Novel copepod luciferase mutant and application thereof

Abstract

The invention relates to a novel copepod luciferase mutant and application thereof. The luciferase mutant takes an amino acid sequence of wild type Gaussian luciferase or an amino acid sequence of luciferase having at least 70% homology with the Gaussian luciferase SEQ ID NO: 3 as a reference, and compared with the amino acid sequence shown as SEQ ID NO: 3, the amino acid sequence of the luciferase mutant has the advantages that the amino acid sequence of the wild type Gaussian luciferase mutant is more than the amino acid sequence shown as SEQ ID NO: 3; at least one of the following positions is mutated: a tenth position, a 12th position, a 19th position, a 20th position, a 21st position, a 22nd position, a 23rd position, a 35th position, a 44th position, a 45th position, a 59th position, a 62nd position, a 67th position, a 78th position, a 85th position, a 86th position, a 87th position, a 93th position, a 107th position, a 144th position and a 121th position.

Description

Novel Rectpoda Luciferase Mutants and Their Applications Technical Field

[0001] This invention relates to the field of biotechnology, specifically to luciferase mutants and their applications, and particularly to copepod luciferase mutants and their applications. Background Technology

[0002] Bioluminescence is a widespread phenomenon in nature, primarily found in organisms such as insects, bacteria, fungi, and marine life. To date, more than 40 bioluminescent systems have been discovered in nature, but only a dozen or so luciferases and related substrates, such as luciferin, have been elucidated. Generally, bioluminescent reactions require the presence of luciferase, substrates (luciferin and analogues), and oxygen atoms (molecular oxygen, etc.).

[0003] Luciferases are a class of enzymes that catalyze the oxidation of luciferin or fatty aldehydes to produce light. Widely found in insects, bacteria, fungi, and marine organisms, they have become an important tool in scientific research, widely used in life sciences, genome sequencing and analysis, clinical medicine and forensic testing, drug screening, environmental monitoring, and enzyme-linked immunosorbent assays (ELISA). Utilizing their self-luminescent properties, luciferases are commonly used in live cell detection, protein-protein interaction analysis, protein localization, small interfering RNA silencing, and high-throughput drug screening. In the field of biomonitoring, luciferases can be used to detect the presence of chemical pollutants. Furthermore, they have broad application prospects in immunoassay and biochemical diagnostics. As reporter genes for detecting the expression intensity of exogenous genes under different promoters and for studying transcriptional regulation, multiple luciferases with similar self-luminescence brightness are needed, allowing for combined use with luciferases that catalyze different substrates.

[0004] Currently, the luciferases with relatively good research and development mainly include: firefly luciferase (FLuc), bacterial luciferase (Lux), and luciferases extracted from sea kidney (Renilla luciferase, RLuc), deep-sea shrimp (Oplophorus luciferase, OLuc), and marine animals such as Gaussia princeps (Gaussia luciferase, GLuc). Among these, FLuc requires ATP, O2, and Mg. 2+ The expression of Lux is non-secretory and requires cofactors such as flavin mononucleotide (FMN), long-chain fatty aldehyde, oxygen, and reduced nicotinamide adenine dinucleotide (NADH). Rluc does not require ATP for luminescence, but its fluorescence intensity is weak and it is also not secretory.

[0005] Gaussian luciferase (Gluc) effectively overcomes the shortcomings of firefly luciferase and Renilla luciferase, as it does not require ATP and Mg for luminescence. 2+ Cofactors are also involved. Gaussian luciferase is a luciferase secreted by a marine copepod. It is one of the smallest luciferases discovered to date and possesses a signal peptide, allowing it to be secreted extracellularly for easy activity detection. In the presence of oxygen, Gaussian luciferase spontaneously catalyzes the oxidation of the substrate coelenterate to produce luminescence. However, it currently faces bottlenecks in achieving high brightness and large-scale production.

[0006] Summary of the Invention

[0007] The present invention aims to solve at least one of the technical problems existing in the prior art.

[0008] To enhance the activity of Gaussian luciferase towards its substrate coelenterate (CTZ) and increase its expression yield, the inventors conducted directed protein evolution on Gaussian luciferase Gluc to obtain a mutant Gaussian luciferase with the highest catalytic activity towards CTZ, exceeding that of the wild-type Gaussian luciferase by more than nine times. Furthermore, this mutant luciferase exhibits low molecular weight, high stability, and can be expressed in both prokaryotes and eukaryotes. Its purification and detection methods are simple, facilitating large-scale production. It shows broad application prospects in immunoassay, biochemical diagnostics, and sequencing applications.

[0009] Therefore, in one aspect of the present invention, a luciferase mutant is provided. According to an embodiment of the present invention, the luciferase mutant refers to a luciferase mutant having a mutation at at least one of the following positions, with reference to the amino acid sequence of wild-type Gaussian luciferase or the amino acid sequence of a luciferase having at least 70% homology with said Gaussian luciferase:

[0010] Positions 10, 12, 19, 20, 21, 22, 23, 35, 44, 45, 59, 62, 67, 78, 79, 85, 86, 87, 93, 107, 114, and 121. It should be noted that the "luciferase with at least 70% homology to the Gaussian luciferase" mentioned in this application refers to a luciferase that is highly conserved and homologous to wild-type Gaussian luciferase during evolution, and the amino acid positions in the amino acid sequence of this luciferase are located with reference to the amino acid positions in the amino acid sequence of wild-type luciferase. The Gaussian luciferase mutant according to embodiments of the present invention exhibits higher catalytic activity for substrates (coelenterin and its analogues) and can be expressed in soluble form in prokaryotes or eukaryotes for large-scale production.

[0011] According to embodiments of the present invention, the above-mentioned luciferase mutant may further include at least one of the following additional technical features:

[0012] According to an embodiment of the present invention, the wild-type Gaussian luciferase without a signal peptide has the amino acid sequence shown in SEQ ID NO:3:

[0013] According to an embodiment of the present invention, the Gaussian luciferase having at least 70% homology is a copepod luciferase.

[0014] According to embodiments of the present invention, the copepod luciferase comprises Mluc, Maluc, Pxluc, and Mpluc. At least one of them. The inventors discovered that copepod luciferases are a highly conserved class of luciferases, and that mutations in the activity-related sites on Gluc have a similar activity-enhancing effect on other copepod luciferases such as Mluc, Maluc, Pxluc, and Mpluc.

[0015] According to an embodiment of the present invention, the mutant luciferase mutant, compared to the amino acid sequence shown in SEQ ID NO:3, has any one or more combinations of the following (1)-(22) mutations:

[0016] (1) The N at the 10th position mutates to S;

[0017] (2) The V at position 12 mutates to A or S;

[0018] (3) The A at position 19 mutates to V;

[0019] (4) The T mutation at position 20 is replaced by V or A;

[0020] (5) The T at position 21 mutates to A;

[0021] (6) The D at position 22 mutates into E or G;

[0022] (7) The L at position 23 mutates to A, T, or I;

[0023] (8) The L at position 35 mutates to F;

[0024] (9) The E at position 44 mutates into G;

[0025] (10) The A at position 45 mutates to V;

[0026] (11) The C at position 59 mutates to S;

[0027] (12) The H at position 62 mutates to K, Q, or N;

[0028] (13) The P at position 67 is mutated to L, K, or A;

[0029] (14) The H at position 78 mutates to A;

[0030] (15) The T mutation at position 79 is changed to H, K, or P;

[0031] (16) The E at position 85 mutates to S or D;

[0032] (17) The S at position 86 is mutated to T or I;

[0033] (18) The A at position 87 mutates into G;

[0034] (19) The E at position 93 mutates into P, A, S, or T;

[0035] (20) The L at position 107 mutates to M;

[0036] (21) The I at position 114 mutates to M;

[0037] (22) The V at position 121 is mutated to D or E.

[0038] Different mutation sites have different activities for substrates (coelenterin and its analogues), which can provide more options for actual production needs.

