Copepod luciferase mutant and use thereof

Abstract

The present invention belongs to the technical field of genetic engineering, and specifically relates to a copepod luciferase mutant and the use thereof. The mutant is obtained by means of performing one or more mutations on a wild-type copepod luciferase with the signal peptide removed, and can be expressed in a prokaryotic expression system, resulting in an increased luminescence half-life or a signal rise within a certain time range, and the luminescence pattern of the luciferase is converted to glow-type luminescence or luminescence with a sustained increase in luminescence intensity. Some copepod luciferase mutants exhibit enhanced luminescence intensity. The mutant is widely used in biological research for real-time tracking and continuous monitoring, such as for labeling and tracking activities or processes in living organisms. On the basis of the advantage of increased luminescence half-life of the copepod luciferase mutant, the mutant can be used as and / or in the preparation of a glow-type luciferase. The copepod luciferase mutant is more suitable as a reporter gene for in-vivo detection in scientific research, and is conjugated with avidin, a digoxin antibody, etc., for sequencing, thereby shortening the sequencing time and improving the sequencing quality.

Description

Amphipod luciferase mutants and uses thereof TECHNICAL FIELD

[0001] The present application belongs to the technical field of genetic engineering, and specifically relates to amphipod luciferase mutants and uses thereof. BACKGROUND

[0002] Luciferase is a general term for a class of enzymes that catalyze the oxidation of luciferin or aldehyde to emit light in living organisms, and is usually found in lower animals. The commonly used luciferases at present include firefly luciferase, sea pansy luciferase, and Gauss luciferase. The principle of luciferase catalyzing the oxidation of the substrate to emit light under aerobic conditions is as follows: luciferase can catalyze the reaction of the substrate luciferin and oxygen, converting them into oxidized luciferin and carbon dioxide, while releasing energy, which is transferred to the luciferin molecule, making it in an excited state. In the excited state, the luciferin molecule emits fluorescent photons, thereby producing visible light self-luminous. The intensity of the fluorescent signal produced by luciferase is proportional to the concentration of the substrate luciferin, so the fluorescent signal produced by the reaction can be detected by adding the substrate luciferin and using a fluorescence detector. Luciferase usually exhibits different light emission modes during light emission, two common modes are "glow" and "flash". Flash refers to the sudden generation of strong fluorescent signal by luciferase in a short time, and then it is quickly extinguished. This mode is usually used for instantaneous detection or reporting the occurrence of a specific event, such as in the study of gene expression regulation, flash mode can be used to monitor the start or stop of a certain gene. Glow refers to the continuous production of fluorescence by luciferase, usually showing a continuous, slow-decaying fluorescent signal, rather than intermittent, burst light emission, which is often used to mark and track activities or processes in living organisms, such as the expression, distribution and movement of proteins in cells. Glow mode is suitable for experiments that require continuous monitoring. In biological research, different light emission modes make luciferase a powerful tool for real-time tracking and studying various biological processes in living organisms.

[0003] Gaussia luciferase (Gluc), also known as Gaussia luciferase, is a luciferase derived from Gaussia princeps, a marine copepod. It is one of the smallest secreted luciferases with a molecular weight of about 19.9 kDa, consisting of 185 amino acids. Gaussia luciferase can catalyze the enzymatic oxidation of luciferin substrates such as coelenterazine and its derivatives, and emit fluorescence with a wavelength of 460-480 nm. Depending on the substrate, the wavelength of the light photon is also different. Compared with other commonly used luciferases, Gaussia luciferase can produce stronger light signals. Moreover, unlike the firefly luciferase system, Gaussia luciferase does not require high-energy molecules such as ATP and coenzyme A. Instead, it only requires coelenterazine and O2, greatly simplifying its use in a variety of reporter applications. Therefore, Gaussia luciferase is often used as a reporter gene and is widely used in life science research, drug screening, environmental monitoring, enzyme-linked detection, and sequencing.

[0004] In sequencing or other research based on Gaussia luciferase as a signal factor, in order to meet the application requirements, the luciferase is required to have high brightness and both glow and flash modes. The wild-type Gaussia luciferase has unsatisfactory brightness and light duration, which greatly limits its application.

[0005] Therefore, it is of great application value to develop a copepod luciferase (Gaussia luciferase) with high brightness and / or long light half-life.

[0006] SUMMARY

[0007] In a first aspect, the present application provides a copepod luciferase mutant, which is (a1) or (a2):

[0008] (a1) Compared with the amino acid sequence of the wild-type copepod luciferase without signal peptide, the amino acid sequence of the mutant comprises a mutation at one or more of the following positions: 43, 67, 72, 78, 79, 80, 86, 87, 93, 110, 119, 121, 146, 153, 157;

[0009] (a2) The amino acid sequence of the mutant comprises the mutation of the mutant of (a1), and has at least 80% identity with the amino acid sequence of the mutant of (a1).

[0010] Preferably, the amphipod luciferase comprises at least one of Gaussia luciferase (Gluc), Maluc (Metridia asymmetrica Luciferase, BAN91823.1), Mpluc (Metridia pacifica Luciferase, BAD93333.1), and Mluc7 (Metridia longa Luciferase, AJC98141.1).

[0011] Preferably, the amphipod luciferase is Gaussia luciferase.

[0012] In a second aspect of the present application, there is provided a recombinant protein comprising a modification moiety and the amphipod luciferase mutant of the first aspect of the present application.

[0013] Preferably, the modification moiety is a protein tag.

[0014] Preferably, the modification moiety is located at the N-terminus and / or the C-terminus of the amphipod luciferase mutant.

[0015] In a third aspect of the present application, there is provided a fusion protein comprising a glow-type luciferase and a flash-type luciferase, the glow-type luciferase comprising the amphipod luciferase mutant of the first aspect of the present application, and / or the recombinant protein of the second aspect of the present application.

[0016] In a fourth aspect of the present application, there is provided a biological material related to the amphipod luciferase mutant of the first aspect of the present application, the recombinant protein of the second aspect of the present application, or the fusion protein of the third aspect of the present application, the biological material comprising any one of d1) to d12):

[0017] d1) a nucleic acid molecule encoding the amphipod luciferase mutant of the first aspect of the present application, the recombinant protein of the second aspect of the present application, or the fusion protein of the third aspect of the present application;

[0018] d2) an expression cassette comprising the nucleic acid molecule of d1);

[0019] d3) a vector comprising the nucleic acid molecule of d1);

[0020] d4) a vector comprising the expression cassette of d2);

[0021] d5) a transgenic cell line comprising the nucleic acid molecule of d1);

[0022] d6) a transgenic cell line comprising the expression cassette of d2);

[0023] d7) a transgenic cell line comprising the vector of d3);

[0024] d8) a transgenic cell line comprising the vector of d4);

[0025] d9) a recombinant bacterium comprising the nucleic acid molecule of d1);

[0026] d10) a recombinant bacterium comprising the expression cassette of d2);

[0027] d11) a recombinant bacterium comprising the vector of d3);

[0028] d12) a recombinant bacterium comprising the vector of d4).

[0029] In a fifth aspect of the present application, there is provided a conjugate or conjugate comprising (a) and (b):

[0030] (a) a conjugation or conjugation moiety;

[0031] (b) at least one of the mutant copepod luciferase of the first aspect of the present application, the recombinant protein of the second aspect of the present application, or the fusion protein of the third aspect of the present application;

[0032] The conjugation or conjugation moiety comprises at least one of a small molecule compound, a biological macromolecule.

[0033] In a sixth aspect of the present application, there is provided a kit.

[0034] A nucleic acid sequencing kit comprising any one of i1) to i4):

[0035] i1) the mutant copepod luciferase of the first aspect of the present application;

[0036] i2) the recombinant protein of the second aspect of the present application;

[0037] i3) the fusion protein of the third aspect of the present application;

[0038] i4) the conjugate or conjugate of the fifth aspect of the present application, wherein the conjugation or conjugation moiety comprises at least one of avidin, digoxin antibody, nucleotide antibody, nucleotide.

[0039] A kit for detecting a target molecule comprising any one of j1) to j4):

[0040] j1) the mutant copepod luciferase of the first aspect of the present application;

[0041] j2) the recombinant protein of the second aspect of the present application;

[0042] j3) the fusion protein of the third aspect of the present application;

[0043] j4) the conjugate or the complex of the fifth aspect of the present application, wherein the conjugate or the complex part comprises a substance that specifically binds to the target molecule.

[0044] In a seventh aspect of the present application, a nucleic acid sequencing method is provided, comprising the step of detecting using the nucleic acid sequencing kit of the sixth aspect of the present application.

[0045] Preferably, the nucleic acid sequencing method comprises the following steps:

[0046] (1) monitoring the sequential incorporation of nucleotides in the complementary strand of the nucleic acid to be detected, wherein the nucleotides are each attached to a luminescent label that initiates a different luminescence kinetics or luminescence type;

[0047] (2) identifying each incorporated nucleotide by detecting the photokinetics curve or luminescence type or light intensity of the luminescence reaction in which the luminescent label is involved.

[0048] In an eighth aspect of the present application, a method for detecting a target molecule is provided, comprising the step of detecting using the kit for detecting a target molecule of the sixth aspect of the present application.

[0049] Preferably, the method for detecting a target molecule comprises the following steps: contacting j4) in the kit for detecting a target molecule of the sixth aspect of the present application with a sample to be detected, then adding a substrate for luciferase, and determining the presence or absence or content of the target molecule according to the fluorescent signal emitted by the reaction of the luciferase with the substrate for luciferase; the substrate for luciferase comprises the substrate for the copepod luciferase.

[0050] Preferably, when the j4) comprises the fusion protein of the third aspect of the present application, the substrate for luciferase further comprises the substrate for the flash-type luciferase.

[0051] Preferably, the substrate for the copepod luciferase comprises at least one of coelenterazine or a coelenterazine derivative.

[0052] In a ninth aspect of the present application, a method for screening a substrate for copepod luciferase is provided, wherein at least one of the copepod luciferase mutant of the first aspect of the present application, the recombinant protein of the second aspect of the present application, the fusion protein of the third aspect of the present application, the conjugate or the complex of the fifth aspect of the present application is mixed with a substrate to be screened, and whether the mixture obtained by mixing emits a fluorescent signal is determined to judge whether the substrate to be screened is a substrate for copepod luciferase.

[0053] In the tenth aspect of the present application, a method for preparing the mutant of the firefly luciferase of the first aspect of the present application, the recombinant protein of the second aspect of the present application, and the fusion protein of the third aspect of the present application is provided, which is obtained by culturing the transgenic cell line of the fourth aspect of the present application and / or the recombinant bacteria.

[0054] In the eleventh aspect of the present application, any of the following applications is provided:

[0055] (1) the use of the mutant of the firefly luciferase of the first aspect of the present application, the recombinant protein of the second aspect of the present application, and / or the biological material of the fourth aspect of the present application in the preparation and / or as a glow-type luciferase;

[0056] (2) the use of the mutant of the firefly luciferase of the first aspect of the present application, the recombinant protein of the second aspect of the present application, the fusion protein of the third aspect of the present application, the biological material of the fourth aspect of the present application, and / or the conjugate or the complex of the fifth aspect of the present application in k1) to k7):

[0057] k1) sequencing;

[0058] k2) detecting a target molecule for non-diagnostic purposes;

[0059] k3) drug screening;

[0060] k4) life science research;

[0061] k5) environmental monitoring;

[0062] k6) as a reporter gene;

[0063] k7) preparing a product for any of k1) to k6).

[0064] Preferably, when the product is for k1), the product further comprises a flash-type luciferase.

[0065] Preferably, the product is a reagent, a kit, a chip, or a system.

[0066] The beneficial effects of the present application are: the inventors have found through research that the mutation sites (positions 43, 67, 72, 78, 79, 80, 86, 87, 93, 110, 119, 121, 146, 153, 157) are important sites affecting the glow-type luminescence of wild-type copepod luciferase with signal peptide removed, based on which the inventors provide a copepod luciferase mutant, which is obtained by mutating one or more of the above-mentioned sites of wild-type copepod luciferase with signal peptide removed, the copepod luciferase mutant can be expressed in a prokaryotic expression system, the half-life of the luminescence generated by catalyzing the substrate is increased or the signal rises within a certain time range, the luciferase luminescence mode changes to a more persistent glow-type luminescence or a luminescence intensity continuously rising type luminescence; and the luminescence intensity generated by part of the copepod luciferase mutant is enhanced; the copepod luciferase mutant is widely used in biological research for real-time tracking and continuous monitoring, such as being used for marking and tracking activities or processes in vivo, such as the expression, distribution and movement of intracellular proteins; based on the advantage of the increased half-life of the luminescence generated by the copepod luciferase mutant, the copepod luciferase mutant can be used as and / or prepared as a glow-type luciferase; it is more suitable as a reporter gene and is applied to in vivo detection for scientific research; and it is coupled with avidin (such as streptavidin SA), digoxin antibody and the like for sequencing (used in combination with flash-type luciferase), which shortens the sequencing time and improves the sequencing quality. BRIEF DESCRIPTION OF DRAWINGS

[0067] Fig. 1 is a schematic diagram of the sequence alignment results of Gaussia luciferase and other homologous copepod luciferases.

[0068] Fig. 2 is a map of the plasmid (pCold-Gluc-NO SP) expressing C-terminal fusion His-tagged wild-type Gaussia luciferase with signal peptide removed in Example 1.

[0069] Fig. 3 is an SDS-PAGE characterization result diagram of the C-terminal fusion His-tagged wild-type Gaussia luciferase with signal peptide removed or its mutant purified in Example 1: wherein 1-43 respectively represent WT-NS Gluc, Gluc-NO SP-1, Gluc-NO SP-42 in Table 3.

