Luciferase mutant and application thereof

Abstract

The invention provides a luciferase mutant and application thereof, and particularly provides a copepoda luciferase mutant and application thereof. Pxluc is subjected to protein directed evolution to obtain a mutant of which the substrate specificity to ZS26 / F-CTZ is improved by more than 2 times and a mutant of which the substrate specificity to ZS2 / F-CTZ is improved by more than 4 times. The luciferase can be expressed in prokaryotic expression, the purification process is simple, and large-scale production is facilitated; the luciferase has the luminance similar to that of Gluc, is easy to detect, and has wide application prospects in the fields of basic scientific research, biological monitoring, biochemical diagnosis and the like.

Description

Luciferase mutants and their applications Technical Field

[0001] The present invention relates to the field of biotechnology, and specifically, to a luciferase mutant and applications thereof, and particularly to a copepod luciferase mutant and applications thereof. Background Art

[0002] Luciferases are enzymes that catalyze the oxidative luminescence of luciferin or fatty aldehydes. Found widely in insects, bacteria, fungi, and marine organisms, they have become essential tools in scientific research, widely used in life science research, genome sequencing and analysis, clinical medicine and forensic testing, drug screening, environmental monitoring, and enzyme-linked assays. Leveraging their self-luminescence, luciferases are often used in live cell assays, protein-protein interactions, protein localization, small interfering RNA silencing, and high-throughput drug screening. In biomonitoring technology, luciferases can be used to detect the presence of chemical pollutants. They also have broad application prospects in immunoassays and biochemical diagnostics. Furthermore, as reporter genes for detecting the expression intensity of exogenous genes from different promoters and studying transcriptional regulation, multiple luciferases with similar self-luminescence brightness and catalyzing different substrates are required.

[0003] At present, the luciferases that have been well studied and developed mainly include: firefly luciferase (FLuc), bacterial luciferase (Lux), and luciferases extracted from sea renilla (RLuc), deep-sea shrimp (OLuc), and marine animal Gaussia princeps (GLuc). Among them, FLuc requires ATP, O2 and Mg 2+ Lux fluorescence reactions mostly require molecules such as flavin mononucleotide (FMN), long-chain fatty aldehydes, oxygen and reduced nicotinamide adenine dinucleotide (NADH); Rluc luminescence does not require ATP, but its fluorescence intensity is weak and it cannot be secreted.

[0004] Although more than 40 bioluminescent systems have been discovered in nature, only a limited number can be developed and utilized.

[0005] Summary of the Invention

[0006] The present invention aims to solve one of the technical problems in the related art to at least a certain extent. To this end, one object of the present invention is to provide a mutant of copepod luciferase Pxluc (Pleuromamma xiphia, also known as sword-shaped water flea luciferase). The inventors carried out protein directed evolution on Pxluc to obtain a mutant whose substrate specificity for ZS26 / F-CTZ was improved by more than 2 times, and a mutant whose substrate specificity for ZS2 / F-CTZ was improved by more than 4 times. This luciferase can be expressed in prokaryotic and eukaryotic cells, and the purification process is simple, which is conducive to large-scale production; it has a luminescence brightness similar to Gluc and is easy to detect. This luciferase will have broad application prospects in many fields such as basic scientific research, biological monitoring and biochemical diagnosis.

[0007] To this end, the first aspect of the present invention provides a mutant luciferase. According to one embodiment of the present invention, compared to the amino acid sequence shown in SEQ ID NO: 2, the mutant luciferase has at least one of the following mutation sites: positions 98, 99, 100, and 101, and may or may not include a signal peptide amino acid sequence. The mutant luciferase according to one embodiment of the present invention has strong catalytic activity for substrates such as coelenterazine, fluorocoelenterazine, and coelenterazine derivatives. Compared with the existing Daphnia pulex luciferase, it has a wider substrate spectrum, stronger substrate selection specificity, and significantly enhanced luminescence brightness. It can be used in basic scientific research, biological detection technology, immunoassay, biochemical detection or diagnosis, and has broad application prospects in the fields of basic scientific research, biological detection technology, immunoassay, biochemical detection or diagnosis, in which the mutant luciferase is applied for luminescence detection.

[0008] The second aspect of the present invention provides a nucleic acid molecule. According to one embodiment of the present invention, the nucleic acid molecule encodes the mutant luciferase described in the first aspect. The mutant (mutant luciferase) encoded by the nucleic acid molecule according to one embodiment of the present invention has strong catalytic activity for substrates such as coelenterazine, fluorocoelenterazine and coelenterazine derivatives. Compared with the existing Daphnia pulex luciferase, it has a wider substrate spectrum or stronger substrate selection specificity and significantly enhanced luminescence brightness. It can be used in basic scientific research, biological detection technology, immunoassay, biochemical detection or diagnosis in the field of luminescence detection of the protein, and has broad application prospects.

[0009] The third aspect of the present invention provides an expression vector. According to one embodiment of the present invention, it comprises the nucleic acid molecule described in the second aspect. The expression vector may include an optional control sequence, and the control sequence is operably connected to the nucleic acid molecule. Wherein, the control sequence is one or more control sequences that can guide the expression of the nucleic acid molecule in the host. The expression vector proposed in one embodiment of the present invention can efficiently express proteins in suitable host cells, and the obtained protein has strong catalytic activity on substrates such as coelenterazine, fluorocoelenterazine and coelenterazine derivatives. Compared with the existing Daphnia pulex luciferase substrate spectrum, the substrate selection specificity is wider or the luminescence brightness is significantly enhanced. It can be used in basic scientific research, biological detection technology, immunoassay, biochemical detection or diagnosis in which the protein is applied for luminescence detection, and has broad application prospects.

[0010] The fourth aspect of the present invention provides a recombinant cell. According to one embodiment of the present invention, it carries the nucleic acid molecule described in the second aspect or the expression vector described in the third aspect. The recombinant cell is obtained by transfecting or transforming the expression vector. According to one embodiment of the present invention, the recombinant cell can efficiently express the above-mentioned mutant under appropriate conditions, and the mutant has strong catalytic activity on substrates such as coelenterazine, fluorocoelenterazine and coelenterazine derivatives. Compared with the existing Daphnia pulex luciferase substrate spectrum, the substrate selection specificity is wider or the luminescence brightness is significantly enhanced. It can be used in basic scientific research, biological detection technology, immunoassay, biochemical detection or diagnosis in which the protein is applied for luminescence detection, and has broad application prospects.

[0011] A fifth aspect of the present invention provides a method for producing mutant luciferase. According to one embodiment of the present invention, the method comprises: introducing the expression vector described in the third aspect into a recombinant cell, culturing and propagating the recombinant cell, and collecting the cultured and propagated product to extract or purify the mutant luciferase.

[0012] A sixth aspect of the present invention provides a method for detecting a nucleic acid sequence using the mutant luciferase described in the first aspect. According to one embodiment of the present invention, the method for detecting a nucleic acid sequence comprises the following steps:

[0013] A) using the mutant luciferase described in the first aspect to form a first mutant luciferase complex with a first specific recognition protein and the mutant luciferase by chemical coupling, biological coupling, or in the form of a fusion protein; using the second luciferase as a signal protein, using the second specific recognition protein and the second luciferase by chemical coupling, biological coupling, or in the form of a fusion protein to form a second luciferase complex;

[0014] B) the first mutant luciferase complex can react with a first substrate to generate a first luminescent signal; the second luciferase complex can react with a second substrate to generate a second luminescent signal; the first mutant luciferase complex does not have a significant cross-substrate reaction with the second substrate, and the second luciferase complex does not have a significant cross-substrate reaction with the first substrate, the first specific recognition protein recognizes and specifically binds to the first substrate, and the second specific recognition protein recognizes and specifically binds to the second substrate;

[0015] C) detecting the fluorescent signals and signal combinations of the mutant luciferase and the second luciferase self-luminescence system to distinguish the four bases A, T, G, and C for target nucleic acid sequencing.

[0016] According to one embodiment of the present invention, when the mutant luciferase and the second luciferase are the same, the first substrate and the second substrate are the same;

[0017] When the mutant luciferase and the second luciferase are different, the first substrate and the second substrate are different.