[0039] According to an embodiment of the present invention, the luciferase mutant has the following mutations compared to the amino acid sequence shown in SEQ ID NO:3:

[0040] (1) The H at position 62 mutates to K, the P at position 67 mutates to L, the H at position 78 mutates to A, the E at position 85 mutates to S, the S at position 86 mutates to T, the A at position 87 mutates to G, the E at position 93 mutates to S, the L at position 107 mutates to M, and the V at position 121 mutates to E; or

[0041] (2) The H at position 62 mutates to K, the P at position 67 mutates to L, the T at position 79 mutates to P, the E at position 85 mutates to S, the S at position 86 mutates to T, the A at position 87 mutates to G, the E at position 93 mutates to S, the L at position 107 mutates to M, and the V at position 121 mutates to E; or

[0042] (3) The 10th N mutates to S, and the 12th V mutates to A; or

[0043] (4) The V at position 12 mutates to A, and the T at position 79 mutates to H; or

[0044] (5) The V mutation at position 12 is changed to A, and the T mutation at position 79 is changed to K; or

[0045] (6) The N at position 10 mutates to S, and the V at position 12 mutates to S; or

[0046] (7) The V at position 12 mutates to A, the H at position 62 mutates to K, the P at position 67 mutates to L, the E at position 85 mutates to S, the S at position 86 mutates to T, the A at position 87 mutates to G, the E at position 93 mutates to P, the L at position 107 mutates to M, and the V at position 121 mutates to E; or

[0047] (8) The T mutation at position 20 is changed to A, and the I mutation at position 114 is changed to M; or

[0048] (9) The L at position 23 mutates to T, the H at position 62 mutates to K, the P at position 67 mutates to L, the E at position 85 mutates to S, the S at position 86 mutates to T, the A at position 87 mutates to G, the E at position 93 mutates to T, the L at position 107 mutates to M, and the V at position 121 mutates to E; or

[0049] (10) The H at position 62 mutates to K, the E at position 85 mutates to D, the S at position 86 mutates to T, the A at position 87 mutates to G, the L at position 107 mutates to M, and the V at position 121 mutates to E; or

[0050] (11) The H at position 62 mutates to K, the P at position 67 mutates to A, the E at position 85 mutates to D, the S at position 86 mutates to T, the A at position 87 mutates to G, and the L at position 107 mutates to M; or

[0051] (12) The H at position 62 mutates to K, the P at position 67 mutates to A, the E at position 85 mutates to D, the S at position 86 mutates to T, the A at position 87 mutates to G, and the V at position 121 mutates to E; or

[0052] (13) The H at position 62 mutates to K, the P at position 67 mutates to K, the E at position 85 mutates to D, the S at position 86 mutates to T, the A at position 87 mutates to G, the L at position 107 mutates to M, and the V at position 121 mutates to E; or

[0053] (14) The H at position 62 mutates to K, the P at position 67 mutates to A, the E at position 85 mutates to D, the S at position 86 mutates to T, the A at position 87 mutates to G, the E at position 93 mutates to A, the L at position 107 mutates to M, and the V at position 121 mutates to E; or

[0054] (15) The H at position 62 is mutated to K, the P at position 67 is mutated to A, the E at position 85 is mutated to D, the S at position 86 is mutated to I, the A at position 87 is mutated to G, the L at position 107 is mutated to M, and the V at position 121 is mutated to E. or

[0055] (16) The P at position 67 mutates to A, the E at position 85 mutates to D, the S at position 86 mutates to T, the A at position 87 mutates to G, the L at position 107 mutates to M, and the V at position 121 mutates to E; or

[0056] (17) The H at position 62 mutates to Q, the E at position 85 mutates to S, the S at position 86 mutates to T, the A at position 87 mutates to G, the E at position 93 mutates to A, the L at position 107 mutates to M, and the V at position 121 mutates to E; or

[0057] (18) The L at position 23 mutates to A, the H at position 62 mutates to K, the P at position 67 mutates to L, the E at position 85 mutates to S, the S at position 86 mutates to T, the A at position 87 mutates to G, the E at position 93 mutates to S, the L at position 107 mutates to M, and the V at position 121 mutates to E; or

[0058] (19) The H at position 62 mutates to K, the P at position 67 mutates to A, the E at position 85 mutates to D, the S at position 86 mutates to T, the A at position 87 mutates to G, the L at position 107 mutates to M, and the V at position 121 mutates to D; or

[0059] (20) The H at position 62 mutates to K, the P at position 67 mutates to A, the E at position 85 mutates to D, the S at position 86 mutates to T, the A at position 87 mutates to G, and the L at position 107 mutates to M; or

[0060] (21) The T mutation at position 20 becomes A, the H mutation at position 62 becomes K, the P mutation at position 67 becomes A, the E mutation at position 85 becomes D, the S mutation at position 86 becomes T, the A mutation at position 87 becomes G, the L mutation at position 107 becomes M, and the V mutation at position 121 becomes E; or

[0061] (22) The T mutation at position 21 becomes A, the H mutation at position 62 becomes K, the P mutation at position 67 becomes K, the E mutation at position 85 becomes D, the S mutation at position 86 becomes T, the A mutation at position 87 becomes G, the L mutation at position 107 becomes M, and the V mutation at position 121 becomes E; or

[0062] (23) The H at position 62 mutates to K, the P at position 67 mutates to K, the E at position 85 mutates to D, the S at position 86 mutates to T, the A at position 87 mutates to G, the L at position 107 mutates to M, and the V at position 121 mutates to D; or

[0063] (24) The T at position 20 mutates to V, the T at position 21 mutates to A, the D at position 22 mutates to E, the H at position 62 mutates to K, the P at position 67 mutates to A, the E at position 85 mutates to D, the S at position 86 mutates to T, the A at position 87 mutates to G, the L at position 107 mutates to M, and the V at position 121 mutates to D; or

[0064] (25) The T mutation at position 21 becomes A, the H mutation at position 62 becomes K, the P mutation at position 67 becomes A, the E mutation at position 85 becomes D, the S mutation at position 86 becomes T, the A mutation at position 87 becomes G, the L mutation at position 107 becomes M, and the V mutation at position 121 becomes E; or

[0065] (26) The T mutation at position 20 becomes V, the T mutation at position 21 becomes A, the D mutation at position 22 becomes G, the H mutation at position 62 becomes K, the P mutation at position 67 becomes A, the T mutation at position 79 becomes P, and the E mutation at position 85 becomes D. The S at position 86 mutates to I, the A at position 87 mutates to G, the L at position 107 mutates to M, and the V at position 121 mutates to E; or

[0066] (27) The H at position 62 mutates to K, the P at position 67 mutates to A, the E at position 85 mutates to D, the S at position 86 mutates to I, the A at position 87 mutates to G, the L at position 107 mutates to M, and the V at position 121 mutates to D; or

[0067] (28) The E at position 44 mutates to G, the H at position 62 mutates to N, the P at position 67 mutates to A, the E at position 85 mutates to D, the S at position 86 mutates to T, the A at position 87 mutates to G, the L at position 107 mutates to M, and the V at position 121 mutates to D; or

[0068] (29) The T mutation at position 21 is changed to A, the P mutation at position 67 is changed to A, the E mutation at position 85 is changed to D, the S mutation at position 86 is changed to T, the A mutation at position 87 is changed to G, the L mutation at position 107 is changed to M, and the V mutation at position 121 is changed to D; or

[0069] (30) The P at position 67 mutates to A, the E at position 85 mutates to D, the S at position 86 mutates to T, the A at position 87 mutates to G, the L at position 107 mutates to M, and the V at position 121 mutates to D; or

[0070] (31) The H at position 62 mutates to Q, the E at position 85 mutates to D, the S at position 86 mutates to T, the A at position 87 mutates to G, the L at position 107 mutates to M, and the V at position 121 mutates to E; or

[0071] (32) The V at position 12 mutates to A, the T at position 21 mutates to A, the D at position 22 mutates to G, the L at position 35 mutates to F, the A at position 45 mutates to V, the C at position 59 mutates to S, the H at position 62 mutates to K, the P at position 67 mutates to L, the E at position 85 mutates to D, the S at position 86 mutates to T, the A at position 87 mutates to G, the E at position 93 mutates to S, the L at position 107 mutates to M, and the V at position 121 mutates to E; or

[0072] (33) The V at position 12 mutates to A, the T at position 20 mutates to A, the T at position 21 mutates to A, the D at position 22 mutates to G, the L at position 35 mutates to F, the A at position 45 mutates to V, the C at position 59 mutates to S, the H at position 62 mutates to K, the P at position 67 mutates to L, the E at position 85 mutates to D, the S at position 86 mutates to T, the A at position 87 mutates to G, the E at position 93 mutates to S, the L at position 107 mutates to M, and the V at position 121 mutates to E.

