Copepod luciferase mutant and use thereof

Abstract

The present invention relates to the technical field of genetic engineering, and in particular to a Copepod luciferase mutant and a use thereof. The mutant is obtained by performing one or more mutations on a wild-type Copepod luciferase from which a signal peptide has been removed, can be solubly expressed in a prokaryotic expression system and exhibits a reduced luminescence half-life, and some Copepod luciferase mutants show enhanced luminescence intensity. The Copepod luciferase mutant is widely used in terms of sequencing, non-diagnostic detection of target molecules, drug screening, life science research, environmental monitoring, serving as a reporter gene, etc. On the basis of the advantage of the reduced luminescence half-life exhibited by the Copepod luciferase mutant, the Copepod luciferase mutant can be used as and / or to prepare a flash-type luciferase; the Copepod luciferase mutant is more suitable as a reporter gene for use in promoter detection; and the Copepod luciferase mutant is conjugated with avidin, a digoxin antibody, etc., for sequencing, thereby shortening sequencing time and improving 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 being "glow" and "flash". Glow refers to the fluorescent signal emitted by luciferase that can be maintained for a long time, with a long fluorescent half-life. Flash refers to the sudden production of strong fluorescent signal by luciferase in a short time, which is then rapidly 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, the flash mode can be used to monitor the start or stop of a certain gene. 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, and is composed 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, and only requires coelenterazine and O2 to participate in the reaction, 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 work.

[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 extremely important application value to develop a copepod luciferase (Gaussia luciferase) with high brightness and / or short 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) the amino acid sequence of the mutant comprises a mutation at one or more of the following positions: position 23, position 31, position 45, position 48, position 55, position 56, position 62, position 67, position 71, position 72, position 73, position 79, position 81, position 85, position 89, position 93, position 95, position 96, position 102, position 121, position 135, position 146, position 161, position 165, position 168, compared with the amino acid sequence of the wild-type copepod luciferase from which the signal peptide is removed;

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

[0010] Preferably, the copepod luciferase comprises at least one selected from Gaussian luciferase (Gluc), Maluc (Metridia asymmetrica Luciferase, BAN91823.1), Mpluc (Metridia pacifica Luciferase, BAD93333.1), and Mluc7 (Metridia longa Luciferase, AJC98141.1). Preferably, the copepod luciferase is Gaussian luciferase.

[0011] A second aspect of the invention provides a recombinant protein comprising a modified portion and a copepod-like luciferase mutant from the first aspect of the invention. Preferably, the modified portion is a protein tag.

[0012] Preferably, the modified portion is located at the N-terminus and / or C-terminus of the copepod luciferase mutant.

[0013] A third aspect of the present invention provides a fusion protein comprising a flash luciferase and a glow luciferase, wherein the flash luciferase comprises a copepod luciferase mutant of the first aspect of the present invention and / or a recombinant protein of the second aspect of the present invention.

[0014] A fourth aspect of the present invention provides biological materials related to the copepod luciferase mutant of the first aspect of the present invention, the recombinant protein of the second aspect of the present invention, or the fusion protein of the third aspect of the present invention, said biological materials comprising any one of d1) to d12):

[0015] d1) A nucleic acid molecule encoding a copepod luciferase mutant of the first aspect of the present invention, a recombinant protein of the second aspect of the present invention, or a fusion protein of the third aspect of the present invention;

[0016] d2) An expression cassette containing the nucleic acid molecule described in d1);

[0017] d3) A carrier containing the nucleic acid molecules described in d1);

[0018] d4) A carrier containing the expression box described in d2);

[0019] d5) Transgenic cell lines containing the nucleic acid molecules described in d1);

[0020] d6) Transgenic cell lines containing the expression cassette described in d2);

[0021] d7) Transgenic cell lines containing the vector described in d3);

[0022] d8) Transgenic cell lines containing the vector described in d4);

[0023] d9) a recombinant bacterium comprising the nucleic acid molecule of d1);

[0024] d10) a recombinant bacterium comprising the expression cassette of d2);

[0025] d11) a recombinant bacterium comprising the vector of d3);

[0026] d12) a recombinant bacterium comprising the vector of d4);

[0027] In a fifth aspect of the present application, there is provided a conjugate or a conjugate comprising (a) and (b):

[0028] (a) a conjugate or a conjugate moiety;

[0029] (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;

[0030] The conjugate or conjugate moiety comprises at least one of a small molecule compound, a biological macromolecule.

[0031] In a sixth aspect of the present application, there is provided a kit.

[0032] A nucleic acid sequencing kit comprising any one of i1) to i4):

[0033] i1) the mutant copepod luciferase of the first aspect of the present application;

[0034] i2) the recombinant protein of the second aspect of the present application;

[0035] i3) the fusion protein of the third aspect of the present application;

[0036] 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, digoxin antibody, nucleotide antibody, nucleotide.

[0037] A kit for detecting a target molecule comprising any one of j1) to j4):

[0038] j1) the mutant copepod luciferase of the first aspect of the present application;

[0039] j2) the recombinant protein of the second aspect of the present application;

[0040] j3) the fusion protein of the third aspect of the present application;

[0041] j4) the conjugate or conjugate of the fifth aspect of the present application, wherein the conjugate or conjugate moiety comprises a substance that specifically binds to the target molecule.

[0042] In a seventh aspect, the present application provides a nucleic acid sequencing method, comprising the step of detecting using the nucleic acid sequencing kit according to the sixth aspect of the present application.

[0043] Preferably, the nucleic acid sequencing method comprises the steps of: (1) monitoring the sequential incorporation of nucleotides in the complementary strand of the nucleic acid to be detected, the nucleotides being all or part of which are each attached to a luminescent label that initiates different luminescence kinetics or luminescence types; and (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 participates.

[0044] In an eighth aspect, the present application provides a method for detecting a target molecule, comprising the step of detecting using the kit for detecting a target molecule according to the sixth aspect of the present application.

[0045] Preferably, the method for detecting a target molecule comprises the steps of: contacting j4) in the kit for detecting a target molecule according to the sixth aspect of the present application with a sample to be detected, and 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.

[0046] The substrate for luciferase comprises the substrate for the copepod luciferase.

[0047] Preferably, when j4) comprises the fusion protein according to the third aspect of the present application, the substrate for luciferase further comprises the substrate for the photoprotein luciferase.

[0048] Preferably, the substrate for the copepod luciferase comprises at least one of coelenterazine, a coelenterazine derivative.

[0049] In a ninth aspect, the present application provides a method for screening a substrate for copepod luciferase, mixing at least one of the copepod luciferase mutant according to the first aspect of the present application, the recombinant protein according to the second aspect of the present application, the fusion protein according to the third aspect of the present application, the conjugate or the complex according to 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.

[0050] In a tenth aspect, the present application provides a method for preparing the copepod luciferase mutant according to the first aspect of the present application, the recombinant protein according to the second aspect of the present application, and the fusion protein according to the third aspect of the present application, by culturing the transgenic cell line and / or the recombinant bacteria according to the fourth aspect of the present application.

[0051] In an eleventh aspect, the present application provides any one of the following uses:

[0052] (1) use of the mutant of the luciferase of the order of the copepoda according to the first aspect of the present invention, the recombinant protein according to the second aspect of the present invention, and / or the biomaterial according to the fourth aspect of the present invention in the production and / or as a flash-type luciferase;

[0053] (2) use of the mutant of the luciferase of the order of the copepoda according to the first aspect of the present invention, the recombinant protein according to the second aspect of the present invention, the fusion protein according to the third aspect of the present invention, the biomaterial according to the fourth aspect of the present invention, and / or the conjugate or the complex according to the fifth aspect of the present invention in k1) to k7):

[0054] k1) sequencing;

[0055] k2) detecting a target molecule for a non-diagnostic purpose;

[0056] k3) drug screening;

[0057] k4) life science research;

[0058] k5) environmental monitoring;

[0059] k6) as a reporter gene;

[0060] k7) production of a product for any one of k1) to k6).

[0061] Preferably, when the product is for k1), the product further comprises a glow-type luciferase.

[0062] Preferably, the product is a reagent, a kit, a chip, or a system.

