no background, and when it is transfected There are no similar endogenous expression products in the cells; 3.
  • the expression products can be quantitatively measured; when used, including but not limited to the following methods of use: fusing the reporter gene and the gene expression regulatory sequence to form a chimeric gene, Or it can be fused with other target genes to express nucleic acids under the control of regulatory sequences, so that its expression products can be used to detect the expression regulation of target genes and study nucleic acids.
  • the luciferase reporter gene detection system is also called a dual-luciferase luminescence system, which includes the luciferase composition and substrate of the present invention.
  • the luciferase composition and the substrate exist independently and emit light after being mixed with the detection system during detection.
  • the present invention screens the substrate, and the luciferase reporter gene detection system finally obtained can have higher fluorescence intensity, thereby improving the sensitivity of detection.
  • the detection reagents include substances that enable the test substance to overexpress Gaussia luciferase and Pleuromamma xiphias luciferase.
  • the substances that enable the test substance to overexpress Gaussia luciferase and Pleuromamma xiphias luciferase include, but are not limited to, plasmid vectors containing nucleic acids encoding Gaussia luciferase and/or nucleic acids encoding Pleuromamma xiphias luciferase, or viruses. carrier.
  • polynucleotide refers to deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or analogs thereof. Polynucleotides may be single-stranded, double-stranded, or contain both single- and double-stranded sequences. Both. Polynucleotide molecules can be derived from double-stranded DNA (dsDNA) forms (e.g., genomic DNA, PCR and amplification products, etc.), or can be derived from single-stranded forms of DNA (ssDNA) or RNA and can be converted to dsDNA form, and vice versa. The exact sequence of the polynucleotide molecule may be known or unknown.
  • dsDNA double-stranded DNA
  • ssDNA single-stranded forms of DNA
  • RNA can be converted to dsDNA form
  • genes or gene fragments e.g., probes, primers, EST or SAGE tags
  • genomic DNA genomic DNA fragment
  • exon intron
  • messenger RNA mRNA
  • transfer RNA transfer RNA
  • ribosomal RNA ribozyme
  • cDNA recombinant polynucleotide
  • synthetic polynucleotide branched polynucleotide
  • plasmid vector, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, primers or amplified copies of any of the above sequences.
  • Polynucleotides may include nucleotides or nucleotide analogs. Nucleotides usually 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.
  • Nucleotides usually 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.
  • 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 (d
  • Nucleotide analogs containing modified bases may also be used in the methods described herein.
  • Exemplary modified bases that may be included in polynucleotides, whether with a native backbone or similar structures include, for example, inosine, xathanine, hypoxathanine, isocytosine, heteroguan Purine, 2-aminopurine, 5-methylcytosine, 5-hydroxymethylcytosine, 2-aminoadenine, 6-methyladenine, 6-methylguanine, 2-propylguanine, 2 -Propyladenine, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 15-halogenated uracil, 15-halogenated cytosine, 5-propynyluracil, 5-propyne Cytosine, 6-Azouracil, 6-Azocytosine, 6-Azothymine, 5-uracil, 4-thiouracil, 8-haloadenine or guanine, 8-aminoa
  • nucleotides include nucleotides A, C, G, T or U.
  • nucleotide A refers to a nucleotide containing adenine (A) or a modification or analog thereof, such as ATP, dATP.
  • Nucleotide G refers to nucleotides containing guanine (G) or its modifications or analogs, such as GTP and dGTP.
  • Nucleotide C refers to nucleotides containing cytosine (C) or its modifications or analogs, such as CTP and dCTP.
  • Nucleotide T refers to nucleotides containing thymine (T) or its modifications or analogs, such as TTP and dTTP.
  • Nucleotide U refers to nucleotides containing uracil (U) or its modifications or analogs, such as UTP and dUTP.
  • the present invention involves labeling nucleotides with different labels individually or in combination, thereby allowing different luciferase enzymes to be attached to the nucleotides.
  • the molecular tag used to label a nucleotide and the tag that specifically binds thereto may be any pairing of molecules capable of specifically binding to each other. Specific binding between pairing members enables the linkage of nucleotides to luciferase.
  • Exemplary pair members include, but are not limited to: (a) haptens or antigenic compounds in combination with corresponding antibodies or binding portions or fragments thereof, e.g., digoxin-digoxin antibodies, N3G-N3G antibodies, FITC-FITC antibodies; ; (b) Nucleic acid aptamers and proteins; (c) Non-immune binding pairs (such as biotin-avidin, biotin-streptavidin, biotin-neutral avidin); (d) hormones - Hormone binding proteins; (e) receptors - receptor agonists or antagonists; (f) homolectins - carbohydrates; (g) enzymes - enzyme cofactors and (h) enzymes - enzyme inhibitors;.
  • haptens or antigenic compounds in combination with corresponding antibodies or binding portions or fragments thereof, e.g., digoxin-digoxin antibodies, N3G-N3G antibodies, FITC-FITC antibodies; ;
  • the first and second molecular markers 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.
  • the first molecular marker is biotin
  • the first luciferase can be streptavidin-labeled luciferase
  • the second molecular marker is digoxigenin
  • the luciferase may be a luciferase different from the first luciferase labeled with a digoxigenin antibody.
  • the first luciferase is the Gaussia luciferase of the present invention or a mutant thereof
  • the second luciferase is the Pleuromamma xiphias luciferase of the present invention or a mutant thereof.
  • a first compound has a first molecular label attached means that all of the first compounds have a first molecular label attached, or that some of the first compounds have a first molecular label attached but the remaining first compounds do not. Molecular markers.
  • the expression "the second compound is connected to a second molecular label” means that all the second compounds are connected to the second molecular label, or that some of the second compounds are connected to the second molecular label but the remaining second compounds are not connected to the molecular label.
  • the third compound is connected to the first molecular label and the second molecular label.
  • all the third compounds are connected to the first molecular label and the second molecular label, or some of the third compounds are connected to the first molecular label and the second molecular label.
  • Molecular tag but the remaining third compound has no attached molecular tag.
  • the dual-luciferase reporter gene detection system of the present invention is suitable for sequencing by synthesis.
  • Sequencing by synthesis is a variety of sequencing by synthesis methods well known in the art. Basically, sequencing by synthesis involves first hybridizing a sequenced nucleic acid molecule to a sequencing primer, followed by polymerizing a nucleoside as described herein at the 3' end of the sequencing primer using the sequenced nucleic acid molecule as a template in the presence of a polymerase Acid or nucleotide analogs. After polymerization, the nucleotide molecule type is identified by detecting the fluorescence signal emitted by the luciferase enzyme. After luciferase is removed from the labeled nucleotides, the next cycle of polymerization sequencing is performed.
  • the method for determining the sequence of a target polynucleotide can be performed by denaturing the sequence of the target polynucleotide, contacting the target polynucleotide with different nucleotides respectively to form a complement of the target nucleotide, and detecting Incorporation of the nucleotides.
  • the method utilizes polymerization such that the polymerase extends the complementary strand by incorporating the correct nucleotides complementary to the target.
  • the polymerization reaction also requires special primers to initiate the polymerization.
  • the incorporation of the nucleotide is carried out by the polymerase, and the incorporation events are subsequently measured.
  • polymerase Many different polymerases exist, and determining the most suitable polymerase is readily apparent to one of ordinary 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 can also be used.
  • the sequencing method is preferably performed on target polynucleotides arranged on a solid support.
  • Multiple target polynucleotides can be immobilized on the solid support via linker molecules, or can be attached to particles such as microspheres, which can also be attached to a solid support material.
  • the polynucleotide can be attached to the solid support by a variety of methods, including the use of biotin-streptavidin interaction.
  • Methods for immobilizing polynucleotides on solid supports are well known in the art and include lithography techniques and spotting each polynucleotide at a specific location 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.
  • the support is usually flat, although microbeads (microspheres) can also be used, and the latter can also be attached to other solid supports by known methods.
  • the microspheres may be of any suitable size, typically ranging from 10 to 100 nanometers in diameter.
  • the polynucleotide is attached directly to a flat surface, preferably to a flat glass surface.
  • the connection is preferably in the form of a covalent bond.
  • the array used is preferably a single molecule array comprising polynucleotides located in unique optically resolvable regions, for example as described in International Application No. WO00/06770.
  • a primer sequence that is recognized by the polymerase and serves as the starting site for subsequent extension of the complementary strand usually must first anneal to the target polynucleotide. role.
  • the primer sequence can be added as a separate component relative to the target polynucleotide.
  • the primer and the target polynucleotide may respectively be part of a single-stranded molecule, and the primer part and part of the target form an intramolecular duplex, that is, a hairpin loop structure.
  • the structure can be immobilized on the solid support at any location on the molecule.
  • Other conditions necessary to carry out the polymerase reaction are well known to those skilled in the art and include temperature, pH and buffer composition.
  • nucleotides or nucleotide analogs of the invention are contacted with the target polynucleotide to enable polymerization.
  • the nucleotides or nucleotide analogs may be added sequentially, i.e. each type of nucleotide (A, C, G or T/U) separately, or multiple types of nucleotides (A , C, G or T/U).
  • the polymerization step is allowed to proceed for a time sufficient to incorporate one nucleotide.
  • Unincorporated nucleotides are then removed, for example, by removing the solution phase of the reaction system from the previous step, leaving the duplex attached to the support.
  • Two luciferases containing different luciferases can then be added to perform a binding reaction.
  • the two luciferases can specifically bind to the molecular tags on the nucleotide, thereby realizing the interaction between the luciferase and the incorporated nucleic acid.
  • the connection of nucleotides. Identification of the incorporated nucleotides is then achieved by adding the corresponding luciferase substrate and detecting the fluorescence signal.
  • deoxyribonucleotide analogs are labeled with different small molecule markers biotin (abbreviated as B) and digoxigenin (abbreviated as D), for example, nucleotide A is labeled with B, nucleoside The acid C labels B and D, the nucleotide T labels D, and the nucleotide G is not labeled.
  • B biotin
  • D digoxigenin
  • nucleotide A is labeled with B
  • the nucleotide T labels D the nucleotide G is not labeled.
  • the 3’-terminal hydroxyl groups of the above four deoxyribonucleotide analogues labeled with different small molecules are all blocked to ensure that only one deoxyribonucleotide is combined in each sequencing reaction.
  • the four labeled deoxyribonucleotide analogs and the sequencing polymerase mixture are first introduced. Under the action of the polymerase, according to the principle of complementary base pairing, one deoxyribonucleotide analog is incorporated into the sequence. into the 3' end of the growing nucleic acid strand. Unbound deoxyribonucleotide analogs can be removed by removing the solution phase of the reaction system from the previous step, leaving the duplex attached to the support. Then, two luciferases containing different luciferases are added. The first luciferase is labeled with streptavidin, which binds to nucleotide A or nucleotide C labeled with small molecule B.
  • the second luciferase is labeled with streptavidin.
  • the enzyme is labeled with a digoxigenin antibody, which binds to nucleotide C or nucleotide T labeled with a small molecule D.
  • a digoxigenin antibody which binds to nucleotide C or nucleotide T labeled with a small molecule D.
  • the linkage of two luciferases containing different luciferases (Gaussia luciferase or mutants thereof, and Pleuromamma xiphias luciferase or mutants thereof) to labeled nucleotides and the signal Testing can be done separately.
  • the first luciferase is added, which is labeled with streptavidin and binds to nucleotide A or nucleotide C labeled with small molecule B.
  • a substrate for the first luciferase is added, the nucleotide connected to the first luciferase emits light, and a detector is used to detect the signal.
  • a detector is used to detect the signal.
