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WO2024000408A1 - 荧光素酶突变体及其应用 - Google Patents

荧光素酶突变体及其应用 Download PDF

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Publication number
WO2024000408A1
WO2024000408A1 PCT/CN2022/102827 CN2022102827W WO2024000408A1 WO 2024000408 A1 WO2024000408 A1 WO 2024000408A1 CN 2022102827 W CN2022102827 W CN 2022102827W WO 2024000408 A1 WO2024000408 A1 WO 2024000408A1
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luciferase
mutates
mutant
substrate
mutated
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PCT/CN2022/102827
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English (en)
French (fr)
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潘璐璐
倪鸣
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青岛华大智造普惠科技有限公司
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Priority to CN202280095644.6A priority Critical patent/CN119156449A/zh
Priority to PCT/CN2022/102827 priority patent/WO2024000408A1/zh
Priority to JP2024576560A priority patent/JP2025522599A/ja
Priority to EP22948513.1A priority patent/EP4549570A1/en
Publication of WO2024000408A1 publication Critical patent/WO2024000408A1/zh
Priority to US19/005,452 priority patent/US20250207107A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0069—Oxidoreductases (1.) acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/66—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving luciferase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y113/00—Oxidoreductases acting on single donors with incorporation of molecular oxygen (oxygenases) (1.13)
    • C12Y113/12—Oxidoreductases acting on single donors with incorporation of molecular oxygen (oxygenases) (1.13) with incorporation of one atom of oxygen (internal monooxygenases or internal mixed function oxidases)(1.13.12)
    • C12Y113/12007—Photinus-luciferin 4-monooxygenase (ATP-hydrolysing) (1.13.12.7), i.e. firefly-luciferase

Definitions

  • the present invention relates to the field of biotechnology. Specifically, the present invention relates to luciferase mutants and their applications, particularly to copepod luciferase mutants and their applications.
  • Luciferase is a type of enzyme that can catalyze the oxidation of luciferin or fatty aldehydes to produce light. It is widely found in insects, bacteria, fungi and marine organisms. It has become an important tool for scientific research and is widely used in life science research, genome sequencing and Analytical technology, clinical medicine and forensic testing, drug screening, environmental monitoring and enzyme-linked testing. Taking advantage of the self-luminescence characteristics of luciferase, luciferase is often used in fields such as live cell detection, protein-protein interaction, protein localization, small interfering RNA silencing technology, and high-throughput drug screening. In the field of biomonitoring technology, luciferase can be used to detect the presence of chemical contaminants.
  • luciferases with good research and development mainly include: firefly luciferase (FLuc), bacterial luciferase (Lux), and those from Renilla luciferase (RLuc), Luciferase extracted from deep-sea shrimp (Oplophorus luciferase, OLuc), marine animal Gaussia princeps (Gaussia luciferase, GLuc), etc.
  • FLuc firefly luciferase
  • Lux bacterial luciferase
  • RLuc Renilla luciferase
  • Luciferase extracted from deep-sea shrimp Oplophorus luciferase, OLuc
  • Gaussia princeps Gaussia luciferase, GLuc
  • Fluc requires cofactors such as ATP, O 2 and Mg 2+ and is non-secreted expression; most of the fluorescence reactions of Lux require flavin mononucleotide (FMN), long-chain aliphatic aldehydes, oxygen and reduced nicotinamide adenine dinucleus Molecules such as nucleotide (NADH); Rluc does not require ATP to emit light, but its fluorescence intensity is weak and it cannot be secreted.
  • FMN flavin mononucleotide
  • NADH nucleotide
  • Rluc does not require ATP to emit light, but its fluorescence intensity is weak and it cannot be secreted.
  • bioluminescent systems Although more than 40 bioluminescent systems have been discovered in nature, only a limited number of them can be exploited.
  • one object of the present invention is to provide a mutant of copepod luciferase Pxluc (Pleuromamma xiphia, also known as Daphnia luciferase).
  • Pxluc copepod luciferase Pxluc
  • the inventors obtained mutants whose substrate specificity for ZS26/F-CTZ was increased by more than 2 times, and mutants whose substrate specificity for ZS2/F-CTZ was increased by more than 4 times.
  • This luciferase can be expressed in prokaryotic and eukaryotic cells.
  • the purification process is simple and is conducive to large-scale production. It has a luminescence brightness similar to Gluc and is easy to detect.
  • This luciferase will be used in many fields such as basic scientific research, biological monitoring and biochemical diagnosis. have a broad vision of application.
  • the first aspect of the present invention provides a mutant luciferase.
  • the mutant luciferase compared with the amino acid sequence shown in SEQ ID NO:2, the mutant luciferase has at least one of the following mutation sites: position 98, position 99, position 100 and position 101 position, and may or may not include a signal peptide amino acid sequence.
  • the mutant luciferase according to one embodiment of the present invention has stronger catalytic activity towards substrates such as coelenterazine, fluorocoelenterazine and coelenterazine derivatives.
  • substrate spectrum is wider, the substrate selection specificity is stronger, and the luminescence brightness is significantly enhanced. It is applicable to the fields of basic scientific research, biological detection technology, immunoassay, biochemical detection or diagnosis when applying the mutant luciferase for luminescence detection. It can be used and has broad application prospects.
  • a second aspect of the invention provides a nucleic acid molecule.
  • 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 stronger catalytic activity towards substrates such as coelenterazine, fluorocoelenterazine and coelenterazine derivatives, and is compared with existing Daphnia luciferase 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, immune detection, and biochemical detection when applying the protein to luminescence detection. It can be used in various fields such as medicine or diagnosis, and has broad application prospects.
  • a third aspect of the invention provides an expression vector.
  • the nucleic acid molecule described in the second aspect is included.
  • the expression vector may include optional control sequences operably linked to the nucleic acid molecule.
  • the control sequence is one or more control sequences that can direct 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 towards substrates such as coelenterazine, fluorocoelenterazine, and coelenterazine derivatives.
  • a fourth aspect of the invention provides a recombinant cell.
  • the nucleic acid molecule described in the second aspect or the expression vector described in the third aspect is carried.
  • the recombinant cells are obtained by transfection or transformation of the expression vector.
  • the recombinant cells can efficiently express the above-mentioned mutant under appropriate conditions, and the mutant has relatively high catalytic activity towards substrates such as coelenterazine, fluorocoelenterazine, and coelenterazine derivatives.
  • a fifth aspect of the present invention provides a method for producing mutant luciferase.
  • the method includes: introducing the expression vector described in the third aspect into recombinant cells, cultivating and propagating the recombinant cells, collecting the cultured propagation products, and extracting or purifying mutant luciferase.
  • a sixth aspect of the present invention provides a method for detecting nucleic acid sequences using the mutant luciferase described in the first aspect.
  • the method for detecting nucleic acid sequences includes the following steps:
  • the second luciferase as a signaling protein, the second specific recognition protein and the second luciferase form a second luciferase complex through chemical coupling, biological coupling or fusion protein;
  • the first mutant luciferase complex can react with the first substrate to generate a first luminescent signal; the second luciferase complex can react with the second substrate to generate a second luminescent signal; the third A 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, and the A first specific recognition protein recognizes the first substrate and specifically binds to it, and the second specific recognition protein recognizes and specifically binds to the second substrate;
  • the four bases A, T, G, and C are distinguished for target nucleic acid sequencing.
  • the mutant luciferase and the second luciferase are the same, the first substrate and the second substrate are the same;
  • the mutant luciferase and the second luciferase are different, the first substrate and the second substrate are different.
  • the method for detecting nucleic acid sequences includes the following steps:
  • a seventh aspect of the present invention provides a nucleic acid sequencing kit.
  • the kit includes the mutant luciferase or luciferase complex described in the first aspect.
  • An eighth aspect of the present invention provides a method for detecting the content of a analyte. According to one embodiment of the invention, the method includes the following steps:
  • the mutant luciferase described in the first aspect as a signaling protein, the specific recognition molecule of the test object is formed with the mutant luciferase through chemical coupling, biological coupling or fusion protein Complex;
  • the mutant can be used as a signaling protein to detect the analyte, for example, by chemical coupling or forming a fusion protein on the mutant and capable of specificity
  • the protein that recognizes the analyte is coupled or fused.