[0073] (34) The V at position 12 mutates to A, the A at position 19 mutates to V, the T at position 20 mutates to A, the T at position 21 mutates to A, the D at position 22 mutates to G, the L at position 23 mutates to I, the L at position 35 mutates to F, the C at position 59 mutates to S, the H at position 62 mutates to K, the P at position 67 mutates to L, the E at position 85 mutates to D, the S at position 86 mutates to T, the A at position 87 mutates to G, the E at position 93 mutates to S, the L at position 107 mutates to M, and the V at position 121 mutates to E.

[0074] (35) The T at position 21 mutates to A, the H at position 62 mutates to K, the P at position 67 mutates to L, the E at position 85 mutates to D, the S at position 86 mutates to T, the A at position 87 mutates to G, and the E at position 93 mutates to P. The L at position 107 mutates to M, and the V at position 121 mutates to E.

[0075] According to some specific embodiments of the present invention, when the amino acid sequence shown in SEQ ID NO:3 has the above-mentioned mutation, the obtained protein has strong catalytic activity for substrates such as coelenterate and its luminescence brightness is significantly enhanced compared to existing Gaussian luciferase.

[0076] According to an embodiment of the present invention, the luciferase mutant further includes a signal peptide disposed at the N-terminus of the luciferase mutant. This signal peptide is used to guide the secretion of the luciferase mutant from the expressing cell.

[0077] According to an embodiment of the present invention, the mutant luciferase further includes a polyhistidine sequence disposed at the C-terminus of the luciferase mutant. This is used for affinity purification of the aforementioned mutant luciferase mutant.

[0078] In a second aspect, the present invention provides a nucleic acid molecule. According to embodiments of the present invention, the nucleic acid molecule encodes the luciferase mutant described in the first aspect of the present invention.

[0079] In a third aspect, the present invention provides an expression vector. According to embodiments of the present invention, the expression vector comprises the nucleic acid molecule described in the second aspect of the present invention.

[0080] According to an embodiment of the present invention, the expression vector further includes a promoter.

[0081] According to an embodiment of the present invention, the promoter is operatively linked to the nucleic acid molecule.

[0082] In a fourth aspect, the present invention provides a recombinant cell. According to embodiments of the present invention, the recombinant cell carries the nucleic acid molecule described in the second aspect of the present invention and the expression vector described in the third aspect of the present invention. It then expresses or secretes the luciferase mutant described in the first aspect of the present invention.

[0083] According to an embodiment of the present invention, the recombinant cells are selected from Escherichia coli, yeast, or mammalian cells.

[0084] In a fifth aspect, the present invention provides a method for obtaining luciferase. According to an embodiment of the invention, the recombinant cells described in the fourth aspect of the invention are cultured under conditions suitable for protein expression to obtain the luciferase.

[0085] In a sixth aspect, the present invention provides a conjugate. According to embodiments of the present invention, the conjugate comprises the luciferase mutant described in the first aspect of the present invention and a small molecule compound or macromolecule, wherein the luciferase mutant and the small molecule compound or macromolecule are coupled by chemical bonds.

[0086] According to embodiments of the present invention, the small molecule compound or macromolecule includes streptavidin (SA), digoxin, antibodies, dNTPs or dNTP analogs, etc.

[0087] In a seventh aspect of the invention, a method for nucleic acid sequencing is provided. According to an embodiment of the invention, the sequencing method includes: using the nucleic acid to be tested as a template, sequentially adding a polymerase capable of binding the luciferase mutant described in the first aspect and / or the conjugate described in the sixth aspect and / or dNTPs or dNTP analogs or modified dNTPs to perform a polymerization reaction; determining the nucleic acid sequence of the nucleic acid to be tested based on the fluorescence signal emitted by the reaction of the luciferase mutant with a substrate or substrate analog of Gaussian luciferase. According to a specific embodiment of the invention, the method includes subsequently using the nucleic acid to be tested as a template... The plate is infused with various labeled dNTPs for polymerization. Then, a luciferase mutant or conjugate with different reactivity to the substrate is added to recognize the different labeled dNTPs. Finally, the substrate is added. Based on the various fluorescent signals emitted by the reaction of the luciferase mutant with the substrate or substrate analog of Gaussian luciferase, the nucleic acid sequence of the nucleic acid to be tested is finally obtained. The above polymerization and enzymatic fluorescence reactions can be repeated multiple times depending on the length of the sequencing template.

[0088] In an eighth aspect, the present invention provides a nucleic acid sequencing kit. According to embodiments of the present invention, the nucleic acid sequencing kit comprises the luciferase mutant described in the first aspect or the conjugate described in the sixth aspect.

[0089] According to embodiments of the present invention, the kit further comprises dNTPs or dNTP analogs, universal primers, PCR polymerase or coenzyme or coenzyme analogs.

[0090] In a ninth aspect of the invention, a method for detecting the content of an analyte is provided. According to an embodiment of the invention, the method for detecting the content of the analyte comprises: contacting an analyte incorporating a luciferase mutant as described in the first aspect of the invention or a conjugate as described in the sixth aspect of the invention with a substrate of Gaussian luciferase or an analogue of the substrate, and finally determining the content of the analyte based on the intensity of the fluorescence signal after the contact treatment.

[0091] According to embodiments of the present invention, the substrate or substrate analog of the Gaussian luciferase includes at least one selected from coenzyme and coenzyme derivatives.

[0092] According to embodiments of the present invention, the coelenterin derivative is as shown in formula (I) or formula (II), or a salt or isomer thereof.

[0093] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0094] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0095] Figure 1 shows the map of the wild-type Gaussian luciferase prokaryotic expression plasmid pCold-Gluc-WT-NS plasmid without the signal peptide;

[0096] Figure 2 shows the map of the wild-type Gaussian luciferase eukaryotic expression plasmid pEE12.4-Gluc WT containing the signal peptide;

[0097] Figure 3 shows the structure of the substrate coelenterin (CTZ) and its derivatives F-CTZ and ZS26;

[0098] Figure 4 shows the electrophoresis diagram of the purified protein from prokaryotic expression of the Gaussian luciferase mutant;

[0099] Figure 5 shows the results of the activity assay of the prokaryotically expressed Gaussian luciferase mutant on the substrate coelenterin protein level;

[0100] Figure 6 shows the electrophoresis diagram of purified proteins from eukaryotic expression of some Gaussian luciferase mutants;

[0101] Figure 7 shows the results of activity assays on the substrate coelenterin protein by partially eukaryotically expressed Gaussian luciferase mutants.

[0102] Figure 8 shows the electrophoresis diagram of some Gaussian luciferase mutant biotin-coupled proteins;

[0103] Figure 9 shows the protein level activity of some Gaussian luciferase mutant biotin-coupled proteins against the substrates CTZ, F-CTZ, and ZS26.

[0104] Figure 10 shows the signal values ​​of some Gaussian luciferase mutant biotin-coupled proteins on the sequencer DNBSEQ E5. Detailed Implementation

[0105] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0106] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0107] In basic scientific research, luciferase genes have been widely used as reporter genes in studies of the expression intensity and transcriptional regulation of exogenous genes under different promoters. In the field of biomonitoring technology, luciferase can be used to detect the presence or absence of chemical pollutants. Furthermore, it also has broad application prospects in immunoassay and biochemical diagnostics.