[0018] According to another embodiment of the present invention, the method for detecting nucleic acid sequences comprises the following steps:

[0019] 1) different bases labeled with affinity tags and reversible blocking modifications and a test template are polymerized under the action of a polymerase; 2) multiple luciferase complexes from step A are added, and the multiple luciferase complexes are coupled to different bases by specifically recognizing different affinity tags; 3) different substrates are added, and the type of polymerized base is determined by detecting the optical signal of the substrate or a combination thereof; 4) a removal reagent is added to remove the blocking group and the linking group, preparing for the next round of polymerization reaction.

[0020] A seventh aspect of the present invention provides a nucleic acid sequencing kit. According to one embodiment of the present invention, the kit comprises the mutant luciferase or luciferase complex described in the first aspect.

[0021] The eighth aspect of the present invention provides a method for detecting the content of an analyte. According to one embodiment of the present invention, the method comprises the following steps:

[0022] a) using the mutant luciferase described in the first aspect as a signal protein, forming a complex between the specific recognition molecule of the analyte and the mutant luciferase by chemical coupling, biological coupling or in the form of a fusion protein;

[0023] b) contacting the analyte with the complex;

[0024] c) adding a substrate of Daphnia lappa luciferase or a substrate analogue to the reaction system;

[0025] d) determining the content of the analyte based on the fluorescence intensity of the reaction system detected after adding the substrate of Daphnia leucophylla luciferase or a substrate analogue.

[0026] By utilizing the properties of the mutants that react upon binding to substrates, the mutants can be used as signal proteins to detect analytes. For example, by chemically coupling or forming a fusion protein with a protein that specifically recognizes the analyte, the mutant can be coupled or fused to the mutant. The protein that specifically recognizes the analyte will bind to the analyte, and the mutant will catalyze its substrate and emit spontaneous light. The intensity of the bioluminescence released during the mutant's catalysis of its substrate can be measured using a chemiluminescence microplate reader. The intensity of the bioluminescence can reflect the activity of the mutant, and the level of the analyte can be determined based on the level of its activity. Therefore, a kit containing the mutant can be used to accurately detect the level of the analyte.

[0027] According to a specific embodiment of the present invention, the analyte may be a nucleic acid. The mutant provided by the present invention has a strong catalytic activity on substrates such as coelenterazine, fluorocoelenterazine, and coelenterazine derivatives. Compared with the existing Daphnia pulex luciferase, the substrate spectrum is wider or the substrate selection specificity is stronger, and the luminescence brightness is significantly enhanced. Therefore, the method can be used to detect the expression of RNA or protein by nucleic acid, such as the expression amount of RNA or protein, the localization or tracing of protein. The accuracy and sensitivity of the nucleic acid detection method are significantly higher than those of the existing Gaussia luciferase, and the results obtained are more accurate.

[0028] In a ninth aspect of the present invention, the present invention provides a method for screening a substrate of Daphnia lappa luciferase, comprising:

[0029] I) contacting the mutant luciferase described in the first aspect with a substrate to be screened to obtain a reaction mixture;

[0030] II) Determining whether the substrate to be screened is the target substrate based on whether the reaction mixture obtained in step I) emits a chemiluminescent signal. Utilizing the property of the mutant to produce bioluminescence upon binding to the target substrate, the substrate of interest to be screened is contacted with the mutant. The mutant releases bioluminescence during catalysis of the substrate to be screened. A chemiluminescence microplate reader is used to determine whether the mutant can catalyze chemiluminescence from the substrate to be screened, thereby determining whether the substrate to be screened is the target substrate. Therefore, the method according to one embodiment of the present invention can accurately screen for target substrates.

[0031] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail 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 listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments with reference to the accompanying drawings, in which:

[0033] FIG1 shows the plasmid map of the prokaryotic expression plasmid pET28a-Pxluc WT of the wild-type Daphnia pulex luciferase containing a signal peptide;

[0034] FIG2 shows the plasmid map of the prokaryotic expression plasmid pCold WT Pxluc of the wild-type Daphnia pulex luciferase containing a signal peptide;

[0035] FIG3 shows the plasmid map of the prokaryotic expression plasmid pCold WT NS Pxluc of the wild-type Daphnia pulex luciferase without a signal peptide;

[0036] FIG4 shows the expression and purification results of wild-type Daphnia radiata luciferase in different competent cells, where the arrows indicate the target protein bands;

[0037] Figure 5 shows the chemical structural formulas of A substrate coelenterazine, B fluorocoelenterazine, C coelenterazine derivative ZS2, and D coelenterazine derivative ZS26(D);

[0038] FIG6 shows the results of prokaryotic expression of Daphnia pulex luciferase and Gaussia luciferase protein activity test results;

[0039] FIG7 is the activity test result of the dominant mutant of Daphnia leucophylla luciferase to substrate-specific ZS26 / F-CTZ protein level;

[0040] FIG8 is the activity test result of the dominant mutant of Daphnia leucophylla luciferase to substrate-specific ZS2 / F-CTZ protein level;

[0041] FIG9 shows the plasmid map of the wild-type Daphnia pulex luciferase eukaryotic expression plasmid pEE12.4-Pxluc WT containing a signal peptide;

[0042] FIG10 is a diagram showing the expression and purification results of Daphnia pulex luciferase in eukaryotic cells, wherein the arrows indicate the target protein bands;

[0043] Figure 11 shows the results of luciferase coupled to SA protein and before and after purification;

[0044] FIG12 shows the activity detection results of eukaryotic expression of luciferase pEE12.4 Pxluc WT and mutant pEE12.4 Pxluc P26-95, and coupled SA-Pxluc WT and SA Pxluc P26-95 against substrate-specific ZS26 / F-CTZ and ZS2 / F-CTZ protein levels.

[0045] Detailed Description of the Invention

[0046] The embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to be used to explain the present invention, but should not be understood as limiting the present invention.

[0047] 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 the technical features being referred to. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In the description of the present invention, "plurality" means at least two, such as two, three, etc., unless otherwise specifically defined.

[0048] In basic scientific research, the luciferase gene has been widely used as a reporter gene to study the expression intensity and transcriptional regulation of exogenous genes under different promoters. In biomonitoring technology, luciferase can be used to detect the presence of chemical pollutants. It also has broad application prospects in immunoassays and biochemical diagnostics.

[0049] mutant

[0050] In one aspect of the present invention, the present invention provides a luciferase mutant, which has any one or more combinations of the following mutation sites compared to the amino acid sequence shown in SEQ ID NO: 2: positions 98, 99, 100 and 101, and may or may not include a signal peptide amino acid sequence.

[0051] According to the mutation site of one embodiment of the present invention, the above-mentioned amino acid sequence is modified, and the obtained mutant has strong catalytic activity for substrates such as coelenterazine, fluorocoelenterazine, and coelenterazine derivatives ZS2 and ZS26. Compared with the existing Daphnia pulex luciferase substrate spectrum, specificity is stronger, and luminescence brightness is significantly enhanced. It can be used in basic scientific research, biological detection technology, immunoassay, biochemical detection or diagnosis in which the mutant is applied for luminescence detection, and has broad application prospects. For example, it can be used as a reporter gene for quantitative detection of DNA, RNA, transcription factors, proteins or cells, etc.; it can be used as a luminescent signal protein in a fusion protein for quantitative detection of target small molecules, proteins, etc.

[0052] In this article, "reporter gene" is a molecular biology concept that refers to a type of gene that is expressed in cells, tissues / organs, or individuals under specific conditions and causes them to produce easily detectable traits that would not otherwise be produced by the experimental material. In other words, it is a gene that encodes a detectable protein or enzyme. As a reporter gene, it must meet the following conditions in genetic selection and screening: 1. It must have been cloned and its entire sequence determined; 2. The expression product must not exist in the recipient cell, that is, there is no background, and there are no similar endogenous expression products in the transfected cells; 3. Its expression product can be quantitatively measured. When used, it includes but is not limited to the following methods: fusing the reporter gene with a gene expression regulatory sequence to form a chimeric gene, or fusing it with other target genes to express the nucleic acid 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.