  • the protein that can specifically recognize the analyte will bind to the analyte.
  • the mutant will catalyze its substrate and self-luminesce.
  • a luminescent microplate reader measures the intensity of bioluminescence released when the mutant catalyzes its substrate.
  • 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. Therefore, the kit containing the mutant can be used to accurately detect the content of the analyte.
  • the analyte may be nucleic acid.
  • the mutant provided by the invention has strong catalytic activity towards substrates such as coelenterazine, fluorocoelenterazine and coelenterazine derivatives, and has a wider substrate spectrum than the existing Daphnia luciferase luciferase Or the substrate selection specificity is stronger and the luminescence brightness is significantly enhanced.
  • the method can be used to detect the expression of RNA or protein by nucleic acid, such as the expression level of RNA or protein, the positioning or tracing of protein, and the detection of nucleic acid
  • nucleic acid such as the expression level of RNA or protein
  • the accuracy and sensitivity of the method are significantly higher than the existing Gaussian luciferase, and the results obtained are more accurate.
  • the present invention proposes a method for screening substrates for Daphnia luciferase, including:
  • step II) Based on whether the reaction mixture obtained in step I) emits a chemical light signal, determine whether the substrate to be screened is the target substrate. Utilizing the characteristics of the mutant to produce bioluminescence when combined with the target substrate, the substrate to be screened of interest is brought into contact with the mutant. The mutant will release bioluminescence during the process of catalyzing the substrate to be screened. A chemiluminescent microplate reader determines whether the mutant can catalyze the substrate to be screened to emit chemiluminescence, 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 target substrates.
  • Figure 1 shows the plasmid map of the wild-type Daphnia luciferase prokaryotic expression plasmid pET28a-Pxluc WT containing the signal peptide;
  • Figure 2 shows the plasmid map of the wild-type Daphnia luciferase prokaryotic expression plasmid pCold WT Pxluc containing the signal peptide;
  • Figure 3 shows the plasmid map of the wild-type Daphnia luciferase prokaryotic expression plasmid pCold WT NS Pxluc without signal peptide;
  • Figure 4 shows the results of expression and purification of wild-type Daphnia luciferase in different competent cells.
  • the arrow in the figure points to the target protein band;
  • Figure 5 shows the chemical structural formulas of A substrate coelenterazine, B fluorocoelenterazine, C coelenterazine derivative ZS2, and D coelenterazine derivative ZS26 (D);
  • Figure 6 shows the results of protein level activity test of prokaryotic expressed Daphnia luciferase and Gaussian luciferase
  • Figure 7 shows the detection results of the substrate-specific ZS26/F-CTZ protein level activity of the Daphnia luciferase dominant mutant
  • Figure 8 shows the detection results of the substrate-specific ZS2/F-CTZ protein level activity of the Daphnia luciferase dominant mutant
  • Figure 9 shows the plasmid map of the wild-type Daphnia luciferase eukaryotic expression plasmid pEE12.4-Pxluc WT containing the signal peptide;
  • Figure 10 is a diagram showing the results of expression and purification of Daphnia luciferase in eukaryotic cells.
  • the arrow in the figure points to the target protein band;
  • Figure 11 shows the results of rapula luciferase coupling to SA protein before and after purification
  • Figure 12 shows the substrate specificity ZS26 of the eukaryotic expression of sword-point luciferase pEE12.4 Pxluc WT and mutant pEE12.4 Pxluc P26-95, and the coupled SA-Pxluc WT and SA Pxluc P26-95. /F-CTZ and ZS2/F-CTZ protein level activity detection results.
  • first and second are used for descriptive purposes only and cannot be understood as indicating or implying relative importance or implicitly indicating the quantity of indicated technical features. Therefore, features defined as “first” and “second” may explicitly or implicitly include at least one of these features.
  • “plurality” means at least two, such as two, three, etc., unless otherwise expressly and specifically limited.
  • luciferase genes have been widely used as reporter genes to study the expression intensity and transcriptional regulation of foreign genes under different promoters.
  • luciferase can be used to detect the presence of chemical contaminants.
  • it also has broad application prospects in fields such as immunoassay and biochemical diagnosis.
  • the invention proposes a luciferase mutant. Compared with the amino acid sequence shown in SEQ ID NO:2, the mutant has any one or more combinations of the following mutation sites : Position 98, 99, 100 and 101, and may or may not include the signal peptide amino acid sequence.
  • the above amino acid sequence is modified according to the mutation site of one embodiment of the present invention, 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 luciferase substrate spectrum, the substrate spectrum is broader, the specificity is stronger, and the luminescence brightness is significantly enhanced. In basic scientific research, biological detection technology, and immune detection of the mutant, it is It can be used in detection, biochemical detection or diagnosis and other fields, and has broad application prospects.
  • it can be used as a reporter gene to quantitatively detect DNA, RNA, transcription factors, proteins or cells, etc.; it can be used as a luminescent signal protein in fusion proteins to quantitatively detect DNA, RNA, transcription factors, proteins or cells. Detect target small molecules, proteins, etc.
  • reporter gene is a molecular biology concept, which refers to a type of gene that is expressed in cells, tissues/organs, or individuals under specific circumstances and causes them to produce traits that are easy to detect and would not otherwise be produced by experimental materials. , a gene that codes for a protein or enzyme that can be detected. As a reporter gene, it must meet the following conditions in terms of genetic selection and screening detection: 1. It has been cloned and the full sequence has been determined; 2. The expression product does not exist in the recipient cells, that is, there is no background, and when it is transfected There are no similar endogenous expression products in the cells; 3.
  • the expression products can be quantitatively measured; when used, including but not limited to the following methods of use: fusing the reporter gene and the gene expression regulatory sequence to form a chimeric gene, Or it can be fused with other target genes to express nucleic acids under the control of regulatory sequences, so that its expression products can be used to detect the expression regulation of target genes and study nucleic acids.
  • nucleic acid expression may refer to the expression of DNA into RNA; or the expression of DNA into RNA, which is further expressed into protein; or the expression of RNA into protein, that is, the product of nucleic acid expression in this article may be RNA or protein.
  • “Chemiluminescence” is also called cold light. It is light radiation produced by chemical reactions without any light, heat or electric field excitation. There is also chemical luminescence in living systems, called bioluminescence, such as the light emitted by fireflies, certain bacteria or fungi, protozoa, worms, and crustaceans. In this application, the mutant can perform self-luminescence, that is, chemiluminescence.
  • the above-mentioned mutants may further include at least one of the following additional technical features:
  • the gene sequence of wild-type Daphnia luciferase (WT Pxluc) containing a signal peptide is as shown in SEQ ID NO: 1, and its encoded amino acid is as shown in SEQ ID NO: 2
  • amino acids 1-17 are signal peptides (bold)
  • the gene sequence of wild-type Daphnia luciferase (WT no signal peptide Pxluc: WT-NS Pxluc) without signal peptide is as SEQ ID NO:3 is shown, and the amino acid encoded by it is shown as SEQ ID NO:4.
  • SEQ ID NO:1-4 sequence details are as follows:
  • the mutant compared to the amino acid sequence shown in SEQ ID NO:2, the mutant has any one or more combinations of the following (1)-(4) mutations:
  • the G at position 98 is mutated to L or P or Q or S or T;
  • Q at position 99 is mutated to R or W or I or Y or A or L or F or V or P or E or M;
  • the G at position 100 is mutated to S or Q or R or W or T or A or L;
  • the G at position 101 is mutated to F or R or S or C or Y or L or I or K or V or P.
  • the mutant luciferase compared to the amino acid sequence shown in SEQ ID NO: 2, has any one or two of the following mutation sites: position 98, position 99, Positions 100 and 101, and may or may not include the signal peptide amino acid sequence.