[0108] The luciferase mutant proposed in this invention, compared to the amino acid sequence shown in SEQ ID NO:1 or the amino acid sequence of a luciferase (i.e., a copepod luciferase) having at least 70% homology with the Gaussian luciferase, has, compared to the amino acid sequence shown in SEQ ID NO:3, any one or more combinations of the following mutation sites: positions 10, 12, 19, 20, 21, 22, 23, 35, 44, 45, 59, 62, 67, 78, 79, 85, 86, 87, 93, 107, 114, and 121, and may or may not include the signal peptide amino acid sequence.

[0109] According to one embodiment of the present invention, the amino acid sequence of wild-type Gaussian luciferase without a signal peptide is modified by a mutation site. The resulting mutant exhibits strong catalytic activity towards substrates such as coenzyme, fluorocoenzyme, and the coenzyme derivative ZS26, showing a broader substrate spectrum, higher specificity, and significantly enhanced luminescence brightness compared to existing Gaussian luciferases. In applications, the mutant can be used in basic scientific research, biodetection technology, immunoassay, biochemical detection, and diagnostics, demonstrating broad application prospects. Furthermore, it can be used in reporter genes and for the quantitative detection of DNA, RNA, and... It plays an important role in transcription factors, proteins, and cells. It can also serve as a luminescent signal protein in fusion proteins for the quantitative detection of target small molecules.

[0110] According to some specific embodiments of the present invention, the present invention provides a nucleic acid molecule that encodes the aforementioned mutant.

[0111] It should be noted that those skilled in the art should understand that the nucleic acids mentioned in this specification and claims actually include any one or both of the complementary double strands. For convenience, although only one strand is given in most cases in this specification and claims, the other complementary strand is actually disclosed as well. In addition, the nucleic acid sequences in this application include DNA or RNA forms; disclosure of one means that the other is also disclosed.

[0112] This invention proposes an expression vector comprising the aforementioned nucleic acid molecule. The type of expression vector is not particularly limited, as long as it can replicate and express the corresponding mutant in a host cell. The expression vector may include an optional control sequence operatively linked to the nucleic acid molecule. The control sequence is one or more control sequences that direct the expression of the nucleic acid molecule in the host. The expression vectors proposed in some specific embodiments of this invention can efficiently express proteins in suitable host cells. The obtained proteins exhibit strong catalytic activity towards substrates such as coelentrin, fluorocoelentrin, and coelentrin derivatives. Compared to existing Gaussian luciferases, they have a broader substrate spectrum, higher specificity, and significantly enhanced luminescence brightness. They can be used in basic scientific research, biodetection technology, immunoassay, biochemical detection, or diagnostics for the application of these proteins, demonstrating broad application prospects.

[0113] This invention proposes a recombinant cell carrying the aforementioned nucleic acid molecules, expression vectors, or mutants. The recombinant cell is obtained by transfecting or transforming the expression vector. According to some specific embodiments of the invention, the recombinant cell can efficiently express the aforementioned mutants under suitable conditions. These mutants exhibit strong catalytic activity towards substrates such as coelentrin, fluorocoelentrin, and coelentrin derivatives, and compared to existing Gaussian luciferases, they have a broader substrate spectrum, higher specificity, and significantly enhanced luminescence brightness. They can be used in basic scientific research, biodetection technology, immunoassay, biochemical detection, or diagnostics for the application of these proteins, demonstrating broad application prospects.

[0114] The present invention proposes a method for nucleic acid sequencing, which involves using the nucleic acid to be tested as a template, sequentially adding dNTPs conjugated with the aforementioned luciferase mutant or the aforementioned conjugate; and using the fluorescence signal and signal combination to distinguish the four bases A, T, G, and C for target nucleic acid sequencing, and finally obtaining the nucleic acid sequence to be tested.

[0115] Specifically, for ease of understanding, the technical solution of this application will be explained and described in detail below. A method for detecting nucleic acid sequences includes the following steps:

[0116] A) Using the mutant luciferase described in the first aspect, a first specific recognition protein is formed with the mutant luciferase through chemical coupling, bio-coupling, or fusion protein to form a first mutant luciferase complex; a second specific recognition protein is formed with the second luciferase to form a second luciferase complex;

[0117] B) The first mutant luciferase complex can react with the first substrate to generate a first luminescent signal; the second luminescent... The luciferase complex reacts with a second substrate to generate a second luminescent signal; the first mutant luciferase complex does not exhibit significant cross-substrate reaction with the second substrate, and the second luciferase complex does not exhibit significant cross-substrate reaction with the first substrate. The first and second substrates are the luminescent substrates for luciferase. The first mutant luciferase complex and the second luciferase complex can bind to different modified bases, respectively.

[0118] C) By detecting the fluorescence signals and signal combinations of the first mutant luciferase and the second luciferase autoluminescent system, the four bases A, T, G, and C are distinguished for target nucleic acid sequencing.

[0119] According to another embodiment of the present invention, a method for detecting nucleic acid sequences includes the following steps:

[0120] 1) Different bases labeled with affinity tags and reversible blocking modifications are polymerized with the template under the action of polymerase;

[0121] 2) Add multiple luciferase complexes from step A, and couple multiple luciferase complexes to different bases by specifically recognizing different affinity tags;

[0122] 3) Add different substrates and determine the type of bases that polymerize by detecting the optical signals of the substrates or combinations thereof;

[0123] 4) Add the excision reagent to excise the blocking and linking groups, in preparation for the next round of polymerization.

[0124] Another aspect of the present invention provides a method for detecting the content of an analyte, comprising the following steps:

[0125] 1) The analyte, the specific recognition protein of the analyte, the aforementioned mutant, and the substrate of Gaussian luciferase are contacted, wherein the analyte is adapted to form a complex with the mutant.

[0126] 2) Determine the content of the analyte based on the change in fluorescence intensity of the mutant before and after the formation of the complex;

[0127] According to embodiments of the present invention, the substrate of the Gaussian luciferase includes coelentrin, fluorocoelentrin, or a coelentrin derivative.

[0128] According to embodiments of the present invention, the coelenterin derivative includes coelenterin derivative F-CTZ or coelenterin derivative ZS26.

[0129] In this article, "reporter gene" is a molecular biology concept, referring to a class of genes that are expressed under specific conditions in cells, tissues / organs, or individuals, resulting in easily detectable traits that would not normally be produced in the experimental material. In other words, it is a gene encoding a detectable protein or enzyme. For genetic selection and screening, a reporter gene must meet the following conditions: 1. It has been cloned and its full sequence has been determined; 2. The expression product is not present in the recipient cells, i.e., there is no background, and no similar endogenous expression product exists in the transfected cells; 3. Its expression product can be quantitatively measured. Its use includes, but is not limited to, the following: fusing the reporter gene with a gene expression regulatory sequence to form a chimeric gene, or fusing it with other target genes, allowing nucleic acid expression under the control of the regulatory sequence, thereby using its expression product to detect the expression regulation of the target gene and study nucleic acids.

[0130] Luciferase is a class of enzymes that catalyze luminescent chemical reactions. The substrate of this enzyme is called luciferin. In the presence of ATP, light emission occurs when luciferin undergoes a chemical reaction catalyzed by luciferase. In this article, "fluorescence" is also referred to as chemical fluorescence. Bioluminescence is the emission of light produced by a chemical reaction in the absence of any excitation from light, heat, or electric fields. Living systems also exhibit fluorescence, known as bioluminescence, such as the light emitted by fireflies, certain bacteria or fungi, protozoa, worms, and crustaceans. In this application, the mutant catalyzes the oxidation of the substrate coelenterate to produce fluorescence.