[0053] Herein, "nucleic acid expression" may refer to DNA being expressed as RNA; or DNA being expressed as RNA, which is further expressed as protein; or RNA being expressed as protein, that is, the product of nucleic acid expression herein may be either RNA or protein.

[0054] Herein, "chemiluminescence," also known as cold light, is light radiation produced by chemical reactions in the absence of any stimulation, such as light, heat, or electric fields. Living systems also exhibit chemiluminescence, known as bioluminescence, such as that produced by fireflies, certain bacteria and fungi, protozoa, worms, and crustaceans. In this application, the mutants described herein are capable of spontaneous illumination, i.e., chemiluminescence.

[0055] According to some specific embodiments of the present invention, the mutant may further include at least one of the following additional technical features:

[0056] According to some specific embodiments of the present invention, the gene sequence of the wild-type Daphnia pulex luciferase containing a signal peptide (WT Pxluc) is shown in SEQ ID NO: 1, and the amino acids encoded therein are shown in SEQ ID NO: 2, wherein amino acids 1-17 are the signal peptide (bold). The gene sequence of the wild-type Daphnia pulex luciferase without a signal peptide (WT no signal peptide Pxluc: WT-NS Pxluc) is shown in SEQ ID NO: 3, and the amino acids encoded therein are shown in SEQ ID NO: 4. The sequences of SEQ ID NOs: 1-4 are as follows:

[0057]

[0058] According to some specific embodiments of the present invention, compared to the amino acid sequence shown in SEQ ID NO: 2, the mutant has any one or more combinations of the following mutations (1) to (4):

[0059] (1) G at position 98 mutated to L, P, Q, S, or T;

[0060] (2) Q at position 99 is mutated to R, W, I, Y, A, L, F, V, P, E, or M;

[0061] (3) G at position 100 is mutated to S, Q, R, W, T, A, or L;

[0062] (4) The G at position 101 is mutated to F, R, S, C, Y, L, I, K, V, or P.

[0063] According to other specific embodiments of the present invention, compared with the amino acid sequence shown in SEQ ID NO: 2, the mutant luciferase has any one or two of the following mutation sites: position 98, position 99, position 100 and position 101, and may or may not include a signal peptide amino acid sequence.

[0064] According to other specific embodiments of the present invention, compared to the amino acid sequence shown in SEQ ID NO: 2, the mutant has any one or two of the following mutations (1) to (4):

[0065] (1) G at position 98 mutated to L, P, Q, S, or T;

[0066] (2) Q at position 99 is mutated to R, W, I, Y, A, L, F, V, P, E, or M;

[0067] (3) G at position 100 is mutated to S, Q, R, W, T, A, or L;

[0068] (4) G at position 101 is mutated to F, R, S, C, Y, L, I, K, V, or P. According to some specific embodiments of the present invention, compared with the amino acid sequence shown in SEQ ID NO: 2, the mutant has the following mutations:

[0069] 1) G at position 98 is mutated to L, and Q at position 99 is mutated to R; or

[0070] 2) G at position 98 is mutated to P; or

[0071] 3) G at position 98 is mutated to Q; or

[0072] 4) G at position 98 is mutated to S, and Q at position 99 is mutated to W; or

[0073] 5) the Q at position 98 is mutated to I; or

[0074] 6) Q at position 99 is mutated to Y; or

[0075] 7) The Q at position 99 is mutated to A; or

[0076] 8) The Q at position 99 is mutated to L; or

[0077] 9) Q at position 99 is mutated to F; or

[0078] 10) G at position 98 is mutated to L, and Q at position 99 is mutated to V; or

[0079] 11) G at position 98 is mutated to T, and Q at position 99 is mutated to P; or

[0080] 12) G at position 98 is mutated to L, and Q at position 99 is mutated to E; or

[0081] 13) Q at position 99 is mutated to M, and G at position 100 is mutated to S; or

[0082] 14) G at position 100 is mutated to Q, and G at position 101 is mutated to F; or

[0083] 15) G at position 100 is mutated to R, and G at position 101 is mutated to R; or

[0084] 16) G at position 100 is mutated to W, and G at position 101 is mutated to F; or

[0085] 17) G at position 100 is mutated to S; or

[0086] 18) G at position 100 is mutated to T, and G at position 101 is mutated to S; or

[0087] 19) G at position 100 is mutated to R, and G at position 101 is mutated to C; or

[0088] 20) G at position 100 is mutated to A, and G at position 101 is mutated to R; or

[0089] 21) G at position 100 is mutated to L, and G at position 101 is mutated to Y; or

[0090] 22) G at position 100 is mutated to S, and G at position 101 is mutated to L; or

[0091] 23) G at position 101 is mutated to I; or

[0092] 24) G at position 100 is mutated to L, and G at position 101 is mutated to K; or

[0093] 25) G at position 100 is mutated to T; or

[0094] 26) G at position 100 is mutated to A, and G at position 101 is mutated to V; or

[0095] 27) The G at position 100 mutated to A, and the G at position 101 mutated to P.

[0096] According to some specific embodiments of the present invention, when the amino acid sequence shown in SEQ ID NO: 2 has the above-mentioned mutation, the obtained protein has strong catalytic activity for substrates such as coelenterazine, fluorocoelenterazine and coelenterazine derivatives. Compared with the existing Daphnia pulex luciferase, the substrate spectrum is wider and the luminescence brightness is significantly enhanced. In practical applications, the detection accuracy is significantly improved. It can be used in basic scientific research, biological detection technology, immunoassay, biochemical detection or diagnosis in which the protein is applied for luminescence detection, and has broad application prospects.

[0097] According to some specific embodiments of the present invention, the mutant luciferase does not contain a signal peptide sequence.

[0098] According to some specific embodiments of the present invention, the mutant luciferase may be a luciferase having a mutation at any one or more combination sites of positions 98, 99, 100 and 101 (for example, any one of the four mutation sites, or a combination of any two sites, or a combination of any three sites, or all four sites are mutated) compared to the amino acid sequence shown in SEQ ID NO: 2.

[0099] According to some specific embodiments of the present invention, the mutant luciferase may also have any one or more combination sites at positions 98, 99, 100 and 101 compared to the amino acid sequence shown in SEQ ID NO: 2, and the mutant luciferase does not contain a signal peptide amino acid sequence.

[0100] The amino acids 1-17 in the amino acid sequence shown in SEQ ID NO: 2 are signal peptides. The inventors have found that, whether it is the complete SEQ ID NO: 2 containing a signal peptide or a sequence without a signal peptide, as long as a mutation occurs at any one or more combination sites of positions 98, 99, 100, and 101 (these sites are numbered based on the SEQ ID NO: 2 sequence), the mutant luciferase having this amino acid sequence can have strong catalytic activity for substrates such as coelenterazine, fluorocoelenterazine, and the coelenterazine derivative ZS2, and has a wider substrate spectrum, stronger specificity, and significantly enhanced luminescence brightness compared to the existing Daphnia pulex luciferase. According to some specific embodiments of the present invention, the mutant is a non-secreted protein or a secreted protein.

[0101] According to some specific embodiments of the present invention, the present invention provides a nucleic acid molecule encoding the aforementioned mutant.

[0102] It should be noted that, for nucleic acids mentioned in the present specification and claims, those skilled in the art will understand that they actually include any one or both of the complementary double strands. For convenience, although only one strand is provided in most cases in this specification and claims, the other complementary strand is also disclosed. In addition, the nucleic acid sequences in this application include DNA or RNA forms, and disclosure of one of them means that the other is also disclosed.

[0103] On the other hand, the present invention proposes an expression vector comprising the aforementioned nucleic acid molecule. The type of expression vector here is not particularly limited, as long as it can replicate and express the corresponding mutant in the host cell. The expression vector may include an optional control sequence, which is operably connected to the nucleic acid molecule. Wherein, the control sequence is one or more control sequences that can guide the expression of the nucleic acid molecule in the host. The expression vectors proposed in some specific embodiments of the present invention can efficiently express proteins in suitable host cells, and the obtained proteins have strong catalytic activity on substrates such as coelenterazine, fluorocoelenterazine and coelenterazine derivatives. Compared with the existing sword-shaped water flea luciferase substrate spectrum, the specificity is stronger, and the luminescence brightness is significantly enhanced. It can be used in basic scientific research, biological detection technology, immunoassay, biochemical detection or diagnosis, etc. applied to the protein for luminescence detection, and has broad application prospects.