[0070] Fig. 4 is a structural diagram of coelenterazine.

[0071] Fig. 5 is a comparison result diagram of the catalytic activity of the C-terminal fusion His-tagged wild-type Gaussia luciferase with signal peptide removed or its mutant on the substrate in Example 2.

[0072] Figure 6 is a graph showing the comparison results of the luminescence half-life of the substrate catalyzed by wild-type Gaussian luciferase or its mutants (WT-NS Gluc, Gluc-NO SP-1~Gluc-NO SP-17) with the C-terminal fusion of His tag and the removal of the signal peptide in Example 2.

[0073] Figure 7 is a luminescence kinetics curve of the substrate catalyzed by wild-type Gaussian luciferase or its mutants (WT-NS Gluc, Gluc-NO SP-18~Gluc-NO SP-27) with the C-terminal fusion of His tag and the removal of the signal peptide in Example 2, wherein the abscissa is time and the ordinate is brightness.

[0074] Figure 8 is a luminescence kinetics curve of the substrate catalyzed by wild-type Gaussian luciferase or its mutants (WT-NS Gluc, Gluc-NO SP-28~Gluc-NO SP-40) with the C-terminal fusion of His tag and the removal of the signal peptide in Example 2, wherein the abscissa is time and the ordinate is brightness.

[0075] Figure 9 is a map of the plasmid (pCold-Maluc-NO SP) of wild-type copepod luciferase Maluc with the C-terminal fusion of His tag and the removal of the signal peptide in Example 3.

[0076] Figure 10 is a map of the plasmid (pCold-Mpluc-NO SP) of wild-type copepod luciferase Mpluc with the C-terminal fusion of His tag and the removal of the signal peptide in Example 3.

[0077] Figure 11 is a map of the plasmid (pCold-Mluc7-NO SP) of wild-type copepod luciferase Mluc7 with the C-terminal fusion of His tag and the removal of the signal peptide in Example 3.

[0078] Figure 12 is a graph showing the comparison results of the catalytic activity of the substrate of wild-type copepod luciferase Maluc / Mpluc / Mluc7 or its mutants with the C-terminal fusion of His tag and the removal of the signal peptide in Example 4.

[0079] Figure 13 is a graph showing the comparison results of the luminescence half-life of the substrate catalyzed by wild-type copepod luciferase Maluc / Mpluc / Mluc7 or its mutants with the C-terminal fusion of His tag and the removal of the signal peptide in Example 4.

[0080] Figure 14 is a graph showing the SDS-PAGE characterization results of the luciferase-digoxin antibody fusion protein purified in Example 5.

[0081] Figure 15 is a luminescence kinetics curve of the luciferase-digoxin antibody fusion protein in Example 5. DETAILED DESCRIPTION

[0082] In the present application, unless otherwise specified, the term "amino acid" is represented by a single letter or a three letter code, and has the following meaning: A: Ala (alanine); R: Arg (arginine); N: Asn (asparagine); D: Asp (aspartic acid); C: Cys (cysteine); Q: Gin (glutamine); E: Glu (glutamic acid); G: Gly (glycine); H: His (histidine); I: Ile (isoleucine); L: Leu (leucine); K: Lys (lysine); M: Met (methionine); F: Phe (phenylalanine); P: Pro (proline); S: Ser (serine); T: Thr (threonine); W: Trp (tryptophan); Y: Tyr (tyrosine); V: Val (valine).

[0083] In the present application, unless otherwise specified, the nucleotide sequence is represented in the 5' end→3' end direction.

[0084] In the present application, unless otherwise specified, the amino acid sequence is represented in the N-terminal→C-terminal direction.

[0085] In the present application, unless otherwise specified, "identity" has the meaning conventionally used in the art, and refers to "homology" between two nucleic acid or amino acid sequences, which is a percentage indicating the statistical percentage of identical nucleotides or amino acid residues between two sequences to be compared, obtained after best alignment, the difference between the two sequences being randomly distributed over the entire length.

[0086] In a first aspect of the present application, there is provided a mutant of copepod luciferase, which is (a1) or (a2):

[0087] (a1) the amino acid sequence of the mutant comprises a mutation at one or more of the following positions: position 43, position 67, position 72, position 78, position 79, position 80, position 86, position 87, position 93, position 110, position 119, position 121, position 146, position 153, position 157, compared to the amino acid sequence of wild-type copepod luciferase from which the signal peptide is removed;

[0088] (a2) the amino acid sequence of the mutant comprises the mutation of (a1) and has at least 80% identity to the amino acid sequence of (a1).

[0089] In some embodiments, the mutation at the above-mentioned positions can be mutated to any natural or unnatural amino acid.

[0090] It should be noted that at least 80% identity includes 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity.

[0091] The inventors found through research that the above mutation sites (positions 43, 67, 72, 78, 79, 80, 86, 87, 93, 110, 119, 121, 146, 153, 157) are important sites affecting the glow-type luminescence of wild-type copepod luciferase with signal peptide removed. By mutating one or more of the above sites of wild-type copepod luciferase with signal peptide removed, the luminescence half-life of the mutant copepod luciferase produced by the mutation can be increased or the signal can be increased within a certain time range, the luciferase luminescence pattern changes to a more persistent glow-type luminescence or a luminescence intensity continuously rising type luminescence; and the luminescence intensity of part of the copepod luciferase mutants is enhanced.

[0092] Flash refers to the sudden production of strong fluorescent signal by luciferase in a short time, and then quickly extinguished. This mode is usually used for instantaneous detection or reporting the occurrence of a specific event, such as in the study of gene expression regulation, flash mode can be used to monitor the start or stop of a certain gene. Glow refers to the continuous production of fluorescence by luciferase, usually showing a continuous, slow-decaying fluorescent signal, rather than intermittent, burst luminescence, which is often used to mark and track activities or processes in living organisms, such as the expression, distribution and movement of proteins in cells. Glow mode is suitable for experiments that require continuous monitoring.

[0093] In application, the above-mentioned mutant luciferases of copepods are widely used in the fields of life science research (e.g. in vivo detection: such as for marking and tracing activities or processes in organisms, such as the expression, distribution and movement of intracellular proteins), medical detection, drug screening, environmental monitoring and enzyme-linked detection, etc., and are applied to genomic sequencing and analysis technology, playing a role in the fields of clinical medicine, forensic detection and customs, etc. Using the self-luminescent characteristics of the mutant luciferases of copepods, the mutant luciferases of copepods are often applied to the fields of live cell detection, protein-protein interaction, protein localization, small interfering RNA silencing technology, high-throughput drug screening, etc. In the field of biological monitoring technology, the mutant luciferases of copepods can be used to detect the presence or absence of chemical pollutants. In addition, they also have broad application prospects in the fields of immunodetection, biochemical diagnosis, etc. The above-mentioned mutant luciferases of copepods are also widely used in biological research for real-time tracking and continuous monitoring, such as for marking and tracing activities or processes in organisms, such as the expression, distribution and movement of intracellular proteins. In addition, they can also be used as reporter genes for the study of the expression strength and transcriptional regulation of exogenous genes under different promoters, and play an important role in the quantitative detection of DNA, RNA, transcription factors, proteins or cells, etc. Based on the advantages of the above-mentioned mutant luciferases of copepods in increasing the luminescent half-life or signal rising within a certain time range, the mutant luciferases of copepods can be used as and / or prepared into glow-type luciferase; are more suitable as reporter genes for in vivo detection in scientific research; and are coupled with avidin (such as streptavidin SA), digoxin antibody, etc. for sequencing, shortening the sequencing time and improving the sequencing quality.

[0094] In some embodiments, the mutant luciferase of copepods comprises at least one of Gluc, Maluc (Metridia asymmetrica Luciferase, BAN91823.1), Mpluc (Metridia pacifica Luciferase, BAD93333.1), and Mluc7 (Metridia longa Luciferase, AJC98141.1).

[0095] In some embodiments, the mutant luciferase of copepods is Gluc.

[0096] In some embodiments, the mutant comprises any one mutation or a combination of multiple mutations in (b1)~(b15) compared to the amino acid sequence of wild-type Gaussia luciferase without signal peptide: (b1) M at position 43 is mutated to L; (b2) P at position 67 is mutated to L; (b3) F at position 72 is mutated to Y; (b4) H at position 78 is mutated to E; (b5) T at position 79 is mutated to P; (b6) Y at position 80 is mutated to W, or F; (b7) S at position 86 is mutated to T; (b8) A at position 87 is mutated to G; (b9) E at position 93 is mutated to P; (b10) M at position 110 is mutated to L; (b11) L at position 119 is mutated to R; (b12) V at position 121 is mutated to E; (b13) Q at position 146 is mutated to G; (b14) S at position 153 is mutated to D; (b15) G at position 157 is mutated to S, or A.

[0097] In some embodiments, the Cephalopod luciferase mutant does not comprise the Gaussia luciferase dominant mutants in Table 3 of patent document WO2023109981A3 (i.e. Gaussia luciferase dominant mutants numbered as D6, B6, 4-C12, 9-2, 11-2, 16-2, 17-1, 18-2, 19-1, 20-3, 21-1, 22-1, 23-1, 24-2, 26-1, 27-2, 30-3, 33-1, 34-1, 35-1, 36-1, 1-1, 2-3, 4-1, 5-1, 6-1, 8-3, 12-2, 13-2, 28-1, 31-1, 32-3, A2-2, A2-3, A3-1, A5-1, A5-2, A7-1, A7-2, A8-1, A8-2, A12-1, A12-4, A12-5, A13-3, A14-1, A25-1, A28-1, A28-2, A28-3, A29-3, A32-2).

[0098] In some embodiments, the mutant has any one of (c1)-(c42) compared to the amino acid sequence of wild-type copepod luciferase with the signal peptide removed: (c1) S153D; (c2) G157S; (c3) M110L; (c4) Q146G; (c5) G157A; (c6) L119R; (c7) V121E; (c8) Y80W; (c9) E93P; (c10) P67L; (c11) Y80F; (c12) S86T; (c13) H78E; (c14) F72Y; (c15) M43L; (c16) T79P; (c17) A87G; (c18) L23A / D24E / D26E / K41I / R48Q / M43L / H62K / P67L / F72Y / H78E / T79P / Y80W / E85S / S86T / A87G / E93P / L107M / M110L / L119R / V121E / Q146G / S153D / G157A; (c19) M43L / H62K / P67L / T79P / Y80W / E85S / S86T / A87G / E93P / L107M / M110L / L119R / V121E / S153D / G157A; (c20) D24E / D26E / K41I / R48Q / M43L / H62K / P67L / F72Y / H78E / T79P / Y80W / E85S / S86T / A87G / E93P / L107M / M110L / L119R / V121E / Q146G / S153D / G157A; (c21) H62K / P67L / F72Y / E85S / S86T / A87G / E93P / L107M / L119R / V121E / S153D; (c22) H62K / P67L / F72Y / T79P / E85S / S86T / A87G / E93P / L107M / L119R / V121E / S153D; (c23) M43L / H62K / P67L / F72Y / T79P / E85S / S86T / A87G / E93P / L107M / L119R / V121E / S153D; (c24) M43L / H62K / P67L / F72Y / T79P / E85S / S86T / A87G / E93P / L107M / M110L / L119R / V121E / S153D; (c25) M43L / H62K / P67L / F72Y / T79P / E85S / S86T / A87G / E93P / L107M / M110L / L119R / V121E / S153D / G157A;(c26) M43L / H62K / P67L / F72Y / T79P / Y80F / E85S / S86T / A87G / E93P / L107M / M110L / L119R / V121E / S153D / G157A; (c27) M43L / H62K / P67L / F72Y / T79P / Y80F / E85S / S86T / A87G / E93P / L107M / M110L / L119R / V121E / Q146G / S153D / G157A; (c28) M43L / H62K / P67L / F72Y / H78E / T79P / Y80F / E85S / S86T / A87G / E93P / L107M / M110L / L119R / V121E / Q146G / S153D / G157A; (c29) H62K / P67L / T79P / E85S / S86T / A87G / E93P / L107M / V121E; (c30) M43L / H62K / P67L / T79P / E85S / S86T / A87G / E93P / L107M / V121E; (c31) M43L / H62K / P67L / T79P / E85S / S86T / A87G / E93P / L107M / M110L / V121E; (c32) M43L / H62K / P67L / T79P / E85S / S86T / A87G / E93P / L107M / M110L / L119R / V121E / S153D; (c33) M43L / H62K / P67L / T79P / E85S / S86T / A87G / E93P / L107M / M110L / L119R / V121E / S153D / G157A; (c34) H62K / P67L / F72Y / E85S / S86T / A87G / E93P / L107M / V121E; (c35) D24E / M43L / H62K / P67L / T79P / Y80F / E85S / S86T / A87G / E93P / L107M / M110L / L119R / V121E / Q146G / S153D / G157A; (c36) H62K / F72Y / E85S / S86T / A87G / E93P / L107M / V121E; (c37) K41I / R48Q / M43L / H62K / P67L / H78E / T79P / Y80F / E85S / S86T / A87G / E93P / L107M / M110L / L119R / V121E / Q146G / S153D / G157A;(c38) D24E / K41I / R48Q / M43L / H62K / P67L / H78E / T79P / Y80F / E85S / S86T / A87G / E93P / L107M / M110L / L119R / V121E / Q146G / S153D / G157A; (c39) K41I / R48Q / M43L / H62K / P67L / F72Y / H78E / T79P / Y80F / E85S / S86T / A87G / E93P / L107M / M110L / L119R / V121E / Q146G / S153D / G157A; (c40) D26E / K41I / R48Q / M43L / H62K / P67L / F72Y / H78E / T79P / Y80F / E85S / S86T / A87G / E93P / L107M / M110L / L119R / V121E / Q146G / S153D / G157A; (c41) H62K / F72Y / E85S / S86T / A87G / E93P / L107M / L119R / V121E / S153D; (c42) K41I / M43L / H62K / P67L / H78E / T79P / Y80F / E85S / S86T / A87G / E93P / L107M / M110L / L119R / V121E / Q146G / S153D / G157A.