[0063] The beneficial effects of the present application are: the inventors have found through research that the mutation sites (positions 23, 31, 45, 48, 55, 56, 62, 67, 71, 72, 73, 79, 81, 85, 89, 93, 95, 96, 102, 121, 135, 146, 161, 165, 168) are important sites affecting the removal of signal peptide of wild-type copepod luciferase flash-type luminescence, based on this, the inventors provide a copepod luciferase mutant, which is obtained by mutating one or more of the above-mentioned sites of the wild-type copepod luciferase with the signal peptide removed, the copepod luciferase mutant can be expressed in a prokaryotic expression system, the half-life of the luminescence produced is reduced, and the luminescence intensity produced by part of the copepod luciferase mutant is enhanced; the copepod luciferase mutant is widely used in sequencing, detecting target molecules for non-diagnostic purposes, drug screening, life science research, environmental monitoring, as a reporter gene and the like, based on the advantage that the half-life of the luminescence produced by the copepod luciferase mutant is reduced, the copepod luciferase mutant can be used as and / or prepared as a flash-type luciferase; it is more suitable as a reporter gene and is applied to promoter detection; and is coupled with avidin (such as streptavidin SA), digoxin antibody and the like for sequencing (used in combination with glow-type luciferase), which shortens the sequencing time and improves the sequencing quality. BRIEF DESCRIPTION OF DRAWINGS

[0064] Fig. 1 is a schematic diagram of the sequence alignment results of Gaussia luciferase and other homologous copepod luciferases.

[0065] Fig. 2 is a map of the plasmid (pCold-Gluc-NO SP) expressing C-terminal fusion His-tagged wild-type Gaussia luciferase with the signal peptide removed in Example 1.

[0066] Fig. 3 is an SDS-PAGE characterization result diagram of the C-terminal fusion His-tagged wild-type Gaussia luciferase with the signal peptide removed or its mutant purified in Example 1: wherein 1-29 respectively represent WT-NS Gluc, Gluc-NO SP-1-Gluc-NO SP-28 in Table 3.

[0067] Fig. 4 is a structure diagram of coelenterazine.

[0068] Fig. 5 is a comparison result diagram of the catalytic activity of the C-terminal fusion His-tagged wild-type Gaussia luciferase with the signal peptide removed or its mutant on the substrate in Example 2.

[0069] Fig. 6 is a comparison result diagram of the catalytic luminescence half-life of the C-terminal fusion His-tagged wild-type Gaussia luciferase with the signal peptide removed or its mutant on the substrate in Example 2.

[0070] Figure 7 is a map of the plasmid (pCold-Maluc-NO SP) of the wild-type copepod luciferase Maluc with a C-terminal fusion His tag and without a signal peptide in Example 3.

[0071] Figure 8 is a map of the plasmid (pCold-Mpluc-NO SP) of the wild-type copepod luciferase Mpluc with a C-terminal fusion His tag and without a signal peptide in Example 3.

[0072] Figure 9 is a map of the plasmid (pCold-Mluc7-NO SP) of the wild-type copepod luciferase Mluc7 with a C-terminal fusion His tag and without a signal peptide in Example 3.

[0073] Figure 10 is a graph of the comparison results of the catalytic activity of the wild-type copepod luciferase Maluc / Mpluc / Mluc7 or its mutant with a C-terminal fusion His tag and without a signal peptide on a substrate in Example 4.

[0074] Figure 11 is a graph of the comparison results of the catalytic luminescence half-life of the wild-type copepod luciferase Maluc / Mpluc / Mluc7 or its mutant with a C-terminal fusion His tag and without a signal peptide on a substrate in Example 4.

[0075] Figure 12 is a graph of the SDS-PAGE characterization results of the luciferase coupled with SA in Example 5.

[0076] Figure 13 is a graph of the SDS-PAGE characterization results of the luciferase-SA coupling complex purified in Example 5.

[0077] Figure 14 is a graph of the comparison results of the catalytic activity of the luciferase-SA coupling complex on a substrate in Example 5.

[0078] Figure 15 is a graph of the comparison results of the catalytic luminescence half-life of the luciferase-SA coupling complex on a substrate in Example 5. DETAILED DESCRIPTION

[0079] 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).

[0080] In the present application, unless otherwise specified, the nucleotide sequence is represented in the 5' end→3' end direction.

[0081] In the present application, unless otherwise specified, the amino acid sequence is represented in the N-terminal→C-terminal direction.

[0082] 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.

[0083] In a first aspect of the present application, there is provided a mutant of copepod luciferase, which is (a1) or (a2):

[0084] (a1) the amino acid sequence of the mutant comprises a mutation at one or more of the following positions: position 23, position 31, position 45, position 48, position 55, position 56, position 62, position 67, position 71, position 72, position 73, position 79, position 81, position 85, position 89, position 93, position 95, position 96, position 102, position 121, position 135, position 146, position 161, position 165, position 168, compared to the amino acid sequence of wild-type copepod luciferase from which the signal peptide is removed (not included);

[0085] (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).

[0086] In some embodiments, the mutation at the above-mentioned positions can be mutated to any natural or unnatural amino acid.

[0087] 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.

[0088] The inventors found through research that the above-mentioned mutation sites (positions 23, 31, 45, 48, 55, 56, 62, 67, 71, 72, 73, 79, 81, 85, 89, 93, 95, 96, 102, 121, 135, 146, 161, 165, 168) are important sites affecting the flash-type luminescence of wild-type copepod luciferase with signal peptide removed. By mutating one or more of the above-mentioned 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 reduced, and the luminescence intensity of some mutant copepod luciferases can be enhanced.

[0089] In application, the above-mentioned copepod luciferase mutants are widely used in the fields of life science research (such as in vivo detection: for example, for labeling and tracking activities or processes in living organisms, such as the expression, distribution, and movement of intracellular proteins), medical detection, drug screening, environmental monitoring, and enzyme-linked detection, etc. At the same time, they are applied to genomic sequencing and analysis technology, and play a role in the fields of clinical medicine, forensic detection, and customs, etc. Taking advantage of the self-luminescence characteristics of copepod luciferase mutants, copepod luciferase mutants are often used in 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, copepod luciferase mutants 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. 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 advantage of reduced luminescence half-life of the above-mentioned copepod luciferase mutants, the copepod luciferase mutants can be used as and / or prepared into flash-type luciferase; they are more suitable as reporter genes for promoter monitoring; and they can be coupled with avidin (such as streptavidin SA), digoxin antibody, etc. for sequencing, shortening the sequencing time, and improving the sequencing quality.

[0090] 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).

[0091] In some embodiments, the amphipod luciferase is Gaussia luciferase.

[0092] In some embodiments, the mutant comprises any one mutation or a combination of multiple mutations of (b1) to (b18) compared to the amino acid sequence of wild-type Gaussia luciferase without signal peptide: (b1) L at position 23 is mutated to A; (b2) R at position 48 is mutated to Q; (b3) H at position 62 is mutated to A, or K; (b4) P at position 67 is mutated to A; (b5) F at position 72 is mutated to W; (b6) I at position 73 is mutated to L; (b7) T at position 79 is mutated to D, or S; (b8) E at position 81 is mutated to G; (b9) E at position 85 is mutated to K, D, or S; (b10) G at position 89 is mutated to M; (b11) E at position 93 is mutated to G, S, or A; (b12) P at position 102 is mutated to S; (b13) V at position 121 is mutated to A; (b14) Q at position 135 is mutated to K; (b15) Q at position 146 is mutated to S; (b16) K at position 161 is mutated to T, or E; (b17) A at position 165 is mutated to L; (b18) D at position 168 is mutated to S.

[0093] In some embodiments, the mutant does not comprise the Gaussia luciferase superior mutants of Table 3 of patent document WO2023109981A3 (i.e., Gaussia luciferase superior mutants numbered 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).