[0131] This invention relates to a nucleic acid containing a base sequence encoding a luciferase mutant according to embodiments of the present invention. Specifically, the nucleic acid contains a luciferase gene derived from the marine copepod *Dichroa febrifuga*. Nucleic acid refers to DNA or RNA. The "gene" of luciferase primarily refers to the region transcribed from mRNA, i.e., it refers to a structural gene.

[0132] Unless otherwise stated, isomers of the structures shown in formula (I) or formula (II) described in this application include all isomer forms (e.g., enantiomers, diastereomeric atropisomers, and geometric (or conformational) forms); for example, R and S configurations of each asymmetric center, (Z) and (E) double bond isomers, and (Z) and (E) conformational isomers. Therefore, individual stereochemical isomers of the compounds of this application, as well as mixtures of enantiomers, diastereomeric mixtures, and mixtures of geometric isomers (or conformational isomers), are all within the scope of this application.

[0133] Salts of formula (I) or formula (II) used in this application refer to both organic and inorganic salts of the compound, as described in the literature (SMBerge et al., describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66: 1-19.). Pharmaceutically acceptable salts formed from non-toxic acids include, but are not limited to, inorganic acid salts formed by reactions with amino groups, such as hydrochlorides, hydrobromic acids, phosphates, sulfates, and perchlorates, and organic acid salts such as acetates, oxalates, maleates, tartrates, citrates, succinates, malonates, or salts obtained by other methods described in the literature, such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, cyclopentylpropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, transbutenedioic acid, glucono-p-ethyl, glycerol phosphate, gluconate, hemisulfate, heptaate, hexanoate, hydroiodate, 2-hydroxy-ethanesulfonate, lacturonate, lactate, laurate, lauryl sulfate, malate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, palmitate, pyruvate, pectinate, persulfate, 3-phenylpropionate, picrate, pentanoate, propionate, stearate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, etc. Salts obtained by means of appropriate bases include alkali metals, alkaline earth metals, ammonium, and N+(C1-4 alkyl)4 salts. This application also envisions the formation of quaternary ammonium salts from any compound containing an N-group. Water-soluble or oil-soluble or dispersed products can be obtained via quaternization. Alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc. Pharmaceutically acceptable salts further include suitable, non-toxic ammonium, quaternary ammonium salts, and amine cations resistant to the formation of equilibrium ions, such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, C1-8 sulfonates, and aromatic sulfonates.

[0134] When detecting luciferase luminescence using imaging equipment, well-known detection methods can be applied. For example, by detecting the fluorescence... Luciferin, ATP and Mg 2+Ions or the like are appropriately added to cells expressing a fusion protein containing luciferase to induce a luminescent response in the luciferase, and the emitted light can be detected by an imaging device. The imaging device is, for example, a microscope equipped with a filter for capturing the emitted light. Based on information obtained by identifying the luminescent location within the cell, the microscope can be used to pinpoint the protein's location. As an imaging device, a microscope capable of taking time-series images can be used, and time-series observation can be achieved with this microscope.

[0135] The present invention will now be described with reference to specific embodiments. It should be noted that these embodiments are merely descriptive and do not limit the present invention in any way.

[0136] Example 1: Plasmid Design and Construction for Prokaryotic Expression of Gaussian Luciferase Mutant

[0137] In this embodiment, wild-type and mutant Gaussian luciferases without signal peptides were constructed to compare the activity differences between the mutant and wild-type Gaussian luciferases.

[0138] The nucleotide sequence of wild-type Gaussian luciferase (Gluc WT) is SEQ ID NO:2, and its encoded amino acid sequence is SEQ ID NO:1. The nucleotide sequence of wild-type Gaussian luciferase without a signal peptide (Gluc WT-NS:Gluc WT no signal peptide) is SEQ ID NO:4, and its encoded amino acid sequence is SEQ ID NO:3. The pCold-Gluc WT-NS plasmid (Figure 1) was synthesized using a whole-genome synthesis method. It contains the amino acid sequence of wild-type Gaussian luciferase without a signal peptide (SEQ ID NO:5) and the nucleotide sequence (SEQ ID NO:6). A purification tag containing six histidine residues (6x His) is fused to its C-terminus to facilitate protein purification. The restriction enzyme sites at both ends are Nde I and EcoRI. Using a whole-genome synthesis method, the following Gaussian luciferase-active mutants of the pCold vector without the signal peptide were synthesized: B6 (H62K,P67L,E85S,S86T,A87G,L107M,V121E), D6 (H62K,P67A,E85D,S86T,A87G,L107M,V121E), and 4-C12 (H62K,E85S,S86T,A87G,E93P,L107M,V121E). Using Gluc WT-NS, B6, D6, and 4-C12 as templates, a random mutant library was constructed using multiplex PCR, comprising the following loci: N10, V12, A19, T20, T21, D22, L23, L35, E44, A45, C59, H62, P67, K71, H78, T79, E85, S86, A87, G89, E93, I95, L107, I114, and V121. The mutation sites compared to Gluc WT-NS are shown in Table 3. The PCR reaction system is shown in Table 1, and the PCR reaction conditions are shown in Table 2.

[0139] Table 1: PCR reaction system:

[0140] Table 2: PCR reaction conditions

[0141] Add 0.5 μL of DpnI enzyme to the PCR system after the reaction, incubate at 37°C for 3 hours to digest the template, and then perform gel recovery of the product of about 4800 bp, which is the combined mutant library at each site.

[0142] 2.5 μL of the above reaction product was transformed into DH5α competent cells and plated on ampicillin-resistant plates containing a final concentration of 100 μg / mL. The plates were incubated overnight at 37°C. The next day, single colonies were picked from the plates, cultured by shaking, and plasmids were extracted. Sequencing was performed to ensure correct amplification by multiplex PCR. The obtained plasmids were the wild-type Gaussian luciferase pCold Gluc WT-NS, B6, D6, 4-C12, and a library of combined mutants at each locus, excluding the signal peptide.

[0143] Example 2: Prokaryotic expression and purification of Gaussian luciferase mutant

[0144] This embodiment describes the prokaryotic expression and protein purification of the constructed pCold Gluc WT-NS, B6, D6, 4-C12 and combined mutant libraries at various loci. The specific experimental procedure is as follows:

[0145] The expression plasmid pCold Gluc WT-NS and the mutant obtained in Example 1 were transformed into OrigamiB(DE3), respectively. Chemically Competent Cells (Weidi Bio, EC1020S) were plated onto agar plates containing ampicillin (100 μg / mL). Single colonies were picked from the plates and incubated overnight at 37°C. The following day, the cells were diluted 1:100 and transferred to 3 mL of fresh LB medium containing ampicillin (100 μg / mL). The medium was incubated at 37°C with shaking at 200 rpm until OD600 ≈ 0.5–0.6. After incubation, the cells were cooled on ice for 1 hour. IPTG was added to a final concentration of 1 mM, and the cells were induced overnight at 16°C.

[0146] The induced bacterial precipitate was collected by centrifugation at 8000 rpm / min for 10 min. 600 μL of binding buffer (50 mM Tris-HCl, pH 8.0, 250 mM NaCl) was added, and the mixture was lysed on ice for 30 min. The mixture was then sonicated (2 s on, 3 s off, 60% power) for 30 min. The supernatant (cell lysate) and precipitate were separated by centrifugation at 12000 rpm for 30 min at 4 °C.

[0147] After washing with 500 μL of deionized water in a 96-well purification plate (GE), equilibrate with 500 μL of wash buffer (50 mM Tris-HCl, pH 8.0, 250 mM NaCl, 10 mM imidazole), and then add filtered cell lysis buffer. Wash 10 times (200 μL / wash) with wash buffer (50 mM Tris-HCl, pH 8.0, 250 mM NaCl, 10 mM imidazole), and then elute the protein with 100 μL of elution buffer (50 mM Tris-HCl, pH 8.0, 250 mM NaCl, 300 mM imidazole). Collect the eluted protein; a partial protein electrophoresis image is shown in Figure 4.