[0104] In another aspect of the present invention, the present invention proposes a recombinant cell carrying the aforementioned nucleic acid molecule, expression vector or mutant. The recombinant cell is obtained by transfecting or transforming the expression vector. According to some specific embodiments of the present invention, the recombinant cell can efficiently express the above-mentioned mutant under appropriate conditions, and the mutant has strong catalytic activity on substrates such as coelenterazine, fluorocoelenterazine and coelenterazine derivatives. Compared with the existing Daphnia pulex luciferase substrate spectrum, the specificity is stronger, and the luminescence brightness is significantly enhanced. It can be used in basic scientific research, biological detection technology, immunoassay, biochemical detection or diagnosis in the field of luminescence detection of the protein, and has broad application prospects.

[0105] According to some specific embodiments of the present invention, the above-mentioned recombinant cell may further include at least one of the following additional technical features:

[0106] According to some specific embodiments of the present invention, the recombinant cell is Escherichia coli, yeast or mammalian cells. According to some specific embodiments of the present invention, the recombinant cell is not particularly limited, and any cell capable of expressing the mutant in a nucleic acid or vector (such as a plasmid) can be used, such as yeast cells, bacteria or mammalian cells such as human embryonic kidney cells.

[0107] According to some specific embodiments of the present invention, the recombinant cells do not include animal germ cells, fertilized eggs or embryonic stem cells.

[0108] According to some specific embodiments of the present invention, the present invention provides a method for producing a mutant luciferase, comprising introducing the aforementioned expression vector into a recombinant cell, culturing and propagating the recombinant cell, and collecting the culture and propagation product to extract or purify the mutant luciferase. According to some specific embodiments of the present invention, the present invention provides the use of the aforementioned mutant luciferase and a corresponding substrate in detecting a nucleic acid sequence.

[0109] It should be noted that, in the present invention, "mutant luciferase", "luciferase mutant", and "mutant" can be replaced with each other equally.

[0110] Kit and use of kit preparation

[0111] In one aspect of the present invention, a kit is provided for detecting the content of an analyte, wherein the kit comprises the aforementioned luciferase mutant, and the specific recognition protein of the analyte is suitable for forming a complex with the mutant.

[0112] According to some specific embodiments of the present invention, the kit further comprises a substrate of Daphnia lappa luciferase or an analog of the substrate.

[0113] According to some specific embodiments of the present invention, the substrate is selected from at least one of coelenterazine, fluorocoelenterazine or a coelenterazine derivative.

[0114] According to some specific embodiments of the present invention, the coelenterazine derivative is selected from the coelenterazine derivative ZS2 or the coelenterazine derivative ZS26.

[0115]

[0116] According to some specific embodiments of the present invention, the kit further comprises a specific recognition protein of the analyte.

[0117] The present invention provides a nucleic acid sequencing kit, which comprises the mutant luciferase described above.

[0118] In another aspect of the present invention, the present invention proposes the use of the aforementioned mutant in preparing a test kit, wherein the test kit is used to detect the content of an analyte, and the specific recognition protein of the analyte is suitable for forming a complex with the mutant. Utilizing the characteristic of the mutant reacting in combination with a substrate, the mutant can be used as a signal protein to detect the analyte, such as by chemical coupling or forming a fusion protein on the mutant and coupling or fusion with a protein that can specifically recognize the analyte. When the mutant and the substrate of the mutant are in the same system, the protein that specifically recognizes the analyte will bind to the analyte, and the mutant will catalyze its substrate and emit light spontaneously. The intensity of the bioluminescence released during the mutant's catalysis of its substrate is measured by a chemiluminescence microplate reader. The intensity of the bioluminescence can reflect the activity of the mutant, and the content of the analyte can be judged by the level of its activity. Before the substrate binds to the mutant, the non-specific binding protein in the system needs to be washed off to eliminate the interference of other factors on the test result. Therefore, the mutant can be used to prepare a test kit and accurately detect the content of the analyte.

[0119] According to some specific embodiments of the present invention, the above-mentioned use may further include at least one of the following additional technical features:

[0120] According to some specific embodiments of the present invention, the kit further comprises a substrate of Daphnia lappa luciferase or an analog of the substrate.

[0121] According to some specific embodiments of the present invention, the substrate is selected from at least one of coelenterazine, fluorocoelenterazine or a coelenterazine derivative.

[0122] According to some specific embodiments of the present invention, the substrate is not particularly limited, and all substances that can chemically react with the mutant are included in this scope, and are not limited to chemiluminescent reactions. Those skilled in the art can replace different substrates according to experimental needs.

[0123] According to some specific embodiments of the present invention, the coelenterazine derivative is selected from the coelenterazine derivative ZS2 or the coelenterazine derivative ZS26.

[0124] According to some specific embodiments of the present invention, the kit further comprises a specific recognition protein of the analyte.

[0125] method

[0126] In one aspect of the present invention, the present invention provides a method for detecting the content of a substance, comprising the following steps:

[0127] i) forming a complex between the specific recognition protein of the analyte and the aforementioned mutant;

[0128] ii) contacting the analyte with the complex;

[0129] iii) adding a substrate of Daphnia lappa luciferase or a substrate analogue to the reaction system;

[0130] iv) determining the content of the analyte based on the fluorescence intensity of the reaction system after adding the substrate of Daphnia lappa luciferase or a substrate analogue.

[0131] According to some specific embodiments of the present invention, the present invention provides a method for detecting the content of an analyte, comprising the following steps:

[0132] a) using the aforementioned mutant luciferase as a signal protein, forming a complex between the specific recognition protein of the analyte and the mutant luciferase by chemical coupling, biological coupling or in the form of a fusion protein;

[0133] b) contacting the analyte with the complex;

[0134] c) adding a substrate of Daphnia lappa luciferase or a substrate analogue to the reaction system;

[0135] d) determining the content of the analyte based on the fluorescence intensity of the reaction system detected after adding a substrate or substrate analog of the Daphnia rapa luciferase. According to some specific embodiments of the present invention, the substrate is selected from at least one of coelenterazine, fluorocoelenterazine, or a coelenterazine derivative.

[0136] According to some specific embodiments of the present invention, the coelenterazine derivative is selected from the coelenterazine derivative ZS2 or the coelenterazine derivative ZS26.

[0137] In another aspect of the present invention, the present invention provides a method for screening a substrate of Daphnia pulex luciferase, comprising:

[0138] Ⅰ) contacting the aforementioned mutant with a substrate to be screened to obtain a reaction mixture;

[0139] II) judging whether the substrate to be screened is a target substrate based on whether the reaction mixture obtained in step I) emits a chemical light signal.

[0140] According to some specific embodiments of the present invention, the reaction mixture obtained in step I) emits a chemical light signal as an indication that the substrate to be screened is a target substrate.

[0141] The present invention is described below with reference to specific examples. It should be noted that these examples are merely illustrative and do not limit the present invention in any way.

[0142] Example 1 Design and Construction of Prokaryotic Expression Plasmid for Wild-Type Daphnia pilosa Luciferase

[0143] In this example, wild-type Daphnia lappa luciferases containing and not containing a signal peptide were constructed to compare the effects of the signal peptide on the wild-type Daphnia lappa luciferase.

[0144] The gene sequence of the wild-type Daphnia pulex luciferase containing a signal peptide (WT Pxluc) is shown in SEQ ID NO: 1, and the amino acids encoded by it are shown in SEQ ID NO: 2, wherein amino acids 1-17 are a signal peptide (bold). The gene sequence of the wild-type Daphnia pulex luciferase without a signal peptide (WT no signal peptide Pxluc: WT-NS Pxluc) is shown in SEQ ID NO: 3, and the amino acids encoded by it are shown in SEQ ID NO: 4.