  • add the second luciferase labeled with digoxigenin antibody, which binds to the nucleotide C or nucleotide T labeled with the small molecule D and then use an elution buffer to remove the unbound second luciferase.
  • Luciferase add the substrate of the second luciferase, the base connected to the second luciferase emits light, and use a detector to detect the signal. From this, the luminescence situation shown in the above table can be obtained, and the base can be determined. Identify.
  • Means of detecting fluorescent signals are well known in the art. For example, this can be achieved by a device that detects the wavelength of fluorescence.
  • a device that detects the wavelength of fluorescence.
  • Such devices are well known in the art.
  • a device may be a confocal scanning microscope that scans the surface of a solid support with a laser to image fluorophores that are directly bound to the nucleic acid molecules being sequenced.
  • each signal generated can be observed, for example, with a sensitive 2-D detector, such as a charge-coupled detector (CCD).
  • CCD charge-coupled detector
  • Other techniques such as scanning near-field optical microscopy (SNOM) may also be used, for example.
  • Gaussia luciferase (GenBank: AY015993.1) used in the examples was prepared from the E. coli expression system, and the expression vector is pET28a.
  • Pleuromamma xiphias luciferase (PMID: 23886588) was also prepared from the E. coli expression system, and the expression vector is pET28a.
  • Wild-type Gaussia luciferase (WT-Gluc) has the amino acid sequence shown below:
  • the wild-type Gaussia luciferase (WT-Gluc) gene has the nucleotide sequence shown below:
  • Wild-type Pleuromamma xiphias luciferase (WT-Pxluc) has the amino acid sequence shown below:
  • Wild-type Pleuromamma xiphias luciferase (WT-Pxluc) plus a signal peptide has the following amino acid sequence:
  • the wild-type Pleuromamma xiphias luciferase (WT-Pxluc) gene has the nucleotide sequence shown below:
  • the protein eluted from the Ni column was dialyzed with dialysis buffer (25mM Tris, pH 8.0, 250mM NaCl) overnight at 4°C.
  • concentration of luciferase was accurately determined using a BCA quantification kit (Thermo Scientific TM Pierce TM BCA Protein Assay Kit).
  • diluent 50mM Tris-HCl pH 8.0, 100mM NaCl, 0.1% (v/v) Tween-20
  • dilute both luciferases to 1 ⁇ g/mL, and add 10 ⁇ L each to a black 96-well plate.
  • Dilute the above substrate to 100 ⁇ M with the same diluent add 90 ⁇ L into the above 96-well plate containing luciferase (90 ⁇ L/well), and read the luminescence intensity using the self-luminescence module of a microplate reader.
  • the test data of Gaussia luciferase and Pleuromamma xiphias luciferase on different substrates are shown in Table 1.
  • the relative intensity is the percentage relative to the activity of wild-type Pleuromamma xiphias luciferase and compound N0.
  • the results showed that the fluorescence intensity of Gaussia luciferase combined with the substrate f-CTZ was the highest, and the fluorescence intensity of Pleuromamma xiphias luciferase combined with the substrate N0 was the highest.
  • the substrate f-CTZ is used to detect Gaussia luciferase
  • the substrate N0 is used to detect Pleuromamma xiphias luciferase, which is obtained after screening with high fluorescence brightness and low cross-interference. Best luminous combination.
  • E1-A3 based on SEQ ID NO:1, mutate the following sites: F26R, V29F, A32V, S33E, A36V, L40I, K66P, H79K, P84L, E102S, S103T, A104G, E110P, L124M, V138E;
  • the gene of the E1-A3 mutant has the nucleotide sequence shown below:
  • G2-F11 based on SEQ ID NO:1, mutate 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 of the G2-F11 mutant has the nucleotide sequence shown below:
  • 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.
  • the gene of the G2-F8 mutant has the nucleotide sequence shown below:
  • the gene of the P26-95 mutant has the nucleotide sequence shown below:
  • Example 1 construct an expression vector containing the above-mentioned Gaussia luciferase mutant gene and Pleuromamma xiphias luciferase mutant gene, construct recombinant bacteria, express the mutant luciferase, and conduct substrate and fluorescence Detection of luminescence intensity of enzyme.
  • Gaussia luciferase mutants E1-A3, G2-F11 and G2-F8 have the highest fluorescence intensity when combined with the substrate f-CTZ
  • Pleuromamma xiphias luciferase mutant P26-95 has the highest fluorescence intensity when combined with the substrate N0.
  • the substrate f-CTZ is used to detect the Gaussia luciferase mutant E1-A3, G2-F11 or G2-F8, and the substrate N0 is used to detect the Pleuromamma xiphias luciferase mutant.
  • P26-95 is the best luminescent combination with high fluorescence brightness and low cross-interference obtained after screening.
  • WT-Gluc wild-type Gaussia luciferase
  • WT-Pxluc wild-type Pleuromamma xiphias luciferase

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Abstract

一种双荧光素酶报告基因检测系统及其应用。所述检测系统包含Gaussia荧光素酶、Pleuromamma xiphias荧光素酶和底物，并且所述底物为腔肠素或其衍生物。相对于其他双荧光素酶报告基因检测系统，所述双荧光素酶报告基因检测系统信号更强，且反应条件简单。

Description

一种双荧光素酶报告基因检测系统及其应用 技术领域
本发明涉及分子生物学技术领域，特别涉及一种双荧光素酶报告基因检测系统及其应用。
背景技术
荧光素酶(Luciferase)是自然界中能够产生生物荧光的酶的统称，荧光素酶报告系统(Luciferase reporter assay)指的是以荧光素为底物来检测荧光素酶活性的检测系统，在细胞成像、氨基酸标记、蛋白标记及定位、抗体的特异性识别、核酸标记、构建双报告基因及基因测序等方面有着广泛的应用。
腔肠素是自然界中资源最丰富的天然荧光素。腔肠素可以作为许多荧光素酶的底物，如海肾荧光素酶、Gaussia分泌型荧光素酶，以及水母发光蛋白。与甲虫荧光素/荧光素酶系统不同，腔肠素/荧光素酶系统不需要三磷酸腺苷(ATP)，更便于体内生物荧光的研究，并且发光亮度更高。因此，腔肠素常用作基于荧光分析的报告基因检测以及活体动物检测的发光底物。
不同的腔肠素衍生物其发光波长，细胞膜渗透性，光量子效率上有所差异，使得其在相同应用上表现出不同的实验效果。由于不同荧光素酶的发光光谱特性和底物有所差异，因此，这为理论上构建低交叉干扰、高特异性的双荧光素酶发光系统提供了依据。
双荧光素酶发光系统在基因测序，构建双报告基因，以及蛋白定位等领域有着广泛的应用。该方法有着灵敏度高，干扰小，检测方便，检测范围广等优点。相较于单荧光素酶检测，多重检测更适用于以下方面：同时研究多个基因的表达调控；降低脱靶效应；识别两个或多个信号通路间的相互作用；将实验体系产生的“假象”归一化。
目前现有的双荧光素酶发光系统主要为萤火虫荧光素酶和海肾荧光素酶组合成的双色检测。萤火虫荧光素酶需要荧光素、氧气、ATP和镁离子同时存在的情况下才能发光；而海肾荧光素酶的发光仅需要腔肠素和氧气。两种荧光素酶系统反应条件的差异性限制了其在同一个场景上的应用。
发明内容
有鉴于此，本发明提供了一种双荧光素酶报告基因检测系统及其应用。
本发明提供双荧光素酶报告基因检测系统，包含Gaussia荧光素酶、Pleuromamma xiphias荧光素酶和底物，
并且所述底物为如式(I)所示的化合物，或式(I)所示的化合物的立体异构体、几何异构体、互变异构体、盐、氮氧化物、水合物或溶剂化物：
Figure PCTCN2022111058-appb-000001
其中，R 1、R 2分别独立地为H、D、F、Cl、Br、I、OH、NH 2、NO 2、CN、N 3、C 1-6烷基、C 2-6烯基、C 2-6炔基、C 1-6卤代烷基、C 1-6烷氧基、C 1-6羟基烷基或C 1-6烷基氨基；
R 3为任选取代的芳基、杂环基或杂芳基；
a 1为0或1-6的任意一个整数，
a 2为0或1-6的任意一个整数，
b为0或1，
c为0、1或2。
本发明中，所述Gaussia荧光素酶为如下I)～III)中任一种：
I)、所述Gaussia荧光素酶的氨基酸序列如SEQ ID NO:1所示；
II)、与I)所述Gaussia荧光素酶的氨基酸序列具有至少85％同源性且与I)所述Gaussia荧光素酶具有相同或相似功能的荧光素酶；
III)、如I)所述的Gaussia荧光素酶的氨基酸序列经修饰、取代、缺失或添加一个或多个氨基酸残基且仍具有Gaussia荧光素酶活性的荧光素酶。
一些实施例中，所述Gaussia荧光素酶未经优化，其来自Gaussia Luciferase，氨基酸序列为 GenBank登录号为AY015993.1的序列(如SEQ ID NO:1所示)。
一些实施例中，与i)所述Gaussia荧光素酶的氨基酸序列具有至少85％同源性，例如至少86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同源性，且与i)所述Gaussia荧光素酶具有相同或相似功能的荧光素酶。
一些实施例中，所述Gaussia荧光素酶为经过优化的突变体，即Gaussia荧光素酶突变体，相较于SEQ ID NO:1所示的氨基酸序列，所述Gaussia荧光素突变体具有如下突变位点中的至少之一：第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和183位。
一些实施例中，所述Gaussia荧光素突变体具有如下突变中的至少之一：
1)第24位的E突变为K；
2)第26位的F突变为R或L；
3)第27位的N突变为D；
4)第29位的V突变为F或L；
5)第30位的A突变为G或D；
6)第31位的V突变为I；
7)第32位的A突变为V；
8)第33位的S突变为E、R或K；
9)第36位的A突变为V或I；
10)第37位的T突变为N或E；
11)第40位的L突变为I或T；
12)第66位的K突变为P、S、I、R或N；
13)第79位的H突变为K；
14)第84位的P突变为A、L、K或V；
15)第88位的K突变为R；
16)第102位的E突变为D、A、S、K或N；
17)第103位的S突变为T；
18)第104位的A突变为G；
19)第110位的E突变为P、G或A；
20)第123位的D突变为N；
21)第124位的L突变为M、G或I；
22)第138位的V突变为E或D；
23)第152位的Q突变为R或H；
24)第163位的Q突变为D；
25)第170位的S突变为N或T；
26)第174位的G突变为K；
27)第175位的Q突变为E；
28)第178位的K突变为T；
29)第182位的A突变为M；
30)第183位的G突变为N或A。
所述突变体相较于野生型Gaussia荧光素酶，其底物谱更广、特异性更强、发光亮度显著增强，在实际应用中检测准确性得到显著提高。
一些实施例中，所述Gaussia荧光素酶可以包括或者不包括信号肽氨基酸序列。
本发明中，所述Pleuromamma xiphias荧光素酶为如下i)～iii)中任一种：
i)、所述Pleuromamma xiphias荧光素酶的氨基酸序列如SEQ ID NO:3所示；
ii)、与i)所述Pleuromamma xiphias荧光素酶的氨基酸序列具有至少85％同源性，且与i)所述Pleuromamma xiphias荧光素酶具有相同或相似功能的荧光素酶；
iii)、如i)所述的Pleuromamma xiphias荧光素酶的氨基酸序列经修饰、取代、缺失或添加一个或多个氨基酸残基且仍具有Pleuromamma xiphias荧光素酶活性的荧光素酶。
一些实施例中，所述Pleuromamma xiphias荧光素酶未经优化，其来自Pleuromamma xiphias，氨基酸序列为PMID号为23886588的序列(如SEQ ID NO:3所示)。
一些实施例中，与i)所述Pleuromamma xiphias荧光素酶的氨基酸序列具有至少85％同源性，例如至少86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同源性，且与i)所述Pleuromamma xiphias荧光素酶具有相同或相似功能的荧光素酶。
一些实施例中，所述Pleuromamma xiphias荧光素酶为经过优化的突变体，即Pleuromamma xiphias荧光素酶突变体，相较于SEQ ID NO:3所示的氨基酸序列，所述Pleuromamma xiphias荧光素突变体具有如下突变位点中的至少之一：第81位、82位、83位和84位。
一些实施例中，所述Pleuromamma xiphias荧光素突变体具有如下突变中的至少之一：
(1)第81位的G突变为L或P或Q或S或T；
(2)第82位的Q突变为R或W或I或Y或A或L或F或V或P或E或M；
(3)第83位的G突变为S或Q或R或W或T或A或L；
(4)第84位的G突变为F或R或S或C或Y或L或I或K或V或P。
所述突变体相较于野生型Pleuromamma xiphias荧光素酶，其底物谱更广、特异性更强、发光亮度显著增强，在实际应用中检测准确性得到显著提高。
一些实施例中，所述Pleuromamma xiphias荧光素酶可以包括或者不包括信号肽氨基酸序列。
一些实施例中，所述Pleuromamma xiphias荧光素酶包括信号肽氨基酸序列；优选地，所述包括信号肽的Pleuromamma xiphias荧光素酶氨基酸序列如SEQ ID NO:4所示.