[0094] In some embodiments, the mutant has any one of the mutations (c1)~(c28) compared to the amino acid sequence of the wild-type Gaussia luciferase without signal peptide: (c1) I73L; (c2) H62A; (c3) T79D; (c4) T79S; (c5) E93G; (c6) F72W; (c7) E93S; (c8) K161T; (c9) R48Q; (c10) H62K; (c11) E81G; (c12) K161E; (c13) A165L; (c14) E93A; (c15) E85K; (c16) L23A; (c17) Q146S; (c18) D168S; (c19) Q135K; (c20) P102S; (c21) H62A / I73L / E85S / S86T / A87G / G89M / E93P / L107M / V121E; (c22) H62A / I73L / E85S / S86T / A87G / G89M / E93P / P102S / L107M / V121E; (c23) H62A / P67A / I73L / T79D / E85D / S86T / A87G / E93G / L107M / V121E; (c24) H62A / P67A / I73L / T79D / E85D / S86T / A87G / P102S / L107M / V121E; (c25) H62A / P67A / I73L / E85D / S86T / A87G / V121E; (c26) H62A / E81G / A87G / G89M / E93P / L107M / V121E; (c27) H62A / P67L / E81G / A87G / G89M / E93P / P102S / L107M / V121E; (c28) H62A / P67L / E81G / S86T / A87G / G89M / E93P / P102S / L107M / V121E.

[0095] In some embodiments, the mutant has any one of the mutations (c1), (c5), (c6), (c9), (c11), (c12), (c14)~(c17), (c19)~(c28) compared to the amino acid sequence of the wild-type Gaussia luciferase without signal peptide.

[0096] In some embodiments, the mutant has any one of the mutations (c1), (c5), (c11), (c21)~(c28) compared to the amino acid sequence of the wild-type Gaussia luciferase without signal peptide.

[0097] In some embodiments, the amino acid sequence of the wild-type Ctenocephalides luciferase without signal peptide is shown in SEQ ID NO: 4.

[0098] In some embodiments, the mutant further comprises a signal peptide.

[0099] In some embodiments, the signal peptide is located at the N-terminus of the mutant.

[0100] In some embodiments, the amino acid sequence of the signal peptide is set forth in SEQ ID NO: 3.

[0101] In some embodiments, the copepod luciferase is Maluc.

[0102] In some embodiments, the amino acid sequence of the mutant comprises a mutation at one or more of the following positions: position 48, position 62, position 72, or position 73, as compared to the amino acid sequence of a wild-type Maluc without a signal peptide.

[0103] In some embodiments, the mutant comprises any one mutation or a combination of multiple mutations at the following positions: V at position 48 is mutated to Q, K at position 62 is mutated to A, Y at position 72 is mutated to W, or I at position 73 is mutated to L, as compared to the amino acid sequence of a wild-type Maluc without a signal peptide.

[0104] In some embodiments, the amino acid sequence of the wild-type Maluc without a signal peptide is set forth in SEQ ID NO: 51.

[0105] In some embodiments, the mutant further comprises a signal peptide.

[0106] In some embodiments, the signal peptide is located at the N-terminus of the mutant.

[0107] In some embodiments, the amino acid sequence of the signal peptide is set forth in SEQ ID NO: 50.

[0108] In some embodiments, the copepod luciferase is Mpluc.

[0109] In some embodiments, the amino acid sequence of the mutant comprises a mutation at one or more of the following positions: position 71, position 85, position 95, or position 96, as compared to the amino acid sequence of a wild-type Mpluc without a signal peptide.

[0110] In some embodiments, the mutant comprises any one mutation or a combination of multiple mutations at the following positions: R at position 71 is mutated to Q, K at position 85 is mutated to A, Y at position 95 is mutated to W, or I at position 96 is mutated to L, as compared to the amino acid sequence of a wild-type Mpluc without a signal peptide.

[0111] In some embodiments, the amino acid sequence of the wild-type Mpluc without signal peptide is set forth in SEQ ID NO: 54.

[0112] In some embodiments, the mutant further comprises a signal peptide.

[0113] In some embodiments, the signal peptide is located at the N-terminus of the mutant.

[0114] In some embodiments, the amino acid sequence of the signal peptide is set forth in SEQ ID NO: 53.

[0115] In some embodiments, the amphipod luciferase is Mluc7.

[0116] In some embodiments, the amino acid sequence of the mutant comprises one or more mutations at position 31, 45, 55, or 56, as compared to the amino acid sequence of the wild-type Mluc7 without signal peptide.

[0117] In some embodiments, the mutant comprises any one mutation or a combination of multiple mutations at position 31 (F to Q), 45 (K to A), 55 (Y to W), or 56 (I to L), as compared to the amino acid sequence of the wild-type Mluc7 without signal peptide.

[0118] In some embodiments, the amino acid sequence of the wild-type Mluc7 without signal peptide is set forth in SEQ ID NO: 57.

[0119] In some embodiments, the mutant further comprises a signal peptide.

[0120] In some embodiments, the signal peptide is located at the N-terminus of the mutant.

[0121] In some embodiments, the amino acid sequence of the signal peptide is set forth in SEQ ID NO: 56.

[0122] The radopholus luciferin is a conserved luciferase, and the important site of Gluc affecting flash light type luminescence is similar to the corresponding site 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 Figure 1, wherein the mutation sites of the Maluc mutant (compared with the amino acid sequence of the wild-type Maluc without signal peptide) V48Q, K62A, Y72W and I73L, the mutation sites of the Mpluc mutant (compared with the amino acid sequence of the wild-type Mpluc without signal peptide) R71Q, K85A, Y95W and I96L, the mutation sites of the Mluc7 mutant (compared with the amino acid sequence of the wild-type Mluc7 without signal peptide) F31Q, K45A, Y55W and I56L, respectively correspond to the mutation sites of the Gluc mutant (compared with the amino acid sequence of the wild-type Gluc without signal peptide) R48Q, H62A, F72W and I73L. By mutating the above-mentioned sites, the luminescence half-life of the luciferase mutant obtained by mutation can be reduced, and the luminescence intensity of part of the mutant can be enhanced.

[0123] 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.

[0124] In some embodiments, the modification moiety is a protein tag.

[0125] 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).

[0126] In some embodiments, the modification moiety is located at the N-terminus and / or C-terminus of the radopholus luciferase mutant.

[0127] In some embodiments, the modification moiety is located at the C-terminus of the radopholus luciferase mutant.

[0128] In some embodiments, the modification moiety further comprises a connecting peptide between the radopholus luciferase mutant.

[0129] In some embodiments, the recombinant protein comprises: TEE site-radopholus luciferase mutant-TEV site-His tag (N-terminus→C-terminus).

[0130] In a third aspect of the present application, a fusion protein comprising a flash luciferase and a glow luciferase is provided: the flash luciferase comprises the mutant of the copepod luciferase according to the first aspect of the present application, and / or the recombinant protein according to the second aspect of the present application.

[0131] In the present application, the "glow luciferase" refers to a type of luciferase having a higher luminescence half-life than the flash luciferase; preferably, the luminescence half-life of the glow luciferase is 10s higher than that of the flash luciferase, more preferably 25s higher. As an example, the glow luciferase can be Aluc (artificial luciferase), turboluc, Nanoluc, or other commercially available glow luciferases, which are not limited herein.

[0132] In some embodiments, the flash luciferase is located at the C-terminal and / or N-terminal of the glow luciferase.

[0133] In some embodiments, the flash luciferase and the glow luciferase are connected by a linker peptide.

[0134] In a fourth aspect of the present application, a biological material related to the mutant of the copepod luciferase according to the first aspect of the present application, the recombinant protein according to the second aspect of the present application, or the fusion protein according to the third aspect of the present application is provided, which comprises any one of d1) to d12):

[0135] d1) a nucleic acid molecule encoding the mutant of the copepod luciferase according to the first aspect of the present application, the recombinant protein according to the second aspect of the present application, or the fusion protein according to the third aspect of the present application;

[0136] d2) an expression cassette comprising the nucleic acid molecule of d1);

[0137] d3) a vector comprising the nucleic acid molecule of d1);

[0138] d4) a vector comprising the expression cassette of d2);

[0139] d5) a transgenic cell line comprising the nucleic acid molecule of d1);

[0140] d6) a transgenic cell line comprising the expression cassette of d2);

[0141] d7) a transgenic cell line comprising the vector of d3);

[0142] d8) a transgenic cell line comprising the vector of d4);

[0143] d9) a recombinant bacterium comprising the nucleic acid molecule of d1);

[0144] d10) a recombinant bacterium comprising the expression cassette of d2);

[0145] d11) a recombinant bacterium comprising the vector of d3);

[0146] d12) a recombinant bacterium comprising the vector of d4).

[0147] In some embodiments, the transgenic cell line in d5) to d8) does not comprise reproductive material.