  • the mutant compared with the amino acid sequence shown in SEQ ID NO:2, the mutant has any one or two of the following (1)-(4) mutations:
  • the G at position 98 is mutated to L or P or Q or S or T;
  • Q at position 99 is mutated to R or W or I or Y or A or L or F or V or P or E or M;
  • the G at position 100 is mutated to S or Q or R or W or T or A or L;
  • the G at position 101 is mutated to F or R or S or C or Y or L or I or K or V or P. According to some specific embodiments of the invention, compared to the amino acid sequence shown in SEQ ID NO: 2, the mutant has the following mutations:
  • the G at position 100 is mutated to L, and the G at position 101 is mutated to K; or
  • the catalytic activity of the obtained protein towards substrates such as coelenterazine, fluorocoelenterazine and coelenterazine derivatives Stronger, compared with the existing Daphnia luciferase substrate spectrum, the substrate spectrum is wider and the luminescence brightness is significantly enhanced. In practical applications, the detection accuracy is significantly improved.
  • substrates such as coelenterazine, fluorocoelenterazine and coelenterazine derivatives Stronger, compared with the existing Daphnia luciferase substrate spectrum, the substrate spectrum is wider and the luminescence brightness is significantly enhanced. In practical applications, the detection accuracy is significantly improved.
  • the protein to luminescence detection It can be used in scientific research, biological detection technology, immune detection, biochemical detection or diagnosis and other fields, and has broad application prospects.
  • the mutant luciferase does not contain a signal peptide sequence.
  • the mutant luciferase may have any one or more of positions 98, 99, 100 and 101 compared to the amino acid sequence shown in SEQ ID NO:2. Luciferase mutated at a combination of sites (for example, it can be 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).
  • the mutant luciferase may also have any one of positions 98, 99, 100 and 101 compared to the amino acid sequence shown in SEQ ID NO: 2, or Multiple combination sites, and the mutant luciferase does not contain a signal peptide amino acid sequence.
  • amino acids 1-17 are signal peptides.
  • the inventor found that whether it is the complete SEQ ID NO:2 containing the signal peptide or the sequence without the signal peptide, as long as it contains Mutation occurs at any one or more combination sites at positions 98, 99, 100 and 101 (these site numbers are based on the SEQ ID NO:2 sequence), and the mutant fluorescence with this amino acid sequence is obtained
  • All luciferases have strong catalytic activity for substrates such as coelenterazine, fluorocoelenterazine and coelenterazine derivative ZS2, and have a higher substrate spectrum than the existing Daphnia luciferase. Broader, stronger specificity, and significantly enhanced luminescence brightness.
  • the mutant is a non-secreted protein or a secreted protein.
  • the invention provides a nucleic acid molecule encoding the aforementioned mutant.
  • nucleic acids mentioned in the description and claims of the present invention actually include either or both complementary double strands.
  • nucleic acid sequence in this application includes DNA form or RNA form, and disclosing one of them means that the other one is also disclosed.
  • the invention provides 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 optional control sequences operably linked to the nucleic acid molecule. Wherein, the control sequence is one or more control sequences that can direct 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 towards substrates such as coelenterazine, fluorocoelenterazine, and coelenterazine derivatives.
  • the present invention provides a recombinant cell carrying the aforementioned nucleic acid molecule, expression vector or mutant.
  • the recombinant cells are obtained by transfection or transformation of the expression vector.
  • the recombinant cells can efficiently express the above-mentioned mutant under appropriate conditions, and the catalytic activity of the mutant on substrates such as coelenterazine, fluorocoelenterazine, and coelenterazine derivatives Stronger.
  • substrates such as coelenterazine, fluorocoelenterazine, and coelenterazine derivatives Stronger.
  • substrates such as coelenterazine, fluorocoelenterazine, and coelenterazine derivatives Stronger.
  • the existing Daphnia luciferase substrate it has a wider substrate spectrum, stronger specificity, and significantly enhanced luminescence brightness. It is suitable for basic scientific research and biological detection technology when applying the protein to luminescence detection. It can be used in fields such as
  • the above-mentioned recombinant cells may further include at least one of the following additional technical features:
  • the recombinant cells are E. coli, yeast or mammalian cells.
  • the recombinant cells are 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 human embryonic kidney cells. and other mammalian cells.
  • the recombinant cells do not include animal germ cells, fertilized eggs or embryonic stem cells.
  • the present invention provides a method for producing mutant luciferase, which includes introducing the aforementioned expression vector into recombinant cells, cultivating and propagating the recombinant cells, collecting the cultured propagation products, and extracting or purifying the mutant luciferase. .
  • the present invention provides the use of the aforementioned mutant luciferase and corresponding substrates in detecting nucleic acid sequences.
  • mutant luciferase and “luciferase mutant” or “mutant” can be equivalently replaced.
  • test kits and preparation kits Use of test kits and preparation kits
  • the present invention proposes a kit for detecting the content of a substance to be tested, wherein the kit contains the luciferase mutant described above, and the kit is used to detect the content of a substance to be tested.
  • the specific recognition protein of the substance is suitable for forming a complex with the mutant.
  • the kit further includes a substrate or an analog of the substrate of Daphnia daphnida luciferase.
  • the substrate is selected from at least one of coelenterazine, fluorocoelenterazine or coelenterazine derivatives.
  • the coelenterazine derivative is selected from coelenterazine derivative ZS2 or coelenterazine derivative ZS26.
  • the kit further includes a specific recognition protein of the test substance.
  • the present invention provides a nucleic acid sequencing kit, which includes the aforementioned mutant luciferase.
  • the present invention proposes the use of the aforementioned mutant in preparing a kit for detecting the content of a substance to be tested, and the specific recognition protein of the substance to be tested is suitable for to form complexes with the mutants.
  • the mutant can be used as a signaling protein to detect the analyte, for example, by chemical coupling or forming a fusion protein on the mutant and capable of specificity
  • the protein that recognizes the analyte is coupled or fused. When it is in the same system as the substrate of the analyte or mutant, the protein that specifically recognizes the analyte will interact with the analyte.
  • the mutant When the analyte binds, the mutant will catalyze its substrate and perform self-luminescence.
  • the intensity of the bioluminescence released during the process of the mutant catalyzing its substrate is measured by a chemiluminescence microplate reader.
  • the intensity of the bioluminescence can reflect the intensity of the bioluminescence.
  • the content of the analyte is judged by the level of its activity.
  • the mutant Before the substrate binds to the mutant, the non-specific binding protein in the system needs to be eluted to eliminate the interference of other factors on the detection results. Therefore, the mutant can be used to prepare a kit and accurately detect the content of the test substance.
  • the above-mentioned uses may further include at least one of the following additional technical features:
  • the kit further includes a substrate or an analogue of the substrate of Daphnia luciferase.
  • the substrate is selected from at least one of coelenterazine, fluorocoelenterazine or coelenterazine derivatives.
  • the substrate is not particularly limited.
  • Substances that can chemically react with the mutant are included in this range and are not limited to chemiluminescence reactions. Those skilled in the art can Experiments require changing different substrates.
  • the coelenterazine derivative is selected from coelenterazine derivative ZS2 or coelenterazine derivative ZS26.
  • the kit further includes a specific recognition protein of the test substance.
  • the invention proposes a method for detecting substance content, which includes the following steps:
  • the present invention provides a method for detecting the content of a analyte, which includes the following steps:
  • mutant luciferase as a signaling protein, form a complex with the specific recognition protein of the test object and the mutant luciferase through chemical coupling, biological coupling or fusion protein ;
  • the substrate is selected from at least one of coelenterazine, fluorocoelenterazine or coelenterazine derivatives.
  • the coelenterazine derivative is selected from coelenterazine derivative ZS2 or coelenterazine derivative ZS26.
  • the present invention proposes a method for screening substrates for Daphnia luciferase, including:
  • step II) Based on whether the reaction mixture obtained in step I) emits a chemical light signal, determine whether the substrate to be screened is the target substrate.
  • the reaction mixture obtained in step I) emits a chemical light signal, which is an indication that the substrate to be screened is a target substrate.
  • wild-type Daphnia luciferase containing a signal peptide and one without a signal peptide were constructed to compare the effect of the signal peptide on the wild-type Daphnia luciferase luciferase.
  • the gene sequence of the wild-type Daphnia luciferase (WT Pxluc) containing the signal peptide is as shown in SEQ ID NO: 1, and its encoded amino acids are as shown in SEQ ID NO: 2, with positions 1-17 The amino acid is the signal peptide (bold).
  • the gene sequence of wild-type Daphnia luciferase (WT no signal peptide Pxluc: WT-NS Pxluc) without signal peptide is shown in SEQ ID NO:3, and its encoding The amino acid is shown in SEQ ID NO: 4.