本发明实施例中，式(I)所示化合物还可以进一步包括如下附加技术特征至少之一：
一些具体实施例中，所述R 1为H。
一些具体实施例中，所述R 2为H、NH 2、OH或C 1-6烷基氨基，优选为H、NH 2、3-OH、4-OH或二甲基氨基。
一些具体实施例中，所述R 3为取代的芳基，优选为OH-C 6H 5或F-C 6H 5，更优选为4-OH-C 6H 5或4-F-C 6H 5。
一些具体实施例中，所述a 1为0或1。
一些具体实施例中，所述a 2为0或1。
一些具体实施例中，当a 1为0时，a 2为1；当a 1为1时，a 2为0；或者当a 1为0时，a 2为0。
一些具体实施例中，所述b为1。
一些具体实施例中，所述c为1。
一些实施例中，所述式(I)所示化合物包括如下任意两种：
Figure PCTCN2022111058-appb-000002
Figure PCTCN2022111058-appb-000003
一些实施例中，所述Gaussia荧光素酶的底物为
Figure PCTCN2022111058-appb-000004
一些实施例中，所述Pleuromamma xiphias荧光素酶的底物为
Figure PCTCN2022111058-appb-000005
一些实施例中，所述Gaussia荧光素酶和Pleuromamma xiphias荧光素酶的摩尔比为1:(0.01-100)；
一些实施例中，所述Gaussia荧光素酶与底物的摩尔比为1:(1-1000)；
一些实施例中，所述Pleuromamma xiphias荧光素酶与底物的摩尔比为1:(1-1000)。
本发明中所述的双荧光素酶报告基因检测系统在制备检测试剂中的用途。
本发明中，所述检测试剂包括：细胞成像试剂、氨基酸标记试剂、蛋白标记及定位试剂、抗体的特异性识别试剂、核酸标记试剂或基因测序试剂。
本发明还提供了荧光检测试剂盒，其包括本发明所述的双荧光素酶报告基因检测系统。
本发明中，所述荧光检测试剂还包括反应缓冲液，所述反应缓冲液包括水和Tris-HCl、NaCl和Tween-20。
本发明还提供了一种荧光素酶发光信号的检测方法，其包括将本发明所述双荧光素酶报告基因检测系统中的底物与样品接触，检测由所述Gaussia荧光素酶和Pleuromamma xiphias荧光素酶产生的发光信号；所述样品中含有Gaussia荧光素酶和Pleuromamma xiphias荧光素酶。
本发明中，所述样品为核酸、核酸类似物、细胞、含有细胞的培养物或由含有细胞的培养物制得的产品。所述细胞为细菌细胞、真菌细胞、动物细胞、植物细胞或人体细胞。所述核酸为DNA或RNA，本发明对此不做限定。
一些实施例中，所述样品为表达本发明所述双荧光素酶报告基因检测系统中所述的Gaussia荧光素酶和Pleuromamma xiphias荧光素酶的细胞、含有细胞的培养物或者细胞裂解物。
另一些实施例中，所述样品为特异性结合本发明所述双荧光素酶报告基因检测系统中的Gaussia荧光素酶和Pleuromamma xiphias荧光素酶的核苷酸类似物，所述核苷酸类似物与荧光素酶与通过亲核试剂特异性结合。
所述亲核试剂为抗原-抗体复合物、生物素-链霉素亲和素或者地高辛-地高辛抗体。
所述核苷酸类似物由含有四种不同碱基类型的核苷酸分子组成，其中第一种核苷酸分子能够与Gaussia荧光素酶特异性结合，第二种核苷酸分子能够与Pleuromamma xiphias荧光素酶特异性结合，第三种核苷酸分子能够与Gaussia荧光素酶和Pleuromamma xiphias荧光素酶特异性结合，第四种核苷酸分子既不与Gaussia荧光素酶结合，也不与Pleuromamma xiphias荧光素酶结合。
本发明中，所述方法还包括：通过所述发光信号判断所述核苷酸分子的类型。
进一步的，本发明还提供了一种对核酸分子进行测序的方法，其包括以下步骤：
(1)提供连接于支持物上的待测序的核酸分子，或者将待测序的核酸分子连接于支持物上；
(2)添加用于起始核苷酸聚合反应的引物，用于进行核苷酸聚合反应的聚合酶，以及四种化合物，从而形成含有溶液相和固相的反应体系；其中，所述四种化合物分别为核苷酸A，(T/U)、C和G的衍生物，并且具有碱基互补配对能力；并且，所述四种化合物的核糖或脱氧核糖的3’位置处的羟基(-OH)被保护基团保护；并且，
第一化合物连接有第一分子标记，
第二化合物连接有第二分子标记，
第三化合物连接有第一分子标记和第二分子标记，或部分第三化合物连接有第一分子标记且 另外一部分第三化合物连接有第二分子标记，第四化合物没有连接分子标记；
(3)使引物退火至待测序的核酸分子上，所述引物作为起始的生长的核酸链，与所述待测序的核酸分子一起形成连接于支持物上的双链体；
(4)在允许聚合酶进行核苷酸聚合反应的条件下，使用聚合酶进行核苷酸聚合反应，从而将所述四种化合物中的一种掺入生长的核酸链的3’端；
(5)使上一步骤中的双链体与本发明所述双荧光素酶报告基因检测系统中Gaussia荧光素酶和Pleuromamma xiphias荧光素酶接触并进行结合反应，所述Gaussia荧光素酶和Pleuromamma xiphias荧光素酶分别能够特异性结合所述第一分子标记和第二分子标记，然后使所述荧光素酶在底物存在的情况下发生荧光反应，检测发出的荧光信号；
(6)去除所掺入的核苷酸的保护基团以及分子标记；
(7)任选地重复步骤(4)-(6)或(3)-(6)一次或多次，从而获得所述核酸分子的序列信息。
在本发明的实施方案中，通过荧光素酶与核苷酸衍生物的连接，实现了待测序核苷酸的自发光检测，从而不需要额外的激发光源。在具体实施方案中，通过荧光素酶上的标记物与核苷酸衍生物上的对应的标记物的特异性结合，实现了荧光素酶与核苷酸的连接。在具体的实施方案中，第一核苷酸连接上第一荧光素酶，第二核苷酸连接上第二荧光素酶，第三核苷酸连接上第一荧光素酶和第二荧光素酶，第四核苷酸不连接任何荧光素酶。然后分别通入两种荧光素酶的对应底物来检测四种碱基的发光信号；当通入第一荧光素酶的底物时，第一核苷酸和第三核苷酸发光，当通入第二荧光素酶的底物时，第二核苷酸和第三核苷酸发光；而通入第一荧光素酶的底物和第二荧光素酶的底物时，第四核苷酸均不发光。因而根据四种核苷酸的发光情况可以进行碱基的识别。
更进一步的，本发明所述的对核酸分子进行测序的方法，包括：
(1)提供连接于支持物上的待测序的核酸分子，或者将待测序的核酸分子连接于支持物上；
(2)添加用于起始核苷酸聚合反应的引物，用于进行核苷酸聚合反应的聚合酶，以及四种化合物，从而形成含有溶液相和固相的反应体系；其中，所述四种化合物分别为核苷酸A，(T/U)、C和G的衍生物，并且具有碱基互补配对能力；并且，所述四种化合物的核糖或脱氧核糖的3’位置处的羟基(-OH)被保护基团保护；并且，
第一化合物连接有第一分子标记，
第二化合物连接有第二分子标记，
第三化合物连接有第一分子标记和第二分子标记，或部分第三化合物连接有第一分子标记且 另外一部分第三化合物连接有第二分子标记，第四化合物没有连接分子标记；
(3)使引物退火至待测序的核酸分子上，所述引物作为起始的生长的核酸链，与所述待测序的核酸分子一起形成连接于支持物上的双链体；
(4)在允许聚合酶进行核苷酸聚合反应的条件下，使用聚合酶进行核苷酸聚合反应，从而将所述四种化合物中的一种掺入生长的核酸链的3’端；
(5)移除前一步骤的反应体系的溶液相，保留连接于支持物上的双链体，并加入两种不同的荧光素酶，进行结合反应，所述两种荧光素酶分别能够特异性结合所述第一分子标记和第二分子标记；
(6)用洗脱缓冲液去除未结合的荧光素酶；
(7)加入第一荧光素酶的底物，检测第一荧光信号；
(8)移除上一步反应的溶液；
(9)加入第二荧光素酶的底物，检测第二荧光信号；
(10)移除上一步反应的溶液；
(11)去除所掺入的核苷酸的保护基团以及分子标记；
(12)任选地移除上一步反应的溶液；
(13)任选地重复步骤(3)-(12)或(4)-(11)一次或多次，从而获得所述核酸分子的序列信息。
本发明将野生型/突变型Gaussia荧光素酶(Gaussia Luciferase)和野生型/突变型Pleuromamma xiphias荧光素酶(Pleuromamma xiphias,AB716975)进行组合。通过筛选不同的腔肠素衍生物与Gaussia荧光素酶和Pleuromamma xiphias荧光素酶的发光作用，得到低交叉干扰的双荧光素酶报告基因检测系统。相对于其他双荧光素酶的组合，该双荧光素酶组合反应条件简单，仅需要腔肠素和氧气，操作更简单。
具体实施方式
本发明公开了双荧光素酶报告基因检测系统及其应用，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
定义和一般术语
在本发明的描述中，除非另有说明，“多个”的含义是两个或两个以上。
本发明将会把确定的具体化的内容所对应的文献详细列出，实施例都伴随有结构式和化学式的图解。本发明有预期地涵盖所有的选择余地、变体和同等物，这些可能像权利要求所定义的那样包含在现有发明领域。所属领域的技术人员将识别许多类似或等同于在此所描述的方法和物质，这些可以应用于本发明的实践中去。本发明绝非限于方法和物质的描述。有很多文献和相似的物质与本发明申请相区别或抵触，其中包括但绝不限于术语的定义，术语的用法，描述的技术，或像本发明申请所控制的范围。
本发明将应用以下定义除非其他方面表明。根据本发明的目的，化学元素根据元素周期表，CAS版本和化学药品手册，75，thEd，1994来定义。另外，有机化学一般原理见"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，因此所有的内容都融合了参考文献。
术语“包含”为开放式表达，即包括本发明所指明的内容，但并不排除其他方面的内容。