[0148] Example 3 Activity detection of Gaussian luciferase mutant

[0149] This embodiment uses the BCA quantitative reagent kit (Thermo Scientific). TM Pierce TMThe BCA Protein Assay Kit was used to accurately determine the protein concentration. The purified luciferase obtained in Example 2 was 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 was added to a black 96-well plate. Then, 90 μL of the substrate coelomic glycoside (MGI, Figure 3) diluted to 100 μM with the same solution was added, and the luminescence intensity was read using the ELISA reader's self-luminescent module (Figure 5). The dominant mutant relative to SEQ ID... Table 3 shows the activity test results of wild-type Gaussian luciferase without the signal peptide catalyzing the substrate coelenterate, as indicated by sequence NO:3. Among them, the following 33 mutant combinations showed an increase of more than 2.5-fold in catalytic activity against coelenterate: 17-1 (H62K, P67L, H78A, E85S, S86T, A87G, E93S, L107M, V121E), 18-2 (H62K, P67L, T79P, E85S, S86T, A87G, E93S, L107M, V121E), and 18-2 (H62K, P67L, T79P, E85S, S86T, A87G, E93S, L107M, V121E). 21E), 20-3(N10S,V12A), 21-1(V12A,T79H), 23-1(V12A,T79K), 24-2(N10S,V12S), 27-2(V12A,H62K,P67L,E 85S,S86T,A87G,E93P,L107M,V121E), 30-3(T20A,I114M), 36-1(L23T,H62K,P67L,E85S,S86T,A87G,E93T,L1 07M, V121E), 1-1 (H62K, E85D, S86T, A87G, L107M, V121E), 2-3 (H62K, P67A, E85D, S86T, A87G, L107M), 4-1 (H6 2K,P67A,E85D,S86T,A87G,V121E), 5-1(H62K,P67K,E85D,S86T,A87G,L107M,V121E), 6-1(H62K,P67A,E85D, S86T, A87G, E93A, L107M, V121E), 8-3 (H62K, P67A, E85D, S86I, A87G, L107M, V121E), 12-2 (P67A, E85D, S86T, A87G,L107M,V121E), 13-2(H62Q,E85S,S86T,A87G,E93A,L107M,V121E), 28-1(L23A,H62K,P67L,E85S,S86T, A87G, E93S, L107M, V121E), A2-2 (H62K, P67A, E85D, S86T, A87G, L107M, V121D), A2-3 (H62K, P67A, E85D, S86T, A87G, L107M), A3-1 (T20A, H62 K,P67A,E85D,S86T,A87G,L107M,V121E), A5-1(T21A,H62K,P67K,E85D,S86T,A87G,L107M,V121E), A5-2(H62K,P67K,E85D,S86T,A87G,L10 7M, V121E), A7-1 (T20V, T21A, D22E, H62K, P67A, E85D, S86T, A87G, L10 7M,V121D),A7-2(T21A,H62K,P67A,E85D,S86T,A87G,L107M,V121E), A8-1(T20V,T21A,D22G,H62K,P67A,E85D,S86I,A87G,T79P,L107M,V1 21E),A8-2(H62K,P67A,E85D,S86I,A87G,L107M,V121D),A12-1(E44G, H62N, P67A, E85D, S86T, A87G, L107M, V121D), A12-4 (T21A, P67A, E85D, S86T, A87G, L107M, V121D), A12-5 (P67A, E85D, S86T, A87G, L107M, V1 21D),A14-1(H62Q,E85D,S86T,A87G,L107M,V121E),A28-1(V12A,T21 A,D22G,L35F,A45V,C59S,H62K,P67L,E85D,S86T,A87G,E93S,L107M,V 121E), A28-2 (V12A, T20A, T21A, D22G, L35F, A45V, C59S, H62K, P67L, E 85D,S86T,A87G,E93S,L107M,V121E),A28-3(V12A,A19V,T20A,T21A, D22G, L23I, L35F, C59S, H62K, P67L, E85D, S86T, A87G, E93S, L107M, V1 21E), A29-3 (T21A, H62K, P67L, E85D, S86T, A87G, E93P, L107M, V121E).

[0150] Table 3: Summary of mutation sites and activity results of dominant Gaussian luciferase mutants

[0151] Example 4: Design and Construction of Eukaryotic Gaussian Luciferase Plasmid

[0152] Wild-type Gaussian luciferase containing a signal peptide (pEE12.4-Gluc WT) of the pEE12.4 vector was synthesized using a whole-genome synthesis method (Figure 2). The amino acid sequence of the wild-type Gaussian luciferase containing the signal peptide is SEQ ID NO:8, and the gene sequence is SEQ ID NO:7. A purification tag containing 6 histidines (6x His) is fused to the N-terminus after the signal peptide to facilitate protein purification, and an Avi-tag for biotinylation is fused to the C-terminus. The Gaussian luciferase plasmid for eukaryotic expression was constructed using the prokaryotic mutant pCold plasmids 4-1, 6-1, A2-2, A2-3, A8-2, and A12-5 (without the signal peptide) as templates. The primer sequences used for PCR are shown in Table 4, the reaction system is shown in Table 5, and the reaction conditions are shown in Table 6.

[0153] Table 4: PCR reaction primer sequences

[0154] Table 5: Reaction System

[0155] Table 6: Reaction Conditions

[0156] Add 0.5 μL of DpnI enzyme to the reaction system, incubate at 37°C for 3 hours to digest the template, and then recover the product of about 543 bp by gel extraction, which is the insert fragment.

[0157] Using the pEE12.4 vector as a template, which has a histidine tag for purification at the N-terminus and an Avitag for biotinylation at the C-terminus, the vector was linearized by PCR to facilitate recombination with the insert fragment. KOD FX neo enzyme was used, and the PCR reaction system was prepared and the PCR reaction was performed according to its instructions. The primer sequences used are shown in Table 7, the PCR reaction system is shown in Table 8, and the PCR reaction conditions are shown in Table 9.

[0158] Table 7: PCR reaction primer sequences

[0159] Table 8: Reaction System

[0160] Table 9: Reaction Conditions

[0161] Add 0.5 μL of DpnI enzyme to the reaction system and incubate at 37°C for 3 hours to digest the template. Then, recover the product of approximately 7600 bp using a gel, which is the linearized vector. Recombinate the insert and vector obtained in this example using the Takara In-Fusion Cloning kit according to the reaction system shown in Table 10, with an incubation period of 50°C for 15 minutes.

[0162] Table 10: Recombination Reaction System

[0163] 2.5 μL of the above reaction product was transformed into DH5α competent cells and plated on an Amp antibody plate containing a final concentration of 100 μg / mL. The next day, single colonies were picked from the plate, and plasmids were extracted. Sequencing was performed to ensure that the target fragment was correctly inserted into the vector. The obtained plasmid is the Gluc mutant for eukaryotic expression.

[0164] Example 5: Eukaryotic expression, purification, and enzyme activity detection of Gaussian luciferase mutant

[0165] The Gluc mutant plasmid of the pEE12.4 vector obtained in Example 4 and the fully synthesized pEE12.4-Gluc WT were subjected to plasmid extraction and then transfected into 30 mL of HEK293E cells (cell density: 4 × 10⁻⁶) using PEI. 6 Cells / ml (viability ≥ 95%), after 5 days of transfection, the cell viability was less than 90%, and the supernatant was collected by centrifugation at 8000 rpm for 10 min at 4℃.

[0166] Add 2 mL of HisTrap Excel packing material to each manual column (purchased from Sangon Biotech, model F506607-0001#, empty column) and rinse sequentially with 20 mL of deionized water, 10 mL of elution buffer I (50 mM Tris-HCl, pH 8.0, 250 mM NaCl, 1 M imidazole), and 20 mL of binding buffer (50 mM Tris-HCl, pH 8.0, 250 mM NaCl). Add approximately 30 mL of filtered cell supernatant to the equilibrated packing material. Rinse with wash buffer (50 mM Tris-HCl, ... After washing 10 times (10 mL / wash) with pH 8.0, 250 mM NaCl, and 10 mM imidazole, the protein was eluted 4-5 times with 500 μL of elution buffer (50 mM Tris-HCl, pH 8.0, 250 mM NaCl, and 300 mM imidazole). The eluted protein was collected. The purified protein was analyzed by 12% SDS-PAGE. The purification results are shown in Figure 6.