[0145] The Daphnia leucophylla luciferase gene sequence (SEQ ID NO: 5) containing a signal peptide (pET28a Pxluc WT) for use in the pET28a vector was synthesized by whole gene synthesis technology. The plasmid map is shown in Figure 1. A purification tag containing 6 histidines (6x His) was fused to its C-terminus to facilitate protein purification, and the restriction enzyme sites at both ends are BamHI and NotI.

[0146] Using PCR technology, the gene sequence SEQ ID NO: 6 of the Daphnia pulex luciferase protein containing a signal peptide for use in the pCold vector was synthesized, and a purification tag containing 6 histidines (6x His) was fused to its C-terminus to facilitate protein purification; using PCR technology, the gene sequence SEQ ID NO: 7 of the Daphnia pulex luciferase protein without the signal peptide was synthesized for use in the pCold vector, and a purification tag containing 6 histidines (6x His) was fused to its C-terminus to facilitate protein purification.

[0147]

[0148]

[0149] 1. Get the insert fragment

[0150] A fully synthesized wild-type Daphnia leucophylla luciferase containing a signal peptide (pET28a Pxluc WT) was dissolved in deionized water and diluted to 10 ng / μl as template DNA. The insert was prepared using KOD FXD Neo enzyme, followed by PCR reaction preparation and PCR reaction according to the manufacturer's instructions.

[0151] The primer sequences used in the PCR reactions of wild-type Daphnia pulex luciferase containing a signal peptide (WT-Pxluc) and wild-type Daphnia pulex luciferase without a signal peptide (WT-NS Pxluc) are shown in Table 1, the PCR reaction system is shown in Table 2, the reaction conditions are shown in Table 3, and the number of PCR cycles is 30.

[0152] Table 1

[0153]

[0154] Table 2

[0155] Component Volume (μL) 2x KOD FX Neo PCR Buffer 25 2mM dNTPs 10 10μM Primer insert-F 1.5 10μM Primer insert-R 1.5 Template DNA (10ng / μL) 2.5KOD FX Neo 1 PCR-grade water 8.5 Total Volume 50

[0156] Table 3

[0157]

[0158] Add 0.5 μL of DpnI enzyme to the reaction system and incubate at 37°C for 3 hours to digest the template. Then, gel-recover products of approximately 586 bp and 531 bp, which are the insert fragments. The 586 bp gel-recovered fragment is the insert containing the signal peptide, and the 531 bp gel-recovered fragment is the insert without the signal peptide.

[0159] 2. Obtain linearized vector

[0160] Using the pCold vector as a template, the vector was linearized by PCR to facilitate recombination with the insert. The PCR reaction system and PCR reaction were prepared using KOD FX neo enzyme according to the manufacturer's instructions.

[0161] The primer sequences used in the PCR reaction are shown in Table 4, the PCR reaction system is shown in Table 5, the reaction conditions are shown in Table 6, and the number of PCR cycles is 30.

[0162] Table 4

[0163]

[0164] Table 5

[0165] Component Volume (μL) 2x KOD FX Neo PCR Buffer 25 2mM dNTPs 10 10μM Primer vector-F 1.5 10μM Primer vector-R 1.5 pCold (10ng / μL) 2.5KOD FX Neo enzyme 1 PCR-grade water 8.5 Total volume 50

[0166] Table 6

[0167]

[0168] 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 approximately 4433 bp from the gel, which is the linearized vector vector.

[0169] The insert and vector were recombined using the In-Fusion Cloning Kit (TAKARA) according to the reaction system shown in Table 7 and incubated at 50°C for 15 minutes.

[0170] Table 7

[0171] Component volume (μL) Insert (10 ng / μL) 5 Linearized vector (10 ng / μL) 3 Premix 2 Total volume 10

[0172] 2.5 μL of the above reaction product was transformed into DH5α competent cells and spread on an ampicillin-resistant plate containing a final concentration of 100 μg / mL. The next day, single colonies were picked from the plate, and the plasmids were extracted for sequencing to ensure that the target fragment was correctly inserted into the vector. The obtained plasmids were the wild-type Daphnia pulex luciferase pCold-WT Pxluc containing a signal peptide (Figure 2) and the wild-type Daphnia pulex luciferase pCold WT NS Pxluc without a signal peptide (Figure 3), which were consistent with experimental expectations.

[0173] Example 2 Prokaryotic expression, purification and protein activity assay of Daphnia rapa luciferase

[0174] 1. Prokaryotic expression of Daphnia pulex luciferase

[0175] The expression plasmid pET28a-Pxluc WT was transformed into BL21(DE3) competent cells, and the expression plasmids pCold WT Pxluc and pCold WT-NS Pxluc were transformed into OrigamiB(DE3) Chemically Competent Cell competent cells (Weidi Biotechnology, EC1020S). Plates were spread, and single colonies were picked from the plates and cultured overnight at 37°C. The next day, the cells were diluted 1:100 and transferred to 300 ml of fresh LB medium containing kanamycin (50 μg / ml) or ampicillin (100 μg / ml), respectively. The cultures were shaken at 37°C, 200 rpm, and cultured to an OD600 of ≈0.5-0.6. The cells were then cooled on ice for 1 hour. The inducer IPTG was added to a final concentration of 1 mM and induced overnight at 16°C.

[0176] 2. Purification of Daphnia pulex luciferase

[0177] The induced bacterial precipitate was collected by centrifugation at 8000 rpm / min for 10 min, 30 ml of binding buffer (50 mM Tris, 250 mM NaCl, pH 8.0) and 300 μL of lysozyme were added, and the cells were lysed on ice for 30 min. Ultrasonication (2 s on 3 s off, 60% power) was used for disruption for 30 min, and the cells were centrifuged at 12000 rpm at 4°C for 30 min to separate the supernatant (cell lysate) and the precipitate.

[0178] Add 2 mL of HisTrap FF filler to a manual column (purchased from Sangon, model F506607-0001# affinity chromatography empty column). Rinse the equilibrated filler with 30 mL of binding buffer. Then, add approximately 30 mL of filtered cell lysate. Rinse 10 times (10 mL / wash) with rinse buffer (50 mM Tris, pH 8.0, 250 mM NaCl, 10 mM imidazole). Then, elute the protein 4-5 times with 500 μL of elution buffer (50 mM Tris, pH 8.0, 250 mM NaCl, 300 mM imidazole). Collect the eluted protein and dialyze overnight at 4°C. Protein concentration and purity were determined using a BCA assay kit and SDS-PAGE.

[0179] The eluted protein was determined by SDS-PAGE. The purification results are shown in Figure 4, indicating that the target protein was successfully expressed in BL21 (DE3) cells transformed with the pET28a-Pxluc WT plasmid; the target protein was also successfully expressed in OrigamiB (DE3) cells transformed with the expression plasmids pCold WT Pxluc and pCold WT-NS Pxluc, respectively.

[0180] 3. Determination of luciferase protein activity in Daphnia pulex

[0181] BCA quantification kit (Thermo Scientific TM Pierce TM The protein concentration was accurately determined using a BCA Protein Assay Kit. The purified luciferase obtained above was diluted with a diluent (50 mM Tris-HCl pH 8.0, 100 mM NaCl, 0.1% (v / v) Tween-20). The protein expressed by the pET28a-Pxluc WT plasmid was diluted to 12 μg / ml, and the proteins expressed by the pCold WT Pxluc and pCold WT-NS Pxluc plasmids were diluted to 1 μg / ml. 10 μL of each was added to a black 96-well plate. Then, 90 μL each of the substrate coelenterazine (purchased from Biolabs), fluorocoelenterazine, and coelenterazine derivatives ZS2 and ZS26 (the structural formulas of each compound are shown in Figure 5) diluted to 100 μM with the same solution were added, and the luminescence intensity was read using a microplate reader self-luminescence module. The activity test results of Daphnia pulex luciferase and CTZ (coelenterazine) are shown in Figure 6. It is known that Gluc expressed in OrigamiB (DE3) competent cells has good luminescence activity, which was used as a control group. The results in Figure 6 show that the luminescence value of OrigamiB (DE3) competent cells containing the plasmid pCold WT NS Pxluc is the highest, which is comparable to the luminescence value of OrigamiB (DE3) competent cells containing the plasmid pCold Gluc. Therefore, the luminescence activity of Daphnia pulex luciferase without a signal peptide towards coelenterazine is similar to that of Gluc towards coelenterazine. Compared with the Daphnia pulex luciferase containing a signal peptide, the Daphnia pulex luciferase without a signal peptide has higher activity, and the Daphnia pulex luciferase without a signal peptide was used in subsequent experiments.