像这里所描述的化合物可以任选地被一个或多个取代基所取代，如本发明中的通式化合物，或者像实施例里面特殊的例子，子类，和本发明所包含的一类化合物。应了解“任选取代的”这个术语与“取代或非取代的”这个术语可以交换使用。一般而言，术语“任选地”不论是否位于术语“取代的”之前，表示所给结构中的一个或多个氢原子被具体取代基所取代。除非其他方面表明，一个任选的取代基团可以有一个取代基在基团的各个可取代的位置进行取代。当所给出的结构式中不止一个位置能被选自具体基团的一个或多个取代基所取代，那么取代基可以相同或不同地在各个位置取代。其中所述的取代基可以是，但并不限于氢、F、Cl、Br、I、硝基、氰基、氧代(＝O)、羟基、烷基、羟基烷基、烷氨基、氨基烷基、卤代烷氧基、环烷基、氨基、芳基、杂环基、杂芳基、烯基、炔基、环烷基氧基、烷氧基、烷氧基烷基、卤代烷基、-COOH、-亚烷基-C(＝O)O-烷基、-亚烷基-S(＝O) 2-烷基、-亚烷基-S(＝O) 2-氨基、-S(＝O) 2-烷基、-S(＝O) 2-氨基、-S(＝O) 2OH、-O-亚烷基-C(＝O)O-烷基、-O-亚烷基-S(＝O) 2-烷基、-O-亚烷基-S(＝O) 2-氨基、-O-亚烷基-S(＝O) 2OH、-C(＝O)NH 2、-C(＝O)NH-烷基、-C(＝O)N(烷基)-烷基、-C(＝O)NHS(＝O) 2-烷基、-C(＝O)NHS(＝O) 2-氨基、-C(＝O)NHS(＝O) 2OH、-N(卤代烷基)-烷基、-N(烷基)-S(＝O) 2-烷基、-NHS(＝O) 2-烷基、-NHS(＝O) 2-卤代烷基、-N(烷基)S(＝O) 2-卤代烷基、-N(烷基)S(＝O) 2-烷氨基、-NHC(＝O)-烷基、 -NHC(＝O)-卤代烷基、-N(烷基)C(＝O)-卤代烷基、-N(烷基)C(＝O)-烷氨基、-N(烷基)C(＝O)O-烷基、-NHC(＝O)O-烷基、-NHC(＝O)O-卤代烷基、-N(烷基)C(＝O)O-卤代烷基、-N(烷基)C(＝O)O-氨基烷基、-NHC(＝O)-NH 2、-NHC(＝O)NH-(烷基)、-NHC(＝O)NH(卤代烷基)、-NHC(＝O)N(烷基)-烷基、-OC(＝O)-烷基、-OC(＝O)-氨基、-OC(＝O)-烷氨基、-OC(＝O)-氨基烷基、-OC(＝O)-烷氧基、-C(＝O)N(烷基)S(＝O) 2-烷基、-C(＝O)N(烷基)S(＝O) 2-氨基、-C(＝O)NH-S(＝O) 2OH、-C(＝NH)NH 2、-C(＝NH)NH-烷基、-C(＝NH)N(烷基)-烷基、-C(＝N-烷基)-NH 2、-C(＝O)NH-亚烷基-S(＝O) 2OH、-C(＝O)NHC(＝O)OH、-C(＝O)NHC(＝O)O-烷基、-C(＝O)N(烷基)C(＝O)O-烷基、-C(＝O)NH-亚烷基-C(＝O)OH和-C(＝O)NH-亚烷基-C(＝O)O-烷基，等等。
本发明使用的术语“烷基”包括1-20个碳原子，或1-10个碳原子，或1-6个碳原子，或1-4个碳原子，或1-3个碳原子，或1-2个碳原子饱和直链或支链的单价烃基，其中烷基可以独立任选地被一个或多个本发明所描述的取代基所取代。烷基基团更进一步的实例包括，但并不限于甲基(Me，-CH 3)、乙基(Et，-CH 2CH 3)、正丙基(n-Pr，-CH 2CH 2CH 3)、异丙基(i-Pr，-CH(CH 3) 2)、正丁基(n-Bu，-CH 2CH 2CH 2CH 3)、异丁基(i-Bu，-CH 2CH(CH 3) 2)、仲丁基(s-Bu，-CH(CH 3)CH 2CH 3)、叔丁基(t-Bu，-C(CH 3) 3)、正戊基(-CH 2CH 2CH 2CH 2CH 3)、2-戊基(-CH(CH 3)CH 2CH 2CH 3)、3-戊基(-CH(CH 2CH 3) 2)、2-甲基-2-丁基(-C(CH 3) 2CH 2CH 3)、3-甲基-2-丁基(-CH(CH 3)CH(CH 3) 2)、3-甲基-1-丁基(-CH 2CH 2CH(CH 3) 2)、2-甲基-1-丁基(-CH 2CH(CH 3)CH 2CH 3)、正己基(-CH 2CH 2CH 2CH 2CH 2CH 3)、2-己基(-CH(CH 3)CH 2CH 2CH 2CH 3)、3-己基(-CH(CH 2CH 3)(CH 2CH 2CH 3))、2-甲基-2-戊基(-C(CH 3) 2CH 2CH 2CH 3)、3-甲基-2-戊基(-CH(CH 3)CH(CH 3)CH 2CH 3)、4-甲基-2-戊基(-CH(CH 3)CH 2CH(CH 3) 2)、3-甲基-3-戊基(-C(CH 3)(CH 2CH 3) 2)、2-甲基-3-戊基(-CH(CH 2CH 3)CH(CH 3) 2)、2,3-二甲基-2-丁基(-C(CH 3) 2CH(CH 3) 2)、3,3-二甲基-2-丁基(-CH(CH 3)C(CH 3) 3)、正庚基和正辛基等等。术语“烷基”和其前缀“烷”在此处使用，都包含直链和支链的饱和碳链。
术语“烯基”表示2-12个碳原子，或2-8个碳原子，或2-6个碳原子，或2-4个碳原子直链或支链的一价烃基，其中至少一个位置为不饱和状态，即一个碳-碳为sp 2双键，其中烯基的基团可以独立任选地被一个或多个本发明所描述的取代基所取代，包括基团有“反”“正”或"E""Z"的定位，其中具体的实例包括，但并不限于乙烯基(-CH＝CH 2)、烯丙基(-CH 2CH＝CH 2)和烯丁基(-CH 2CH 2CH＝CH 2)等等。
术语“炔基”表示2-12个碳原子，或2-8个碳原子，或2-6个碳原子，或2-4个碳原子直链或支链 的一价烃基，其中至少一个位置为不饱和状态，即一个碳-碳为sp三键，其中炔基基团可以独立任选地被一个或多个本发明所描述的取代基所取代，具体的实例包括，但并不限于乙炔基(-C≡CH)和炔丙基(-CH 2C≡CH)。
术语“卤素”是指F、Cl、Br或I。
在本发明中所使用的术语“不饱和的”表示部分含有一个或多个不饱和度。
本发明中所使用的术语“烷氧基”或“烷基氧基”，涉及到烷基，像本发明所定义的，通过氧原子连接到化合物分子的其它部分上，一些实施例中，烷氧基为C 1-4烷氧基；这样的实例包括，但并不限于甲氧基、乙氧基、丙氧基和丁氧基等。并且所述烷氧基可以独立地未被取代或被一个或多个本发明所描述的取代基所取代。
术语“卤代烷基”“卤代烯基”和“卤代烷基氧基”表示烷基，烯基或烷基氧基可以被一个或多个卤素原子所取代的情况，一些实施例中，卤代烷基为卤代C 1-6烷基。另一些实施例中，卤代烷基为卤代C 1-3烷基。一些实施例中，卤代烷基氧基或卤代烷氧基为卤代C 1-6烷基氧基或卤代C 1-6烷氧基。另一些实施例中，卤代烷基氧基或卤代烷氧基为卤代C 1-3烷基氧基或卤代C 1-3烷氧基。这样的实例包括，但并不限于三氟甲基、2-氯-乙烯基、2,2-二氟乙基和三氟甲氧基等。所述的“卤代烷基”“卤代烯基”和“卤代烷基氧基”基团可以独立任选地被一个或多个本发明所描述的取代基所取代。
术语“环烷基”或“环烷烃”表示含有3-12个碳原子的，单价或多价的饱和单环，双环或三环碳环体系，但绝不包含芳香环。在一实施方案中，环烷基包含3-12个碳原子；在另一实施方案中，环烷基包含3-8个碳原子；在又一实施方案中，环烷基包含3-6个碳原子。这样的实例包括，但并不限于环丙基、环丁基、环戊基和环己基等。所述环烷基基团可以独立地未被取代或被一个或多个本发明所描述的取代基所取代。
术语“杂环基”和“杂环”在此处可交换使用，都是指包含3-12个环原子的饱和或部分不饱和的单环、双环或三环，绝不包含芳香环，其中至少一个环原子为杂原子。除非另外说明，杂环基可以是碳基或氮基，杂原子具有如本发明所述的含义。杂环基的实例包括，但不限于：环氧乙烷基、氮杂环丁基、氧杂环丁基、硫杂环丁基、吡咯烷基、2-吡咯啉基、3-吡咯啉基、吡唑啉基、吡唑烷基、咪唑啉基、咪唑烷基、四氢呋喃基、二氢呋喃基、四氢噻吩基、二氢噻吩基、1,3-二氧环戊基、二硫环戊基、四氢吡喃基、二氢吡喃基、2H-吡喃基、4H-吡喃基、四氢噻喃基、哌啶基、吗啉基、硫代吗啉基、哌嗪基、二噁烷基、二噻烷基、噻噁烷基、高哌嗪基、高哌啶基、氧杂环 庚烷基、硫杂环庚烷基、氧氮杂
Figure PCTCN2022111058-appb-000006
基、二氮杂
Figure PCTCN2022111058-appb-000007
基、硫氮杂
Figure PCTCN2022111058-appb-000008
基和2-氧杂-5-氮杂双环[2.2.1]庚-5-基。杂环基中-CH 2-基团被-C(＝O)-取代的实例包括，但不限于：2-氧代吡咯烷基、氧代-1,3-噻唑烷基、2-哌啶酮基、3,5-二氧代哌啶基和嘧啶二酮基。杂环基中硫原子被氧化的实例包括，但不限于环丁砜基和1,1-二氧代硫代吗啉基。所述的杂环基基团可以任选地被一个或多个本发明所描述的取代基所取代。
术语“杂芳基”表示含有5-12个环原子，或5-10个环原子，或5-6个环原子的单环、双环和三环体系，其中至少一个环体系是芳香环，且至少一个环体系包含一个或多个杂原子，其中每一个环包含5-7个原子组成的环，且有一个或多个附着点与分子其余部分相连。术语“杂芳基”可以与术语“杂芳环”或“杂芳族化合物”交换使用。所述杂芳基基团任选地被一个或多个本发明所描述的取代基所取代。在一实施方案中，5-10个原子组成的杂芳基包含1、2、3或4个独立选自O，S和N的杂原子，其中氮原子可以被进一步氧化。
杂芳基基团的实例包括，但并不限于：呋喃基、咪唑基(如N-咪唑基、2-咪唑基、4-咪唑基、5-咪唑基)、异噁唑基、恶唑基(如2-噁唑基、4-噁唑基、5-噁唑基)、吡咯基(如N-吡咯基、2-吡咯基、3-吡咯基)、吡啶基、嘧啶基(如2-嘧啶基、4-嘧啶基、5-嘧啶基)、哒嗪基、噻唑基(如2-噻唑基、4-噻唑基、5-噻唑基)、四唑基(如5-四唑基)、三唑基、噻吩基(如2-噻吩基、3-噻吩基)、吡唑基、异噻唑基、1,2,3-噁二唑基、1,2,5-噁二唑基、1,2,4-噁二唑基、1,2,3-三唑基、1,2,3-硫代二唑基、1,3,4-硫代二唑基、1,2,5-硫代二唑基、吡嗪基、1,3,5-三嗪基；也包括以下的双环，但绝不限于这些双环：苯并咪唑基、苯并呋喃基、苯并噻吩基、吲哚基(如2-吲哚基)、嘌呤基、喹啉基(如2-喹啉基，3-喹啉基，4-喹啉基)、1,2,3,4-四氢异喹啉基、1,3-苯并二噁茂基、吲哚啉基、异喹啉基(如1-异喹啉基、3-异喹啉基或4-异喹啉基)、咪唑并[1,2-a]吡啶基、吡唑并[1,5-a]吡啶基、吡唑并[1,5-a]嘧啶基、咪唑并[1,2-b]哒嗪基、[1,2,4]三唑并[4,3-b]哒嗪基、[1,2,4]三唑并[1,5-a]嘧啶基和[1,2,4]三唑并[1,5-a]吡啶基，等等。
术语“氨基烷基”包括被一个或多个氨基所取代的C 1-10直链或支链烷基基团。其中一些实施例是，氨基烷基是被一个或多个氨基基团所取代的C 1-6烷基，这样的实例包括，但并不限于：氨甲基、氨乙基、氨丙基、氨丁基和氨己基。所述氨基烷基基团可以任选地被一个或多个本发明所描述的取代基所取代。
术语“羟基烷基”表示烷基基团被一个或多个羟基基团所取代，其中烷基基团具有本发明所述的含义。这样的实例包含，但并不限于羟甲基、羟乙基和1,2-二羟基乙基等等。
像本发明所描述的，取代基画一个键连接到中心的环上形成的环体系代表取代基在该环上任何可取代的位置都可以取代。例如，式(a)代表取代基R o可以在E环上任何可能被取代的位置上单取代或多取代。
Figure PCTCN2022111058-appb-000009
另外，需要说明的是，除非以其他方式明确指出，在本文中通篇采用的描述方式“各…和…独立地为”、“…和…各自独立地为”和“…和…分别独立地为”可以互换，应做广义理解，其既可以是指在不同基团中，相同符号之间所表达的具体选项之间互相不影响，也可以表示在相同的基团中，相同符号之间所表达的具体选项之间互相不影响。