[0148] In some embodiments, the vector in d3), d4) can further comprise a promoter operably linked to the nucleic acid molecule.

[0149] In some embodiments, the vector in d3), d4) is independently selected from the group consisting of a non-pathogenic viral vector and a non-viral vector.

[0150] In some embodiments, the non-pathogenic viral vector comprises an adenoviral vector or a retroviral vector.

[0151] In some embodiments, the non-viral vector comprises, but is not limited to, a plasmid vector.

[0152] In some embodiments, the recombinant bacterium in d9) to d12) comprises, but is not limited to, Escherichia coli.

[0153] In a fifth aspect of the present application, there is provided a conjugate or a conjugate comprising (a) and (b):

[0154] (a) a conjugate or a conjugate moiety;

[0155] (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;

[0156] The conjugate or conjugate moiety comprises at least one (preferably any one) of a small molecule compound, a biological macromolecule.

[0157] In some embodiments, the conjugate or conjugate moiety comprises at least one of avidin (e.g. streptavidin), digoxin antibody, nucleotide antibody, nucleotide, a substance that specifically binds to a target molecule.

[0158] 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).

[0159] 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.

[0160] In some embodiments, the nucleotide is a 3'-OH end modified nucleotide.

[0161] In some embodiments, the 3'-OH end modified nucleotide is modified by adding a blocking group at the 3'-OH end of the nucleotide.

[0162] In some embodiments, the blocking group comprises at least one of O-amino, O-allyl, O-azido, O-phosphate group.

[0163] In a sixth aspect of the present application, a kit is provided.

[0164] In some embodiments, a nucleic acid sequencing kit is provided, comprising any one of i1) to i4):

[0165] i1) the mutant of copepod luciferase of the first aspect of the present application;

[0166] i2) the recombinant protein of the second aspect of the present application;

[0167] i3) the fusion protein of the third aspect of the present application;

[0168] 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.

[0169] In some embodiments, the nucleotide is the nucleotide of the fifth aspect of the present application.

[0170] In some embodiments, the nucleic acid sequencing kit further comprises at least one of a substrate of the mutant of copepod luciferase, a polymerase (preferably a DNA polymerase), a PCR buffer.

[0171] In some embodiments, the substrate of the mutant of copepod luciferase comprises at least one of coelenterazine, a coelenterazine derivative.

[0172] 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.

[0173] In some embodiments, the nucleic acid sequencing kit further comprises a substrate of the luciferase when i3) is present.

[0174] In some embodiments, the nucleic acid sequencing kit further comprises a nucleotide when the coupling or conjugation moiety is an avidin (e.g., streptavidin), a digoxin antibody, or a nucleotide antibody.

[0175] In some embodiments, the nucleotide is the nucleotide of the fifth aspect of the present application.

[0176] In some embodiments, a kit for detecting a target molecule is provided, which comprises any one of j1) to j4):

[0177] j1) the mutant of the luciferase of the first aspect of the present application;

[0178] j2) the recombinant protein of the second aspect of the present application;

[0179] j3) the fusion protein of the third aspect of the present application;

[0180] j4) the conjugate or conjugate of the fifth aspect of the present application, wherein the coupling or conjugation moiety comprises a substance that specifically binds to the target molecule.

[0181] In some embodiments, the kit for detecting a target molecule further comprises a substrate of the luciferase.

[0182] In some embodiments, the substrate of the luciferase comprises at least one of coelenterazine, a coelenterazine derivative.

[0183] 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.

[0184] In some embodiments, the kit for detecting a target molecule comprises j3), the kit further comprises a substrate of the glow-type luciferase.

[0185] 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.

[0186] In some embodiments, the nucleic acid sequencing method comprises the following steps:

[0187] (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; and (2) identifying each incorporated nucleotide by detecting the luminescence kinetics curve or luminescence type or light intensity of the luminescence reaction in which the luminescent label is involved.

[0188] In some embodiments, the incorporated nucleotide carries a blocking modification group.

[0189] In some embodiments, the luminescent label comprises the copepod luciferase mutant of the first aspect of the present application.

[0190] 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 from the copepod luciferase mutant.

[0191] In some embodiments, the nucleic acid sequencing method further comprises the following step: (3) removing the blocking modification group carried by the incorporated nucleotide so that the complementary strand can continue to extend, and removing the luminescent label.

[0192] 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.

[0193] 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.

[0194] 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.

[0195] 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.

[0196] 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 flash type luciferases, which comprise the Copepod luciferase mutant of the first aspect of the present application or the recombinant protein of the second aspect of the present application.

[0197] In some embodiments, the light-emitting marker comprises two luciferases that initiate different light emission kinetics or light emission types.

[0198] 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.

[0199] 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.

[0200] In some embodiments, the light emission type includes flash type, glow type and mixed type.

[0201] 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.

[0202] In some embodiments, the substrates of the light-emitting markers can be the same or different.

[0203] In some embodiments, the nucleotide comprises at least one of the nucleotides A, C, G, T / U (i.e. the nucleotide comprises at least one of the nucleotides A, C, G, T; or the nucleotide comprises at least one of the nucleotides A, C, G, U); further comprising the nucleotides A, C, G, and T / U.

[0204] In some embodiments, the nucleotide comprises at least one of the nucleotides ATP, dATP, GTP, dGTP, CTP, dCTP, TTP, dTTP, UTP, dUTP; further comprising the nucleotides dATP, dGTP, dCTP, and dTTP; or ATP, GTP, CTP, and UTP.

[0205] In some embodiments, the nucleotide is a 3'-OH end modified nucleotide.

[0206] In some embodiments, the 3'-OH end modified nucleotide is modified by adding a blocking group to the 3'-OH end of the nucleotide.

[0207] In some embodiments, the blocking group comprises at least one of O-amino, O-allyl, O-azido, O-phosphate group.

[0208] In some embodiments, the light emission type or the photokinetics curve of the luminescence reaction involving the Copepod luciferase mutant is flash type; and the light emission type or the photokinetics curve of the luminescence reaction involving the second luminescent marker is glow type.

[0209] In some embodiments, each incorporated nucleotide is identified by detecting the photokinetics curve or the light emission type or the light intensity of the luminescence reaction involving the luminescent marker;

[0210] The luminescent marker is selected from luciferases that initiate different light emission types;

[0211] The luciferases that initiate different light emission types comprise flash type luciferases, which comprise the Copepod luciferase mutant of the first aspect of the present application or the recombinant protein of the second aspect of the present application.

[0212] In some embodiments, the luciferases that initiate different light emission types further comprise glow type luciferases.

[0213] In some embodiments, in the nucleotide, the first nucleotide is attached to the Copepod luciferase mutant of the first aspect of the present application, the second nucleotide is attached to the second luminescent marker, the third nucleotide is attached to both the Copepod luciferase mutant of the first aspect of the present application and the second luminescent marker, and the fourth nucleotide is not attached to a luminescent marker.

[0214] In some embodiments, the attachment between the nucleotide and the luminescent label comprises attachment mediated by an affinity interaction.

[0215] In some embodiments, the affinity interaction comprises an antigen-antibody interaction and a biotin-avidin (e.g., streptavidin) interaction.

[0216] 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.

[0217] In some embodiments, the member linked to the nucleotide is biotin and the member linked to the luminescent label is avidin (e.g., streptavidin).

[0218] In some embodiments, the member linked to the nucleotide is digoxigenin and the member linked to the luminescent label is an anti-digoxigenin antibody.

[0219] 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.

[0220] 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 of skill 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.

[0221] 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.

[0222] 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.

[0223] 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.

[0224] 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.

[0225] 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.

[0226] 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.

[0227] 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.

[0228] 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.

[0229] 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.

[0230] 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.

[0231] 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.

[0232] 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.

[0233] In some embodiments, the substrate for luciferase comprises a substrate for the copepod luciferase.

[0234] In some embodiments, the substrate for the copepod luciferase comprises at least one of coelenterazine, a coelenterazine derivative.

[0235] 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.

[0236] 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.

[0237] 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.

[0238] 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.

[0239] 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 flash-type luciferase is provided.

[0240] 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) ~ k7) is provided:

[0241] k1) sequencing;

[0242] k2) detecting a target molecule for non-diagnostic purposes;

[0243] k3) drug screening;

[0244] k4) life science research;

[0245] k5) environmental monitoring;

[0246] k6) as a reporter gene;

[0247] k7) preparing a product for any one of k1) ~ k6).