  • the gene sequence SEQ ID NO: 6 of Daphnia luciferase protein containing signal peptide for pCold vector was synthesized, and its C-terminus was fused with a purified protein containing 6 histidines (6x His) tag to facilitate protein purification;
  • the gene sequence of Daphnia luciferase protein with the signal peptide removed for the pCold vector was synthesized, SEQ ID NO: 7, and its C-terminus was fused with 6 histidines. (6x His) purification tag to facilitate protein purification.
  • the fully gene-synthesized wild-type Daphnia luciferase (pET28a Pxluc WT) containing signal peptide was dissolved in deionized water and diluted to 10ng/ ⁇ l as template DNA.
  • KOD FXD Neo enzyme Use KOD FXD Neo enzyme and follow its instructions to prepare the PCR reaction system and perform the PCR reaction to prepare the insert.
  • the primer sequences used in the PCR reaction of the wild-type Daphnia japonica luciferase containing the signal peptide (WT-Pxluc) and the wild-type Daphnia japonica luciferase without the signal peptide (WT-NS Pxluc) are as follows: Table 1 is shown, 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.
  • the 586 bp gel recovery fragment is an insert fragment containing the signal peptide
  • the 531 bp gel recovery fragment is an insert fragment not containing the signal peptide
  • the vector was linearized by PCR to facilitate recombination with the inserted fragment.
  • KOD FX neo enzyme and follow its instructions to prepare the PCR reaction system and carry out the PCR reaction.
  • 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.
  • TAKARA Use In-Fusion Cloning Kit
  • 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 (WEB, EC1020S), respectively.
  • Spread a plate pick a single colony from the plate, culture it overnight at 37°C, dilute it at a ratio of 1:100 the next day, and transfer it to fresh 300ml of kanamycin (50 ⁇ g/ml) or ampicillin (100 ⁇ g/ml). ) in LB medium, culture at 37°C with shaking at 200 rpm until OD600 ⁇ 0.5-0.6, and cool on ice for 1 hour. Add the inducer IPTG at a final concentration of 1 mM and induce overnight at 16°C.
  • the eluted protein was measured by SDS-PAGE.
  • the purification results are shown in Figure 4, indicating that the BL21 (DE3) cells transformed with pET28a-Pxluc WT plasmid successfully expressed the target protein; the transformed expression plasmids pCold WT Pxluc and pCold WT-NS Pxluc OrigamiB(DE3) also successfully expressed the target protein respectively.
  • the concentration of the protein was accurately determined using a BCA quantification kit (Thermo Scientific TM Pierce TM BCA Protein Assay Kit), and the purified luciferase obtained above was diluted with diluent (50mM Tris-HCl pH 8.0, 100mM NaCl, 0.1% (v /v)Tween-20), dilute the protein expressed by pET28a-Pxluc WT plasmid to 12 ⁇ g/ml, dilute the protein expressed by pCold WT Pxluc and pCold WT-NS Pxluc plasmid to 1 ⁇ g/ml, take 10 ⁇ L of each and add black 96 orifice plate.
  • diluent 50mM Tris-HCl pH 8.0, 100mM NaCl, 0.1% (v /v)Tween-20
  • each of the substrate coelenterazine purchased from Biolab
  • fluorocoelenterazine fluorocoelenterazine
  • coelenterazine derivatives ZS2 and ZS26 diluted to 100 ⁇ M with the same solution (the structural formula of each compound is shown in Figure 5)
  • the activity test results of Daphnia luciferase and CTZ (coelenterazine) are shown in Figure 6. It is known that the Gluc expressed in OrigamiB (DE3) competent cells has better luminescence activity and is used as a control group.
  • mutant library 1 is a site-directed saturation mutant library (L1) at amino acid position G98
  • mutant library 2 is a site-directed saturation mutant library (L2) at amino acid position Q99
  • mutant library 3 is a site-directed saturation mutant library at amino acid position G98 (L2).
  • mutant library 4 is a site-directed saturation mutant library (L4) at site G101
  • mutant library 5 is a combined mutant library at amino acid sites G98 and Q99 ( L5)
  • mutant library 6 is a combined mutant library at amino acid positions G100 and G101 (L6)
  • mutant library 7 is a combined mutant library at amino acid positions G98, Q99, G100 and G101 (L7) .
  • N stands for A/C/G/T
  • K stands for G/T
  • M stands for A/C
  • Table 8 shows the reaction system for error-prone PCR
  • Table 9 shows the reaction conditions
  • the number of PCR cycles is 30.
  • CTZ substrate coelenterazine
  • ZS2 fluorocoelenterazine
  • ZS26 fluorocoelenterazine
  • Screening standard screen based on the ratio of the 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 is greater than the ratio of the wild type to the luminescence intensity of the two substrates, the mutant is considered dominant).
  • the mutant bacterial liquid selected from the mutant library pCold-no sp Pxluc L1, pCold-no sp Pxluc L2, pCold-no sp Pxluc L3, pCold-no sp Pxluc L4, pCold-no sp Pxluc L5 was cultured overnight at 37°C. The next day, dilute the solution at a ratio of 1:100, transfer it to 15 ml of fresh LB medium containing ampicillin (100 ⁇ g/ml), and culture with shaking at 37°C and 200 rpm until OD600 ⁇ 0.5-0.6, and cool on ice for 1 hour. Add the inducer IPTG at a final concentration of 1 mM and induce overnight at 16°C.
  • the protein eluted from the Ni column was dialyzed with dialysis buffer (25mM Tris, pH 8.0, 250mM NaCl) overnight at 4°C. Protein concentration and purity distribution were determined by BCA quantitative kit method (Thermo Scientific TM Pierce TM BCA Protein Assay Kit) and SDS-PAGE method.
  • the concentration of the protein was accurately determined using a BCA quantification kit (Thermo Scientific TM Pierce TM BCA Protein Assay Kit), and luciferase was diluted with diluent (50mM Tris-HCl pH 8.0, 100mM NaCl, 0.1% (v/v) Tween-20 ) dilute to 1 ⁇ g/ml, add 10 ⁇ L to a black 96-well plate. Then add 90 ⁇ L each of the substrate coelenterazine (purchased from Biolab), fluorocoelenterazine, and coelenterazine derivatives ZS2 and ZS26 diluted with the same solution to 100 ⁇ M, and read them with the self-luminescence module of a microplate reader.
  • diluent 50mM Tris-HCl pH 8.0, 100mM NaCl, 0.1% (v/v) Tween-20 ) dilute to 1 ⁇ g/ml, add 10 ⁇ L to a black 96-well plate. Then add 90
  • Luminescence intensity, and the substrate specificity of the mutants was compared with the activity ratio of ZS2/F-CTZ and ZS26/F-CTZ.
  • the activity test results of the dominant specific mutants are shown in Figure 7 and Figure 8.
  • Table 10 below shows the mutation sites of the dominant mutants of Daphnia luciferase and the results of substrate specificity of ZS26/F-CTZ and ZS2/F-CTZ.
  • the results in Table 10 and Figures 7 and 8 show that the substrate specificity of wild-type Daphnia luciferase without signal peptide for ZS26/F-CTZ is approximately 1.77 times that of ZS2/F-CTZ.
  • the substrate specificity is about 3.56 times; the dominant mutant obtained by modifying the enzyme has an increased specificity for the substrate ZS26/F-CTZ, up to about 74.857 times; its specificity for the substrate ZS2/F- CTZ specificity increased, up to approximately 24.750 times.
  • Daphnia luciferase containing signal peptide for pEE12.4 vector was synthesized.
  • the gene sequence is shown in SEQ ID NO: 28. Its N-terminal signal peptide is followed by histidine for purification.
  • the Daphnia luciferase plasmid used for eukaryotic expression uses the prokaryotic expression plasmid pET28a Pxluc WT containing the signal peptide as the template DNA.
  • the primer sequences used in the PCR reaction to construct the expression plasmid insert are shown in Table 11, and the reaction system is as follows As shown in Table 12, the reaction conditions are as shown in Table 13.
  • the vector was linearized by PCR to facilitate recombination with the inserted fragment.
  • 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.