除非其他方面表明，本发明所描述的结构式包括所有的同分异构形式(如对映异构，非对映异构，几何异构或构象异构)：例如含有不对称中心的R、S构型，双键的(Z)、(E)异构体，和(Z)、(E)的构象异构体。因此，本发明的化合物的单个立体化学异构体或其对映异构体、非对映异构体、几何异构体或构象异构体的混合物都属于本发明的范围。
除非其他方面表明，本发明所描述的结构式和所述的化合物包括所有的同分异构形式(如对映异构，非对映异构，几何异构或构象异构)、盐、氮氧化物、水合物或溶剂化物。因此，本发明的化合物的单个立体化学异构体、对映异构体、非对映异构体、几何异构体、构象异构体、盐、氮氧化物、水合物、溶剂化物的化合物也属于本发明的范围。另外，除非其他方面表明，本发明所描述的化合物的结构式包括一个或多个不同的原子的富集同位素。
本发明中立体化学的定义和惯例的使用通常参考以下文献：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.本发明的化合物可以包含不对称中心或手性中心，因此存在不同的立体异构体。本发明的化合物所有的立体异构形式，包括但绝不限于，非对映体，对映异构体，阻转异构体，和它们的混合物，如外消旋混合物，组成了本发明的一部分。很多有机化合物都以光学活性形式存在，即它们有能力旋转平面偏振光的平面。在描述光学活性化合物时，前缀D、L或R、S用来表示分子手性中心的绝对构型。前缀d、l或(+)、(-)用来命名化合物平面偏振光旋转的符号，(-)或l是指化合物是左旋的，前缀(+)或d是指化合物是右旋的。这些立体异构体的化学结构是相同的，但是它们的立体结构不一样。特定的立体异构体可以是对映体，异构体的混合物通常称为对映异构体混合物。50：50的对映体混 合物被称为外消旋混合物或外消旋体，这可能导致化学反应过程中没有立体选择性或立体定向性。术语“外消旋混合物”和“外消旋体”是指等摩尔的两个对映异构体的混合物，缺乏光学活性。
术语“互变异构体”或“互变异构的形式”是指不同能量的结构的同分异构体可以通过低能垒互相转化。例如质子互变异构体(即质子移变的互变异构体)包括通过质子迁移的互变，如酮式-烯醇式和亚胺-烯胺的同分异构化作用。原子价(化合价)互变异构体包括重组成键电子的互变。
本发明的“水合物”是指溶剂分子是水所形成的缔合物。
本发明的“溶剂化物”是指一个或多个溶剂分子与本发明的化合物所形成的缔合物。形成溶剂化物的溶剂包括，但并不限于：水、异丙醇、乙醇、甲醇、二甲亚砜、乙酸乙酯、乙酸、氨基乙醇。
本发明的“酯”是指含有羟基的式(I)化合物形成体内可水解的酯。这样的酯是例如在人或动物体内水解产生母体醇的药学上可接受的酯。含有羟基的式(I)化合物体内可水解的酯的基团包括，但不限于：磷酸基、乙酰氧基甲氧基、2,2-二甲基丙酰氧基甲氧基、烷酰基、苯甲酰基、苯乙酰基、烷氧基羰基、二烷基氨基甲酰基和N-(二烷基氨基乙基)-N-烷基氨基甲酰基等。
本发明的“氮氧化物”是指当化合物含几个胺官能团时，可将1个或大于1个的氮原子氧化形成N-氧化物。N-氧化物的特殊实例是叔胺的N-氧化物或含氮杂环氮原子的N-氧化物。可用氧化剂例如过氧化氢或过酸(例如过氧羧酸)处理相应的胺形成N-氧化物(参见Advanced Organic Chemistry,Wiley Interscience,第4版,Jerry March,pages)。尤其是，N-氧化物可用L.W.Deady的方法制备(Syn.Comm.1977,7,509-514)，例如在惰性溶剂(例如二氯甲烷中)，使胺化合物与间-氯过氧苯甲酸(MCPBA)反应。
在本说明书的描述中，参考术语“一个实施例”、“本发明实施例”“一些实施例”、“示意性实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不一定指的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任何的一个或多个实施例或示例中以合适的方式结合。
除非另有定义，本文使用的所有科技术语具有本领域普通技术人员所理解的相同含义。关于本领域的定义及术语，专业人员具体可参考Current Protocols in Molecular Biology(Ausubel)。氨基酸残基的缩写是本领域中所用的指代20个常用L-氨基酸之一的标准3字母和/或1字母代码。
本发明提供的所述荧光素酶组合中，包含两种荧光素酶，其一为Gaussia荧光素酶，另一为 Pleuromamma xiphias荧光素酶。Gaussia荧光素酶也称“高斯荧光素酶”，是一种分子量为20kDa的蛋白质，其来源于海洋桡脚类的动物高斯基质(Gaussia princeps)的生物发光酶，高斯荧光素酶受体分子非常小，不需要ATP的辅助，在氧分子存在条件下，催化腔肠素氧化发光。Pleuromamma xiphias荧光素酶是一种桡足类荧光素酶，也称为剑乳点水蚤荧光素酶，它可在氧分子存在条件下，催化腔肠素氧化发光。本发明对荧光酶的组合进行了筛选，结果表明，相对于其他的荧光酶组合能够产生更强的荧光，且不产生交叉干扰。
本发明所述的荧光酶报告基因检测系统中，所述荧光素酶的存在形式可以为编码的核酸、表达模块、表达载体、重组菌株、重组菌株的表达产物或经纯化的表达产物。
以编码的核酸为例，本发明所述的检测系统包括为编码Gaussia荧光素酶的核酸和编码Pleuromamma xiphias荧光素酶的核酸的混合物、也可为两个独立存在的编码Gaussia荧光素酶的核酸和编码Pleuromamma xiphias荧光素酶的核酸、也可为编码Gaussia荧光素酶的核酸和编码Pleuromamma xiphias荧光素酶的核酸形成的融合片段，本发明对此不做限定。
以表达模块为例，所述表达模块包括启动子、编码核酸和终止子。所述编码Gaussia荧光素酶的核酸和编码Pleuromamma xiphias荧光素酶的核酸在同一表达模块中，也可在两个不同的表达模块中。两个编码核酸所在的两个表达模块的启动子可以相同也可不同，本发明对此不做限定。
作为举例，所述表达载体包括骨架载体和编码核酸。所述表达载体包括但不限于质粒载体或者病毒载体。编码Gaussia荧光素酶的核酸和编码Pleuromamma xiphias荧光素酶的核酸可以在同一表达载体表达，也可以在不同的表达载体上表达，本发明对此不做限定，当核酸在不同的载体表达时，骨架载体可以相同也可以不同。
本发明所述的表达菌株亦称为重组宿主，其可以表达Gaussia荧光素酶和/或Pleuromamma xiphias荧光素酶，即Gaussia荧光素酶和Pleuromamma xiphias荧光素酶以同一宿主表达，也可以在不同的宿主中表达，本发明对此不做限定。所述的重组宿主包括原核宿主或真核宿主，其可以通过电转的方式构建，也可通过病毒侵染的方式构建。其可为大肠杆菌、酵母菌或动物细胞或人体细胞，本发明对此不做限定。在两个不同的宿主中表达两荧光素酶可以采用相同的宿主细胞也可采用不同的宿主细胞。
本发明重组菌株的表达产物或经纯化的表达产物，其来自前述表达菌株或重组宿主，其可为两个表达荧光酶的菌株分别表达后的混合物，可以为表达两个荧光酶的菌株表达后的产物。
本发明中，“报告基因”是一个分子生物学概念，是指一类在细胞、组织/器官或个体处于特 定情况下会表达并使得他们产生易于检测、且实验材料原本不会产生的性状的基因，即是一种编码可被检测的蛋白质或酶的基因。作为报告基因，在遗传选择和筛选检测方面必须具有以下几个条件：1、已被克隆和全序列已测定；2、表达产物在受体细胞中本不存在，即无背景，在被转染的细胞中无相似的内源性表达产物；3、其表达产物能进行定量测定；在使用时，包括但并不限于如下使用方式：把报告基因和基因表达调节序列相融合形成嵌合基因，或与其它目的基因相融合，在调控序列的控制下进行核酸表达，从而利用它的表达产物来检测目的基因的表达调控，研究核酸。
本发明中，所述的荧光素酶报告基因检测系统，也称为双荧光素酶发光系统，其包括本发明所述的荧光酶组合物和底物。所述荧光酶组合物与底物分别独立存在，在进行检测时于检测体系混合后发光。本发明对底物进行了筛选，最终获得的荧光素酶报告基因检测系统能够具有更高的荧光强度，从而提高检测的灵敏性。
所述检测试剂中，包括能够使待测物过表达Gaussia荧光素酶和Pleuromamma xiphias荧光素酶的物质。所述能够使待测物过表达Gaussia荧光素酶和Pleuromamma xiphias荧光素酶的物质包括但不限于含有编码Gaussia荧光素酶的核酸和/或编码Pleuromamma xiphias荧光素酶的核酸的质粒载体，或者病毒载体。
如本文所用，术语“多核苷酸”是指脱氧核糖核酸((DNA)、核糖核酸(RNA)或其类似物。多核苷酸可以是单链的、双链的或含有单链和双链序列两者。多核苷酸分子可以来源于双链DNA(dsDNA)形式(例如，基因组DNA,PCR和扩增产物等)，或者可以来源于单链形式的DNA(ssDNA)或RNA并且其可以转化为dsDNA形式，并且反之亦然。多核苷酸分子的准确序列可以是己知的或未知的。以下是多核苷酸的示例性实例:基因或基因片段(例如，探针、引物、EST或SAGE标签)、基因组DNA、基因组DNA片段、外显子、内含子、信使RNA(mRNA)、转运RNA、核糖体RNA、核糖酶、cDNA，重组多核苷酸、合成多核苷酸、分枝多核苷酸、质粒、载体、任何序列的分离的DNA，任何序列的分离的RNA、任何上述序列的核酸探针、引物或扩增拷贝。
多核苷酸可以包括核苷酸或核苷酸类似物。核苷酸通常含有糖(如核糖或脱氧核糖)、碱基和至少一个磷酸基。核苷酸包括脱氧核糖核苷酸、修饰的脱氧核糖核苷酸、核糖核苷酸、修饰的核糖核苷酸、修饰磷酸盐糖主链核苷酸及其混合物。核苷酸的实例包括(例如)腺苷一磷酸((AMP)、腺苷二磷酸((ADP)、腺苷三磷酸((ATP)、胸苷一磷酸(TMP)、胸苷二磷酸(TDP)、胸苷三磷酸(TTP)、胞苷一磷酸(CMP)、胞苷二磷酸(CDP)、胞苷三磷酸(CTP)、鸟苷一磷酸(GMP)、鸟苷二磷酸(GDP)、 鸟苷三磷酸(GTP)、尿苷一磷酸(UMP)、尿苷二磷酸(UDP)、尿苷三磷酸(UTP}、脱氧腺苷一磷酸(dAMP)、脱氧腺苷二磷酸(dADP)、脱氧腺苷三磷酸(dATP)、脱氧胸苷一磷酸(dTMP)、脱氧胸苷二磷酸(dTDP)、脱氧胸苷三磷酸(dTTP)、脱氧胞苷一磷酸(dCMP)、脱氧胞苷二磷酸(dCDP)、脱氧胞苷三磷酸(dCTP)、脱氧鸟苷一磷酸((dGMP)、脱氧鸟苷二磷酸((dGDP)、脱氧鸟苷三磷酸(dGTP)、脱氧尿苷一磷酸(BUMP)、脱氧尿苷二磷酸(dUDP)和脱氧尿苷三磷酸(dUTP)。