[0182] Example 3 Design and Construction of Daphnia rapa luciferase mutant library

[0183] By PCR technology, using the pCold WT-NS Pxluc plasmid sequence as a template, a site-directed saturation mutant library at the G98, Q99, G100, and G101 sites of SEQ ID NO: 2 was synthesized (specific amino acids were mutated to any other amino acids), wherein mutant library 1 was a site-directed saturation mutant library at amino acid site G98 (L1), mutant library 2 was a site-directed saturation mutant library at the Q99 site (L2), mutant library 3 was a site-directed saturation mutant library at amino acid site G100 (L3), mutant library 4 was a site-directed saturation mutant library at the G101 site (L4), mutant library 5 was a combined mutant library at amino acid sites G98 and Q99 (L5), mutant library 6 was a combined mutant library at amino acid sites G100 and G101 (L6), and mutant library 7 was a combined mutant library at amino acid sites G98, Q99, G100, and G101 (L7).

[0184] A pair of primers designed for mutant library L1:

[0185] Upstream primer Primer F1:

[0186] 5'-GATAAATCTATTNNKCAGGGAGGCATAGGTGGCCCTATTG-3' (SEQ ID NO: 14)

[0187] Downstream primer Primer R1:

[0188] 5'-TAGGGCCACCTATGCCTCCTTGMNNAATAGATTTATCTCC-3' (SEQ ID NO: 15)

[0189] A pair of primers designed for mutant library L2:

[0190] Upstream primer Primer F2:

[0191] 5'-GATAAATCTATTGGANNKGGAGGCATAGGTGGCCCTATTG-3' (SEQ ID NO: 16)

[0192] Downstream primer Primer R2:

[0193] 5'-TAGGGCCACCTATGCCTCCMNNTCCAATAGATTTATCTCC-3' (SEQ ID NO: 17)

[0194] A pair of primers designed for mutant library L3:

[0195] Upstream primer Primer F3:

[0196] 5'-GATAAATCTATTGGACAGNNKGGCATAGGTGGCCCTATTG-3' (SEQ ID NO: 18)

[0197] Downstream primer Primer R3:

[0198] 5'-TAGGGCCACCTATGCCMNNCTGTCCAATAGATTTATCTCC-3' (SEQ ID NO: 19)

[0199] A pair of primers designed for mutant library L4:

[0200] Upstream primer Primer F4:

[0201] 5'-GATAAATCTATTGGACAGGGANNKATAGGTGGCCCTATTG-3' (SEQ ID NO: 20)

[0202] Downstream primer Primer R4:

[0203] 5'-TAGGGCCACCTATMNNTCCCTGTCCAATAGATTTATCTCC-3' (SEQ ID NO: 21)

[0204] A pair of primers designed for mutant library L5:

[0205] Upstream primer Primer F5:

[0206] 5'-GATAAATCTATTNNKNNKGGAGGCATAGGTGGCCCTATTG-3' (SEQ ID NO: 22)

[0207] Downstream primer Primer R5:

[0208] 5'-TAGGGCCACCTATGCCTCCMNNMNNAATAGATTTATCTCC-3' (SEQ ID NO: 23)

[0209] A pair of primers designed for mutant library L6:

[0210] Upstream primer Primer F6:

[0211] 5'-GATAAATCTATTGGACAGNNKNNKATAGGTGGCCCTATTG-3' (SEQ ID NO: 24)

[0212] Downstream primer Primer R6:

[0213] 5'-TAGGGCCACCTATMNNMNNCTGTCCAATAGATTTATCTCC-3' (SEQ ID NO: 25)

[0214] A pair of primers designed for mutant library L7:

[0215] Upstream primer Primer F7:

[0216] 5'-GAAGGAGATAAATCTATTNNKNNKNNKNNKATAGGTGGCCCTATTGTTGATA-3' (SEQ ID NO: 26)

[0217] Downstream primer Primer R7:

[0218] 5'-CAATAGGGCCACCTATMNNMNNMNNMNNAATAGATTTATCTCCTTCATAAGT-3' (SEQ ID NO: 27)

[0219] In the nucleic acid sequence, "N" is A / C / G / T, "K" is G / T, and "M" is A / C.

[0220] Table 8 shows the reaction system for error-prone PCR, Table 9 shows the reaction conditions, and the number of PCR cycles is 30.

[0221] Table 8

[0222]

[0223] Table 9

[0224]

[0225] After the reaction is complete, add 0.5 μL of DpnI enzyme to the reaction system and incubate at 37°C for 3 hours to digest the template. The digested product is then transformed into DH5a competent cells, and 10 ml of the culture is cultured overnight to extract the plasmid. The extracted plasmid is transformed into OrigamiB (DE3) Chemically Competent Cells (Weidi Biotechnology, EC1020S). Plates are then plated, and a single colony is picked from the plate. Culture overnight at 37°C. The next day, the colony is diluted 1:100 and transferred to fresh 0.4 ml of LB medium containing 100 μg / ml ampicillin. Cultivate at 37°C with shaking at 200 rpm to an OD600 of approximately 0.5-0.6, then cool on ice for 1 hour. IPTG is added to a final concentration of 1 mM and induced overnight at 16°C.

[0226] 50 μL of induced bacterial solution was added to a black 96-well plate, and then 10 μL of substrate coelenterazine (CTZ) (purchased from Biolabs), fluorocoelenterazine, ZS2, and ZS26 with a final concentration of 100 μM were added respectively. The luminescence intensity was read using a microplate reader self-luminescence module to screen the bacterial solution for mutants (screening criteria: screening was based on the ratio of luminescence intensity, the ratio of the luminescence intensity of the substrate ZS2 or ZS26 to the luminescence intensity of the substrate F-CTZ. If this ratio was greater than the ratio of the luminescence intensity of the wild type to the two substrates, it was considered a dominant mutant).

[0227] Example 4 Expression and purification of Daphnia rapa luciferase mutant library

[0228] 1. Prokaryotic expression of Daphnia pulex luciferase mutants

[0229] The mutant strains screened from the mutant libraries pCold-no sp Pxluc L1, pCold-no sp Pxluc L2, pCold-no sp Pxluc L3, pCold-no sp Pxluc L4, and pCold-no sp Pxluc L5 were cultured overnight at 37°C. The next day, the strains were diluted 1:100 and transferred to 15 ml of fresh LB medium containing 100 μg / ml ampicillin. The cultures were shaken at 37°C, 200 rpm, and cultured to an OD600 of 0.5-0.6. The cells were then cooled on ice for 1 hour. The inducer IPTG was added to a final concentration of 1 mM and induced overnight at 16°C.

[0230] 2. Purification of Daphnia pulex luciferase mutants

[0231] The induced bacterial precipitate was collected by centrifugation at 8000 rpm / min for 10 min, 600 μL binding buffer (50 mM Tris, pH 8.0, 250 mM NaCl) and 6 μL lysozyme were added, lysed on ice for 30 min, ultrasonically disrupted (2 s on 3 s off, 60% power) for 10 min, and centrifuged at 12000 rpm at 4°C for 30 min to separate the supernatant (cell lysate) and precipitate.

[0232] Add 50 μL of HisTrap FF filler to a manual column (purchased from Sangon, model F506607-0001# affinity chromatography column empty column) and rinse the equilibrated filler with 3 ml of binding buffer. Then add 600 μL of filtered cell lysate. Rinse 10 times (3 ml / time) with rinse buffer (50 mM Tris, pH 8.0, 250 mM NaCl, 10 mM imidazole), then elute the protein with 100 μL of elution buffer (50 mM Tris, pH 8.0, 250 mM NaCl, 300 mM imidazole) and collect the eluted protein.