[0248] In some embodiments, when the product is for k1), the product further comprises a glow-type luciferase.

[0249] In some embodiments, the product is a reagent, a kit, a chip, or a system.

[0250] The present application will be further described in detail by the following specific examples.

[0251] 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.

[0252] 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.

[0253] 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.

[0254] Example 1 Plasmid construction and expression and purification of wild-type Gaussia luciferase without signal peptide and its mutants

[0255] 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.

[0256] (I) Plasmid construction of prokaryotic expression of wild-type Gaussia luciferase without signal peptide or its mutants

[0257] 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).

[0258] A plasmid expressing wild-type Gaussia luciferase (WT no signal peptide Gluc: WT-NS Gluc) without signal peptide C-terminally fused with His tag (6 histidines, 6xHis, for facilitating protein purification) (pCold-Gluc-NO SP, Fig. 2) was constructed by the method of total gene synthesis (the coding gene of wild-type Gaussia luciferase without signal peptide C-terminally fused with 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 the 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 tag (Poly his), FLAG, Strep-Tag II, Poly arg, C-myc, etc.; similarly, the addition or non-addition of the signal peptide, which guides the enzyme into the secretory pathway, enabling it to function in the extracellular environment, also has no effect on the performance of wild-type Gaussia luciferase or Gaussia luciferase mutants. Copepods luciferase is a eukaryotic secretory protein, and contains a signal peptide under natural conditions; however, the signal peptide affects the expression of this type of luciferase in E. coli, so the signal peptide is removed when expressing luciferase in E. coli.The nucleotide sequence of the coding gene of the wild-type Gaussia luciferase with a C-terminal fusion His tag and a removed signal peptide is: ATGAATCACAAAGTGCATATGAAACCAACTGAAAACAATGAAGATTTCAACATTGTAGCTGTAGCTAGCAACTTTGCTACAACGGATCTCGATGCTGACCGTGGTAAATTGCCCGGAAAAAAATTACCACTTGAGGTACTCAAAGAAATGGAAGCCAATGCTAGGAAAGCTGGCTGCACTAGGGGATGTCTGATATGCCTGTCACACATCAAGTGTACACCCAAAATGAAGAAGTTTATCCCAGGAAGATGCCACACCTATGAAGGAGACAAAGAAAGTGCACAGGGAGGAATAGGAGAGGCTATTGTTGACATTCCTGAAATTCCTGGGTTTAAGGATTTGGAACCCATGGAACAATTCATTGCACAAGTTGACCTATGTGTAGACTGCACAACTGGATGCCTCAAAGGTCTTGCCAATGTGCAATGTTCTGATTTACTCAAGAAATGGCTGCCACAAAGATGTGCAACTTTTGCTAGCAAAATTCAAGGCCAAGTGGACAAAATAAAGGGTGCCGGTGGTGATGGTGGCAGTGAGAACCTGTACTTCCAGAGCGGCCACCACCACCACCATCACGGCTCCTGA (5'→3', SEQ ID NO: 5), wherein the nucleotides 1-15 encode a TEE site, the nucleotides 16-21 are a NdeI restriction site, the nucleotides 22-525 encode the amino acids of the wild-type Gaussia luciferase with a removed signal peptide, the nucleotides 535-555 encode a TEV site, the nucleotides 559-576 encode a histone tag sequence, and the nucleotides 526-534, 556-558, and 559-564 are linkers. The NdeI restriction site is located between the TEE and Gluc, and the EcoRI restriction site is located after the stop codon.

[0259] (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.

[0260] A 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 C-terminal fusion His tag without signal peptide 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). The plasmid of Gauss luciferase mutant without signal peptide expressed in a C-terminal fusion His tag (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 (I73L): 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): I73L; Gluc-NO SP-2 (H62A): the amino acid sequence of Gluc-NO SP-2 has the following mutation compared with SEQ ID NO: 4: H62A; Gluc-NO SP-3 (T79D): the amino acid sequence of Gluc-NO SP-3 has the following mutation compared with SEQ ID NO: 4: T79D; Gluc-NO SP-4 (T79S): the amino acid sequence of Gluc-NO SP-4 has the following mutation compared with SEQ ID NO: 4: T79S; Gluc-NO SP-5 (E93G): the amino acid sequence of Gluc-NO SP-5 has the following mutation compared with SEQ ID NO: 4: E93G; Gluc-NO SP-6 (F72W): the amino acid sequence of Gluc-NO SP-6 has the following mutation compared with SEQ ID NO: 4: F72W; Gluc-NO SP-7 (E93S): the amino acid sequence of Gluc-NO SP-7 has the following mutation compared with SEQ ID NO: 4: E93S; Gluc-NO SP-8 (K161T): the amino acid sequence of Gluc-NO SP-8 has the following mutation compared with SEQ ID NO: 4: K161T; Gluc-NO SP-9 (R48Q): the amino acid sequence of Gluc-NO SP-9 has the following mutation compared with SEQ ID NO: 4: R48Q; Gluc-NO SP-10 (H62K): the amino acid sequence of Gluc-NO SP-10 has the following mutation compared with SEQ ID NO: 4: H62K.Gluc-NO SP-11 (E81G): the amino acid sequence of Gluc-NO SP-11 has the following mutation as compared to SEQ ID NO: 4: E81G; Gluc-NO SP-12 (K161E): the amino acid sequence of Gluc-NO SP-12 has the following mutation as compared to SEQ ID NO: 4: K161E; Gluc-NO SP-13 (A165L): the amino acid sequence of Gluc-NO SP-13 has the following mutation as compared to SEQ ID NO: 4: A165L; Gluc-NO SP-14 (E93A): the amino acid sequence of Gluc-NO SP-14 has the following mutation as compared to SEQ ID NO: 4: E93A; Gluc-NO SP-15 (E85K): the amino acid sequence of Gluc-NO SP-15 has the following mutation as compared to SEQ ID NO: 4: E85K; Gluc-NO SP-16 (L23A): the amino acid sequence of Gluc-NO SP-16 has the following mutation as compared to SEQ ID NO: 4: L23A; Gluc-NO SP-17 (Q146S): the amino acid sequence of Gluc-NO SP-17 has the following mutation as compared to SEQ ID NO: 4: Q146S; Gluc-NO SP-18 (D168S): the amino acid sequence of Gluc-NO SP-18 has the following mutation as compared to SEQ ID NO: 4: D168S; Gluc-NO SP-19 (Q135K): the amino acid sequence of Gluc-NO SP-19 has the following mutation as compared to SEQ ID NO: 4: Q135K; Gluc-NO SP-20 (P102S): the amino acid sequence of Gluc-NO SP-20 has the following mutation as compared to SEQ ID NO: 4: P102S; Gluc-NO SP-21 (Seq-22): the amino acid sequence of Gluc-NO SP-21 has the following mutations as compared to SEQ ID NO: 4: H62A, I73L, E85S, S86T, A87G, G89M, E93P, L107M, and V121E; Gluc-NO SP-22 (Seq-23): the amino acid sequence of Gluc-NO SP-22 has the following mutations as compared to SEQ ID NO: 4: H62A, I73L, E85S, S86T, A87G, G89M, E93P, P102S, L107M, and V121E;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: H62A, P67A, I73L, T79D, E85D, S86T, A87G, E93G, L107M, and V121E; 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: H62A, P67A, I73L, T79D, E85D, S86T, A87G, P102S, L107M, and V121E; 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: H62A, P67A, I73L, E85D, S86T, A87G, and V121E; 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: H62A, E81G, A87G, G89M, E93P, L107M, and V121E; 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: H62A, P67L, E81G, A87G, G89M, E93P, P102S, L107M, and V121E; Gluc-NO SP-28 (Seq-29): The amino acid sequence of Gluc-NO SP-28 has the following mutations compared to SEQ ID NO: 4: H62A, P67L, E81G, S86T, A87G, G89M, E93P, P102S, L107M, and V121E.

[0261] Table 1 PCR reaction system

[0262] Table 2 PCR reaction conditions

[0263] Table 3 Primer sequence

[0264] 0.5 μL DpnI enzyme was added to the reaction system, and the template was digested at 37°C for 3 hours, and then about 4800 bp of product was recovered by gel, that is, the plasmid of the mutant of the C-terminal fusion His tag of the Gauss luciferase mutant with the removal of the signal peptide.