还可以在本文所述的方法中使用包含修饰的碱基的核苷酸类似物。无论是具有天然主链还是类似结构，可以包含在多核苷酸中的示例性修饰的碱基包括(例如)肌苷、黄嘌呤(xathanine)、次黄嘌呤(hypoxathanine)、异胞嘧啶、异鸟嘌呤、2-氨基嘌呤、5-甲基胞嘧啶、5-羟甲基胞嘧啶、2-氨基腺嘌呤、6-甲基腺嘌呤、6-甲基鸟嘌呤、2-丙基鸟嘌呤、2-丙基腺嘌呤、2-硫脲嘧啶、2-硫胸腺嘧啶、2-硫胞嘧啶、15-卤代脲嘧啶、15-卤代胞嘧啶、5-丙炔基尿嘧啶、5-丙炔基胞嘧啶、6-偶氮尿嘧啶、6-偶氮胞嘧啶、6-偶氮胸腺嘧啶、5-尿嘧啶、4-硫尿嘧啶、8-卤代腺嘌呤或鸟嘌呤、8-氨基腺嘌呤或鸟嘌呤、8-硫腺嘌呤或鸟嘌呤、8-硫烷基腺嘌呤或鸟嘌呤、8-羟基腺嘌呤或鸟嘌呤、5-卤素取代的尿嘧啶或胞嘧啶、7-甲基鸟嘌呤、7-甲基腺嘌呤、8-氮杂鸟嘌呤、8-氮杂腺嘌呤、7-去氮鸟嘌呤、7-去氮腺嘌呤、3-去氮鸟嘌呤、3-去氮腺嘌呤等。
通常而言，核苷酸包括核苷酸A、C、G、T或U。如本文所用，术语“核苷酸A”是指含有腺嘌呤(A)或其修饰物或类似物的核苷酸，例如ATP、dATP。“核苷酸G”，是指含有鸟嘌呤(G)或其修饰物或类似物的核苷酸，例如GTP、dGTP。“核苷酸C”，是指含有胞嘧啶(C)或其修饰物或类似物的核苷酸，例如CTP、dCTP。“核苷酸T”，是指含有胸腺嘧啶(T)或其修饰物或类似物的核苷酸，例如TTP、dTTP。“核苷酸U”，是指含有尿嘧啶(U)或其修饰物或类似物的核苷酸，例如UTP、dUTP。
核苷酸的标记
本发明涉及用不同的标记物单独或组合地标记核苷酸，从而使得可以在核苷酸上连接不同的荧光素酶。如本文所用，用于标记核苷酸的所述分子标记和与其特异性结合的标记可以是任何能够彼此特异性结合的分子配对。配对成员之间的特异性结合实现核苷酸与荧光素酶的连接。示例性的配对成员包括但不限于:(a)与相应抗体或其结合部分或片段组合的半抗原或抗原性化合物，例如地高辛-地高辛抗体，N3G-N3G抗体，FITC-FITC抗体；(b)核酸适配体和蛋白质；(c)非免疫结合对(例如生物素-抗生物素蛋白、生物素-链霉亲和素、生物素-中性抗生蛋白)；(d)激素-激素结合蛋白；(e)受体-受体激动剂或拮抗剂；(f)均凝集素-碳水化合物；(g)酶-酶辅因子和(h)酶-酶抑制剂；。
在具体的实施方案中，所述第一分子标记和第二分子标记是小分子标记物，其选自生物素、地高辛、N3G或FITC。两种荧光素酶分别能够特异性结合所述第一分子标记和第二分子标记。例如，在一个具体实施方案中，第一分子标记是生物素，则第一荧光素酶可以是经链霉亲和素标记的荧光素酶；第二分子标记是地高辛，则第二荧光素酶可以是经地高辛抗体标记的与所述第一荧光素酶不同的荧光素酶。优选地，所述第一荧光素酶为本发明所述的Gaussia荧光素酶或其突变体，所述第二荧光素酶为本发明所述的Pleuromamma xiphias荧光素酶或其突变体。
如本文中所使用，表述“第一化合物连接有第一分子标记”是指全部第一化合物都连接有第一分子标记，或者部分第一化合物连接有第一分子标记但其余第一化合物没有连接分子标记。同理，表述“第二化合物连接有第二分子标记”是指全部第二化合物都连接有第二分子标记，或者部分第二化合物连接有第二分子标记但其余第二化合物没有连接分子标记。表述“第三化合物连接有第一分子标记和第二分子标记”是指全部第三化合物都连接有第一分子标记和第二分子标记，或者部分第三化合物连接有第一分子标记和第二分子标记但其余第三化合物没有连接分子标记。
多核苷酸的测序
优选地，本发明的双荧光素酶报告基因检测系统适用于合成法测序。如本文所用的合成法测序是本领域熟知的各种合成法测序方法。基本地，合成法测序涉及首先将被测序的核酸分子与测序引物杂交，随后在聚合酶的存在下，以被测序的核酸分子为模板在测序引物的3’端聚合如本文所述的核苷酸或核苷酸类似物。聚合之后，通过检测所述荧光素酶发出的荧光信号来鉴定核苷酸分子类型。从经标记的核苷酸上除去荧光素酶之后，进行下一个聚合测序循环。
用于测定靶多核苷酸序列的方法可以这样进行：使靶多核苷酸序列变性，使靶多核苷酸分别与不同的核苷酸接触，以便形成所述靶核苷酸的互补体，并且检测所述核苷酸的掺入。所述方法利用了聚合，使得聚合酶通过掺入互补于所述靶的正确的核苷酸，以延伸所述互补链。所述聚合反应还需要特殊引物来启动聚合作用。
对每一轮反应来说，所述核苷酸的掺入是通过聚合酶进行的，并随后测定所述掺入事件。存在很多不同的聚合酶，并且对本领域普通技术人员来说容易确定最适合的聚合酶。优选的酶包括DNA聚合酶I,Klenow片段、DNA聚合酶III,T4或T7DNA聚合酶、Taq聚合酶或vent聚合酶。还可以使用通过工程方法改造成具有特定性质的聚合酶。
所述测序方法优选对排列在固体支持物上的靶多核苷酸进行。可以通过接头分子将多个靶多核苷酸固定在所述固体支持物上，或者可以连接在诸如微球体的颗粒上，所述颗粒还可以连接在 固体支持材料上。
可以通过多种方法将所述多核苷酸连接在所述固体支持物上，包括使用生物素-链霉亲和素相互作用。用于将多核苷酸固定在固体支持物上的方法为本领域所公知，并且包括石板印刷技术以及将每一种多核苷酸点样在固体支持物的特定位置上。合适的固体支持物为本领域所公知，并且包括玻璃载玻片和珠、陶瓷和硅表面和塑料材料。
所述支持物通常是平面，尽管也可以使用微珠(微球体)，并且还可以通过已知方法将后者连接在其他固体支持物上。所述微球体可以具有任何合适的大小，其直径通常为10-100纳米。在优选实施方案中，将所述多核苷酸直接连接在平面上，优选连接在平的玻璃表面上。连接优选通过共价键的形式进行。所使用的阵列优选是单分子阵列，它包括位于独特的光学可分辨区域的多核苷酸，例如在国际申请号WO00/06770中所描述的。
进行聚合的必须条件对本领域技术人员来说是熟知的。为了进行所述聚合酶反应，通常首先必须使引物序列与所述靶多核苷酸退火，所述引物序列是由所述聚合酶识别的，并且起着所述互补链随后延伸的起始位点的作用。所述引物序列可以相对所述靶多核苷酸作为独立的成分添加。另外，所述引物和靶多核苷酸可以分别是一个单链分子的一部分，由所述引物部分与所述靶的一部分形成分子内双链体，即发卡环结构。可以在所述分子的任何位点，将该结构固定在所述固体支持物上。进行所述聚合酶反应所必需的其他条件，对本领域技术人员来说是熟知的，这些条件包括温度、pH和缓冲液组成。
随后，使本发明的经标记的核苷酸或核苷酸类似物与所述靶多核苷酸接触，以便能够进行聚合。所述核苷酸或核苷酸类似物可以依次添加，即分别添加每一种类型的核苷酸(A、C、G或T/U)，或同时添加多种类型的核苷酸(A、C、G或T/U)。
使所述聚合步骤进行足以掺入一个核苷酸的时间。
然后除去未掺入的核苷酸，例如，通过移除前一步骤的反应体系的溶液相，保留连接于支持物上的双链体。
随后可以加入含有不同荧光素酶的两种荧光素酶，进行结合反应，所述两种荧光素酶分别能够特异性结合核苷酸上的分子标记，由此实现荧光素酶与掺入的核苷酸的连接。然后通过加入对应的荧光素酶的底物并检测荧光信号而实现掺入的核苷酸的鉴别。
一个具体实施方案中，四种脱氧核糖核苷酸类似物分别被标记了不同的小分子标记物生物素(简称B)和地高辛(简称D)，例如核苷酸A标记B，核苷酸C标记B和D，核苷酸T标记D，核苷酸G不 做标记。上述被标记不同小分子的四种脱氧核糖核苷酸类似物的3’端羟基均作阻断处理，保证每次测序反应仅结合一个脱氧核糖核苷酸。测序反应过程中，先通入所述四种经标记的脱氧核苷酸类似物及测序聚合酶混合物，在聚合酶的作用下，按照碱基互补配对原则，一个脱氧核糖核苷酸类似物掺入到生长的核酸链的3’端。通过移除前一步骤的反应体系的溶液相，保留连接于支持物上的双链体，可去除未结合的脱氧核糖核苷酸类似物。然后，加入含有不同荧光素酶的两种荧光素酶，第一荧光素酶经链霉亲和素标记，其与标记了小分子B的核苷酸A或核苷酸C结合，第二荧光素酶经地高辛抗体标记，其与标记了小分子D的核苷酸C或核苷酸T结合。使用洗脱缓冲液去除未结合的荧光素酶之后，加入第一荧光素酶的底物，连接有第一荧光素酶的核苷酸发光，采用检测器检测信号；加入第二荧光素酶的底物，连接有第二荧光素酶的核苷酸发光，采用检测器检测信号，由此得出下表所示的发光情况，即可进行碱基的识别(1表示检测到荧光信号，0表示未检测到荧光信号)。
  第一次检测 第二次检测
A 1 0
C 1 1
G 0 0
T 0 1
在一个具体实施方案中，含有不同荧光素酶的两种荧光素酶(Gaussia荧光素酶或其突变体，和Pleuromamma xiphias荧光素酶或其突变体)与经标记的核苷酸的连接以及信号检测可以分开进行。先加入第一荧光素酶，其经链霉亲和素标记，与标记了小分子B的核苷酸A或核苷酸C结合。使用洗脱缓冲液去除未结合的第一荧光素酶之后，加入第一荧光素酶的底物，连接有第一荧光素酶的核苷酸发光，采用检测器检测信号。移除反应溶液后加入经地高辛抗体标记的第二荧光素酶，其与标记了小分子D的核苷酸C或核苷酸T结合，然后用洗脱缓冲液去除未结合的第二荧光素酶，加入第二荧光素酶的底物，连接有第二荧光素酶的碱基发光，采用检测器检测信号，由此得出上表所示的发光情况，即可进行碱基的识别。
荧光信号的检测
检测荧光信号的方式是本领域熟知的。例如，可以通过检测荧光的波长的装置来实现。这样的装置是本领域熟知的。例如，这样的装置可以是共焦扫描显微镜，其用激光扫描固体支持物的表面，以便使直接结合被测序的核酸分子上的荧光团成像。另外，可以例如用灵敏的2-D探测器， 如电荷偶连的探测器(CCD)观察所产生的每一种信号。还可以例如使用诸如扫描近场光学显微方法(SNOM)的其他技术。
本发明中所用试剂或仪器均可由市场购得。实施例中使用的Gaussia荧光素酶(GenBank:AY015993.1)由大肠杆菌表达系统制备获得，表达载体为pET28a。Pleuromamma xiphias荧光素酶(PMID:23886588)也由大肠杆菌表达系统制备获得，表达载体为pET28a。
下面结合实施例，进一步阐述本发明。
实施例1双荧光素酶系统(野生型)的配对发光检测
实验目的：通过测试不同底物对野生型Gaussia荧光素酶和野生型Pleuromamma xiphias荧光素酶的发光强度，筛选出高亮度、低交叉干扰的发光组合。
实验步骤：
(1)野生型Gaussia荧光素酶(WT-Gluc)具有如下所示的氨基酸序列：
Figure PCTCN2022111058-appb-000010
野生型Gaussia荧光素酶(WT-Gluc)基因具有如下所示的核苷酸序列：
Figure PCTCN2022111058-appb-000011
野生型Pleuromamma xiphias荧光素酶(WT-Pxluc)具有如下所示的氨基酸序列：
Figure PCTCN2022111058-appb-000012
野生型Pleuromamma xiphias荧光素酶(WT-Pxluc)加上信号肽后具有如下所示的氨基酸序列：