[0233] The protein eluted from the Ni column was dialyzed with dialysis buffer (25 mM Tris, pH 8.0, 250 mM NaCl) at 4°C overnight. Protein concentration and purity were determined by BCA quantitative kit (Thermo Scientific TM Pierce TM The protein was determined by BCA Protein Assay Kit) and SDS-PAGE.

[0234] Example 5 Substrate Specificity Test of Dominant Mutants of Daphnia lanceolata Luciferase

[0235] BCA quantification kit (Thermo Scientific TM Pierce TM The protein concentration was accurately determined using a BCA Protein Assay Kit. Luciferase was diluted to 1 μg / ml with a diluent (50 mM Tris-HCl pH 8.0, 100 mM NaCl, 0.1% (v / v) Tween-20), and 10 μL was added to a black 96-well plate. Then, 90 μL each of the substrates coelenterazine (purchased from Biolabs), fluorocoelenterazine, and coelenterazine derivatives ZS2 and ZS26, diluted to 100 μM with the same solution, were added. Luminescence intensity was read using a microplate reader with a self-luminescence module, and the substrate specificity of the mutants was compared using the activity ratios of ZS2 / F-CTZ and ZS26 / F-CTZ. The activity test results of the dominant specific mutants are shown in Figures 7 and 8. Table 10 below shows the mutation sites of the dominant Daphnia lanceolata luciferase mutants and the substrate specificity of ZS26 / F-CTZ and ZS2 / F-CTZ.

[0236] Table 10

[0237]

[0238]

[0239] The results in Table 10 and Figures 7 and 8 show that the wild-type Daphnia pulex luciferase without a signal peptide has a substrate specificity of approximately 1.77 times for ZS26 / F-CTZ and a substrate specificity of approximately 3.56 times for ZS2 / F-CTZ; the dominant mutant obtained by modifying the enzyme has an increased specificity for the substrate ZS26 / F-CTZ, with a maximum of approximately 74.857 times; and an increased specificity for the substrate ZS2 / F-CTZ, with a maximum of approximately 24.750 times.

[0240] Example 6 Design and Construction of a Eukaryotic Expression Plasmid for Daphnia pilosula luciferase

[0241] The Daphnia rapa luciferase containing a signal peptide was synthesized by PCR for use in the pEE12.4 vector. The gene sequence is shown in SEQ ID NO: 28. The N-terminal signal peptide is followed by a histidine tag (6x His) for purification, and the C-terminal Avitag for biotinylation is provided:

[0242]

[0243] The Daphnia pulex luciferase plasmid for eukaryotic expression used the prokaryotic expression plasmid pET28a Pxluc WT containing a signal peptide as template DNA. The primer sequences used for the PCR reaction to construct the expression plasmid insert were shown in Table 11, the reaction system was shown in Table 12, and the reaction conditions were shown in Table 13.

[0244] Table 11

[0245]

[0246] Table 12

[0247] Component Volume (μL) 2x KOD FX Neo PCR Buffer 25 2mM dNTPs 10 10μM Primer insert-F 1.5 10μM Primer insert-R 1.5 Template DNA pET28a Pxluc WT (10ng / μL) 2.5 KOD FX Neo enzyme 1 PCR-grade water 8.5 Total volume 50

[0248] Table 13

[0249]

[0250] 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 approximately 528 bp from the gel, which is the insert fragment.

[0251] Using the pEE12.4 vector as a template, the vector was linearized by PCR to facilitate recombination with the insert. The PCR reaction system and PCR reaction were prepared using KOD FX neo enzyme according to the manufacturer's instructions. The primer sequences used are shown in Table 14, the PCR reaction system is shown in Table 15, and the PCR reaction conditions are shown in Table 16.

[0252] Table 14:

[0253]

[0254] Table 15:

[0255] Component Volume (μL) 2x KOD FX Neo PCR Buffer 25 2mM dNTPs 10 10μM Primer vector-F 1.5 10μM Primer vector-R 1.5 pEE12.4 plasmid (10ng / μL) 2.5KOD FX Neo 1 PCR-grade water 8.5

[0256] Total volume 50

[0257] Table 16:

[0258]

[0259] 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 7712 bp from the gel, which is the linearized vector.

[0260] The insert and vector obtained in this example were recombined using the Takara In-Fusion Cloning Kit according to the reaction system shown in Table 17. The reaction conditions were incubation at 50° C. for 15 minutes.

[0261] Table 17:

[0262] Component volume (μL) insert (10 ng / μL) 5 vector (10 ng / μL) 3 Premix 2 Total volume 10

[0263] 2.5 μL of the reaction product was transformed into DH5α competent cells and plated onto plates containing ampicillin at 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 that the target fragment was correctly inserted into the vector. The resulting plasmid was pEE12.4-Pxluc WT, the wild-type luciferase for eukaryotic expression. The plasmid map, shown in Figure 9, was consistent with experimental expectations.

[0264] The mutant plasmid pEE12.4 Pxluc P26-95 for eukaryotic expression was synthesized by PCR using the constructed pEE12.4-Pxluc WT as template DNA; the primer sequences are shown in Table 18, the PCR reaction system is shown in Table 19, and the PCR reaction conditions are the same as those in Table 16.

[0265] Table 18:

[0266]

[0267] Table 19:

[0268] Component Volume (μL) 2x KOD FX Neo PCR Buffer 25 2mM dNTPs 10 10μM Primer vector-F 1.5 10μM Primer vector-R 1.5 pEE12.4 Pxluc P26-95 (10ng / μL) 2.5KOD FX Neo 1 PCR-grade water 8.5 Total volume 50

[0269] After the reaction is complete, add 0.5 μL of DpnI enzyme to the reaction system and incubate at 37°C for 3 hours to digest the template. Take 2.5 μL of the digestion product and transform it into DH5α competent cells. Plate it on plates containing ampicillin at a final concentration of 100 μg / mL. The next day, pick a single colony from the plate and extract the plasmid. Sequencing is performed to ensure the correctness of the mutant sequence. The resulting plasmid is the mutant luciferase pEE12.4 Pxluc P26-95 (a luciferase mutant expression vector containing a signal peptide sequence, with mutations at sites G100A and G101P) for eukaryotic expression.

[0270] Example 7 Eukaryotic Expression and Protein Purification of Daphnia pulex Luciferase

[0271] The pEE12.4 Pxluc WT and mutant pEE12.4 Pxluc P26-95 plasmids obtained in Example 6 were purified by ExpiFectamine TM The cells were transfected into 30 mL of Expi-CHO cells according to the instructions of the CHO transfection kit (Gibco, A29129). Seven days after transfection, the cell viability was measured to be less than 90%. The cells were centrifuged at 8000 rpm for 10 min at 4°C and the supernatant was collected.

[0272] 2 mL of HisTrap FF filler was added to each handmade column (purchased from Sangon, model F506607-0001# affinity chromatography column empty column), and the equilibrated filler was rinsed with 30 mL of binding buffer. Then, approximately 30 mL of filtered cell supernatant was added. The column was rinsed 10 times (10 mL / time) with rinse buffer (50 mM Tris, pH 8.0, 250 mM NaCl, 10 mM imidazole), and then the protein was eluted 4-5 times with 500 μL of elution buffer (50 mM Tris, pH 8.0, 250 mM NaCl, 300 mM imidazole). The eluted protein was collected separately. The eluted protein was detected by 12% SDS-PAGE. The purification results are shown in Figure 10, indicating that a relatively pure Pxluc protein can be obtained.

[0273] Example 8 Coupling and activity test of dominant mutants of luciferase

[0274] The Pxluc protein obtained in Example 7 contains an AviTag tag, namely AviTag-Pxluc-WT and mutant AviTag-Pxluc-P26-95, which can be biotinylated by BirA enzyme (Avidity, BIRA500) to Biotin-Avitag-Pxluc-WT and Biotin-AviTag-Pxluc-P26-95. The biotinylation reaction system is shown in Table 18.