  • the obtained plasmid is the wild-type luciferase pEE12.4-Pxluc WT for eukaryotic expression.
  • the obtained plasmid is detected.
  • the detected plasmid map is shown in Figure 9. In line with experimental expectations.
  • 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 sequence is shown in Table 18, and the PCR reaction system is shown in Table 18 As shown in 19, the PCR reaction conditions are the same as Table 16.
  • the reaction After the reaction is completed, add 0.5 ⁇ L DpnI enzyme to the reaction system and incubate at 37°C for 3 hours to digest the template. Take 2.5 ⁇ L of the enzyme digestion reaction product and transform it into DH5 ⁇ competent cells, and spread it on an ampicillin-resistant plate containing a final concentration of 100 ⁇ g/mL. The next day, single clones are picked from the plate and the plasmid is extracted. Perform sequencing to ensure that the mutant sequence is correct. The obtained plasmid is the mutant luciferase pEE12.4 Pxluc P26-95 for eukaryotic expression (luciferase mutant expression vector containing signal peptide sequence, mutation site G100A, G101P).
  • the pEE12.4 Pxluc WT and mutant pEE12.4 Pxluc P26-95 plasmids obtained in Example 6 were respectively transfected into 30 mL Expi-CHO cells according to the instructions of ExpiFectamine TM CHO transfection kit (Gibco, A29129), and transfected for 7 days. Later, when the cell viability was measured to be less than 90%, the supernatant was collected by centrifugation at 4°C and 8000 rpm for 10 min.
  • the Pxluc protein obtained in Example 7 contains AviTag tags, namely AviTag-Pxluc-WT and mutant AviTag-Pxluc-P26-95, which can be biotinylated by BirA enzyme (Avidity, BIRA500) into Biotin-Avitag-Pxluc-WT and Biotin-AviTag-Pxluc-P26-95, the biotinylation reaction system is shown in Table 18.
  • 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.
  • references to the terms “one embodiment,” “some embodiments,” “an example,” “specific examples,” or “some examples” or the like means that specific features are described in connection with the embodiment or example. , structures, materials or features are included in at least one embodiment or example of the invention. In this specification, the schematic expressions of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the specific features, structures, materials or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, those skilled in the art may combine and combine different embodiments or examples and features of different embodiments or examples described in this specification unless they are inconsistent with each other.

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Abstract

提供了荧光素酶突变体及其应用，具体提供了桡足类荧光素酶突变体及其应用。通过对Pxluc进行蛋白定向进化，获得对ZS26/F-CTZ的底物特异性提高2倍以上的突变体，对ZS2/F-CTZ的底物特异性提高4倍以上的突变体。此荧光素酶可在原核表达，纯化工艺简单，有利于规模生产；具有与Gluc相似的发光亮度，易于检测，该荧光素酶将在基础科研、生物监测及生化诊断等多领域具有广阔的应用前景。

Description

荧光素酶突变体及其应用 技术领域
本发明涉及生物技术领域，具体的，本发明涉及荧光素酶突变体及其应用，特别涉及桡足类荧光素酶突变体及其应用。
背景技术
荧光素酶是能够催化荧光素或脂肪醛氧化发光的一类酶，广泛存在于昆虫、细菌、真菌和海洋生物中，已经成为科学研究的重要工具，被广泛应用于生命科学研究、基因组测序及分析技术、临床医学及法医学检测、药物筛选、环境监测和酶联检测等领域。利用荧光素酶自发光的特点，荧光素酶常应用于活细胞检测、蛋白与蛋白相互作用、蛋白定位、小干扰RNA沉默技术、高通量药物筛选等领域。在生物监测技术领域，荧光素酶可用于检测化学污染物的有无。另外，在免疫检测、生化诊断等领域亦具有广阔的应用前景。且作为检测不同启动子下外源基因表达强度和转录调控研究的报告基因，需要多种自发光亮度相近，且催化底物不同的荧光素酶联合使用。
现阶段，具有较好的研究和开发的荧光素酶主要包括：萤火虫荧光素酶(firefly luciferase，FLuc)、细菌荧光素酶(bacterial luciferase,Lux)，以及从海肾(Renilla luciferase，RLuc)、深海虾(Oplophorus luciferase，OLuc)、海洋动物Gaussia princeps(Gaussia luciferase，GLuc)等中提取的荧光素酶等。其中Fluc需要ATP、O 2和Mg 2+等辅助因子，为非分泌型表达；Lux的荧光反应大多需要黄素单核苷酸(FMN)、长链脂肪醛、氧和还原型烟酰胺腺嘌呤二核苷酸(NADH)等分子；Rluc发光不需要ATP，但其荧光强度较弱，且同样不能分泌型表达。