[0265] The reaction product or 2.5 μL of pCold-Gluc-NO SP obtained above was transformed into DH5α competent cells, and plated on an ampicillin-resistant plate with a final concentration of 100 μg / mL. The next day, a single colony was picked from the plate, and the plasmid was extracted. Sequencing was performed to ensure correct amplification by multiplex PCR, and the obtained plasmid was a plasmid expressing a C-terminal fusion His-tagged signal peptide-removed Gaussia luciferase mutant and a plasmid expressing a C-terminal fusion His-tagged signal peptide-removed wild-type Gaussia luciferase (pCold-Gluc-NO SP).

[0266] (ii) Prokaryotic expression and purification of wild-type Gaussia luciferase or a mutant thereof without a signal peptide

[0267] Transformation: Origami DE3 competent cells were thawed on ice, and aliquoted into 50 μL per tube. 5 μL of the plasmid prepared in step (i) (a plasmid expressing a C-terminal fusion His-tagged signal peptide-removed Gaussia luciferase mutant or a plasmid expressing a C-terminal fusion His-tagged signal peptide-removed wild-type Gaussia luciferase) was added to each tube, and the tubes were gently tapped to mix the contents. The tubes were incubated on ice for 1 h, heat shocked at 42°C for 1 min, and then incubated on ice for 5 min. 500 μL of LB without antibiotics was added, and the cells were recovered at 37°C at 220 rpm for 1 h. Then, 60 μL of the cell solution was uniformly spread on an Amp-resistant LB agar plate, and incubated at 37°C overnight.

[0268] Protein expression and purification: A single colony was picked from the overnight culture plate, and inoculated in an Amp-resistant LB broth at 37°C at 220 rpm until the OD600 reached 0.6. The cells were then inoculated in an Amp LB broth in a ratio of 50:1 to the test volume in a shaking tube or flask, and incubated at 37°C at 200 rpm for 3-4 h until the OD600 was 0.6. The cells were cooled on ice for 15 min, and 1 mM of IPTG was added to a final concentration of 1 uM. The cells were induced to express by overnight incubation at 16°C at 300 rpm. The cells were centrifuged at 4000 g for 10 min, and the supernatant was removed. The cells were resuspended in pre-cooled PBS with a final concentration of 1 mM of PMSF, and sonicated to lyse the cells. The lysed cells were centrifuged at 12000 g at 4°C for 30 min, and the supernatant was stored on ice for later use. The column was equilibrated with 5 CV of sterilized deionized water, 5 CV of 1×PBS, and 5 CV of 1 M imidazole. The column was then equilibrated with 5 CV of 20 mM imidazole. The treated column was added to the treated sample, and gently shaken at 4°C for 30 min. The sample-column mixture was then loaded onto the column, and the column was retained. The column was washed with 5 CV of 1×PBS, and 5 CV of 20 mM imidazole. The column was eluted with 300 mM imidazole, and the eluate was the target protein. The column was washed with 5 CV of 1 mM imidazole, and 5 CV of water. Finally, 20% ethanol was added, and the column was stored at 4°C.

[0269] Ultrafiltration buffer exchange: The purified protein was transferred to a 10k ultrafiltration tube (Merck ), 10 volumes of 4°C pre-cooled 1xPBS was added, centrifuged at 3900g for 15min at 4°C, repeated for 3 times. The purified protein was characterized by 15% SDS-PAGE for molecular weight and purity. The results were shown in Figure 3: the molecular weight of the C-terminal fusion His-tagged signal peptide-removed wild-type Gaussia luciferase mutant and the C-terminal fusion His-tagged signal peptide-removed wild-type Gaussia luciferase were consistent with the expected size.

[0270] Example 2 Activity detection of wild-type Gaussia luciferase or its mutants

[0271] In this example, the concentration of the protein (purified C-terminal fusion His-tagged signal peptide-removed wild-type Gaussia luciferase and C-terminal fusion His-tagged signal peptide-removed Gaussia luciferase mutants obtained in Example 1, corresponding to WT-NS Gluc, Gluc-NO SP-1~Gluc-NO SP-28 in Table 4) was accurately determined by BCA quantitative kit (Thermo Scientific TM Pierce TM BCA Protein Assay Kit). The purified C-terminal fusion His-tagged signal peptide-removed wild-type Gaussia luciferase and C-terminal fusion His-tagged signal peptide-removed Gaussia luciferase mutants obtained in Example 1 were diluted to 1 μg / mL with 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 substrate coelenterazine (purchased from Bioolab, Figure 4) diluted to 100 μM with the same diluent was added, and the luminescence intensity and mode were read by the luminescence module of the enzyme marker. The results were shown in Figure 5, 6 and Table 4: all Gaussia luciferase mutants could catalyze the substrate coelenterazine to produce luminescence intensity; compared with wild-type Gaussia luciferase, the half-life of luminescence produced by Gaussia luciferase mutants was reduced, and the luminescence intensity produced by some Gaussia luciferase mutants was improved.

[0272] Table 4 Luminescence intensity and half-life produced by wild-type Gaussia luciferase or its mutants

[0273] Example 3 Construction of plasmid for prokaryotic expression of wild-type copepod luciferase or its mutants without signal peptide

[0274] 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) R48Q, H62A, F72W and I73L 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.

[0275] 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) R48Q, H62A, F72W and I73L of the Gluc mutant correspond to the mutation sites (compared with the amino acid sequence of wild-type Maluc without signal peptide) V48Q, K62A, Y72W and I73L of the Maluc mutant, the mutation sites (compared with the amino acid sequence of wild-type Mpluc without signal peptide) R71Q, K85A, Y95W and I96L of the Mpluc mutant, and the mutation sites (compared with the amino acid sequence of wild-type Mluc7 without signal peptide) F31Q, K45A, Y55W and I56L of the Mluc7 mutant, respectively.

[0276] The amino acid sequence of wild-type Maluc is:

[0277] DIKVLFALICVAMVQAKATENNDDIDIVGIASTFITTNTDADRGKMPGKRLPLAVLKEMEANAVKAGCSRGCLICLSKIKCTAKMKQYIPGRCHDYGGDKKTGQAAIEGAIDDIPEISGFKEMAPMEQFIAQVDLCADCTTGCLKGLANVKCSELLKKWLPKRCTSFATKMOKEIHNIKGMGGDR (N-terminal to C-terminal, SEQ ID NO: 49; wherein the 1st-16th amino acid in SEQ ID NO: 49 is a signal peptide, which is numbered as SEQ ID NO: 50; the 17th-185th amino acid in SEQ ID NO: 49 is a wild-type Maluc (Maluc WT) without signal peptide, which is numbered as SEQ ID NO: 51), a plasmid (pCold-Maluc-NO SP, FIG. 7) 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-V48Q: the amino acid sequence has the following mutation compared with the wild-type Maluc (Maluc WT, SEQ ID NO: 51) without signal peptide: V48Q; Maluc-K62A: the amino acid sequence has the following mutation compared with the wild-type Maluc (Maluc WT, SEQ ID NO: 51) without signal peptide: K62A; Maluc-Y72W: the amino acid sequence has the following mutation compared with the wild-type Maluc (Maluc WT, SEQ ID NO: 51) without signal peptide: Y72W; Maluc-I73L: the amino acid sequence has the following mutation compared with the wild-type Maluc (Maluc WT, SEQ ID NO: 51) without signal peptide: I73L.