[0099] In some embodiments, the mutant has any one of the (c1), (c3), (c4), (c6), (c7), (c9), (c10), (c12), (c15)-(c27), (c30)-(c42) mutations compared to the amino acid sequence of the wild-type copepod luciferase without signal peptide.

[0100] In some embodiments, the mutant has any one of the (c1), (c3), (c6), (c7), (c9), (c12), (c17)-(c27), (c33)-(c42) mutations compared to the amino acid sequence of the wild-type copepod luciferase without signal peptide.

[0101] In some embodiments, the amino acid sequence of the wild-type copepod luciferase without signal peptide is shown in SEQ ID NO: 4.

[0102] In some embodiments, the mutant further comprises a signal peptide.

[0103] In some embodiments, the signal peptide is located at the N-terminus of the mutant.

[0104] In some embodiments, the amino acid sequence of the signal peptide is set forth in SEQ ID NO: 3.

[0105] In some embodiments, the amphipod luciferase is Maluc.

[0106] In some embodiments, the amino acid sequence of the mutant comprises a mutation at one or more of the following positions: position 78, position 79, position 110, or position 146, as compared to the amino acid sequence of the wild-type Maluc without the signal peptide.

[0107] In some embodiments, the mutant comprises any one mutation or a combination of multiple mutations of: H at position 78 mutated to E, D at position 79 mutated to P, M at position 110 mutated to L, or K at position 146 mutated to G, as compared to the amino acid sequence of the wild-type Maluc without the signal peptide.

[0108] In some embodiments, the amino acid sequence of the wild-type Maluc without the signal peptide is set forth in SEQ ID NO: 49.

[0109] In some embodiments, the mutant further comprises a signal peptide.

[0110] In some embodiments, the signal peptide is located at the N-terminus of the mutant.

[0111] In some embodiments, the amino acid sequence of the signal peptide is set forth in SEQ ID NO: 48.

[0112] In some embodiments, the amphipod luciferase is Mpluc.

[0113] In some embodiments, the amino acid sequence of the mutant comprises a mutation at one or more of the following positions: position 101, position 102, position 133, or position 169, as compared to the amino acid sequence of the wild-type Mpluc without the signal peptide.

[0114] In some embodiments, the mutant comprises any one mutation or a combination of multiple mutations of: H at position 101 mutated to E, D at position 102 mutated to P, M at position 133 mutated to L, or D at position 169 mutated to G, as compared to the amino acid sequence of the wild-type Mpluc without the signal peptide.

[0115] In some embodiments, the amino acid sequence of the wild-type Mpluc without the signal peptide is set forth in SEQ ID NO: 52.

[0116] In some embodiments, the mutant further comprises a signal peptide.

[0117] In some embodiments, the signal peptide is located at the N-terminus of the mutant.

[0118] In some embodiments, the amino acid sequence of the signal peptide is set forth in SEQ ID NO: 51.

[0119] In some embodiments, the signal peptide is located at the N-terminus of the mutant.

[0120] In some embodiments, the amino acid sequence of the signal peptide is set forth in SEQ ID NO: 54.

[0121] In some embodiments, the amino acid sequence of the signal peptide is set forth in SEQ ID NO: 54.

[0122] In some embodiments, the amino acid sequence of the signal peptide is set forth in SEQ ID NO: 54.

[0123] In some embodiments, the signal peptide is located at the N-terminus of the mutant.

[0124] In some embodiments, the signal peptide is located at the N-terminus of the mutant.

[0125] In some embodiments, the amino acid sequence of the signal peptide is set forth in SEQ ID NO: 54.

[0126] The radopholus luciferin is a conserved luciferase, and the important site of Gluc affecting flash-type luminescence is similar to the corresponding sites of other radopholus luciferases such as Maluc, Mpluc, Mluc7, etc. The sequence alignment of the Gaussia luciferase and other homologous radopholus luciferases is shown in FIG. 1, wherein the mutation sites (compared with the amino acid sequence of the wild-type Maluc without signal peptide) of the Maluc mutant are H78E, D79P, M110L and K146G, the mutation sites (compared with the amino acid sequence of the wild-type Mpluc without signal peptide) of the Mpluc mutant are H101E, D102P, M133L and D169G, the mutation sites (compared with the amino acid sequence of the wild-type Mluc7 without signal peptide) of the Mluc7 mutant are H61E, D62P, M93L and D129G, which respectively correspond to the mutation sites (compared with the amino acid sequence of the wild-type Gluc without signal peptide) of the Gluc mutant are H78E, T79P, M110L and Q146G. By mutating the above-mentioned sites, the luminescence half-life of the luciferase mutant obtained by mutation can be increased, and the luminescence intensity of part of the mutant can be enhanced.

[0127] In a second aspect of the present application, a recombinant protein is provided, which comprises a modification moiety and the radopholus luciferase mutant of the first aspect of the present application.

[0128] In some embodiments, the modification moiety is a protein tag.

[0129] In some embodiments, the protein tag is selected from at least one of Poly his (His), FLAG, Strep-Tag II, Poly arg, C-myc, HA, V5, VSV-G, Trx, SUMO, GST, MBP, and NusA; and further is Poly his (His) (such as: 6xHis).

[0130] In some embodiments, the modification moiety is located at the N-terminus and / or the C-terminus of the radopholus luciferase mutant.

[0131] In some embodiments, the modification moiety is located at the C-terminus of the radopholus luciferase mutant.

[0132] In some embodiments, the modification moiety further comprises a connecting peptide between the radopholus luciferase mutant.

[0133] In some embodiments, the recombinant protein comprises: TEE site-radopholus luciferase mutant-TEV site-His tag (N-terminus→C-terminus).

[0134] In a third aspect of the present application, a fusion protein comprising a glow luciferase and a flash luciferase is provided.

[0135] The glow luciferase comprises the copepod luciferase mutant of the first aspect of the present application, and / or the recombinant protein of the second aspect of the present application.

[0136] In the present application, the "flash luciferase" refers to a type of luciferase having a shorter luminescence half-life than the glow luciferase; preferably, the luminescence half-life of the flash luciferase is 10 s shorter than that of the glow luciferase, more preferably 25 s shorter. The flash luciferase can be a commercially available flash luciferase, which is not limited herein.

[0137] In some embodiments, the glow luciferase is located at the C-terminus and / or N-terminus of the flash luciferase.

[0138] In some embodiments, the glow luciferase and the flash luciferase are connected by a linker peptide.

[0139] In a fourth aspect of the present application, a biological material related to the copepod luciferase mutant of the first aspect of the present application, the recombinant protein of the second aspect of the present application, or the fusion protein of the third aspect of the present application is provided, which comprises any one of d1) to d12):

[0140] d1) a nucleic acid molecule encoding the copepod luciferase mutant of the first aspect of the present application, the recombinant protein of the second aspect of the present application, or the fusion protein of the third aspect of the present application;

[0141] d2) an expression cassette comprising the nucleic acid molecule of d1);

[0142] d3) a vector comprising the nucleic acid molecule of d1);

[0143] d4) a vector comprising the expression cassette of d2);

[0144] d5) a transgenic cell line comprising the nucleic acid molecule of d1);

[0145] d6) a transgenic cell line comprising the expression cassette of d2);

[0146] d7) a transgenic cell line comprising the vector of d3);

[0147] d8) a transgenic cell line comprising the vector of d4);

[0148] d9) a recombinant bacterium comprising the nucleic acid molecule of d1);

[0149] d10) a recombinant bacterium comprising the expression cassette of d2);

[0150] d11) a recombinant bacterium comprising the vector of d3);

[0151] d12) a recombinant bacterium comprising the vector of d4).

[0152] In some embodiments, the transgenic cell lines in d5) to d8) do not comprise reproductive material.

[0153] In some embodiments, the vectors in d3), d4) can further comprise a promoter operably linked to the nucleic acid molecule.

[0154] In some embodiments, the vectors in d3), d4) are independently selected from the group consisting of a non-pathogenic viral vector and a non-viral vector.

[0155] In some embodiments, the non-pathogenic viral vector comprises an adenoviral vector or a retroviral vector.

[0156] In some embodiments, the non-viral vector comprises, but is not limited to, a plasmid vector.

[0157] In some embodiments, the recombinant bacterium in d9) to d12) comprises, but is not limited to, Escherichia coli.

[0158] In a fifth aspect of the present application, there is provided a conjugate or a conjugate comprising (a) and (b):

[0159] (a) a conjugating or conjugating moiety;

[0160] (b) at least one (preferably any one) of the mutant copepod luciferase of the first aspect of the present application, the recombinant protein of the second aspect of the present application, or the fusion protein of the third aspect of the present application; the conjugating or conjugating moiety comprises at least one (preferably comprises any one of) a small molecule compound, a biological macromolecule.

[0161] In some embodiments, the conjugating or conjugating moiety comprises at least one of avidin (e.g. streptavidin), digoxin antibody, nucleotide antibody, nucleotide, a substance that specifically binds to a target molecule.

[0162] In some embodiments, the nucleotide comprises at least one of nucleotides A, C, G, T / U (i.e. the nucleotide comprises at least one of nucleotides A, C, G, T; or the nucleotide comprises at least one of nucleotides A, C, G, U).

[0163] In some embodiments, the nucleotide comprises at least one of ATP, dATP, GTP, dGTP, CTP, dCTP, TTP, dTTP, UTP, dUTP; further comprises at least one of dATP, dGTP, dCTP, dTTP.

[0164] In some embodiments, the nucleotide is a 3'-OH end modified nucleotide.

[0165] In some embodiments, the 3'-OH end modified nucleotide is modified by adding a blocking group at the 3'-OH end of the nucleotide.

[0166] In some embodiments, the blocking group comprises at least one of O-amino, O-allyl, O-azido, O-phosphate group.

[0167] In a sixth aspect of the present application, a kit is provided.

[0168] In some embodiments, a nucleic acid sequencing kit is provided, comprising any one of i1) to i4):

[0169] i1) the mutant of the amphiopod luciferase of the first aspect of the present application;

[0170] i2) the recombinant protein of the second aspect of the present application;

[0171] i3) the fusion protein of the third aspect of the present application;

[0172] i4) the conjugate or conjugate of the fifth aspect of the present application, wherein the conjugate or conjugate moiety comprises at least one of avidin (e.g. streptavidin), digoxin antibody, nucleotide antibody, nucleotide.

[0173] In some embodiments, the nucleotide is the nucleotide of the fifth aspect of the present application.

[0174] In some embodiments, the nucleic acid sequencing kit further comprises at least one of a substrate of the amphiopod luciferase, a polymerase (preferably a DNA polymerase), a PCR buffer.

[0175] In some embodiments, the substrate of the amphiopod luciferase comprises at least one of coelenterazine, a coelenterazine derivative.

[0176] In some embodiments, the coelenterazine derivative comprises at least one of coelenterazine h, coelenterazine hcp, coelenterazine 400a, coelenterazine f, coelenterazine cp, coelenterazine n, coelenterazine e.

[0177] In some embodiments, the nucleic acid sequencing kit further comprises a substrate of the flash luciferase, when i3) is included.

[0178] In some embodiments, the nucleic acid sequencing kit further comprises a nucleotide, when the coupling or conjugating moiety is an avidin (e.g., streptavidin), a digoxin antibody, or a nucleotide antibody.

[0179] In some embodiments, the nucleotide is the nucleotide of the fifth aspect of the present application.

[0180] In some embodiments, a kit for detecting a target molecule is provided, which comprises any one of j1) to j4):

[0181] j1) the mutant of the flash luciferase of the first aspect of the present application;

[0182] j2) the recombinant protein of the second aspect of the present application;

[0183] j3) the fusion protein of the third aspect of the present application;

[0184] j4) the conjugate or conjugate of the fifth aspect of the present application, wherein the coupling or conjugating moiety comprises a substance that specifically binds to the target molecule.

[0185] In some embodiments, the kit for detecting a target molecule further comprises a substrate of the flash luciferase.

[0186] In some embodiments, the substrate of the flash luciferase comprises at least one of coelenterazine, a coelenterazine derivative.

[0187] In some embodiments, the coelenterazine derivative comprises at least one of coelenterazine h, coelenterazine hcp, coelenterazine 400a, coelenterazine f, coelenterazine cp, coelenterazine n, coelenterazine e.

[0188] In some embodiments, the kit for detecting a target molecule comprises j3), the kit further comprises a substrate of the flash luciferase.

[0189] In a seventh aspect of the present application, a nucleic acid sequencing method is provided, comprising the step of detecting using the nucleic acid sequencing kit of the sixth aspect of the present application.

[0190] In some embodiments, the nucleic acid sequencing method comprises the following steps:

[0191] (1) monitoring the sequential incorporation of nucleotides in a complementary strand of a nucleic acid to be detected, the nucleotides each being attached to a luminescent label that initiates a different luminescence kinetics or luminescence type;

[0192] (2) identifying each incorporated nucleotide by detecting the luminescence kinetics or luminescence type or light intensity of the luminescence reaction in which the luminescent label is involved.