尽管在自然界中已经发现了40余种生物发光系统，但是能被开发利用的非常有限。
发明内容
本发明旨在至少在一定程度上解决相关技术中的技术问题之一。为此，本发明的一个目的在于提供一种桡足类荧光素酶Pxluc(Pleuromamma xiphia,也称为剑乳点水蚤荧光素酶)的突变体。发明人通过对Pxluc进行蛋白定向进化，获得对ZS26/F-CTZ的底物特异性提高2倍以上的突变体，对ZS2/F-CTZ的底物特异性提高4倍以上的突变体。此荧光素酶可在原核和真核细胞表达，纯化工艺简单，有利于规模生产；具有与Gluc相似的发光亮度，易于检测，该荧光素酶将在基础科研、生物监测及生化诊断等多领域具有广阔的应用前景。
为此，本发明第一方面提供一种突变荧光素酶。根据本发明的一个实施方案，相较于SEQ ID NO:2所示的氨基酸序列，所述突变荧光素酶具有以下突变位点中的至少之一：第98位、99位、100位和101位，并且可以包括或者不包括信号肽氨基酸序列。根据本发明一个实施方案的突变荧光素酶对腔肠素、氟代腔肠素以及腔肠素衍生物等底物的催化活性较强，相较于现有的剑乳点水蚤荧光素酶底物谱更广、底物选择特异性更强、发光亮度显著增强，在应用到所述突变荧光素酶进行发光检测的基础科学研究、生物检测技术、免疫检测、生化检测或诊断等领域均可使用，具有广阔的应用前景。
本发明第二方面提供一种核酸分子。根据本发明的一个实施方案，所述核酸分子编码第一方面所述的突变荧光素酶。根据本发明一个实施方案的核酸分子编码的突变体(突变荧光素酶)对腔肠素、氟代腔肠素以及腔肠素衍生物等底物的催化活性较强，相较于现有的剑乳点水蚤荧光素酶底物谱更广或底物选择特异性更强、发光亮度显著增强，在应用到所述蛋白进行发光检测的基础科学研究、生物检测技术、免疫检测、生化检测或诊断等领域均可使用，具有广阔的应用前景。
本发明第三方面提供一种表达载体。根据本发明的一个实施方案，包含第二方面所述的核酸分子。所述表达载体可包括可选的控制序列，所述控制序列与所述核酸分子可操作地连接。其中，所述控制序列为可指导所述核酸分子在宿主中表达的一个或多个控制序列。本发明的一个实施方案所提出的表达载体可在适合的宿主细胞中高效表达蛋白，获得的蛋白对腔肠素、氟代腔肠素以及腔肠素衍生物等底物的催化活性较强，相较于现有的剑乳点水蚤荧光素酶底物谱更广或底物选择特异性更强、发光亮度显著增强，在应用到所述蛋白进行发光检测的基础科学研究、生物检测技术、免疫检测、生化检测或诊断等领域均可使用，具有广阔的应用前景。
本发明第四方面提供一种重组细胞。根据本发明的一个实施方案，携带第二方面所述的核酸分子或 第三方面所述的表达载体。所述重组细胞是通过转染或者转化所述表达载体获得的。根据本发明的一个实施方案，所述重组细胞在合适条件下可高效表达上述突变体，所述突变体对腔肠素、氟代腔肠素以及腔肠素衍生物等底物的催化活性较强，相较于现有的剑乳点水蚤荧光素酶底物谱更广或底物选择特异性更强、发光亮度显著增强，在应用到所述蛋白进行发光检测的基础科学研究、生物检测技术、免疫检测、生化检测或诊断等领域均可使用，具有广阔的应用前景。
本发明第五方面提供一种产生突变荧光素酶的方法。根据本发明的一个实施方案，包括：将第三方面所述的表达载体导入重组细胞，培养繁殖重组细胞，收集培养繁殖产物提取或纯化突变荧光素酶。
本发明第六方面提供一种利用第一方面所述突变荧光素酶检测核酸序列的方法。根据本发明的一个实施方案，检测核酸序列的方法包括以下步骤：
A)以第一方面所述突变荧光素酶通过化学偶联、生物偶联或融合蛋白的形式，将第一特异性识别蛋白与所述突变荧光素酶形成第一突变荧光素酶复合物；以第二荧光素酶为信号蛋白，通过化学偶联、生物偶联或融合蛋白的形式，将第二特异性识别蛋白与第二荧光素酶形成第二荧光素酶复合物；
B)所述第一突变荧光素酶复合物能与第一底物反应生成第一发光信号；所述第二荧光素酶复合物能与第二底物反应生成第二发光信号；所述第一突变荧光素酶复合物与所述第二底物不具有显著的交叉底物反应，且所述第二荧光素酶复合物与所述第一底物不具有显著的交叉基底反应，所述第一特异性识别蛋白识别所述第一底物并特异性结合，所述第二特异性识别蛋白识别所述第二底物并特异性结合；
C)通过检测所述突变荧光素酶和所述第二荧光素酶自发光体系的荧光信号及信号组合，来分辨A、T、G、C四种碱基进行目标核酸测序。
根据本发明的一个实施方案，当所述突变荧光素酶和所述第二荧光素酶相同时，所述第一底物和所述第二底物相同；
当所述突变荧光素酶和所述第二荧光素酶不同时，所述第一底物和所述第二底物不同。
根据本发明的另一个实施方案，检测核酸序列的方法包括以下步骤：
1)将标记了亲和标记和具有可逆阻断修饰的不同碱基和待测模板在聚合酶的作用下发生聚合反应；2)加入步骤A中的多种荧光素酶复合物，通过特异性识别不同的亲和标记，使多种荧光素酶复合物偶联在不同碱基上；3)加入不同底物，通过检测底物的光学信号或其组合确定聚合的碱基类型；4)加入切除试剂，切除阻断基团和连接基团，准备进行下一轮聚合反应。
本发明第七方面提供一种核酸测序试剂盒。根据本发明的一个实施方案，所述试剂盒包括第一方面所述的突变荧光素酶或荧光素酶复合物。
本发明第八方面提供一种检测待测物含量的方法。根据本发明的一个实施方案，所述方法包括以下步骤：
a)以第一方面所述的突变荧光素酶为信号蛋白，通过化学偶联、生物偶联或融合蛋白的形式，将所述待测物的特异性识别分子与所述突变荧光素酶形成复合物；
b)将所述待测物与所述复合物接触；
c)向反应体系中加入剑乳点水蚤荧光素酶的底物或底物的类似物；
d)基于加入所述剑乳点水蚤荧光素酶的底物或底物的类似物后，检测的所述反应体系的荧光强度，确定所述待测物的含量。
利用所述突变体与底物结合发生反应的特性，可以将所述突变体作为信号蛋白，用以检测待测物，如在突变体上通过化学偶联或形成融合蛋白的方式与能够特异性识别所述待测物的蛋白进行偶联或融合，能够特异性识别所述待测物的蛋白将会与待测物结合，所述突变体将催化其底物，并进行自发光，通过化学发光酶标仪测定突变体催化其底物过程中释放的生物发光的强度，所述生物发光的强度可以反映所述突变体的活性，通过其活性的高低判断待测物的含量。因此，包含所述突变体的试剂盒可用于准确检测待测物的含量。
根据本发明的具体实施方案，所述待测物可以是核酸。本发明提供的突变体对腔肠素、氟代腔肠素以及腔肠素衍生物等底物的催化活性较强，相较于现有的剑乳点水蚤荧光素酶底物谱更广或底物选择特异性更强、发光亮度显著增强，因此，所述方法可以用于检测核酸表达RNA或蛋白的情况，如RNA或蛋白的表达量、蛋白的定位或示踪，所述检测核酸方法的准确性和灵敏度均显著高于现有的高斯荧光素酶，获得的结果更加准确。
在本发明的第九方面，本发明提出了一种筛选剑乳点水蚤荧光素酶的底物的方法，包括：
Ⅰ)将第一方面所述的突变荧光素酶与待筛选底物进行接触，获得反应混合物；
Ⅱ)基于步骤Ⅰ)获得的所述反应混合物是否发出化学光信号，判断所述待筛选底物是否为目标底物。利用所述突变体与目标底物结合发生生物发光的特性，把感兴趣的待筛选底物与所述突变体进行接 触，所述突变体催化待筛选底物的过程中会释放生物发光，通过化学发光酶标仪测定所述突变体是否可以催化所述待筛选底物发出化学发光，从而判断所述待筛选底物是否为目标底物。因此，根据本发明一个实施方案的方法可以准确筛选目标底物。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施方案)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中：
图1显示了含信号肽的野生型剑乳点水蚤荧光素酶原核表达质粒pET28a-Pxluc WT的质粒图谱；
图2显示了含信号肽的野生型剑乳点水蚤荧光素酶原核表达质粒pCold WT Pxluc的质粒图谱；
图3显示了不含信号肽的野生型剑乳点水蚤荧光素酶原核表达质粒pCold WT NS Pxluc的质粒图谱；
图4显示了野生型剑乳点水蚤荧光素酶在不同感受态细胞中表达与纯化的结果图，图中箭头所指为目的蛋白条带；
图5显示了A底物腔肠素、B氟代腔肠素、C腔肠素衍生物ZS2、D腔肠素衍生物ZS26(D)的化学结构式；
图6显示了原核表达的剑乳点水蚤荧光素酶和高斯荧光素酶蛋白水平活性测试结果；
图7为剑乳点水蚤荧光素酶优势突变体对底物特异性ZS26/F-CTZ蛋白水平活性检测结果；