[0278] The amino acid sequence of the wild-type Maluc is:

[0279] EIQVLFALICFALVQANPTENKDDIDIVGVEGKFGTTDLETDLFTIVEDMNVISRDTNLANSDADRGKMPGKKLPLEVLIEMEANARKAGCTRGCLICLSKIKCTAKMKVYIPGRCHDYGGDKKTGQAGIVGAIVDIPEISGFKELGPMEQFIAQVDLCADCTTGCLKGLANVKCSALLKKWLPDRCASFADKIQSEVDNIKGLAGDR (N-terminal to C-terminal, SEQ ID NO: 52; wherein, the 1st to 16th amino acids in SEQ ID NO: 52 are a signal peptide, which is numbered as SEQ ID NO: 53; the 17th to 208th amino acids in SEQ ID NO: 52 are a wild-type Mpluc without signal peptide (Mpluc WT), which is numbered as SEQ ID NO: 54), a plasmid (pCold-Mpluc-NO SP, FIG. 8) expressing the wild-type Mpluc without 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 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 signal peptide were as follows: Mpluc-R71Q: the amino acid sequence has the following mutation compared to the wild-type Mpluc without signal peptide (Mpluc WT, SEQ ID NO: 54): R71Q; Mpluc-K85A: the amino acid sequence has the following mutation compared to the wild-type Mpluc without signal peptide (Mpluc WT, SEQ ID NO: 54): K85A; Mpluc-Y95W: the amino acid sequence has the following mutation compared to the wild-type Mpluc without signal peptide (Mpluc WT, SEQ ID NO: 54): Y95W; Mpluc-I96L: the amino acid sequence has the following mutation compared to the wild-type Mpluc without signal peptide (Mpluc WT, SEQ ID NO: 54): I96L.

[0280] The amino acid sequence of the wild-type Mluc7 is:

[0281] DIKFIFALVCIALVQANPTVNNDVNRGKMPGKKLPLEVLIEMEANAFKAGCTRGCLICLSKIKCTAKMKQYIPGRCHDYGGDKKTGQAGIVGAIVDIPEISGFKEMEPMEQFIAQVDLCADCTTGCLKGLANVKCSELLKKWLPDRCASFADKIOKEAHNIKGLAGDR (N-terminal to C-terminal, SEQ ID NO: 55; wherein, the 1st to 16th amino acids in SEQ ID NO: 55 are a signal peptide, which is numbered as SEQ ID NO: 56; the 17th to 168th amino acids in SEQ ID NO: 55 are a wild-type Mluc7 without signal peptide (Mluc7 WT), which is numbered as SEQ ID NO: 57), a plasmid (pCold-Mluc7-NO SP, FIG. 9) expressing the wild-type Mluc7 without signal peptide (Mluc7 WT) C-terminally fused with a His tag (6 histidines, 6xHis, for facilitating protein purification) was constructed by a whole gene synthesis method (the coding gene of the wild-type Mluc7 without signal peptide C-terminally fused with a His tag was inserted between Ndel and EcoRI of the base plasmid pCold II). The Mluc7 mutants without signal peptide were as follows: Mluc7-F31Q: the amino acid sequence has the following mutation compared to the wild-type Mluc7 without signal peptide (Mluc7 WT, SEQ ID NO: 57): F31Q; Mluc7-K45A: the amino acid sequence has the following mutation compared to the wild-type Mluc7 without signal peptide (Mluc7 WT, SEQ ID NO: 57): K45A; Mluc7-Y55W: the amino acid sequence has the following mutation compared to the wild-type Mluc7 without signal peptide (Mluc7 WT, SEQ ID NO: 57): Y55W; Mluc7-I56L: the amino acid sequence has the following mutation compared to the wild-type Mluc7 without signal peptide (Mluc7 WT, SEQ ID NO: 57): I56L.

[0282] 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.

[0283] Example 4 Activity detection of wild-type copepod luciferase or its mutants

[0284] The luminescent intensity and luminescent mode of the purified amphipod luciferase or its mutants obtained in Example 3 were detected respectively, and the detection method was the same as that in Example 2. The results are shown in Figures 10, 11 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 reduced, 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 reduced, 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 reduced, and the luminescent intensity of some Mluc7 mutants is improved.

[0285] Table 5 Luminescent intensity and half-life that can be produced by wild type amphipod luciferase or its mutants

[0286] The above results are consistent with the results of Gaussia luciferase, which shows that Gluc has an important site for influencing flash luminescence, and has a similar effect on the rest of the amphipod luciferase.

[0287] Example 5 Test of SA coupling with Gaussia luciferase mutants

[0288] 1. Coupling method:

[0289] (1) The amount of each component reagent was weighed according to the following Table 6 (100 ml of buffer solution A required for coupling was prepared) (10x Biomix A) (after all components were added, the pH was adjusted to 8.3).

[0290] Table 6

[0291] (2) The amount of each component reagent was weighed according to the following Table 7 (100 ml of buffer solution B required for coupling was prepared) (10x Biomix B) (after all components were added, the pH was adjusted to 8.3).

[0292] Table 7

[0293] (3) The catalytic reaction system was prepared according to the following Table 8, and after mixing uniformly, it was placed at 25°C for 2.5h.

[0294] Table 8 BirA enzyme catalytic reaction system

[0295] (4) After the reaction was completed, 1x PBS buffer was used for dialysis to remove excess biotin in the reaction system, and dialysis was performed for 3 times, 30 min each time.

[0296] (5) According to the molar ratio of Gluc to SA of 1:5, an excess of SA is added to ensure that one molecule of Gluc is combined with one molecule of SA tetramer to form the correct SA-Gluc.

[0297] (6) The SA-Gluc after coupling is detected by 12% SDS-PAGE.

[0298] 2. Coupling product detection

[0299] In this example, six luciferases are selected for SA coupling (WT-NS and Gluc-NO SP-1~SP-5), and it is found that there is a clear SA-Gluc band at about 75 kDa (as shown in Figure 12), indicating that the coupling is successful; the luciferase protein-SA coupling product is purified according to the luciferase purification method (same as Example 1), and the purified coupling product is obtained, and its purity is characterized by 12% SDS-PAGE, and the results are shown in Figure 13.

[0300] 3. Luminescence intensity and half-life test of coupling complex

[0301] The luciferase-SA coupling complex after step 2 is tested for luminescence intensity and half-life (test method same as Example 2), and the results are shown in Figures 14-15. Figure 14 is the luciferase-SA coupling complex catalyzing coelenterazine luminescence half-life test; Figure 15 is the luciferase-SA coupling complex catalyzing coelenterazine luminescence intensity test. From the above results, it can be seen that the luminescence intensity and half-life of the luciferase-SA coupling complex are similar to those of the luciferase monomer protein (compared with Figures 5-6), indicating that the SA coupling experiment does not affect the luminescence intensity and half-life of the luciferase, and can be followed by large-scale coupling.

[0302] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application are equivalent replacement methods, and are all included in the protection scope of the present application.