[0193] In some embodiments, the incorporated nucleotides carry a blocking modification group.

[0194] In some embodiments, the luminescent label comprises the copepod luciferase mutant of the first aspect of the present application.

[0195] In some embodiments, the luminescent label comprises the copepod luciferase mutant of the first aspect of the present application and a second luminescent label that initiates a different luminescence kinetics or luminescence type or light intensity than the copepod luciferase mutant.

[0196] In some embodiments, the nucleic acid sequencing method further comprises the following step: (3) removing the blocking modification group carried by the incorporated nucleotides so that the complementary strand can continue to extend, and removing the luminescent label.

[0197] In some embodiments, the nucleic acid sequencing method further comprises the following step: repeating steps (1) to (3) until the complementary strand is extended completely to determine the sequence of the nucleic acid to be detected.

[0198] In the present application, a "luminescent label" refers to any compound that can be attached to a nucleotide that can generate a detectable light signal without the need for excitation light by contact with a suitable substrate to trigger a chemiluminescent reaction. In general, any component that participates in a chemiluminescent reaction can be used as a luminescent label as described herein, and correspondingly, other components that participate in a chemiluminescent reaction are referred to herein as substrates for the luminescent label. Examples of commonly used suitable luminescent labels include, but are not limited to, peroxidases, alkaline phosphatase, luciferase, aequorin, functionalized iron-porphyrin derivatives, luminol, luminol, isoluminol, acridinium ester, sulfonamide, etc. The substrate for a luminescent label will depend on the specific luminescent label used, for example, the substrate for alkaline phosphatase can be AMPPD (adamantyl 1,2-dioxetane aromatic phosphate), the substrate for luciferase can be luciferin, the substrate for acridinium ester can be a mixture of sodium hydroxide and H2O2, etc. Detailed descriptions of luminescent labels and their corresponding substrates can be found, for example, in Larry J. Kricka, Chemiluminescent and Bioluminescent Techniques, CLIN. CHEM. 37 / 9, 1472-1481 (1991) and Tsuji, A. et al. (Eds.) (2005) Bioluminescence and chemiluminescence: Progress and perspectives. World Scientific: [s.l.]. ISBN 978-981-256-118-3. 596 pp.

[0199] In the present application, the "luminescence kinetics of a luminescent reaction" refers to the characteristic profile of the intensity of light emitted by a chemiluminescent reaction as a function of time. This can be characterized, for example, by plotting the intensity of light emitted by a chemiluminescent reaction as a function of time.

[0200] In the present application, the "light emission type of light emission reaction" is classified according to the duration of light emitted from a chemiluminescence reaction, which generally includes flash type and glow type. The light emission time of flash type is within several seconds, such as acridinium ester system. The light emission time of glow type is longer, which can be several minutes to several tens of minutes or more, such as horseradish peroxidase-luminol system, alkaline phosphatase-AMPPD system, xanthine oxidase-luminol system, etc. In the present application, the light emission type between flash type and glow type is also referred to as mixed type. The light emission of mixed type is usually generated by mixing the light emission of flash type and the light emission of glow type together. For example, when a flash type chemiluminescence marker and a glow type chemiluminescence marker are mixed together and simultaneously contacted with their substrates and emit light, a mixed light emission type between flash type and glow type is generated. Typical examples of the light emission characteristic spectrum of flash type, glow type and mixed type are shown in FIG. 1 of patent document CN112384632A.

[0201] In some embodiments, the light-emitting marker is selected from luciferases that initiate different light emission kinetics or light emission types; the luciferases that initiate different light emission kinetics or light emission types comprise glow type luciferases comprising the copepod luciferase mutant of the first aspect of the present application or the recombinant protein of the second aspect of the present application.

[0202] In some embodiments, the light-emitting marker comprises two luciferases that initiate different light emission kinetics or light emission types.

[0203] In some embodiments, detecting the light emission kinetics of the chemiluminescence reaction in which the light-emitting marker is involved comprises contacting the light-emitting marker with a suitable substrate to trigger a chemiluminescence reaction, and detecting the light emission kinetics of the light thus emitted.

[0204] In some embodiments, detecting the light emission type of the chemiluminescence reaction in which the light-emitting marker is involved comprises contacting the chemiluminescence marker with a suitable substrate to trigger a chemiluminescence reaction, and detecting the light emission type of the light thus emitted.

[0205] In some embodiments, the light emission type includes flash type, glow type and mixed type.

[0206] In some embodiments, the nucleic acid sequencing method further comprises the step of: passing a suitable substrate to contact the light-emitting marker with the substrate.

[0207] In some embodiments, the substrates of the light-emitting markers can be the same or different.

[0208] In some embodiments, the nucleotides comprise at least one of nucleotides A, C, G, T / U (i.e. the nucleotides comprise at least one of nucleotides A, C, G, T; or the nucleotides comprise at least one of nucleotides A, C, G, U); further comprising nucleotides A, C, G, and T / U.

[0209] In some embodiments, the nucleotides comprise at least one of ATP, dATP, GTP, dGTP, CTP, dCTP, TTP, dTTP, UTP, dUTP; further comprising dATP, dGTP, dCTP, and dTTP; or ATP, GTP, CTP, and UTP.

[0210] In some embodiments, the nucleotides are 3’-OH end modified nucleotides.

[0211] In some embodiments, the 3’-OH end modified nucleotides are modified by adding a blocking group to the 3’-OH end of the nucleotides.

[0212] In some embodiments, the blocking group comprises at least one of O-amino, O-allyl, O-azido, O-phosphate group.

[0213] In some embodiments, the light emission type or photokinetic curve of the luminescence reaction involving the amphipod luciferase mutant is glow type; and the light emission type or photokinetic curve of the luminescence reaction involving the second luminescent marker is flash type.

[0214] In some embodiments, each incorporated nucleotide is identified by detecting the photokinetic curve or light emission type or light intensity of the luminescence reaction involving the luminescent marker; the luminescent marker is selected from luciferases that initiate different light emission types; the luciferases that initiate different light emission types comprise a glow type luciferase, which comprises the amphipod luciferase mutant of the first aspect of the present application or the recombinant protein of the second aspect of the present application.

[0215] In some embodiments, the luciferases that initiate different light emission types further comprise a flash type luciferase.

[0216] In some embodiments, in the nucleotides, a first nucleotide is attached to the amphipod luciferase mutant of the first aspect of the present application, a second nucleotide is attached to a second luminescent marker, a third nucleotide is attached to both the amphipod luciferase mutant of the first aspect of the present application and the second luminescent marker, and a fourth nucleotide is not attached to a luminescent marker.

[0217] In some embodiments, the attachment between the nucleotide and the luminescent label comprises attachment mediated by an affinity interaction.

[0218] In some embodiments, the affinity interaction comprises an antigen-antibody interaction and a biotin-avidin (e.g., streptavidin) interaction.

[0219] In some embodiments, the luminescent label is attached to the nucleotide by affinity interaction between members that are linked to the luminescent label and members that are linked to the nucleotide.

[0220] In some embodiments, the member linked to the nucleotide is biotin and the member linked to the luminescent label is avidin (e.g., streptavidin).

[0221] In some embodiments, the member linked to the nucleotide is digoxigenin and the member linked to the luminescent label is an anti-digoxigenin antibody.

[0222] In some embodiments, the member linked to the nucleotide is digoxigenin and the member linked to the luminescent label is avidin (e.g., streptavidin), wherein the digoxigenin and the avidin are affinity bound by an anti-digoxigenin antibody linked to biotin.

[0223] In some embodiments, "labeling a nucleotide with a luminescent label" means attaching a luminescent label to a nucleotide. Specific ways of attaching a luminescent label to a nucleotide are known to those skilled in the art, e.g., see the relevant descriptions in Sambrook et al., Molecular Cloning, A Laboratory Manual, 2 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y, 1989), Chapter 10; U.S. Patent Nos. 4,581,333, 5,283,174, 5,547,842, 5,656,207, and 5,658,737.

[0224] In some embodiments, the luminescent label can be attached directly to the nucleotide by a covalent bond. In another embodiment, the luminescent label can be attached to the nucleotide by a linker group.

[0225] In some embodiments, the luminescent label is attached to the nucleotide by an affinity interaction. As is well known to those skilled in the art, an "affinity interaction" generally refers to a specific interaction between a biomolecule (e.g. a proteinaceous molecule, e.g. an enzyme, an antibody) and a substance to which it specifically recognizes. Affinity interactions can include, for example, antigen-antibody interactions and biotin-avidin (e.g. streptavidin) interactions. An example of an antigen-antibody interaction can be, for example, a digoxin-anti-digoxin antibody interaction. In embodiments utilizing an affinity interaction, a luminescent label can be linked to one of the members involved in the affinity interaction, and a nucleotide is linked to the other member involved in the affinity interaction, thereby attaching the chemiluminescent label to the nucleotide by the affinity interaction between said members. In exemplary embodiments utilizing a digoxin-anti-digoxin antibody interaction, digoxin can be linked to the nucleotide, and a luminescent label is linked to the anti-digoxin antibody, thereby attaching the luminescent label to the nucleotide by the digoxin-anti-digoxin antibody interaction. In exemplary embodiments utilizing a biotin-avidin (e.g. streptavidin) interaction, biotin can be linked to the nucleotide, and a luminescent label is linked to avidin (e.g. streptavidin), thereby attaching the luminescent label to the nucleotide by the biotin-avidin (e.g. streptavidin) interaction.

[0226] In other embodiments, the present application also relates to multiplex labeling of a nucleotide, i.e. attaching more than one luminescent label to the same nucleotide. In preferred embodiments, more than one luminescent label is attached to the same nucleotide by different affinity interactions. This can be achieved, for example, by simultaneously linking members involved in different affinity interactions to the nucleotide, and linking different luminescent labels to be attached to the nucleotide to other members involved in said different affinity interactions, thereby achieving attachment of more than one luminescent label to the nucleotide by said different affinity interactions. In preferred embodiments, two different luminescent labels are attached to the same nucleotide by different affinity interactions.

[0227] In exemplary embodiments in which two different luminescent labels are attached to the nucleotide, the nucleotide can be simultaneously linked to one of the members involved in a first affinity interaction and to one of the members involved in a second affinity interaction, and the first luminescent label is linked to the other member involved in the first affinity interaction, and the second luminescent label is linked to the other member involved in the first affinity interaction, thereby attaching said first luminescent label and second luminescent label to the nucleotide by said first affinity interaction and second affinity interaction.

[0228] In some embodiments, the first affinity interaction is a digoxigenin-anti-digoxigenin antibody interaction, and the second affinity interaction is a biotin-avidin (e.g., streptavidin) interaction. In a specific embodiment, a nucleotide can be linked to both digoxigenin and biotin, a first luminescent label can be linked to an anti-digoxigenin antibody, and a second luminescent label can be linked to avidin (e.g., streptavidin), such that the first and second luminescent labels are attached to the nucleotide via the digoxigenin-anti-digoxigenin antibody interaction and the biotin-avidin (e.g., streptavidin) interaction.

[0229] In another specific embodiment, a nucleotide can be linked to both digoxigenin and biotin, a first luminescent label can be linked to an anti-digoxigenin antibody, and a second luminescent label can be linked to avidin (e.g., streptavidin), such that the first and second luminescent labels are attached to the nucleotide via the digoxigenin-anti-digoxigenin antibody interaction and the biotin-avidin (e.g., streptavidin) interaction.

[0230] In addition, a luminescent label can also be attached to a nucleotide by combining different affinity interactions. In one exemplary embodiment, a nucleotide can be linked to one of the members involved in a first affinity interaction, one of the members involved in a second affinity interaction can be linked to the other member involved in the first affinity interaction, and a luminescent label can be linked to the other member involved in the second affinity interaction, such that the luminescent label is attached to the nucleotide via the first and second affinity interactions.

[0231] In one specific embodiment, a nucleotide can be linked to digoxigenin, biotin can be linked to an anti-digoxigenin antibody, and a luminescent label can be linked to avidin (e.g., streptavidin), such that the luminescent label is attached to the nucleotide via the digoxigenin-anti-digoxigenin antibody interaction and the biotin-avidin (e.g., streptavidin) interaction.

[0232] In the present application, a "linkage" between a luminescent label or a nucleotide and a member involved in an affinity interaction can be any suitable form of linkage known in the art. Such a linkage can include, for example, a direct linkage or an indirect linkage, e.g., via a linker, and can also include, for example, a non-covalent linkage (e.g., a linkage mediated by a hydrogen bond, an affinity interaction, etc.) and a covalent linkage, and can also be achieved, for example, by forming a recombinantly expressed fusion protein.

[0233] In some embodiments, the nucleic acid sequencing method is a method for sequencing a nucleic acid molecule in the patent documents WO2021031109A1, WO2020073274A1 or WO2020227953A1, wherein the luminescent label or luciferase comprises the mutant of the copepod luciferase of the first aspect of the present application, the recombinant protein of the second aspect of the present application or the fusion protein of the third aspect of the present application.

[0234] In the eighth aspect of the present application, a method for detecting a target molecule (preferably a method for detecting a target molecule for non-diagnostic purposes) is provided, which comprises the step of detecting the target molecule using the kit for detecting a target molecule of the sixth aspect of the present application.

[0235] In some embodiments, the method for detecting a target molecule comprises the step of contacting j4) in the kit for detecting a target molecule of the sixth aspect of the present application with a sample to be tested, and then adding a substrate for luciferase, and determining the presence or absence or the amount of the target molecule based on the fluorescent signal emitted by the reaction of the luciferase with the substrate for luciferase.

[0236] In some embodiments, the substrate for luciferase comprises a substrate for the copepod luciferase.