图8为剑乳点水蚤荧光素酶优势突变体对底物特异性ZS2/F-CTZ蛋白水平活性检测结果；
图9显示了含信号肽的野生型剑乳点水蚤荧光素酶真核表达质粒pEE12.4-Pxluc WT的质粒图谱；
图10为剑乳点水蚤荧光素酶在真核细胞中表达与纯化的结果图，图中箭头所指为目的蛋白条带；
图11显示了剑乳点荧光素酶偶联SA蛋白及纯化前后的结果图；
图12显示了真核表达剑乳点荧光素酶pEE12.4 Pxluc WT与突变体pEE12.4 Pxluc P26-95、及偶联后的SA-Pxluc WT与SA Pxluc P26-95对底物特异性ZS26/F-CTZ和ZS2/F-CTZ蛋白水平活性检测结果。
发明详细描述
下面详细描述本发明的实施例，所述实施例的示例在附图中示出。下面通过参考附图描述的实施例是示例性的，旨在用于解释本发明，而不能理解为对本发明的限制。
此外，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括至少一个该特征。在本发明的描述中，“多个”的含义是至少两个，例如两个，三个等，除非另有明确具体的限定。
在基础科研领域，荧光素酶基因已被广泛应用于不同启动子下外源基因表达强度和转录调控的研究的报告基因。在生物监测技术领域，荧光素酶可用于检测化学污染物的有无。另外，在免疫检测、生化诊断等领域亦具有广阔的应用前景。
突变体
在本发明的一个方面，本发明提出了一种荧光素酶突变体，相较于SEQ ID NO:2所示的氨基酸序列，所述突变体具有以下突变位点中的任意一个或者多个组合：第98位、99位、100位和101位，并且可以包括或者不包括信号肽氨基酸序列。
根据本发明一个实施方案的突变位点对上述氨基酸序列进行改造，获得的突变体对腔肠素、氟代腔肠素以及腔肠素衍生物ZS2、ZS26等底物具有较强的催化活性，相较于现有的剑乳点水蚤荧光素酶底物谱更广、特异性更强、发光亮度显著增强，在应用到所述突变体进行发光检测的基础科学研究、生物检测技术、免疫检测、生化检测或诊断等领域均可使用，具有广阔的应用前景，如可用作报告基因，定量检测DNA、RNA、转录因子、蛋白或细胞等；可作为融合蛋白中的发光信号蛋白，定量检测目标小分子、蛋白等。
本文中，“报告基因”是一个分子生物学概念，是指一类在细胞、组织/器官或个体处于特定情况下会表达并使得他们产生易于检测、且实验材料原本不会产生的性状的基因，即是一种编码可被检测的蛋白质或酶的基因。作为报告基因，在遗传选择和筛选检测方面必须具有以下几个条件：1、已被克隆和全序列已测定；2、表达产物在受体细胞中本不存在，即无背景，在被转染的细胞中无相似的内源性表达产 物；3、其表达产物能进行定量测定；在使用时，包括但并不限于如下使用方式：把报告基因和基因表达调节序列相融合形成嵌合基因，或与其它目的基因相融合，在调控序列的控制下进行核酸表达，从而利用它的表达产物来检测目的基因的表达调控，研究核酸。
本文中，“核酸表达”可以指DNA表达为RNA；或DNA表达为RNA，RNA进一步表达为蛋白质；或RNA表达为蛋白质，即本文中核酸表达后的产物可以是RNA也可以是蛋白。
本文中，“化学发光(Chemiluminescence)”又称为冷光(Cold Light)，它是在没有任何光、热或电场等激发的情况下，由化学反应而产生的光辐射。生命系统中也有化学发光，称生物发光(Bioluminescence)，如萤火虫、某些细菌或真菌、原生动物、蠕虫以及甲壳动物等所发射的光。本申请中，所述突变体可进行自发光，即化学发光。
根据本发明一些具体的实施方案，上述突变体还可以进一步包括如下附加技术特征至少之一：
根据本发明一些具体的实施方案，含有信号肽的野生型剑乳点水蚤荧光素酶(WT Pxluc)的基因序列为如SEQ ID NO：1所示，其编码的氨基酸如SEQ ID NO：2所示，其中第1-17位氨基酸为信号肽(粗体)，不含信号肽的野生型剑乳点水蚤荧光素酶(WT no signal peptide Pxluc：WT-NS Pxluc)的基因序列如SEQ ID NO:3所示，其编码的氨基酸如SEQ ID NO：4所示。SEQ ID NO：1-4序列具体如下：
Figure PCTCN2022102827-appb-000001
根据本发明一些具体的实施方案，相较于SEQ ID NO:2所示的氨基酸序列，所述突变体具有以下(1)-(4)突变中的任意一个或者多个组合：
(1)第98位的G突变为L或P或Q或S或T；
(2)第99位的Q突变为R或W或I或Y或A或L或F或V或P或E或M；
(3)第100位的G突变为S或Q或R或W或T或A或L；
(4)第101位的G突变为F或R或S或C或Y或L或I或K或V或P。
根据本发明另一些具体的实施方案，相较于SEQ ID NO:2所示的氨基酸序列，所述突变荧光素酶具有以下突变位点中的任意一个或者两个：第98位、99位、100位和101位，，并且可以包括或者不包括信号肽氨基酸序列。
根据本发明另一些具体的实施方案，相较于SEQ ID NO:2所示的氨基酸序列，所述突变体具有以下 (1)-(4)突变中的任意一个或者两个：
(1)第98位的G突变为L或P或Q或S或T；
(2)第99位的Q突变为R或W或I或Y或A或L或F或V或P或E或M；
(3)第100位的G突变为S或Q或R或W或T或A或L；
(4)第101位的G突变为F或R或S或C或Y或L或I或K或V或P。根据本发明的一些具体的实施方案，相较于SEQ ID NO:2所示的氨基酸序列，所述突变体具有以下突变：
1)第98位的G突变为L，第99位的Q突变为R；或
2)第98位的G突变为P；或
3)第98位的G突变为Q；或
4)第98位的G突变为S，第99位的Q突变为W；或
5)第98位的Q突变为I；或
6)第99位的Q突变为Y；或
7)第99位的Q突变为A；或
8)第99位的Q突变为L；或
9)第99位的Q突变为F；或
10)第98位的G突变为L，第99位的Q突变为V；或
11)第98位的G突变为T，第99位的Q突变为P；或
12)第98位的G突变为L，第99位的Q突变为E；或
13)第99位的Q突变为M，第100位的G突变为S；或
14)第100位的G突变为Q，第101位的G突变为F；或
15)第100位的G突变为R，第101位的G突变为R；或
16)第100位的G突变为W，第101位的G突变为F；或
17)第100位的G突变为S；或
18)第100位的G突变为T，第101位的G突变为S；或
19)第100位的G突变为R，第101位的G突变为C；或
20)第100位的G突变为A，第101位的G突变为R；或
21)第100位的G突变为L，第101位的G突变为Y；或
22)第100位的G突变为S，第101位的G突变为L；或
23)第101位的G突变为I；或
24)第100位的G突变为L，第101位的G突变为K；或
25)第100位的G突变为T；或
26)第100位的G突变为A，第101位的G突变为V；或
27)第100位的G突变为A，第101位的G突变为P。
根据本发明一些具体的实施方案，当SEQ ID NO:2所示的氨基酸序列具有上述突变时，获得的蛋白对腔肠素、氟代腔肠素以及腔肠素衍生物等底物的催化活性较强，相较于现有的剑乳点水蚤荧光素酶底物谱更广、发光亮度显著增强，在实际应用中检测准确性得到显著提高，在应用到所述蛋白进行发光检测的基础科学研究、生物检测技术、免疫检测、生化检测或诊断等领域均可使用，具有广阔的应用前景。
根据本发明一些具体的实施方案，所述突变荧光素酶不含有信号肽序列。
根据本发明一些具体的实施方案，所述突变荧光素酶可以是相较于SEQ ID NO:2所示的氨基酸序列，具有第98位、99位、100位和101位中的任意一个或者多个组合位点(例如可以是四个突变位点中任意一种，或者任意两种位点组合，或者任意三种位点组合，或者四个位点均突变)突变的荧光素酶。
根据本发明一些具体的实施方案，所述突变荧光素酶也可以是相较于SEQ ID NO:2所示的氨基酸序列，具有第98位、99位、100位和101位中的任意一个或者多个组合位点，并且所述突变荧光素酶不含有信号肽氨基酸序列。