Figure PCTCN2022111058-appb-000013
野生型Pleuromamma xiphias荧光素酶(WT-Pxluc)基因具有如下所示的核苷酸序列：
Figure PCTCN2022111058-appb-000014
(2)腔肠素衍生物作为底物：参照专利申请PCT/CN2018/107646公开的方法合成底物f-CTZ、N0、N1、N3、N4、N5、N6、N7和N8。
Figure PCTCN2022111058-appb-000015
(3)按照常规方法，例如PCT/CN2017/082180公开的方法，构建含有上述野生型Gaussia荧光素酶基因和野生型Pleuromamma xiphias荧光素酶基因的表达载体(质粒)。将质粒转化入大 肠杆菌OrigamiB(DE3)中，涂平板，从平板上挑取单菌落，于37℃过夜培养，次日按1:100的比例进行稀释，转接于新鲜的3mL含氨苄抗性(100μg/mL)的LB培养基中，于37℃、200rpm振荡培养4小时，冰上冷却1小时。按照终浓度为1mM的量加入诱导剂IPTG，并于16℃条件下过夜诱导。
次日离心收菌，用500μL裂解液(50mM Tris-HCl pH 8.0,500mM NaCl,0.2％lysozyme,PMSF)重悬，室温裂解20分钟后液氮反复冻融3次，18,000g离心30分钟取上清，用离心型Ni柱纯化。
将Ni柱洗脱后的蛋白用透析缓冲液(25mM Tris，pH 8.0，250mMNaCl)4℃过夜透析。采用BCA定量试剂盒(Thermo Scientific TMPierce TMBCA Protein Assay Kit)精确测定荧光素酶的浓度。
用稀释液(50mM Tris-HCl pH 8.0，100mMNaCl，0.1％(v/v)Tween-20)将两种荧光素酶均稀释至1μg/mL，分别取10μL加入黑色96孔板。用相同的稀释液稀释上述底物至100μM，取90μL加入上述含有荧光素酶的96孔板中(90μL/孔)，用酶标仪自发光模块读取发光强度。
Gaussia荧光素酶和Pleuromamma xiphias荧光素酶对不同底物的测试数据如表1所示，相对强度是相对于野生型Pleuromamma xiphias荧光素酶与化合物N0活性的百分比。结果表明，Gaussia荧光素酶与底物f-CTZ配合的荧光强度最高，Pleuromamma xiphias荧光素酶与底物N0配合的荧光强度最高。即在该双荧光素酶报告基因检测系统中，用底物f-CTZ检测Gaussia荧光素酶，用底物N0检测Pleuromamma xiphias荧光素酶，是经筛选后获得的高荧光亮度、低交叉干扰的最佳发光组合。
表1各种底物与荧光素酶的发光强度
Figure PCTCN2022111058-appb-000016
Figure PCTCN2022111058-appb-000017
实施例2双荧光素酶系统(突变型)的配对发光检测
实验目的：通过测试不同底物对突变型Gaussia荧光素酶和突变型Pleuromamma xiphias荧光素酶的发光强度，筛选出高亮度、低交叉干扰的发光组合。
实验步骤：
(1)突变型Gaussia荧光素酶E1-A3、G2-F11和G2-F8：
a)E1-A3，在SEQ ID NO:1基础上，突变以下位点：F26R,V29F,A32V,S33E,A36V,L40I,K66P,H79K,P84L,E102S,S103T,A104G,E110P,L124M,V138E；
E1-A3突变体的基因具有如下所示的核苷酸序列：
Figure PCTCN2022111058-appb-000018
b)G2-F11，在SEQ ID NO:1基础上，突变以下位点：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；
G2-F11突变体的基因具有如下所示的核苷酸序列：
Figure PCTCN2022111058-appb-000019
Figure PCTCN2022111058-appb-000020
c)G2-F8，在SEQ ID NO:1基础上，突变以下位点：H79K,P84L,E102S,S103T,A104G,E110P,L124M,Q152H,Q163D,T167S,S170T,G174K,A182M,G183A。
G2-F8突变体的基因具有如下所示的核苷酸序列：
Figure PCTCN2022111058-appb-000021
突变型Pleuromamma xiphias荧光素酶P26-95：
d)P26-95，在SEQ ID NO:3基础上，突变以下位点：G83A,G84P。
P26-95突变体的基因具有如下所示的核苷酸序列：
Figure PCTCN2022111058-appb-000022
Figure PCTCN2022111058-appb-000023
按照实施例1所示方法，构建含有上述Gaussia荧光素酶突变体基因、Pleuromamma xiphias荧光素酶突变体基因的表达载体，构建重组菌，表达所述突变型荧光素酶，并进行底物与荧光素酶发光强度检测。
结果表明，Gaussia荧光素酶突变体E1-A3、G2-F11和G2-F8与底物f-CTZ配合的荧光强度最高，Pleuromamma xiphias荧光素酶突变体P26-95与底物N0配合的荧光强度最高。即在该双荧光素酶报告基因检测系统中，用底物f-CTZ检测Gaussia荧光素酶突变体E1-A3、G2-F11或G2-F8，用底物N0检测Pleuromamma xiphias荧光素酶突变体P26-95，是经筛选后获得的高荧光亮度、低交叉干扰的最佳发光组合。
实施例3双荧光素酶报告基因检测系统的使用
构建以野生型Gaussia荧光素酶(WT-Gluc)基因和野生型Pleuromamma xiphias荧光素酶(WT-Pxluc)基因为报告基因的表达质粒，转染至预先铺有0.5x10 4个HEK293细胞的24孔板中，放置于细胞培养箱中，在5％CO 2,37℃条件下过夜培养。
次日，倾去培养板/皿中的培养液，加入足量PBS，轻轻洗涤细胞。完全倾去PBS洗涤液。加入50μL 1x Passive lysis buffer(Promega)裂解细胞。将细胞悬液移入1.5ml离心管，在涡旋振荡器上充分混悬震荡30秒。取裂解细胞悬液用于发光测定，也可100g离心30秒，取上清液做发光测定。
取50μL细胞悬液或上清液加到黑色96孔板中。用稀释液(50mM Tris-HClpH 8.0，100mM NaCl，0.1％(v/v)Tween-20)稀释腔肠素f-CTZ至100μM，取50μL加入到上述含有细胞悬液或上清液的96孔板中(50μL/孔)，用酶标仪自发光模块读取发光强度。测定完毕后，加入1x stop&glo试剂(Promega)终止f-CTZ的发光，并在同体系中加入用相同稀释液稀释至100μM的N0底物，用酶标仪自发光模块读取发光强度。
结果表明，该双荧光素酶报告基因检测系统可以用于检测细胞中表达的荧光素酶。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (22)

  1. 一种双荧光素酶报告基因检测系统，其特征在于，其包含Gaussia荧光素酶、Pleuromamma xiphias荧光素酶和底物，并且所述底物为如式(I)所示的化合物，或式(I)所示的化合物的立体异构体、几何异构体、互变异构体、盐、氮氧化物、水合物或溶剂化物：
    Figure PCTCN2022111058-appb-100001
    其中，R 1、R 2分别独立地为H、D、F、Cl、Br、I、OH、NH 2、NO 2、CN、N 3、C 1-6烷基、C 2-6烯基、C 2-6炔基、C 1-6卤代烷基、C 1-6烷氧基、C 1-6羟基烷基或C 1-6烷基氨基；
    R 3为任选取代的芳基、杂环基或杂芳基；
    a 1为0或1-6的任意一个整数，
    a 2为0或1-6的任意一个整数，b为0或1，
    c为0、1或2。
  2. 根据权利要求1所述的双荧光素酶报告基因检测系统，其特征在于，所述Gaussia荧光素酶为如下I)～III)中任一种：
    I)、所述Gaussia荧光素酶的氨基酸序列如SEQ ID NO:1所示；
    II)、与I)所述Gaussia荧光素酶的氨基酸序列具有至少85％同源性且与I)所述Gaussia荧光素酶具有相同或相似功能的荧光素酶；
    III)、如I)所述的Gaussia荧光素酶的氨基酸序列经修饰、取代、缺失或添加一个或多个氨基酸残基且仍具有Gaussia荧光素酶活性的荧光素酶。
  3. 根据权利要求2所述的双荧光素酶报告基因检测系统，其特征在于，所述Gaussia荧光素酶为经过优化的突变体，相较于SEQ ID NO:1所示的氨基酸序列，所述Gaussia荧光素突变体具有如下突变位点中的至少之一：第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和183位。
  4. 根据权利要求3所述的双荧光素酶报告基因检测系统，其特征在于，所述Gaussia荧光素突变体具有如下突变中的至少之一：
    1)第24位的E突变为K；
    2)第26位的F突变为R或L；
    3)第27位的N突变为D；
    4)第29位的V突变为F或L；
    5)第30位的A突变为G或D；
    6)第31位的V突变为I；
    7)第32位的A突变为V；
    8)第33位的S突变为E、R或K；
    9)第36位的A突变为V或I；
    10)第37位的T突变为N或E；
    11)第40位的L突变为I或T；
    12)第66位的K突变为P、S、I、R或N；
    13)第79位的H突变为K；
    14)第84位的P突变为A、L、K或V；
    15)第88位的K突变为R；
    16)第102位的E突变为D、A、S、K或N；
    17)第103位的S突变为T；
    18)第104位的A突变为G；
    19)第110位的E突变为P、G或A；
    20)第123位的D突变为N；
    21)第124位的L突变为M、G或I；
    22)第138位的V突变为E或D；
    23)第152位的Q突变为R或H；
    24)第163位的Q突变为D；
    25)第170位的S突变为N或T；
    26)第174位的G突变为K；
    27)第175位的Q突变为E；
    28)第178位的K突变为T；
    29)第182位的A突变为M；
    30)第183位的G突变为N或A。
  5. 根据权利要求1所述的双荧光素酶报告基因检测系统，其特征在于，所述Pleuromamma xiphias荧光素酶为如下i)～iii)中任一种：