[0237] In some embodiments, the substrate for the copepod luciferase comprises at least one of coelenterazine, a coelenterazine derivative.

[0238] In some embodiments, the coelenterazine derivative comprises at least one of coelenterazine h, coelenterazine hcp, coelenterazine 400a, coelenterazine f, coelenterazine cp, coelenterazine n, coelenterazine e.

[0239] In the ninth aspect of the present application, a method for screening a substrate for copepod luciferase is provided, which comprises mixing at least one of the mutant of the copepod luciferase of the first aspect of the present application, the recombinant protein of the second aspect of the present application, the fusion protein of the third aspect of the present application, the conjugate or the complex of the fifth aspect of the present application with a substrate to be screened, and determining whether the substrate to be screened is a substrate for copepod luciferase based on whether the mixture obtained by the mixing emits a fluorescent signal.

[0240] In some embodiments, the amphipod luciferase comprises at least one of Gaussia luciferase (Gluc), Maluc (Metridia asymmetrica Luciferase, BAN91823.1), Mpluc (Metridia pacifica Luciferase, BAD93333.1), and Mluc7 (Metridia longa Luciferase, AJC98141.1); further Gaussia luciferase.

[0241] In a tenth aspect of the present application, a method for preparing the amphipod luciferase mutant of the first aspect of the present application, the recombinant protein of the second aspect of the present application, and the fusion protein of the third aspect of the present application is provided, by culturing the transgenic cell line and / or the recombinant bacteria of the fourth aspect of the present application.

[0242] In an eleventh aspect of the present application, the use of the amphipod luciferase mutant of the first aspect of the present application, the recombinant protein of the second aspect of the present application, and / or the biological material of the fourth aspect of the present application (wherein d1 does not comprise a nucleic acid molecule encoding the fusion protein of the third aspect of the present application) in the preparation and / or as a glow-type luciferase is provided.

[0243] The use of the amphipod luciferase mutant of the first aspect of the present application, the recombinant protein of the second aspect of the present application, the fusion protein of the third aspect of the present application, the biological material of the fourth aspect of the present application, and / or the conjugate or conjugate of the fifth aspect of the present application in k1) to k7) is provided:

[0244] k1) sequencing;

[0245] k2) detecting a target molecule for non-diagnostic purposes;

[0246] k3) drug screening;

[0247] k4) life science research;

[0248] k5) environmental monitoring;

[0249] k6) as a reporter gene;

[0250] k7) preparing a product for any one of k1) to k6).

[0251] In some embodiments, when the product is for k1), the product further comprises a flash-type luciferase.

[0252] In some embodiments, the product is a reagent, a kit, a chip, or a system.

[0253] The present application will be further described in detail by the following specific examples.

[0254] It should be understood that the examples are only used for illustrating the present application but not for limiting the scope of the present application.

[0255] The experimental methods in the following examples, unless otherwise specified, were generally carried out according to conventional conditions or according to the conditions recommended by the manufacturers. The materials, reagents and the like used in the present examples, unless otherwise specified, were reagents and materials obtained from commercial channels.

[0256] The present application optimizes the sequence and expression system of Gaussia luciferase, and constructs the Gaussia luciferase without signal peptide in pCold vector, and expresses it in Origami(DE3) competent cells. Further, the present application designs a random mutant library of specific sites of Gaussia luciferase, constructs it in Escherichia coli expression vector pCold, and prepares a mutant library of Gaussia luciferase with a polyhistidine(6x His) tag at the C-terminal. The mutant library is transformed into Origami(DE3) competent cells, and single clones are picked for expression, purification and activity test. The enzyme can be expressed in a soluble form in a prokaryotic expression system, and can produce Gaussia luciferase with different light emission modes.

[0257] Example 1 Plasmid construction and expression and purification of wild-type Gaussia luciferase without signal peptide and its mutants

[0258] In this example, wild-type Gaussia luciferase without signal peptide and its mutants are constructed for comparing the light emission half-life and light emission brightness(activity) of wild-type Gaussia luciferase and its mutants.

[0259] (I) Plasmid construction for prokaryotic expression of wild-type Gaussia luciferase without signal peptide or its mutants

[0260] The gene sequence of wild-type Gaussia luciferase (WT Gluc) is: ATGGGAGTGAAAGTTCTTTTTGCCCTTATTTGTATTGCTGTGGCCGAGGCCAAACCAACTGAAAACAATGAAGATTTCAACATTGTAGCTGTAGCTAGCAACTTTGCTACAACGGATCTCGATGCTGACCGTGGTAAATTGCCCGGAAAAAAATTACCACTTGAGGTACTCAAAGAAATGGAAGCCAATGCTAGGAAAGCTGGCTGCACTAGGGGATGTCTGATATGCCTGTCACACATCAAGTGTACACCCAAAATGAAGAAGTTTATCCCAGGAAGATGCCACACCTATGAAGGAGACAAAGAAAGTGCACAGGGAGGAATAGGAGAGGCTATTGTTGACATTCCTGAAATTCCTGGGTTTAAGGATTTGGAACCCATGGAACAATTCATTGCACAAGTTGACCTATGTGTAGACTGCACAACTGGATGCCTCAAAGGTCTTGCCAATGTGCAATGTTCTGATTTACTCAAGAAATGGCTGCCACAAAGATGTGCAACTTTTGCTAGCAAAATTCAAGGCCAAGTGGACAAAATAAAGGGTGCCGGTGGTGAT (5'→ 3', SEQ ID NO: 1), and the encoded amino acid sequence is: MGVKVLFALICIAVAEAKPTENNEDFNIVAVASNFATTDLDADRGKLPGKKLPLEVLKEMEANARKAGCTRGCLICLSHIKCTPKMKKFIPGRCHTYEGDKESAQGGIGEAIVDIPEIPGFKDLEPMEQFIAQVDLCVDCTTGCLKGLANVQCSDLLKKWLPQRCATFASKIQGQVDKIKGAGGD (N-terminal→ C-terminal, SEQ ID NO: 2; wherein the 1st-17th amino acid in SEQ ID NO: 2 is a signal peptide, which is numbered as SEQ ID NO: 3; the 18th-185th amino acid in SEQ ID NO: 2 is wild-type Gaussia luciferase without signal peptide (WT no signal peptide Gluc: WT-NS Gluc), which is numbered as SEQ ID NO: 4).

[0261] A plasmid (pCold-Gluc-NO SP, FIG. 2) expressing wild-type Gaussia luciferase (WT no signal peptide Gluc: WT-NS Gluc) without signal peptide and with a C-terminal fusion His tag (6 histidines, 6xHis, to facilitate protein purification) was constructed by the method of total gene synthesis (the coding gene of wild-type Gaussia luciferase without signal peptide and with a C-terminal fusion His tag was inserted between Ndel and EcoRI of the base plasmid pCold II), which is well known to those skilled in the art: the addition or non-addition of a tag has no effect on the performance of wild-type Gaussia luciferase or Gaussia luciferase mutants, and the tag can be added to the C-terminus and / or N-terminus of wild-type Gaussia luciferase or Gaussia luciferase mutants, and the tag can also be other His tags (Poly his), FLAG, Strep-Tag II, Poly arg, C-myc, etc.; similarly, the addition or non-addition of a signal peptide also has no effect on the performance of wild-type Gaussia luciferase or Gaussia luciferase mutants, and the signal peptide is contained in the naturally occurring eukaryotic secretory protein Copepod luciferase; however, the signal peptide affects the expression of this type of luciferase in E. coli, so the signal peptide should be removed when expressing luciferase in E. coli. The nucleotide sequence of the coding gene of wild-type Gaussia luciferase without signal peptide and with a C-terminal fusion His tag is:

[0262] ATGAATCACAAAGTGCATATGAAACCAACTGAAAACAATGAAGATTTCAACATTGTAGCTGTAGCTAGCAACTTTGCTACAACGGATCTCGATGCTGACCGTGGTAAATTGCCCGGAAAAAAATTACCACTTGAGGTACTCAAAGAAATGGAAGCCAATGCTAGGAAAGCTGGCTGCACTAGGGGATGTCTGATATGCCTGTCACACATCAAGTGTACACCCAAAATGAAGAAGTTTATCCCAGGAAGATGCCACACCTATGAAGGAGACAAAGAAAGTGCACAGGGAGGAATAGGAGAGGCTATTGTTGACATTCCTGAAATTCCTGGGTTTAAGGATTTGGAACCCATGGAACAATTCATTGCACAAGTTGACCTATGTGTAGACTGCACAACTGGATGCCTCAAAGGTCTTGCCAATGTGCAATGTTCTGATTTACTCAAGAAATGGCTGCCACAAAGATGTGCAACTTTTGCTAGCAAAATTCAAGGCCAAGTGGACAAAATAAAGGGTGCCGGTGGTGATGGTGGCAGTGAGAACCTGTACTTCCAGAGCGGCCACCACCACCACCATCACGGCTCCTGA (5'→ 3', SEQ ID NO: 5), wherein the 1st-15th nucleotides encode a TEE site, the 16th-21st nucleotides are a Ndel enzyme digestion site, the 22nd-525th nucleotides encode amino acids of wild-type Gaussia luciferase without a signal peptide, the 535th-555th nucleotides encode a TEV site, the 559th-576th nucleotides encode a histone tag sequence, the 526th-534th, 556th-558th, and 559th-564th nucleotides are linkers, the Ndel enzyme digestion site is located between the TEE and Gluc, and EcoRI is located after the stop codon; and the amino acids of wild-type Gaussia luciferase without a signal peptide fused with a His tag at the C-terminus are:

[0263] (N-terminal to C-terminal, SEQ ID NO: 6), the N-terminal of which is fused with a TEE site for the purpose of improving expression; the C-terminal of which is fused with a TEV site and a His tag for the purpose of facilitating protein purification; wherein the 1st-5th amino acids (wavy line part) are the amino acid sequence of the TEE site; the 8th-175th amino acids (italic part) are the amino acid sequence of the wild-type Gaussia luciferase without signal peptide, i.e. SEQ ID NO: 4; the 179th-185th amino acids (underlined part) are the amino acid sequence of the TEV site; and the 187th-192nd amino acids (bold part) are the amino acid sequence of the histone tag.

[0264] By site-directed mutagenesis (PCR reaction system for site-directed mutagenesis is shown in Table 1, reaction procedure is shown in Table 2, and primer sequences are shown in Table 3), single or multiple mutation sites were introduced into wild-type Gauss luciferase without signal peptide (WT no signal peptide Gluc: WT-NS Gluc) expressed in a plasmid (pCold-Gluc-NO SP) of wild-type Gauss luciferase without signal peptide fused with His tag at C-terminus, to obtain a plasmid of Gauss luciferase mutant without signal peptide fused with His tag at C-terminus (the difference from pCold-Gluc-NO SP is only that the nucleotide sequence encoding wild-type Gauss luciferase without signal peptide is replaced by the nucleotide sequence encoding Gauss luciferase mutant without signal peptide). Among them, the Gauss luciferase mutant without signal peptide is as follows: Gluc-NO SP-1 (S153D, Seq2): the amino acid sequence of Gluc-NO SP-1 has the following mutation compared with wild-type Gauss luciferase without signal peptide (WT no signal peptide Gluc: WT-NS Gluc, SEQ ID NO: 4): S153D; Gluc-NO SP-2 (G157S, Seq3): the amino acid sequence of Gluc-NO SP-2 has the following mutation compared with SEQ ID NO: 4: G157S; Gluc-NO SP-3 (M110L, Seq4): the amino acid sequence of Gluc-NO SP-3 has the following mutation compared with SEQ ID NO: 4: M110L; Gluc-NO SP-4 (Q146G, Seq5): the amino acid sequence of Gluc-NO SP-4 has the following mutation compared with SEQ ID NO: 4: Q146G; Gluc-NO SP-5 (G157A, Seq6): the amino acid sequence of Gluc-NO SP-5 has the following mutation compared with SEQ ID NO: 4: G157A; Gluc-NO SP-6 (L119R, Seq7): the amino acid sequence of Gluc-NO SP-6 has the following mutation compared with SEQ ID NO: 4: L119R; Gluc-NO SP-7 (V121E, Seq8): the amino acid sequence of Gluc-NO SP-7 has the following mutation compared with SEQ ID NO: 4: V121E; Gluc-NO SP-8 (Y80W, Seq9): the amino acid sequence of Gluc-NO SP-8 has the following mutation compared with SEQ ID NO: 4: Y80W; Gluc-NO SP-9 (E93P, Seq10): the amino acid sequence of Gluc-NO SP-9 has the following mutation compared with SEQ ID NO: 4: E93P.Gluc-NO SP-10 (P67L, Seq 11): the amino acid sequence of Gluc-NO SP-10 has the following mutation compared to SEQ ID NO: 4: P67L; Gluc-NO SP-11 (Y80F, Seq 12): the amino acid sequence of Gluc-NO SP-11 has the following mutation compared to SEQ ID NO: 4: Y80F; Gluc-NO SP-12 (S86T, Seq 13): the amino acid sequence of Gluc-NO SP-12 has the following mutation compared to SEQ ID NO: 4: S86T; Gluc-NO SP-13 (H78E, Seq 14): the amino acid sequence of Gluc-NO SP-13 has the following mutation compared to SEQ ID NO: 4: H78E; Gluc-NO SP-14 (F72Y, Seq 15): the amino acid sequence of Gluc-NO SP-14 has the following mutation compared to SEQ ID NO: 4: F72Y; Gluc-NO SP-15 (M43L, Seq 16): the amino acid sequence of Gluc-NO SP-15 has the following mutation compared to SEQ ID NO: 4: M43L; Gluc-NO SP-16 (T79P, Seq 17): the amino acid sequence of Gluc-NO SP-16 has the following mutation compared to SEQ ID NO: 4: T79P; Gluc-NO SP-17 (A87G, Seq 18): the amino acid sequence of Gluc-NO SP-17 has the following mutation compared to SEQ ID NO: 4: A87G; Gluc-NO SP-18 (Seq 19): the amino acid sequence of Gluc-NO SP-18 has the following mutation compared to SEQ ID NO: 4:

[0265] L23A / D24E / D26E / K41I / R48Q / M43L / H62K / P67L / F72Y / H78E / T79P / Y80W / E85S / S86T / A87G / E93P / L107M / M110L / L119R / V121E / Q146G / S153D / G157A; Gluc-NO SP-19 (Seq 20): the amino acid sequence of Gluc-NO SP-19 has the following mutation compared to SEQ ID NO: 4:

[0266] M43L / H62K / P67L / T79P / Y80W / E85S / S86T / A87G / E93P / L107M / M110L / L119R / V121E / S153D / G157A; Gluc-NO SP-20 (Seq 21): The amino acid sequence of Gluc-NO SP-20 has the following mutations compared to SEQ ID NO: 4:

[0267] D24E / D26E / K41I / R48Q / M43L / H62K / P67L / F72Y / H78E / T79P / Y80W / E85S / S86T / A87G / E93P / L107M / M110L / L119R / V121E / Q146G / S153D / G157A; Gluc-NO SP-21 (Seq-22): The amino acid sequence of Gluc-NO SP-21 has the following mutations compared to SEQ ID NO: 4:

[0268] H62K / P67L / F72Y / T79P / E85S / S86T / A87G / E93P / L107M / L119R / V121E / S153D; Gluc-NO SP-23 (Seq-24): The amino acid sequence of Gluc-NO SP-23 has the following mutations compared to SEQ ID NO: 4:

[0269] M43L / H62K / P67L / F72Y / T79P / E85S / S86T / A87G / E93P / L107M / L119R / V121E / S153D; Gluc-NO SP-24 (Seq-25): The amino acid sequence of Gluc-NO SP-24 has the following mutations compared to SEQ ID NO: 4:

[0270] M43L / H62K / P67L / F72Y / T79P / E85S / S86T / A87G / E93P / L107M / M110L / L119R / V121E / S153D; Gluc-NO SP-25 (Seq-26): The amino acid sequence of Gluc-NO SP-25 has the following mutations compared to SEQ ID NO: 4:

[0271] M43L / H62K / P67L / F72Y / T79P / Y80F / E85S / S86T / A87G / E93P / L107M / M110L / L119R / V121E / S153D / G157A; Gluc-NO SP-27 (Seq-28): The amino acid sequence of Gluc-NO SP-27 has the following mutations compared to SEQ ID NO: 4:

[0272] Gluc-NO SP-26 (Seq-27): The amino acid sequence of Gluc-NO SP-26 has the following mutations compared to SEQ ID NO: 4:

[0273] M43L / H62K / P67L / F72Y / T79P / Y80F / E85S / S86T / A87G / E93P / L107M / M110L / L119R / V121E / S153D / G157A; Gluc-NO SP-27 (Seq-28): The amino acid sequence of Gluc-NO SP-27 has the following mutations compared to SEQ ID NO: 4:

[0274] M43L / H62K / P67L / F72Y / T79P / Y80F / E85S / S86T / A87G / E93P / L107M / M110L / L119R / V121E / S153D / G157A; Gluc-NO SP-27 (Seq-28): The amino acid sequence of Gluc-NO SP-27 has the following mutations compared to SEQ ID NO: 4:

[0275] M43L / H62K / P67L / F72Y / T79P / Y80F / E85S / S86T / A87G / E93P / L107M / M110L / L119R / V121E / S153D / G157A; Gluc-NO SP-27 (Seq-28): The amino acid sequence of Gluc-NO SP-27 has the following mutations compared to SEQ ID NO: 4:

[0276] M43L / H62K / P67L / T79P / E85S / S86T / A87G / E93P / L107M / V121E; Gluc-NO SP-31 (Seq-32): The amino acid sequence of Gluc-NO SP-31 has the following mutations compared to SEQ ID NO: 4:

[0277] M43L / H62K / P67L / T79P / E85S / S86T / A87G / E93P / L107M / M110L / V121E; Gluc-NO SP-32 (Seq-33): The amino acid sequence of Gluc-NO SP-32 has the following mutations compared to SEQ ID NO: 4:

[0278] M43L / H62K / P67L / T79P / E85S / S86T / A87G / E93P / L107M / M110L / L119R / V121E / S153D; Gluc-NO SP-33 (Seq-34): The amino acid sequence of Gluc-NO SP-33 has the following mutations compared to SEQ ID NO: 4:

[0279] M43L / H62K / P67L / T79P / E85S / S86T / A87G / E93P / L107M / M110L / L119R / V121E / S153D / G157A; Gluc-NO SP-34 (Seq-35): The amino acid sequence of Gluc-NO SP-34 has the following mutations compared to SEQ ID NO: 4:

[0280] H62K / P67L / F72Y / E85S / S86T / A87G / E93P / L107M / V121E; Gluc-NO SP-35 (Seq-36): The amino acid sequence of Gluc-NO SP-35 has the following mutations compared to SEQ ID NO: 4:

[0281] D24E / M43L / H62K / P67L / T79P / Y80F / E85S / S86T / A87G / E93P / L107M / M110L / L119R / V121E / Q146G / S153D / G157A; Gluc-NO SP-36 (Seq-37): The amino acid sequence of Gluc-NO SP-36 has the following mutations compared to SEQ ID NO: 4:

[0282] K41I / R48Q / M43L / H62K / P67L / H78E / T79P / Y80F / E85S / S86T / A87G / E93P / L107M / M110L / L119R / V121E / Q146G / S153D / G157A; Gluc-NO SP-38 (Seq-39): The amino acid sequence of Gluc-NO SP-38 has the following mutations compared to SEQ ID NO: 4:

[0283] D24E / K41I / R48Q / M43L / H62K / P67L / H78E / T79P / Y80F / E85S / S86T / A87G / E93P / L107M / M110L / L119R / V121E / Q146G / S153D / G157A; Gluc-NO SP-39 (Seq-40): The amino acid sequence of Gluc-NO SP-39 has the following mutations compared to SEQ ID NO: 4:

[0284] K41I / R48Q / M43L / H62K / P67L / F72Y / H78E / T79P / Y80F / E85S / S86T / A87G / E93P / L107M / M110L / L119R / V121E / Q146G / S153D / G157A; Gluc-NO SP-40 (Seq-41): The amino acid sequence of Gluc-NO SP-40 has the following mutations compared to SEQ ID NO: 4:

[0285] D26E / K41I / R48Q / M43L / H62K / P67L / F72Y / H78E / T79P / Y80F / E85S / S86T / A87G / E93P / L107M / M110L / L119R / V121E / Q146G / S153D / G157A; Gluc-NO SP-41 (Seq-42): The amino acid sequence of Gluc-NO SP-41 has the following mutations compared to SEQ ID NO: 4: H62K / F72Y / E85S / S86T / A87G / E93P / L107M / L119R / V121E / S153D; Gluc-NO SP-42 (Seq-43): The amino acid sequence of Gluc-NO SP-42 has the following mutations compared to SEQ ID NO: 4:

[0286] K41I / M43L / H62K / P67L / H78E / T79P / Y80F / E85S / S86T / A87G / E93P / L107M / M110L / L119R / V121E / Q146G / S153D / G157A.

[0287] Table 1 PCR reaction system

[0288] Table 2 PCR reaction conditions

[0289] Table 3 Primer sequence

[0290] Add 0.5 μL DpnI enzyme to the reaction system, incubate at 37°C for 3 hours to digest the template, and then gel recover about 4800 bp of the product, i.e. the plasmid expressing the C-terminal fusion His-tagged signal peptide-removed Gaussia luciferase mutant.

[0291] Take the above reaction product or pCold-Gluc-NO SP 2.5 μL and transform into DH5α competent cells, spread on an Amp-resistant plate with a final concentration of 100 μg / mL, and the next day pick single colonies from the plate and extract the plasmid. Perform sequencing to ensure correct amplification of the multiplex PCR, and the obtained plasmid is the plasmid expressing the C-terminal fusion His-tagged signal peptide-removed Gaussia luciferase mutant and the plasmid expressing the C-terminal fusion His-tagged signal peptide-removed wild-type Gaussia luciferase (pCold-Gluc-NO SP).

[0292] (ii) Prokaryotic expression and purification of wild-type Gaussia luciferase or its mutant without signal peptide

[0293] Transformation: Take Origami DE3 competent cells, melt on ice, and divide into 50 μL per tube. Add 5 μL of the plasmid prepared in step (i) (plasmid expressing C-terminal fusion His-tagged signal peptide-removed Gaussia luciferase mutant or plasmid expressing C-terminal fusion His-tagged signal peptide-removed wild-type Gaussia luciferase) and gently flick the tube bottom to mix, incubate on ice for 1 h, heat shock at 42°C for 1 min, and incubate on ice for 5 min. Add 500 μL of LB without antibiotics and incubate at 37°C at 220 rpm for 1 h. Then take 60 μL of the bacterial solution and evenly spread on an Amp-resistant LB agar plate, and incubate at 37°C overnight.

[0294] Protein expression and purification: Single colony was picked from an overnight culture plate and inoculated into Amp resistant LB broth at 37°C 220 rpm until OD600 reached 0.6, then expanded into Amp LB in a ratio of 50: 1 into a suitable volume for the experiment in a shaker tube or flask at 37°C 200 rpm for 3-4 h until OD600 reached 0.6, cooled on ice for 15 min, then added 1 mM IPTG to a final concentration of 1 uM, induced expression at 16°C 300 rpm overnight. The bacterial solution was centrifuged at 4000 g for 10 min, the supernatant was removed, and the bacterial solution was resuspended with pre-cooled PBS with a final concentration of 1 mM PMSF, and the bacterial cells were broken by sonication until clear, centrifuged at 12000 g for 30 min at 4°C, and the supernatant was stored on ice for later use. The pre-treatment of the packing material was washed with 5 CV (column volume) of sterilized deionized water, 5 CV of 1x PBS, and 5 CV of 1 M imidazole; the column was equilibrated with 5 CV of 20 mM imidazole; the treated packing material was added to the treated sample, gently shaken at 4°C for 30 min, and then the sample and packing material mixture was loaded onto the column, and the packing material was retained; the packing material was washed with 5 CV of 1x PBS and 5 CV of 20 mM imidazole; elution was performed with 300 mM imidazole, and the eluate was the target protein; the packing material was washed with 5 CV of 1 mM imidazole and 5 CV of water, and 20% ethanol was added for storage at 4°C.

[0295] Ultrafiltration: The purified protein was transferred to a 10 k ultrafiltration tube (Merck ), and 10 volumes of 4°C pre-cooled 1x PBS was added, centrifuged at 3900 g for 15 min at 4°C, and repeated 3 times. The purified protein was characterized by 15% SDS-PAGE for molecular weight and purity. The results are shown in Figure 3: the molecular weight of the C-terminal His-tagged signal peptide-removed Gaussian luciferase mutant and the C-terminal His-tagged signal peptide-removed wild-type Gaussian luciferase was consistent with the expected size.

[0296] Example 2: Activity detection of wild-type Gaussian luciferase or its mutant

[0297] In this example, the BCA quantitative kit (Thermo Scientific TM Pierce TMThe concentrations of the purified wild-type Gaussia luciferase with C-terminal fusion His tag and the signal peptide removed and the Gaussia luciferase mutants with C-terminal fusion His tag and the signal peptide removed obtained in Example 1 were accurately determined by using the BCA Protein Assay Kit. The purified wild-type Gaussia luciferase with C-terminal fusion His tag and the signal peptide removed and the Gaussia luciferase mutants with C-terminal fusion His tag and the signal peptide removed obtained in Example 1 were diluted to 1 μg / mL with a diluent (50 mM Tris-HCl pH 8.0, 100 mM NaCl, 0.1% (v / v) Tween-20), and 10 μL of each was added to a black 96-well plate. Then, 90 μL of the substrate coelenterazine (purchased from Biooly, Figure 4) diluted to 100 μM with the same diluent was added, and the luminescence intensity and mode were read by using a luminometer with a luminescence module. The results were averaged from three replicates, and are shown in Figures 5-8 and Table 4. Figure 5 shows the catalytic activity (i.e., brightness) of some Gaussia luciferase mutants on the substrate coelenterazine, Figure 6 shows the decay half-life of the catalyzed luminescence of some Gaussia luciferase mutants on the substrate coelenterazine, Figures 7 and 8 show the luminescence kinetics curves of some Gaussia luciferase mutants, with the horizontal axis representing time and the vertical axis representing brightness. The curves reflect the decay of the luminescence of the luciferase mutants catalyzed by the oxidation of the substrate over time. The results show that the Gaussia luciferase mutants can catalyze the substrate coelenterazine to produce luminescence intensity. Compared with the wild-type Gaussia luciferase, the Gaussia luciferase mutants have an increased decay half-life of the luminescence, or an increased signal over a certain period of time, and the luminescence mode of the luciferase changes to a more persistent glow-type luminescence or a luminescence intensity continuously increasing-type luminescence. Some Gaussia luciferase mutants produce increased luminescence intensity.