[0167] The activity of Gluc WT and Gluc mutant was tested according to the method described in Example 3, and the results are shown in Figure 7.

[0168] Example 6: Coupling and Enzyme Activity Detection of Gaussian Luciferase Mutant

[0169] The Gluc protein obtained in Example 5 contains an AviTag tag, which can be biotinylated by BirA enzyme (MGI) to form Biotin-Avi-tag-Gluc. The biotinylation reaction system is shown in Table 11.

[0170] Table 11: Reaction System

[0171] After standing at room temperature for 30 minutes, SA was added to the system to prepare SA-Gluc. The histidine tag on the mutant can be used to further purify it to obtain a purer SA-Gluc (mutant). The purification results are shown in Figure 8.

[0172] The activity of SA-Gluc (Gluc wt) and SA-Gluc mutants against the substrate coelenterin and coelenterin derivatives F-CTZ and ZS26 was tested according to the method described in Example 3. The results are shown in Figure 9.

[0173] Example 7: Instrumental Validation of Gaussian Luciferase Mutant

[0174] After conjugation, SA-Gluc (Gluc wt) and SA-Gluc mutant were subjected to activity detection using a DNBSEQ E5 (MGI) sequencer. The results are shown in Figure 10.

[0175] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0176] Although embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary. This should not be construed as a limitation of the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A luciferase mutant, characterized in that, Referring to the amino acid sequence of wild-type Gaussian luciferase or the amino acid sequence of a luciferase having at least 70% homology with the Gaussian luciferase SEQ ID NO:3, the luciferase mutant has a mutation at at least one of the following positions compared to the amino acid sequence shown in SEQ ID NO:3: 10th, 12th, 19th, 20th, 21st, 22nd, 23rd, 35th, 44th, 45th, 59th, 62nd, 67th, 78th, 79th, 85th, 86th, 87th, 93rd, 107th, 114th, 121st.

2. The luciferase mutant according to claim 1, characterized in that, The wild-type Gaussian luciferase has the amino acid sequence shown in SEQ ID NO:

1.

3. The luciferase mutant according to claim 1, characterized in that, The Gaussian luciferase has at least 70% homology with the luciferase being a copepod luciferase.

4. The luciferase mutant according to claim 3, characterized in that, The copepod luciferase includes at least one selected from Mluc, Maluc, Pxluc, and Mpluc.

5. The luciferase mutant according to any one of claims 1 to 4, characterized in that, The mutant luciferase mutant has any one or more combinations of the following mutations (1)-(24): (1) The N at the 10th position mutates to S; (2) The V at position 12 mutates to A or S; (3) The A at position 19 mutates to V; (4) The T mutation at position 20 is replaced by V or A; (5) The T at position 21 mutates to A; (6) The D at position 22 mutates into E or G; (7) The L at position 23 mutates to A, T, or I; (8) The L at position 35 mutates to F; (9) The E at position 44 mutates into G; (10) The A at position 45 mutates to V; (11) The C at position 59 mutates to S; (12) The H at position 62 mutates to K, Q, or N; (13) The P at position 67 is mutated to L, K, or A; (14) The H at position 78 mutates to A; (15) The T mutation at position 79 is changed to H, K, or P; (16) The E at position 85 mutates to S or D; (17) The S at position 86 is mutated to T or I; (18) The A at position 87 mutates into G; (19) The E at position 93 mutates into P, A, S, or T; (20) The L at position 107 mutates to M; (21) The I at position 114 mutates to M; (22) The V at position 121 is mutated to D or E.