Claims

1. A mutant of a copepod luciferase, the mutant being (al) or (a2): (al) the amino acid sequence of the mutant comprises a mutation at one or more of the following positions: 23, 31, 45, 48, 55, 56, 62, 67, 71, 72, 73, 79, 81, 85, 89, 93, 95, 96, 102, 121, 135, 146, 161, 165, 168, compared to the amino acid sequence of wild-type copepod 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 copepod luciferase comprises at least one of Gaussia luciferase, Maluc, Mpluc, and Mluc7; preferably, the copepod luciferase is Gaussia luciferase; 2. The mutant according to claim 1, wherein: preferably, the mutant comprises any one of the mutations of (bl) to (bl8) or a combination of multiple mutations of (bl) to (bl8), compared to the amino acid sequence of wild-type Gaussia luciferase with the signal peptide removed: (bl) L at position 23 is mutated to A; (b2) R at position 48 is mutated to Q; (b3) H at position 62 is mutated to A, or K; (b4) P at position 67 is mutated to A; (b5) F at position 72 is mutated to W; (b6) I at position 73 is mutated to L; (b7) T at position 79 is mutated to D, or S; (b8) E at position 81 is mutated to G; (b9) E at position 85 is mutated to K, D, or S; (blO) G at position 89 is mutated to M; (bl l) E at position 93 is mutated to G, S, or A; (bl2) P at position 102 is mutated to S; (bl3) V at position 121 is mutated to A; (bl4) Q at position 135 is mutated to K; (bl5) Q at position 146 is mutated to S; (bl6) K at position 161 is mutated to T, or E; (bl7) A at position 165 is mutated to L; (bl8) D at position 168 is mutated to S; preferably, the mutant has any one of the mutations of (cl) to (c28), compared to the amino acid sequence of wild-type Gaussia luciferase with the signal peptide removed: (cl) L at position 23 is mutated to A; (c2) R at position 48 is mutated to Q; (c3) H at position 62 is mutated to A, or K; (c4) P at position 67 is mutated to A; (c5) F at position 72 is mutated to W; (c6) I at position 73 is mutated to L; (c7) T at position 79 is mutated to D, or S; (c8) E at position 81 is mutated to G; (c9) E at position 85 is mutated to K, D, or S; (clO) G at position 89 is mutated to M; (cl l) E at position 93 is mutated to G, S, or A; (cl2) P at position 102 is mutated to S; (cl3) V at position 121 is mutated to A; (cl4) Q at position 135 is mutated to K; (cl5) Q at position 146 is mutated to S; (cl6) K at position 161 is mutated to T, or E; (cl7) A at position 165 is mutated to L; (cl8) D at position 168 is mutated to S; ​ ​ ​ (c1) I73L; (c2) H62A; (c3) T79D; (c4) T79S; (c5) E93G; (c6) F72W; (c7) E93S; (c8) K161T; (c9) R48Q; (c10) H62K; (c11) E81G; (c12) K161E; (c13) A165L; (c14) E93A; (c15) E85K; (c16) L23A; (c17) Q146S; (c18) D168S; (c19) Q135K; (c20) P102S; (c21) H62A / I73L / E85S / S86T / A87G / G89M / E93P / L107M / V121E; (c22) H62A / I73L / E85S / S86T / A87G / G89M / E93P / P102S / L107M / V121E; (c23) H62A / P67A / I73L / T79D / E85D / S86T / A87G / E93G / L107M / V121E; (c24) H62A / P67A / I73L / T79D / E85D / S86T / A87G / P102S / L107M / V121E; (c25) H62A / P67A / I73L / E85D / S86T / A87G / V121E; (c26) H62A / E81G / A87G / G89M / E93P / L107M / V121E; (c27) H62A / P67L / E81G / A87G / G89M / E93P / P102S / L107M / V121E; (c28) H62A / P67L / E81G / S86T / A87G / G89M / E93P / P102S / L107M / V121E; Preferably, the mutant has any one of the (c1), (c5), (c6), (c9), (c11), (c12), (c14) to (c17), (c19) to (c28) mutations compared with the amino acid sequence of the wild-type Gaussia luciferase without signal peptide; Preferably, the mutant has any one of the (c1), (c5), (c11), (c21) to (c28) mutations compared with the amino acid sequence of the wild-type Gaussia luciferase without signal peptide; Preferably, the amino acid sequence of the wild-type Gaussia luciferase without signal peptide is shown as 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 Copepoda luciferase comprises at least one of Gaussia luciferase, Maluc, Mpluc, and Mluc7; Preferably, the Copepoda luciferase is Maluc; Preferably, the amino acid sequence of the mutant comprises a mutation at one or more of the following positions: 48th, 62nd, 72nd, or 73rd, compared with the amino acid sequence of the wild-type Maluc without signal peptide; Preferably, the mutant comprises any one mutation or a combination of multiple mutations of V at position 48 mutated to Q, K at position 62 mutated to A, Y at position 72 mutated to W, or I at position 73 mutated to L, compared to the amino acid sequence of wild-type Maluc without signal peptide; Preferably, the copepod luciferase is Mpluc; Preferably, the amino acid sequence of the mutant comprises a mutation at one or more of positions 71, 85, 95, or 96, compared to the amino acid sequence of wild-type Mpluc without signal peptide; Preferably, the mutant comprises any one mutation or a combination of multiple mutations of R at position 71 mutated to Q, K at position 85 mutated to A, Y at position 95 mutated to W, or I at position 96 mutated to L, compared to the amino acid sequence of wild-type Mpluc without signal peptide; Preferably, the copepod luciferase is Mluc7; Preferably, the amino acid sequence of the mutant comprises a mutation at one or more of positions 31, 45, 55, or 56, compared to the amino acid sequence of wild-type Mluc7 without signal peptide; Preferably, the mutant comprises any one mutation or a combination of multiple mutations of F at position 31 mutated to Q, K at position 45 mutated to A, Y at position 55 mutated to W, or I at position 56 mutated to L, compared to the amino acid sequence of wild-type Mluc7 without signal peptide.

4. A recombinant protein comprising a modified moiety and the copepod luciferase mutant of any one of claims 1-3; Preferably, the modified 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 flash luciferase and a glow luciferase, the flash luciferase comprising the copepod luciferase mutant of any one of claims 1-3, and / or the recombinant protein of claim 4.

6. A biological material related to the copepod luciferase mutant of any one of claims 1-3, the recombinant protein of claim 4, or the fusion protein of claim 5, the biological material comprising any one of d1)-d12): d1) a nucleic acid molecule encoding the copepod luciferase mutant of any one of claims 1-3, 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); 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), d4) comprises a promoter operably linked to the nucleic acid molecule. Preferably, the vector in d3), d4) is independently selected from the group consisting of 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 conjugate comprising (a) and (b): (a) a conjugation or conjugation moiety; (b) at least one of the mutant amphipod luciferase of any one of claims 1 to 3, the recombinant protein of claim 4, or the fusion protein of claim 5; The conjugation or conjugation moiety comprises at least one of a small molecule compound, a biological macromolecule.

8. The conjugate or conjugate of claim 7, wherein: The conjugation or conjugation 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-phosphoryl group.

9. A nucleic acid sequencing kit comprising any one of i1) to i4): i1) the mutant amphipod luciferase of any one of claims 1 to 3; i2) the recombinant protein of claim 4; i3) the fusion protein of claim 5; i4) The conjugate or conjugate of any one of claims 7-8, wherein, The conjugation or conjugation moiety comprises at least one of avidin, digoxin antibody, nucleotide antibody, nucleotide.

10. The nucleic acid sequencing kit of claim 9, wherein: The nucleotide is the nucleotide described in claim 8; Preferably, the nucleic acid sequencing kit further comprises at least one of a substrate of the amphipod luciferase, a polymerase, a PCR buffer; Preferably, the substrate of the amphipod luciferase comprises at least one of coelenterazine, a coelenterazine derivative; Preferably, when the nucleic acid sequencing kit comprises i3), the nucleic acid sequencing kit further comprises a substrate of the glow luciferase; Preferably, when the conjugation or conjugation moiety is avidin, digoxin antibody, or nucleotide antibody, the nucleic acid sequencing kit further comprises a nucleotide.

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

12. A nucleic acid sequencing method, comprising the following steps: (1) monitoring the sequential incorporation of nucleotides in a complementary strand of a nucleic acid to be detected, wherein each of the nucleotides is attached to a luminescent label that initiates different luminescence kinetics or luminescence types; (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 participates; the incorporated nucleotide carries a blocking modification group; the luminescent label comprises the amphioxin luciferase mutant of any one of claims 1-3; Preferably, the luminescent label comprises the amphioxin luciferase mutant of any one of claims 1-3 and a second luminescent label that initiates different luminescence kinetics or luminescence types or light intensity from the amphioxin luciferase mutant; Preferably, the nucleic acid sequencing method further comprises the following step: (3) removing the blocking modification group carried by the incorporated nucleotide so that the complementary strand can continue to extend, and removing the luminescent label; Preferably, the nucleic acid sequencing method further comprises the following step: repeating steps (1) to (3) until the complementary strand is completely extended to determine the sequence of the nucleic acid to be detected.

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

14. A method for detecting target molecules, comprising the step of detecting using the kit for detecting target molecules of claim 11.

15. The method of claim 14, wherein: the method for detecting target molecules comprises the following steps: contacting j4) in the kit for detecting target molecules of claim 11 with a sample to be detected, then adding a substrate of luciferase, and determining the presence or absence or content of the target molecules according to the fluorescent signal emitted by the reaction of the luciferase with the substrate of luciferase; the substrate of luciferase comprises the substrate of the amphioxin luciferase; Preferably, the substrate of the amphioxin luciferase comprises at least one of coelenterazine, a coelenterazine derivative.

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

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

18. Use of the copepod luciferase mutant of any one of claims 1-3, the recombinant protein of claim 4, the fusion protein of claim 5, and / or the biomaterial of claim 6 in the manufacture and / or as a flash-type luciferase.

19. Use of the copepod luciferase mutant of any one of claims 1-3, the recombinant protein of claim 4, the fusion protein of claim 5, the biomaterial of claim 6, and / or the conjugate or the complex of any one of claims 7-8 in k1) to 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) to k6); Preferably, when the product is for k1), the product further comprises a glow-type luciferase.

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