[0298] Table 4 Luminescence intensity and decay half-life produced by wild-type Gaussia luciferase or its mutants

[0299] Example 3 Construction of plasmids for prokaryotic expression of wild-type copepod luciferase or its mutants without signal peptide

[0300] Since the luciferases of the genus Amplex are highly conserved, one skilled in the art can expect similar changes in the emission pattern at the corresponding sites of other luciferases of the genus Amplex, such as Maluc, Mpluc, Mluc7, etc., based on the changes in the emission pattern at the relevant sites of Gluc. As a verification, the mutation sites (compared with the amino acid sequence of wild-type Glu without signal peptide) H78E, T79P, M110L and Q146G of the Gluc mutant were used as reference sites, and plasmids for prokaryotic expression of wild-type Amplex luciferases (Maluc, Mpluc, Mluc7) without signal peptide or mutants thereof corresponding to the above-mentioned sites were constructed, and the construction method was the same as that of Example 1.

[0301] Among them, the amino acid sequence alignment of Gluc and other homologous Amplex luciferases (Maluc, Mpluc, Mluc7) is shown in Figure 1, and the mutation sites (compared with the amino acid sequence of wild-type Gluc without signal peptide) H78E, T79P, M110L and Q146G of the Gluc mutant correspond to the mutation sites (compared with the amino acid sequence of wild-type Maluc without signal peptide) H78E, D79P, M110L and K146G of the Maluc mutant, the mutation sites (compared with the amino acid sequence of wild-type Mpluc without signal peptide) H101E, D102P, M133L and D169G of the Mpluc mutant, and the mutation sites (compared with the amino acid sequence of wild-type Mluc7 without signal peptide) H61E, D62P, M93L and D129G of the Mluc7 mutant.

[0302] The amino acid sequence of wild-type Maluc is:

[0303] DIKVLFALICVAMVQAKATENNDDIDIVGIASTFITTNTDADRGKMPGKRLPLAVLKEMEANAVKAGCSRGCLICLSKIKCTAKMKQYIPGRCHDYGGDKKTGQAAIEGAIDDIPEISGFKEMAPMEQFIAQVDLCADCTTGCLKGLANVKCSELLKKWLPKRCTSFATKMOKEIHNIKGMGGDR (N-terminal to C-terminal, SEQ ID NO: 47; wherein the 1st-16th amino acid in SEQ ID NO: 47 is a signal peptide, which is numbered as SEQ ID NO: 48; the 17th-185th amino acid in SEQ ID NO: 47 is a wild-type Maluc (Maluc WT) without signal peptide, which is numbered as SEQ ID NO: 49), a plasmid (pCold-Maluc-NO SP, FIG. 9) for expressing a wild-type Maluc (Maluc WT) without signal peptide C-terminally fused with a His tag (6 histidines, 6xHis, for facilitating protein purification) was constructed by a whole gene synthesis method (the gene encoding the wild-type Maluc without signal peptide C-terminally fused with a His tag was inserted between Ndel and EcoRI of a base plasmid pCold II). The Maluc mutants without signal peptide were as follows: Maluc-H78E: the amino acid sequence was compared with that of the wild-type Maluc (Maluc WT, SEQ ID NO: 49) without signal peptide, and the following mutation was present: H78E; Maluc-D79P: the amino acid sequence was compared with that of the wild-type Maluc (Maluc WT, SEQ ID NO: 49) without signal peptide, and the following mutation was present: D79P; Maluc-M110L: the amino acid sequence was compared with that of the wild-type Maluc (Maluc WT, SEQ ID NO: 49) without signal peptide, and the following mutation was present: M110L; Maluc-K146G: the amino acid sequence was compared with that of the wild-type Maluc (Maluc WT, SEQ ID NO: 49) without signal peptide, and the following mutation was present: K146G.

[0304] The amino acid sequence of the wild-type Maluc is:

[0305] EIQVLFALICFALVQANPTENKDDIDIVGVEGKFGTTDLETDLFTIVEDMNVISRDTNLANSDADRGKMPGKKLPLEVLIEMEANARKAGCTRGCLICLSKIKCTAKMKVYIPGRCHDYGGDKKTGQAGIVGAIVDIPEISGFKELGPMEQFIAQVDLCADCTTGCLKGLANVKCSALLKKWLPDRCASFADKIQSEVDNIKGLAGDR (N-terminal→C-terminal, SEQ ID NO: 50; wherein, the 1st to 16th amino acids in SEQ ID NO: 50 are a signal peptide, which is numbered as SEQ ID NO: 51; the 17th to 208th amino acids in SEQ ID NO: 50 are a wild-type Mpluc without a signal peptide (Mpluc WT), which is numbered as SEQ ID NO: 52), a plasmid (pCold-Mpluc-NO SP, FIG. 10) expressing the wild-type Mpluc without a signal peptide (Mpluc WT) C-terminally fused with a His tag (6 histidines, 6xHis, for facilitating protein purification) was constructed by a whole gene synthesis method (the gene encoding the wild-type Mpluc without a signal peptide C-terminally fused with a His tag was inserted between Ndel and EcoRI of the base plasmid pCold II). The Mpluc mutants without a signal peptide were as follows: Mpluc-H101E: the amino acid sequence has the following mutation compared to the wild-type Mpluc without a signal peptide (Mpluc WT, SEQ ID NO: 52): H101E; Mpluc-D102P: the amino acid sequence has the following mutation compared to the wild-type Mpluc without a signal peptide (Mpluc WT, SEQ ID NO: 52): D102P; Mpluc-M133L: the amino acid sequence has the following mutation compared to the wild-type Mpluc without a signal peptide (Mpluc WT, SEQ ID NO: 52): M133L; Mpluc-D169G: the amino acid sequence has the following mutation compared to the wild-type Mpluc without a signal peptide (Mpluc WT, SEQ ID NO: 52): D169G.

[0306] The amino acid sequence of the wild-type Mluc7 is:

[0307] DIKFIFALVCIALVQANPTVNNDVNRGKMPGKKLPLEVLIEMEANAFKAGCTRGCLICLSKIKCTAKMKQYIPGRCHDYGGDKKTGQAGIVGAIVDIPEISGFKEMEPMEQFIAQVDLCADCTTGCLKGLANVKCSELLKKWLPDRCASFADKIOKEAHNIKGLAGDR (N-terminal to C-terminal, SEQ ID NO: 53; wherein, the 1st to 16th amino acids in SEQ ID NO: 53 are a signal peptide, which is numbered as SEQ ID NO: 54; the 17th to 168th amino acids in SEQ ID NO: 53 are a wild-type Mluc7 without signal peptide (Mluc7 WT), which is numbered as SEQ ID NO: 55), a plasmid expressing Mluc7 WT without signal peptide C-terminally fused with a His-tag (6 histidines, 6xHis, for facilitating protein purification) (pCold-Mluc7-NO SP, FIG. 11) (the gene encoding Mluc7 WT without signal peptide C-terminally fused with a His-tag was inserted between Ndel and EcoRI of the base plasmid pCold II) was constructed by the method of whole gene synthesis. The Mluc7 mutants without signal peptide were as follows: Mluc7-H61E: the amino acid sequence has the following mutation compared with Mluc7 WT without signal peptide (SEQ ID NO: 55): H61E; Mluc7-D62P: the amino acid sequence has the following mutation compared with Mluc7 WT without signal peptide (SEQ ID NO: 55): D62P; Mluc7-M93L: the amino acid sequence has the following mutation compared with Mluc7 WT without signal peptide (SEQ ID NO: 55): M93L; Mluc7-D129G: the amino acid sequence has the following mutation compared with Mluc7 WT without signal peptide (SEQ ID NO: 55): D129G.

[0308] The wild-type copepod luciferase without signal peptide or its mutants constructed above were expressed in prokaryotes and purified, in the same manner as in Example 1, to obtain the purified copepod luciferase or its mutants.

[0309] Example 4 Activity detection of wild-type copepod luciferase or its mutants

[0310] The luminescent intensity and luminescent mode of the purified amphioxus luciferase or its mutants obtained in Example 3 were detected respectively, and the detection method was the same as that in Example 2, and the results are shown in Figures 12, 13 and Table 5: the Maluc mutants can all catalyze the substrate coelenterazine to produce luminescent intensity; at the same time, compared with the wild type Maluc, the luminescent half-life of the Maluc mutants is increased, and the luminescent intensity of some Maluc mutants is improved. The Mpluc mutants can all catalyze the substrate coelenterazine to produce luminescent intensity; at the same time, compared with the wild type Mpluc, the luminescent half-life of the Mpluc mutants is increased, and the luminescent intensity of some Mpluc mutants is improved. The Mluc7 mutants can all catalyze the substrate coelenterazine to produce luminescent intensity; at the same time, compared with the wild type Mluc7, the luminescent half-life of the Mluc7 mutants is increased, and the luminescent intensity of some Mluc7 mutants is improved.

[0311] Table 5 Luminescent intensity and half-life that can be produced by wild type amphioxus luciferase or its mutants

[0312] The above results are consistent with the results of the Gaussia luciferase, which shows that Gluc has an important site for influencing flash luminescence, and has a similar effect on the remaining amphioxus luciferases.

[0313] Example 5 Test of Gaussia luciferase mutants fused with digoxin antibody for expression

[0314] The Gaussia luciferase and its mutants in glow type can be fused with digoxin antibody for expression to meet subsequent on-machine needs. In this embodiment, six luciferases are selected for digoxin antibody fusion expression (WT-NS and Gluc-NO SP-19, Gluc-NO SP-20, Gluc-NO SP-23, Gluc-NO SP-27, Gluc-NO SP-28), wherein the sequence of digoxin antibody, fusion expression, coupling and purification method refer to Chinese patent CN201811475418.2 “Fusion protein of Gaussia luciferase and digoxin single-chain antibody and application thereof” Examples 1-5.

[0315] The purity of the glow-type luciferase-digoxin antibody fusion protein is tested by using 8% SDS-PAGE, and it is found that the purity is >90% (Figure 14). The luminescent kinetic curve of the glow-type luciferase-digoxin antibody fusion protein is shown in Figure 15, and it can be seen from the figure that after fusion expression with digoxin antibody, the luminescent intensity of the luciferase is doubled, and the glow property is unchanged.

[0316] The above embodiments are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above embodiments, and any changes, modifications, substitutions, combinations, simplifications, etc. made without departing from the spirit and principles of the present application should be equivalent replacement manners and should be included in the protection scope of the present application.