6. The luciferase mutant according to claim 1, characterized in that, The mutant luciferase has the following mutations: (1) The H at position 62 mutates to K, the P at position 67 mutates to L, the H at position 78 mutates to A, the E at position 85 mutates to S, the S at position 86 mutates to T, the A at position 87 mutates to G, the E at position 93 mutates to S, the L at position 107 mutates to M, and the V at position 121 mutates to E; or (2) The H at position 62 mutates to K, the P at position 67 mutates to L, the T at position 79 mutates to P, the E at position 85 mutates to S, the S at position 86 mutates to T, the A at position 87 mutates to G, the E at position 93 mutates to S, the L at position 107 mutates to M, and the V at position 121 mutates to E; or (3) The 10th N mutates to S, and the 12th V mutates to A; or (4) The V mutation at position 12 is changed to A, and the T mutation at position 79 is changed to H; or (5) The V mutation at position 12 is changed to A, and the T mutation at position 79 is changed to K; or (6) The N at position 10 mutates to S, and the V at position 12 mutates to S; or (7) The V at position 12 mutates to A, the H at position 62 mutates to K, the P at position 67 mutates to L, the E at position 85 mutates to S, the S at position 86 mutates to T, the A at position 87 mutates to G, the E at position 93 mutates to P, the L at position 107 mutates to M, and the V at position 121 mutates to E; or (8) The T mutation at position 20 is changed to A, and the I mutation at position 114 is changed to M; or (9) The L at position 23 mutates to T, the H at position 62 mutates to K, the P at position 67 mutates to L, the E at position 85 mutates to S, the S at position 86 mutates to T, the A at position 87 mutates to G, the E at position 93 mutates to T, the L at position 107 mutates to M, and the V at position 121 mutates to E; or (10) The H at position 62 mutates to K, the E at position 85 mutates to D, the S at position 86 mutates to T, the A at position 87 mutates to G, the L at position 107 mutates to M, and the V at position 121 mutates to E; or (11) The H at position 62 mutates to K, the P at position 67 mutates to A, the E at position 85 mutates to D, the S at position 86 mutates to T, the A at position 87 mutates to G, and the L at position 107 mutates to M; or (12) The H at position 62 mutates to K, the P at position 67 mutates to A, the E at position 85 mutates to D, and the H at position 86 mutates to K. The S mutation at position 87 is changed to T, the A mutation at position 87 is changed to G, and the V mutation at position 121 is changed to E; or (13) The H at position 62 mutates to K, the P at position 67 mutates to K, the E at position 85 mutates to D, the S at position 86 mutates to T, the A at position 87 mutates to G, the L at position 107 mutates to M, and the V at position 121 mutates to E; or (14) The H at position 62 mutates to K, the P at position 67 mutates to A, the E at position 85 mutates to D, the S at position 86 mutates to T, the A at position 87 mutates to G, the E at position 93 mutates to A, the L at position 107 mutates to M, and the V at position 121 mutates to E; or (15) The H at position 62 mutates to K, the P at position 67 mutates to A, the E at position 85 mutates to D, the S at position 86 mutates to I, the A at position 87 mutates to G, the L at position 107 mutates to M, and the V at position 121 mutates to E; or (16) The P at position 67 mutates to A, the E at position 85 mutates to D, the S at position 86 mutates to T, the A at position 87 mutates to G, the L at position 107 mutates to M, and the V at position 121 mutates to E; or (17) The H at position 62 mutates to Q, the E at position 85 mutates to S, the S at position 86 mutates to T, the A at position 87 mutates to G, the E at position 93 mutates to A, the L at position 107 mutates to M, and the V at position 121 mutates to E; or (18) The L at position 23 mutates to A, the H at position 62 mutates to K, the P at position 67 mutates to L, the E at position 85 mutates to S, the S at position 86 mutates to T, the A at position 87 mutates to G, the E at position 93 mutates to S, the L at position 107 mutates to M, and the V at position 121 mutates to E; or (19) The H at position 62 mutates to K, the P at position 67 mutates to A, the E at position 85 mutates to D, the S at position 86 mutates to T, the A at position 87 mutates to G, the L at position 107 mutates to M, and the V at position 121 mutates to D; or (20) The H at position 62 mutates to K, the P at position 67 mutates to A, the E at position 85 mutates to D, the S at position 86 mutates to T, the A at position 87 mutates to G, and the L at position 107 mutates to M; or (21) The T mutation at position 20 becomes A, the H mutation at position 62 becomes K, the P mutation at position 67 becomes A, the E mutation at position 85 becomes D, the S mutation at position 86 becomes T, the A mutation at position 87 becomes G, the L mutation at position 107 becomes M, and the V mutation at position 121 becomes E; or (22) The T mutation at position 21 becomes A, the H mutation at position 62 becomes K, the P mutation at position 67 becomes K, the E mutation at position 85 becomes D, the S mutation at position 86 becomes T, the A mutation at position 87 becomes G, the L mutation at position 107 becomes M, and the V mutation at position 121 becomes E; or (23) The H at position 62 mutates to K, the P at position 67 mutates to K, the E at position 85 mutates to D, the S at position 86 mutates to T, the A at position 87 mutates to G, the L at position 107 mutates to M, and the V at position 121 mutates to... D; or (24) The T at position 20 mutates to V, the T at position 21 mutates to A, the D at position 22 mutates to E, the H at position 62 mutates to K, the P at position 67 mutates to A, the E at position 85 mutates to D, the S at position 86 mutates to T, the A at position 87 mutates to G, the L at position 107 mutates to M, and the V at position 121 mutates to D; or (25) The T mutation at position 21 becomes A, the H mutation at position 62 becomes K, the P mutation at position 67 becomes A, the E mutation at position 85 becomes D, the S mutation at position 86 becomes T, the A mutation at position 87 becomes G, the L mutation at position 107 becomes M, and the V mutation at position 121 becomes E; or (26) The T mutation at position 20 becomes V, the T mutation at position 21 becomes A, the D mutation at position 22 becomes G, the H mutation at position 62 becomes K, the P mutation at position 67 becomes A, the T mutation at position 79 becomes P, the E mutation at position 85 becomes D, the S mutation at position 86 becomes I, the A mutation at position 87 becomes G, the L mutation at position 107 becomes M, and the V mutation at position 121 becomes E; or (27) The H at position 62 mutates to K, the P at position 67 mutates to A, the E at position 85 mutates to D, the S at position 86 mutates to I, the A at position 87 mutates to G, the L at position 107 mutates to M, and the V at position 121 mutates to D; or (28) The E at position 44 mutates to G, the H at position 62 mutates to N, the P at position 67 mutates to A, the E at position 85 mutates to D, the S at position 86 mutates to T, the A at position 87 mutates to G, the L at position 107 mutates to M, and the V at position 121 mutates to D; or (29) The T mutation at position 21 is changed to A, the P mutation at position 67 is changed to A, the E mutation at position 85 is changed to D, the S mutation at position 86 is changed to T, the A mutation at position 87 is changed to G, the L mutation at position 107 is changed to M, and the V mutation at position 121 is changed to D; or (30) The P at position 67 mutates to A, the E at position 85 mutates to D, the S at position 86 mutates to T, the A at position 87 mutates to G, the L at position 107 mutates to M, and the V at position 121 mutates to D; or (31) The H at position 62 mutates to Q, the E at position 85 mutates to D, the S at position 86 mutates to T, the A at position 87 mutates to G, the L at position 107 mutates to M, and the V at position 121 mutates to E; or (32) The V at position 12 mutates to A, the T at position 21 mutates to A, the D at position 22 mutates to G, the L at position 35 mutates to F, the A at position 45 mutates to V, the C at position 59 mutates to S, the H at position 62 mutates to K, the P at position 67 mutates to L, the E at position 85 mutates to D, the S at position 86 mutates to T, the A at position 87 mutates to G, the E at position 93 mutates to S, the L at position 107 mutates to M, and the V at position 121 mutates to E; or (33) The V at position 12 mutates to A, the T at position 20 mutates to A, the T at position 21 mutates to A, the D at position 22 mutates to G, the L at position 35 mutates to F, the A at position 45 mutates to V, and the C at position 59 mutates to S. The 62nd position H mutates to K, the 67th position P mutates to L, the 85th position E mutates to D, the 86th position S mutates to T, the 87th position A mutates to G, the 93rd position E mutates to S, the 107th position L mutates to M, and the 121st position V mutates to E; or (34) The V at position 12 mutates to A, the A at position 19 mutates to V, the T at position 20 mutates to A, the T at position 21 mutates to A, the D at position 22 mutates to G, the L at position 23 mutates to I, the L at position 35 mutates to F, the C at position 59 mutates to S, the H at position 62 mutates to K, the P at position 67 mutates to L, the E at position 85 mutates to D, the S at position 86 mutates to T, the A at position 87 mutates to G, the E at position 93 mutates to S, the L at position 107 mutates to M, and the V at position 121 mutates to E; or (35) The T at position 21 is mutated to A, the H at position 62 is mutated to K, the P at position 67 is mutated to L, the E at position 85 is mutated to D, the S at position 86 is mutated to T, the A at position 87 is mutated to G, the E at position 93 is mutated to P, the L at position 107 is mutated to M, and the V at position 121 is mutated to E.

7. The luciferase mutant according to claim 1, characterized in that, The mutant luciferase further includes a signal peptide located at the N-terminus of the luciferase mutant.

8. The luciferase mutant according to claim 1, characterized in that, The mutant luciferase further includes a polyhistidine sequence located at the C-terminus of the luciferase mutant.

9. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the luciferase mutant according to any one of claims 1-8.

10. An expression carrier, characterized in that, It includes the nucleic acid molecule as described in claim 9.

11. The expression vector according to claim 10, characterized in that, It further includes a promoter that is operatively linked to the nucleic acid molecule.

12. A recombinant cell, characterized in that, Carrying the nucleic acid molecule of claim 9 or the expression vector of claim 10 or 11.

13. The recombinant cell according to claim 12, characterized in that, The recombinant cells are selected from Escherichia coli, yeast, or mammalian cells.

14. A method for obtaining luciferase, characterized in that, include: The recombinant cells of claim 12 are cultured under conditions suitable for protein expression in order to obtain the luciferase.

15. A conjugate, characterized in that, It includes the luciferase mutant and small molecule compound or macromolecule as described in any one of claims 1 to 8, wherein the luciferase mutant and small molecule compound or macromolecule are coupled by chemical bonds.

16. The conjugate according to claim 15, characterized in that, The small molecule compounds or macromolecules include streptavidin, digoxin, antibodies, dNTPs, or dNTP analogs.

17. A method for nucleic acid sequencing, characterized in that, include: Using the nucleic acid to be tested as a template, a polymerization reaction is carried out on the luciferase mutant described in any one of claims 1 to 8 and / or the conjugate described in any one of claims 15 to 16 and / or dNTP or dNTP analog or modified dNTP. The nucleic acid sequence of the nucleic acid to be tested is determined based on the fluorescence signal emitted by the reaction of the luciferase with the substrate or analogue of the Gaussian luciferase.

18. A nucleic acid sequencing kit, characterized in that, Includes the luciferase mutant of any one of claims 1-8 or the conjugate of claim 15 or 16.

19. The reagent kit according to claim 18, characterized in that, Further includes: dNTPs, universal primers, PCR polymerase, or coencin or coencin analogues.

20. A method for detecting the content of an analyte, characterized in that, include: The analyte, comprising a luciferase mutant according to any one of claims 1-8 or a conjugate according to claim 15 or 16, is contacted with a substrate of Gaussian luciferase or an analogue thereof; and the content of the analyte is determined based on the intensity of the fluorescence signal after contact treatment.

21. The method according to claim 17 or 20, characterized in that, The substrate or analogue of the Gaussian luciferase includes at least one selected from coelentrin and coelentrin derivatives.

22. The method according to claim 21, wherein the coelenterin derivative is as shown in formula (I) or formula (II), or a salt or isomer thereof,

Images