[0275] Table 20:

[0276] Component volume (μL) AviTag-Pxluc (1 mg / mL) 12 BiomixA 10 BiomixB10 BirA (1 mg / mL) 3 H2O 65 Total volume 100

[0277] SA-Pxluc can be prepared by adding SA to the system shown in Table 20. The histidine tag on Pxluc can be used for further purification to obtain purer SA-Pxluc. The purification results are shown in Figure 11.

[0278] According to the method described in Example 5, pEE12.4 Pxluc WT and mutant pEE12.4 Pxluc P26-95, as well as the coupled products SA-Pxluc WT and SA-Pxluc P26-95 were tested for activity. The results are shown in Figure 12, indicating that both the eukaryotic expressed Pxluc WT and the mutant Pxluc P26-95 have catalytic activity against the substrate coelenterazine derivatives ZS2 and ZS26, and compared with Pxluc WT, the mutant Pxluc P26-95 has a significantly improved substrate specificity for ZS26 / F-CTZ, and the substrate specificity for ZS26 / F-CTZ is improved. The coupled product SA-Pxluc P26-95 has a significantly improved substrate specificity for ZS26 / F-CTZ compared to SA-Pxluc WT.

[0279] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0280] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A mutant luciferase, wherein Compared with the amino acid sequence shown in SEQ ID NO: 2, the mutant luciferase has at least one of the following mutation sites: positions 98, 99, 100 and 101, and may or may not include a signal peptide amino acid sequence.

2. The mutant luciferase according to claim 1, wherein Compared to the amino acid sequence shown in SEQ ID NO: 2, the mutant luciferase has any one or more combinations of the following mutations (1) to (4): (1) G at position 98 mutated to L, P, Q, S, or T; (2) Q at position 99 is mutated to R, W, I, Y, A, L, F, V, P, E, or M; (3) G at position 100 is mutated to S, Q, R, W, T, A, or L; (4) The G at position 101 is mutated to F, R, S, C, Y, L, I, K, V, or P.

3. The mutant luciferase according to claim 1, wherein Compared with the amino acid sequence shown in SEQ ID NO: 2, the mutant luciferase has any one or two of the following mutation sites: positions 98, 99, 100 and 101, and may or may not include a signal peptide amino acid sequence.

4. The mutant luciferase according to claim 3, wherein Compared to the amino acid sequence shown in SEQ ID NO: 2, the mutant luciferase has any one or two of the following mutations (1) to (4): (1) G at position 98 mutated to L, P, Q, S, or T; (2) Q at position 99 is mutated to R, W, I, Y, A, L, F, V, P, E, or M; (3) G at position 100 is mutated to S, Q, R, W, T, A, or L; (4) The G at position 101 is mutated to F, R, S, C, Y, L, I, K, V, or P.

5. The mutant luciferase according to any one of claims 1 to 4, wherein Compared to the amino acid sequence shown in SEQ ID NO: 2, the mutant luciferase has the following mutations: 1) G at position 98 is mutated to L, and Q at position 99 is mutated to R; or 2) G at position 98 is mutated to P; or 3) G at position 98 is mutated to Q; or 4) G at position 98 is mutated to S, and Q at position 99 is mutated to W; or 5) the Q at position 99 is mutated to I; or 6) Q at position 99 is mutated to Y; or 7) The Q at position 99 is mutated to A; or 8) The Q at position 99 is mutated to L; or 9) Q at position 99 is mutated to F; or 10) G at position 98 is mutated to L, and Q at position 99 is mutated to V; or 11) G at position 98 is mutated to T, and Q at position 99 is mutated to P; or 12) G at position 98 is mutated to L, and Q at position 99 is mutated to E; or 13) Q at position 99 is mutated to M, and G at position 100 is mutated to S; or 14) G at position 100 is mutated to Q, and G at position 101 is mutated to F; or 15) G at position 100 is mutated to R, and G at position 101 is mutated to R; or 16) G at position 100 is mutated to W, and G at position 101 is mutated to F; or 17) G at position 100 is mutated to S; or 18) G at position 100 is mutated to T, and G at position 101 is mutated to S; or 19) G at position 100 is mutated to R, and G at position 101 is mutated to C; or 20) G at position 100 is mutated to A, and G at position 101 is mutated to R; or 21) G at position 100 is mutated to L, and G at position 101 is mutated to Y; or 22) G at position 100 is mutated to S, and G at position 101 is mutated to L; or 23) G at position 101 is mutated to I; or 24) G at position 100 is mutated to L, and G at position 101 is mutated to K; or 25) G at position 100 is mutated to T; or 26) G at position 100 is mutated to A, and G at position 101 is mutated to V; or 27) The G at position 100 mutated to A, and the G at position 101 mutated to P.

6. The mutant luciferase according to any one of claims 1 to 5, wherein The mutant luciferase does not contain a signal peptide amino acid sequence.

7. A nucleic acid molecule, wherein The nucleic acid molecule encodes the mutant luciferase according to any one of claims 1 to 6.

8. An expression vector, wherein Comprising the nucleic acid molecule of claim 7.

9. A recombinant cell, wherein: Carrying the nucleic acid molecule according to claim 7 or the expression vector according to claim 8.

10. The recombinant cell according to claim 9, wherein The recombinant cell is selected from Escherichia coli, yeast or mammalian cells.

11. A method for producing a mutant luciferase, comprising: The expression vector according to claim 8 is introduced into recombinant cells, the recombinant cells are cultured and propagated, and the culture and propagation products are collected to extract or purify the mutant luciferase.

12. A method for detecting a nucleic acid sequence using the mutant luciferase according to any one of claims 1 to 6, the method comprising the following steps: A) using the mutant luciferase according to any one of claims 1 to 6, by chemical coupling, biological coupling, or in the form of a fusion protein, to form a first mutant luciferase complex with a first specific recognition protein and the mutant luciferase; using a second luciferase as a signal protein, by chemical coupling, biological coupling, or in the form of a fusion protein, to form a second luciferase complex with a second specific recognition protein and the second luciferase; B) the first mutant luciferase complex can react with a first substrate to generate a first luminescent signal; the second luciferase complex can react with a second substrate to generate a second luminescent signal; the first mutant luciferase complex does not have a significant cross-substrate reaction with the second substrate, and the second luciferase complex does not have a significant cross-substrate reaction with the first substrate; C) detecting the fluorescent signals and signal combinations of the mutant luciferase and the second luciferase self-luminescence system to distinguish the four bases A, T, G, and C for target nucleic acid sequencing.

13. The method according to claim 12, wherein: When the mutant luciferase and the second luciferase are the same, the first substrate and the second substrate are the same; When the mutant luciferase and the second luciferase are different, the first substrate and the second substrate are different.

14. A nucleic acid sequencing kit comprising the mutant luciferase according to any one of claims 1 to 6.

15. A method for detecting the content of an analyte, comprising the following steps: a) using the mutant luciferase according to any one of claims 1 to 6 as a signal protein, and forming a complex between the specific recognition protein of the analyte and the mutant luciferase by chemical coupling, biological coupling or in the form of a fusion protein; b) contacting the analyte with the complex; c) adding a substrate of Daphnia lappa luciferase or a substrate analogue to the reaction system; d) determining the content of the analyte based on the fluorescence intensity of the reaction system detected after adding the substrate of Daphnia leucophylla luciferase or a substrate analogue.

16. The method according to claim 15, wherein the substrate of the Daphnia pilosa luciferase is at least one selected from coelenterazine, fluorocoelenterazine, or a coelenterazine derivative; Optionally, the coelenterazine derivative is selected from the coelenterazine derivative ZS2 or the coelenterazine derivative ZS26, 17. A method for screening a substrate of Daphnia pulex luciferase, wherein: include: Ⅰ) contacting the mutant luciferase according to any one of claims 1 to 6 with a substrate to be screened to obtain a reaction mixture; II) judging whether the substrate to be screened is a target substrate based on whether the reaction mixture obtained in step I) emits a chemical light signal.

18. The method according to claim 17, wherein The reaction mixture obtained in step I) emits a chemical light signal as an indication that the substrate to be screened is the target substrate.

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