Claims

1. A mutant of a crustacean luciferase, which is (al) or (a2): (al) the amino acid sequence of the mutant comprises a mutation at one or more of the following positions: 43, 67, 72, 78, 79, 80, 86, 87, 93, 110, 119, 121, 146, 153, 157, compared to the amino acid sequence of wild-type crustacean luciferase with the signal peptide removed; (a2) the amino acid sequence of the mutant comprises the mutations of the mutant of (al), and has at least 80% identity to the amino acid sequence of the mutant of (al). the crustacean luciferase comprises at least one of Gaussia luciferase, Maluc, Mpluc, and Mluc7; preferably, the crustacean luciferase is Gaussia luciferase; 2. The mutant according to claim 1, wherein preferably, the mutant comprises any one of (bl) to (bl5) or a combination of mutations of (bl) to (bl5) compared to the amino acid sequence of wild-type Gaussia luciferase with the signal peptide removed: (bl) M at position 43 is mutated to L; (b2) P at position 67 is mutated to L; (b3) F at position 72 is mutated to Y; (b4) H at position 78 is mutated to E; (b5) T at position 79 is mutated to P; (b6) Y at position 80 is mutated to W, or F; (b7) S at position 86 is mutated to T; (b8) A at position 87 is mutated to G; (b9) E at position 93 is mutated to P; (blO) M at position 110 is mutated to L; (bl l) L at position 119 is mutated to R; (bl2) V at position 121 is mutated to E; (bl3) Q at position 146 is mutated to G; (bl4) S at position 153 is mutated to D; (bl5) G at position 157 is mutated to S, or A; preferably, the mutant has any one of (cl) to (c42) compared to the amino acid sequence of wild-type crustacean luciferase with the signal peptide removed: D26E / K41I / R48Q / M43L / H62K / P67L / F72Y / H78E / T79P / Y80F / E85S / S86T / A87G / E93P / L107M / M110L / L119R / V121E / Q146G / S153D / G157A; (c41) H62K / F72Y / E85S / S86T / A87G / E93P / L107M / L119R / V121E / S153D; (c42) K41I / M43L / H62K / P67L / H78E / T79P / Y80F / E85S / S86T / A87G / E93P / L107M / M110L / L119R / V121E / Q146G / S153D / G157A; ​ (c1) S153D; (c2) G157S; (c3) M110L; (c4) Q146G; (c5) G157A; (c6) L119R; (c7) V121E; (c8) Y80W; (c9) E93P; (c10) P67L; (c11) Y80F; (c12) S86T; (c13) H78E; (c14) F72Y; (c15) M43L; (c16) T79P; (c17) A87G; (c18) L23A / D24E / D26E / K41I / R48Q / M43L / H62K / P67L / F72Y / H78E / T79P / Y80W / E85S / S86T / A87G / E93P / L107M / M110L / L119R / V121E / Q146G / S153D / G157A; (c19) M43L / H62K / P67L / T79P / Y80W / E85S / S86T / A87G / E93P / L107M / M110L / L119R / V121E / S153D / G157A; (c20) D24E / D26E / K41I / R48Q / M43L / H62K / P67L / F72Y / H78E / T79P / Y80W / E85S / S86T / A87G / E93P / L107M / M110L / L119R / V121E / Q146G / S153D / G157A; (c21) H62K / P67L / F72Y / E85S / S86T / A87G / E93P / L107M / L119R / V121E / S153D; (c22) H62K / P67L / F72Y / T79P / E85S / S86T / A87G / E93P / L107M / L119R / V121E / S153D; (c23) M43L / H62K / P67L / F72Y / T79P / E85S / S86T / A87G / E93P / L107M / L119R / V121E / S153D; (c24) M43L / H62K / P67L / F72Y / T79P / E85S / S86T / A87G / E93P / L107M / M110L / L119R / V121E / S153D; (c25) M43L / H62K / P67L / F72Y / T79P / E85S / S86T / A87G / E93P / L107M / M110L / L119R / V121E / S153D / G157A; (c26) M43L / H62K / P67L / F72Y / T79P / Y80F / E85S / S86T / A87G / E93P / L107M / M110L / L119R / V121E / S153D / G157A;(c27) M43L / H62K / P67L / F72Y / T79P / Y80F / E85S / S86T / A87G / E93P / L107M / M110L / L119R / V121E / Q146G / S153D / G157A; (c28) M43L / H62K / P67L / F72Y / H78E / T79P / Y80F / E85S / S86T / A87G / E93P / L107M / M110L / L119R / V121E / Q146G / S153D / G157A; (c29) H62K / P67L / T79P / E85S / S86T / A87G / E93P / L107M / V121E; (c30) M43L / H62K / P67L / T79P / E85S / S86T / A87G / E93P / L107M / V121E; (c31) M43L / H62K / P67L / T79P / E85S / S86T / A87G / E93P / L107M / M110L / V121E; (c32) M43L / H62K / P67L / T79P / E85S / S86T / A87G / E93P / L107M / M110L / L119R / V121E / S153D; (c33) M43L / H62K / P67L / T79P / E85S / S86T / A87G / E93P / L107M / M110L / L119R / V121E / S153D / G157A; (c34) H62K / P67L / F72Y / E85S / S86T / A87G / E93P / L107M / V121E; (c35) D24E / M43L / H62K / P67L / T79P / Y80F / E85S / S86T / A87G / E93P / L107M / M110L / L119R / V121E / Q146G / S153D / G157A; (c36) H62K / F72Y / E85S / S86T / A87G / E93P / L107M / V121E; (c37) K41I / R48Q / M43L / H62K / P67L / H78E / T79P / Y80F / E85S / S86T / A87G / E93P / L107M / M110L / L119R / V121E / Q146G / S153D / G157A; (c38) D24E / K41I / R48Q / M43L / H62K / P67L / H78E / T79P / Y80F / E85S / S86T / A87G / E93P / L107M / M110L / L119R / V121E / Q146G / S153D / G157A;(c39) K41I / R48Q / M43L / H62K / P67L / F72Y / H78E / T79P / Y80F / E85S / S86T / A87G / E93P / L107M / M110L / L119R / V121E / Q146G / S153D / G157A; (c40) ; ​ Preferably, the mutant has any one of the mutations (c1), (c3), (c6), (c7), (c9), (c12), (c17)-(c27), (c33)-(c42) compared with the amino acid sequence of the wild-type copepod luciferase from which the signal peptide is removed; Preferably, the mutant has any one of the mutations (c1), (c3), (c6), (c7), (c9), (c12), (c17)-(c27), (c33)-(c42) compared with the amino acid sequence of the wild-type copepod luciferase from which the signal peptide is removed; Preferably, the amino acid sequence of the wild-type copepod luciferase from which the signal peptide is removed is shown in SEQ ID NO: 4; Preferably, the mutant further comprises a signal peptide.

3. The mutant according to claim 1 or 2, characterized in that, The copepod luciferase comprises at least one of Gaussia luciferase, Maluc, Mpluc, and Mluc7; Preferably, the copepod luciferase is Maluc; Preferably, the amino acid sequence of the mutant comprises a mutation at one or more of the following positions: 78th, 79th, 110th, or 146th compared with the amino acid sequence of the wild-type Maluc from which the signal peptide is removed; Preferably, the mutant comprises any one or a combination of the following mutations: H at the 78th position is mutated to E, D at the 79th position is mutated to P, M at the 110th position is mutated to L, or K at the 146th position is mutated to G compared with the amino acid sequence of the wild-type Maluc from which the signal peptide is removed; Preferably, the copepod luciferase is Mpluc; Preferably, the amino acid sequence of the mutant comprises a mutation at one or more of the following positions: 101st, 102nd, 133rd, or 169th compared with the amino acid sequence of the wild-type Mpluc from which the signal peptide is removed; Preferably, the mutant comprises any one or a combination of the following mutations: H at the 101st position is mutated to E, D at the 102nd position is mutated to P, M at the 133rd position is mutated to L, or D at the 169th position is mutated to G compared with the amino acid sequence of the wild-type Mpluc from which the signal peptide is removed; Preferably, the copepod luciferase is Mluc7; Preferably, the amino acid sequence of the mutant comprises a mutation at one or more of the following positions: 61st, 62nd, 93rd, or 129th compared with the amino acid sequence of the wild-type Mluc7 from which the signal peptide is removed; Preferably, the mutant comprises any one or a combination of the following mutations: H at the 61st position is mutated to E, D at the 62nd position is mutated to P, M at the 93rd position is mutated to L, or D at the 129th position is mutated to G compared with the amino acid sequence of the wild-type Mluc7 from which the signal peptide is removed.

4. A recombinant protein comprising a modification moiety and the mutant copepod luciferase of any one of claims 1-3; Preferably, the modification moiety is a protein tag. Preferably, the protein tag is selected from at least one of Poly his, FLAG, Strep-Tag II, Poly arg, C-myc, HA, V5, VSV-G, Trx, SUMO, GST, MBP, and NusA.

5. A fusion protein comprising a glow-type luciferase and a flash-type luciferase, the glow-type luciferase comprising the copepod luciferase mutant of any one of claims 1 to 3, and / or the recombinant protein of claim 4.

6. A biological material related to the copepod luciferase mutant of any one of claims 1 to 3, the recombinant protein of claim 4, or the fusion protein of claim 5, the biological material comprising any one of d1) to d12): d1) a nucleic acid molecule encoding the copepod luciferase mutant of any one of claims 1 to 3, the recombinant protein of claim 4, or the fusion protein of claim 5; d2) an expression cassette comprising the nucleic acid molecule of d1); d3) a vector comprising the nucleic acid molecule of d1); d4) a vector comprising the expression cassette of d2); d5) a transgenic cell line comprising the nucleic acid molecule of d1); d6) a transgenic cell line comprising the expression cassette of d2); d7) a transgenic cell line comprising the vector of d3); d8) a transgenic cell line comprising the vector of d4); d9) a recombinant bacterium comprising the nucleic acid molecule of d1); d10) a recombinant bacterium comprising the expression cassette of d2); d11) a recombinant bacterium comprising the vector of d3); and d12) a recombinant bacterium comprising the vector of d4). Preferably, the transgenic cell line in d5) to d8) does not comprise reproductive material. Preferably, the vector in d3) and d4) comprises a promoter operably linked to the nucleic acid molecule. Preferably, the vector in d3) and d4) is independently selected from a non-pathogenic viral vector and a non-viral vector. Preferably, the non-pathogenic viral vector comprises an adenoviral vector or a retroviral vector. Preferably, the non-viral vector comprises a plasmid vector. Preferably, the recombinant bacterium in d9) to d12) comprises Escherichia coli.

7. A conjugate or a conjugate comprising (a) and (b): (a) a conjugating or conjugating moiety; (b) at least one of the copepod luciferase mutant of any one of claims 1 to 3, the recombinant protein of claim 4, or the fusion protein of claim 5. The conjugating or conjugating moiety comprises at least one of a small molecule compound, a biological macromolecule. The conjugating or conjugating moiety comprises at least one of avidin, digoxin antibody, nucleotide antibody, nucleotide, a substance that specifically binds to a target molecule. Preferably, the nucleotide is a 3’-OH end modified nucleotide. Preferably, the 3’-OH end modified nucleotide is modified by adding a blocking group to the 3’-OH end of the nucleotide. Preferably, the blocking group comprises at least one of O-amino, O-allyl, O-azido, O-phosphate group. ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 8. The conjugate or conjugate of claim 7, wherein, ​ ​ ​ ​ 9. A nucleic acid sequencing kit comprising any one of i1) to i4): i1) the mutant amphipod luciferase according to any one of claims 1 to 3; i2) the recombinant protein according to claim 4; i3) the fusion protein according to claim 5; i4) The conjugate or conjugate of any one of claims 7-8, wherein, the coupling or conjugation moiety comprises at least one of an avidin, a digoxin antibody, a nucleotide antibody, a nucleotide.

10. The nucleic acid sequencing kit of claim 9, wherein, the nucleotide is the nucleotide according to claim 8; preferably, the nucleic acid sequencing kit further comprises at least one of a substrate of the mutant amphipod luciferase, a polymerase, a PCR buffer; preferably, the substrate of the mutant amphipod luciferase comprises at least one of coelenterazine, a coelenterazine derivative; preferably, the nucleic acid sequencing kit further comprises a substrate of the glow-type luciferase when i3) is comprised; preferably, the nucleic acid sequencing kit further comprises a nucleotide when the coupling or conjugation moiety is an avidin, a digoxin antibody, or a nucleotide antibody.

11. A kit for detecting a target molecule comprising any one of j1) to j4): j1) the mutant amphipod luciferase according to any one of claims 1 to 3; j2) the recombinant protein according to claim 4; j3) the fusion protein according to claim 5; j3) The conjugate or conjugate of any one of claims 7-8, wherein, the coupling or conjugation moiety comprises a substance that specifically binds to the target molecule; preferably, the kit for detecting a target molecule further comprises a substrate of the mutant amphipod luciferase; preferably, the substrate of the mutant amphipod luciferase comprises at least one of coelenterazine, a coelenterazine derivative; preferably, the kit for detecting a target molecule further comprises a substrate of the glow-type luciferase when j3) is comprised.

12. A method for nucleic acid sequencing, the method comprising the steps of: (1) monitoring the sequential incorporation of nucleotides in a complementary strand of a nucleic acid to be sequenced, the nucleotides each being attached to a luminescent label that initiates a luminescence with different kinetics or a luminescence type; (2) identifying each incorporated nucleotide by detecting the luminescence kinetics or luminescence type or light intensity of the luminescence reaction in which the luminescent label is involved; the incorporated nucleotides carry a blocking modification group; the luminescent label comprises the mutant amphipod luciferase according to any one of claims 1 to 3; preferably, the luminescent label comprises the mutant amphipod luciferase according to any one of claims 1 to 3 and a second luminescent label that initiates a luminescence with different kinetics or a luminescence type or light intensity than the mutant amphipod luciferase; preferably, the method for nucleic acid sequencing further comprises the step of (3) removing the blocking modification group carried by the incorporated nucleotides so that the complementary strand can continue to be elongated, and removing the luminescent label; preferably, the method for nucleic acid sequencing further comprises the step of repeating steps (1) to (3) until the complementary strand is completely elongated to determine the sequence of the nucleic acid to be sequenced.

13. The method of claim 12, wherein, In the nucleotide, a first nucleotide is attached to the mutant of the amphipod luciferase according to any one of claims 1-3, a second nucleotide is attached to a second luminescent marker, a third nucleotide is attached to both the mutant of the amphipod luciferase according to any one of claims 1-3 and the second luminescent marker, and a fourth nucleotide is not attached to a luminescent marker; Preferably, the attachment between the nucleotide and the luminescent marker comprises an attachment mediated by an affinity interaction.

14. A method for detecting a target molecule, comprising the step of detecting the target molecule using the kit for detecting a target molecule according to claim 11.

15. The method of claim 14, wherein, The method for detecting a target molecule comprises the steps of contacting j3) in the kit for detecting a target molecule according to claim 11 with a sample to be tested, then adding a substrate for luciferase, and determining the presence or amount of the target molecule based on the fluorescence signal emitted by the reaction of the luciferase with the substrate for luciferase. The substrate for luciferase comprises a substrate for the amphipod luciferase. Preferably, the substrate for the amphipod luciferase comprises at least one of coelenterazine, a coelenterazine derivative.

16. A method for screening a substrate for amphipod luciferase, mixing at least one of the mutant of the amphipod luciferase according to any one of claims 1-3, the recombinant protein according to claim 4, the fusion protein according to claim 5, the conjugate or the complex according to any one of claims 7-8 with a substrate to be screened, and determining whether the substrate to be screened is a substrate for amphipod luciferase based on whether the mixture obtained emits a fluorescence signal. Preferably, the amphipod luciferase comprises at least one of Gaussia luciferase, Maluc, Mpluc, and Mluc7; further, Gaussia luciferase.

17. A method for preparing the mutant of the amphipod luciferase according to any one of claims 1-3, the recombinant protein according to claim 4, the fusion protein according to claim 5, by culturing the transgenic cell line and / or the recombinant bacteria according to claim 6.

18. Use of the mutant of the amphipod luciferase according to any one of claims 1-3, the recombinant protein according to claim 4, the fusion protein according to claim 5, and / or the biomaterial according to claim 6 in the preparation and / or as a glow-type luciferase.

19. Use of the mutant of the amphipod luciferase according to any one of claims 1-3, the recombinant protein according to claim 4, the fusion protein according to claim 5, the biomaterial according to claim 6, and / or the conjugate or the complex according to any one of claims 7-8 in k1)-k7): k1) sequencing; k2) detecting a target molecule for non-diagnostic purposes; k3) drug screening; k4) life science research; k5) environmental monitoring; k6) as a reporter gene; k7) preparing a product for any one of k1)-k6); Preferably, when the product is for k1), the product further comprises a flash-type luciferase.

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