    i)、所述Pleuromamma xiphias荧光素酶的氨基酸序列如SEQ ID NO:3所示；
    ii)、与i)所述Pleuromamma xiphias荧光素酶的氨基酸序列具有至少85％同源性且与i)所述Pleuromammaxiphias荧光素酶具有相同或相似功能的荧光素酶；
    iii)、如i)所述的Pleuromamma xiphias荧光素酶的氨基酸序列经修饰、取代、缺失或添加一个或多个氨基酸残基且仍具有Pleuromamma xiphias荧光素酶活性的荧光素酶。
  6. 根据权利要求5所述的双荧光素酶报告基因检测系统，其特征在于，所述Pleuromamma xiphias荧光素酶为经过优化的突变体，相较于SEQ ID NO:3所示的氨基酸序列，所述Pleuromamma xiphias荧光素突变体具有如下突变位点中的至少之一：第81位、82位、83位和84位。
  7. 根据权利要求6所述的双荧光素酶报告基因检测系统，其特征在于，所述Pleuromamma xiphias荧光素突变体具有如下突变中的至少之一：
    (1)第81位的G突变为L或P或Q或S或T；
    (2)第82位的Q突变为R或W或I或Y或A或L或F或V或P或E或M；
    (3)第83位的G突变为S或Q或R或W或T或A或L；
    (4)第84位的G突变为F或R或S或C或Y或L或I或K或V或P。
  8. 根据权利要求1所述的双荧光素酶报告基因检测系统，其特征在于，式(I)所示化合物还可以进一步包括如下附加技术特征至少之一：
    所述R 1为H；
    所述R 2为H、NH 2、OH或C 1-6烷基氨基；优选为H、NH 2、3-OH、4-OH或二甲基氨基；
    所述R 3为取代的芳基，优选为OH-C 6H 5或F-C 6H 5，更优选为4-OH-C 6H 5或4-F-C 6H 5；
    所述a 1为0或1；
    所述a 2为0或1；
    优选为当a 1为0时，a 2为1；当a 1为1时，a 2为0；或者当a 1为0时，a 2为0；
    所述b为1；
    所述c为1。
  9. 根据权利要求8任一项所述的双荧光素酶报告基因检测系统，其特征在于，式(I)所示化合物包括如下任意两种：
    Figure PCTCN2022111058-appb-100002
  10. 根据权利要求9所述的双荧光素酶报告基因检测系统，其特征在于，
    所述Gaussia荧光素酶的底物为
    Figure PCTCN2022111058-appb-100003
    所述Pleuromamma xiphias荧光素酶的底物为
    Figure PCTCN2022111058-appb-100004
  11. 根据权利要求1所述的双荧光素酶报告基因检测系统，其特征在于，
    所述Gaussia荧光素酶和Pleuromamma xiphias荧光素酶的摩尔比为1:(0.01-100)；
    所述Gaussia荧光素酶与底物的摩尔比为1:(1-1000)；
    所述Pleuromamma xiphias荧光素酶与底物的摩尔比为1:(1-1000)。
  12. 权利要求1-11任一项所述的双荧光素酶报告基因检测系统在制备检测试剂中的用途。
  13. 根据权利要求12所述的用途，其特征在于，所述检测试剂包括：细胞成像试剂、氨基酸标记试剂、蛋白标记及定位试剂、抗体的特异性识别试剂、核酸标记试剂或基因测序试剂。
  14. 一种荧光检测试剂盒，其包括权利要求1-11任一项所述的双荧光素酶报告基因检测系统。
  15. 根据权利要求14所述的荧光检测试剂盒，其特征在于，还包括反应缓冲液，所述反应缓冲液包括水和Tris-HCl、NaCl和Tween-20。
  16. 一种荧光素酶发光信号的检测方法，其包括将权利要求1-11任一项所述的底物与样品接触，检测由所述Gaussia荧光素酶和Pleuromamma xiphias荧光素酶产生的发光信号；所述样品中含有权利要求1-11任一项所述的Gaussia荧光素酶和Pleuromamma xiphias荧光素酶。
  17. 根据权利要求16所述的方法，其特征在于，所述样品为表达权利要求1-11任一项所述的Gaussia荧光素酶和Pleuromamma xiphias荧光素酶的细胞、含有细胞的培养物或者细胞裂解物。
  18. 根据权利要求16所述的方法，其特征在于，所述样品为特异性结合权利要求1-11任一项所述的Gaussia荧光素酶和Pleuromamma xiphias荧光素酶的核苷酸类似物，所述核苷酸类似物与荧光素酶与通过亲核试剂特异性结合。
  19. 根据权利要求18所述的方法，其特征在于，所述亲核试剂为抗原-抗体复合物、生物素-链霉素亲和素或者地高辛-地高辛抗体。
  20. 根据权利要求18或19所述的方法，其特征在于，所述核苷酸类似物由含有四种不同碱基类型的核苷酸分子组成，其中第一种核苷酸分子能够与Gaussia荧光素酶特异性结合，第二种核苷酸分子能够与Pleuromamma xiphias荧光素酶特异性结合，第三种核苷酸分子能够与Gaussia荧光素酶和Pleuromamma xiphias荧光素酶特异性结合，第四种核苷酸分子既不与Gaussia荧光素酶结合，也不与Pleuromamma xiphias荧光素酶结合。
  21. 根据权利要求16～20任一项所述的方法，其特征在于，所述方法还包括：通过所述发光信号判断所述核苷酸分子的类型。
  22. 一种对核酸分子进行测序的方法，其包括以下步骤：
    (1)提供连接于支持物上的待测序的核酸分子，或者将待测序的核酸分子连接于支持物上；
    (2)添加用于起始核苷酸聚合反应的引物，用于进行核苷酸聚合反应的聚合酶，以及四种化合物，从而形成含有溶液相和固相的反应体系；其中，所述四种化合物分别为核苷酸A，(T/U)、C和G的衍生物，并且具有碱基互补配对能力；并且，所述四种化合物的核糖或脱氧核糖的3’位置处的羟基(-OH)被保护基团保护；并且，
    第一化合物连接有第一分子标记，
    第二化合物连接有第二分子标记，
    第三化合物连接有第一分子标记和第二分子标记，或部分第三化合物连接有第一分子标记且另外一部分第三化合物连接有第二分子标记，第四化合物没有连接分子标记；
    (3)使引物退火至待测序的核酸分子上，所述引物作为起始的生长的核酸链，与所述待测序的核酸分子一起形成连接于支持物上的双链体；
    (4)在允许聚合酶进行核苷酸聚合反应的条件下，使用聚合酶进行核苷酸聚合反应，从而将所述四种化合物中的一种掺入生长的核酸链的3’端；
    (5)使上一步骤中的双链体与权利要求1-11任一项所述的Gaussia荧光素酶和Pleuromamma xiphias荧光素酶接触并进行结合反应，所述Gaussia荧光素酶和Pleuromamma xiphias荧光素酶分别能够特异性结合所述第一分子标记和第二分子标记，然后使所述荧光素酶在底物存在的情况下发生荧光反应，检测发出的荧光信号；
    (6)去除所掺入的核苷酸的保护基团以及分子标记；
    (7)任选地重复步骤(4)-(6)或(3)-(6)一次或多次，从而获得所述核酸分子的序列信息。
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101636503A (zh) * 2006-10-24 2010-01-27 基因流股份有限公司 萤光素酶信号增强组合物
CN101636504A (zh) * 2006-12-21 2010-01-27 基因流股份有限公司 利用分泌性荧光素酶的生物发光测定
CN112368397A (zh) * 2018-03-13 2021-02-12 萨摩尔股份有限公司 用于单分子测序的方法
CN114807299A (zh) * 2022-06-27 2022-07-29 启德医药科技（苏州）有限公司 一种用于检测adc药物活性的双荧光素酶法、细胞及试剂盒

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101636503A (zh) * 2006-10-24 2010-01-27 基因流股份有限公司 萤光素酶信号增强组合物
CN101636504A (zh) * 2006-12-21 2010-01-27 基因流股份有限公司 利用分泌性荧光素酶的生物发光测定
CN112368397A (zh) * 2018-03-13 2021-02-12 萨摩尔股份有限公司 用于单分子测序的方法
CN114807299A (zh) * 2022-06-27 2022-07-29 启德医药科技（苏州）有限公司 一种用于检测adc药物活性的双荧光素酶法、细胞及试剂盒

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
COUTANT, E. P ET AL.: "Bioluminescence Profiling of NanoKAZ/NanoLuc Luciferase Using a Chemical Library of Coelenterazine Analogues", CHEMICAL BIOLOGY, vol. 26, 31 December 2020 (2020-12-31), pages 948 - 958, XP071851866, DOI: 10.1002/chem.201904844 *
HEISE, K ET AL.: "Dual Luciferase Assay for Secreted Luciferases Based on Gaussia and NanoLuc", ASSAY AND DRUG DEVELOPMENT TECHNOLOGIES, vol. 11, no. 4, 31 May 2013 (2013-05-31), pages 244 - 252, XP055697314, DOI: 10.1089/adt.2013.509 *
TAKENAKA, Y. ET AL.: "Computational Analysis and Functional Expression of Ancestral Copepod Luciferase", GENE, vol. 528, 23 July 2013 (2013-07-23), pages 201 - 205, XP028700819, DOI: 10.1016/j.gene.2013.07.011 *

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