SEQ ID NO:2所示的氨基酸序列中第1-17位氨基酸为信号肽，发明人发现，不论是含有信号肽的完整的SEQ ID NO:2，还是不含有信号肽的序列，只要在其98位、99位、100位和101位(这些位点编号是以SEQ ID NO:2序列进行的编号)中的任意一个或者多个组合位点处发生突变，获得具有此氨基酸序列的突变荧光素酶均能够对腔肠素、氟代腔肠素以及腔肠素衍生物ZS2等底物具有较强的催化活性，且相较于现有的剑乳点水蚤荧光素酶底物谱更广、特异性更强、发光亮度显著增强。根据本发明一些具体的实施方案，所述突变体为非分泌型蛋白或分泌型蛋白。
根据本发明一些具体的实施方案，本发明提供一种核酸分子，所述核酸分子编码前述的突变体。
需要说明的是，对于本发明说明书和权利要求书中所提及的核酸，本领域技术人员应当理解，实际包括互补双链的任意一条，或者两条。为了方便，在本说明书和权利要求书中，虽然多数情况下只给出了一条链，但实际上也公开了与之互补的另一条链。另外，本申请中的核酸序列包括DNA形式或RNA形式，公开其中一种，意味着另一种也被公开。
在本发明另一方面，本发明提出了一种表达载体，包含前面所述的核酸分子。这里的表达载体的类型并不受特别限制，只要能够在宿主细胞中复制表达相应的突变体就可以。所述表达载体可包括可选的控制序列，所述控制序列与所述核酸分子可操作地连接。其中，所述控制序列为可指导所述核酸分子在宿主中表达的一个或多个控制序列。本发明一些具体的实施方案所提出的表达载体可在适合的宿主细胞中高效表达蛋白，获得的蛋白对腔肠素、氟代腔肠素以及腔肠素衍生物等底物的催化活性较强，相较于现有的剑乳点水蚤荧光素酶底物谱更广、特异性更强、发光亮度显著增强，在应用到所述蛋白进行发光检测的基础科学研究、生物检测技术、免疫检测、生化检测或诊断等领域均可使用，具有广阔的应用前景。
在本发明的再一方面，本发明提出了一种重组细胞，携带前面所述的核酸分子、表达载体或突变体。所述重组细胞是通过转染或者转化所述表达载体获得的。根据本发明一些具体的实施方案，所述重组细胞在合适条件下可高效表达上述突变体，所述突变体对腔肠素、氟代腔肠素以及腔肠素衍生物等底物的催化活性较强，相较于现有的剑乳点水蚤荧光素酶底物谱更广、特异性更强、发光亮度显著增强，在应用到所述蛋白进行发光检测的基础科学研究、生物检测技术、免疫检测、生化检测或诊断等领域均可使用，具有广阔的应用前景。
根据本发明一些具体的实施方案，上述重组细胞还可以进一步包括如下附加技术特征至少之一：
根据本发明一些具体的实施方案，所述重组细胞为大肠杆菌、酵母或哺乳动物细胞。根据本发明一些具体的实施方案，所述重组细胞不受特殊限制，任何能够在核酸或载体(如质粒)中表达所述突变体的细胞均可使用，如酵母细胞、细菌或人胚胎肾细胞等哺乳动物细胞。
根据本发明一些具体的实施方案，所述重组细胞不包括动物生殖细胞、受精卵或胚胎干细胞。
根据本发明一些具体的实施方案，本发明提供一种产生突变荧光素酶的方法，包括将前面所述的表达载体导入重组细胞，培养繁殖重组细胞，收集培养繁殖产物提取或纯化突变荧光素酶。根据本发明一些具体的实施方案，本发明提供前面所述的突变荧光素酶和对应底物在检测核酸序列中的用途。
需要说明的是，在本发明中，“突变荧光素酶”和“荧光素酶突变体”、“突变体”可进行同等替换。
试剂盒及制备试剂盒的用途
在本发明的一个方面，本发明提出了一种试剂盒，所述试剂盒用于检测待测物的含量，其中，所述试剂盒包含前面所述的荧光素酶突变体，所述待测物的特异性识别蛋白适于与所述突变体形成复合物。
根据本发明一些具体的实施方案，所述试剂盒进一步包括剑乳点水蚤荧光素酶的底物或底物的类似物。
根据本发明一些具体的实施方案，所述底物选自腔肠素、氟代腔肠素或腔肠素衍生物中的至少之一。
根据本发明一些具体的实施方案，所述腔肠素衍生物选自腔肠素衍生物ZS2或腔肠素衍生物ZS26。
Figure PCTCN2022102827-appb-000002
根据本发明一些具体的实施方案，所述试剂盒还包括所述待测物的特异性识别蛋白。
本发明提供一种核酸测序试剂盒，其包括前面所述的突变荧光素酶。
在本发明的另一方面，本发明提出了前面所述的突变体在制备试剂盒中的用途，所述试剂盒用于检 测待测物的含量，所述待测物的特异性识别蛋白适于与所述突变体形成复合物。利用所述突变体与底物结合发生反应的特性，可以将所述突变体作为信号蛋白，用以检测待测物，如在突变体上通过化学偶联或形成融合蛋白的方式与能够特异性识别所述待测物的蛋白进行偶联或融合，当其与所述待测物、突变体的底物在同一体系中时，所述特异性识别所述待测物的蛋白将会与待测物结合，所述突变体将催化其底物，并进行自发光，通过化学发光酶标仪测定突变体催化其底物过程中释放的生物发光的强度，所述生物发光的强度可以反映所述突变体的活性，通过其活性的高低判断待测物的含量，在底物与所述突变体结合前需要洗脱掉体系中的非特异性结合蛋白，以排除其它因素对检测结果的干扰。因此，所述突变体可以用于制备试剂盒，并准确检测待测物的含量。
根据本发明一些具体的的实施方案，上述用途还可以进一步包括如下附加技术特征至少之一：
根据本发明一些具体的的实施方案，所述试剂盒进一步包括剑乳点水蚤荧光素酶的底物或底物的类似物。
根据本发明一些具体的的实施方案，所述底物选自腔肠素、氟代腔肠素或腔肠素衍生物中的至少之一。
根据本发明一些具体的实施方案，所述底物不受特别限制，可以与所述突变体发生化学反应的物质均包含在该范围内，并不局限于化学发光反应，本领域技术人员可根据实验需要更换不同的底物。
根据本发明一些具体的实施方案，所述腔肠素衍生物选自腔肠素衍生物ZS2或腔肠素衍生物ZS26。
根据本发明一些具体的实施方案，所述试剂盒进一步包括所述待测物的特异性识别蛋白。
方法
在本发明的一个方面，本发明提出了一种检测物质含量的方法，包括以下步骤：
ⅰ)将待测物的特异性识别蛋白与前面所述的突变体形成复合物；
ⅱ)将待测物与所述复合物接触；
ⅲ)向反应体系中加入剑乳点水蚤荧光素酶的底物或底物的类似物；
ⅳ)基于加入所述剑乳点水蚤荧光素酶的底物或底物的类似物后所述反应体系的荧光强度，确定所述待测物的含量。
根据本发明一些具体的实施方案，本发明提供一种检测待测物含量的方法，包括以下步骤：
a)以前面所述的突变荧光素酶为信号蛋白，通过化学偶联、生物偶联或融合蛋白的形式，将所述待测物的特异性识别蛋白与所述突变荧光素酶形成复合物；
b)将所述待测物与所述复合物接触；
c)向反应体系中加入剑乳点水蚤荧光素酶的底物或底物的类似物；
d)基于加入所述剑乳点水蚤荧光素酶的底物或底物的类似物后，检测的所述反应体系的荧光强度，确定所述待测物的含量。根据本发明一些具体的实施方案，所述底物选自腔肠素、氟代腔肠素或腔肠素衍生物中的至少之一。
根据本发明一些具体的实施方案，所述腔肠素衍生物选自腔肠素衍生物ZS2或腔肠素衍生物ZS26。
在本发明的再一方面，本发明提出了一种筛选剑乳点水蚤荧光素酶的底物的方法，包括：
Ⅰ)将前面所述的突变体与待筛选底物进行接触，获得反应混合物；
Ⅱ)基于步骤Ⅰ)获得的所述反应混合物是否发出化学光信号，判断所述待筛选底物是否为目标底物。
根据本发明一些具体的实施方案，步骤Ⅰ)获得的所述反应混合物发出化学光信号为所述待筛选底物是目标底物的指示。
下面参考具体实施例，对本发明进行描述，需要说明的是，这些实施例仅仅是描述性的，而不以任何方式限制本发明。
实施例1 原核表达野生型剑乳点水蚤荧光素酶质粒的设计与构建
本实施例对含有信号肽和不含有信号肽的野生型剑乳点水蚤荧光素酶进行构建，以比较信号肽对野生型剑乳点水蚤荧光素酶的影响。
含有信号肽的野生型剑乳点水蚤荧光素酶(WT Pxluc)的基因序列为如SEQ ID NO：1所示，其编码的氨基酸如SEQ ID NO：2所示，其中第1-17位氨基酸为信号肽(粗体)，不含信号肽的野生型剑乳点水蚤荧光素酶(WT no signal peptide Pxluc：WT-NS Pxluc)的基因序列如SEQ ID NO:3所示，其编码的氨基酸如SEQ ID NO：4所示。
通过全基因合成技术，合成了用于pET28a载体的含信号肽的剑乳点水蚤荧光素酶(pET28a Pxluc  WT)基因序列SEQ ID NO：5(生工)，质粒图谱如图1所示，其C端融合了含有6个组氨酸(6x His)的纯化标签以利于蛋白纯化，两端的酶切位点为BamHI和NotI；
通过PCR技术，合成了用于pCold载体的含信号肽的剑乳点水蚤荧光素酶蛋白的基因序列SEQ ID NO：6，其C端融合了含有6个组氨酸(6x His)的纯化标签以利于蛋白纯化；通过PCR技术，合成了用于pCold载体的去掉信号肽的剑乳点水蚤荧光素酶蛋白的基因序列SEQ ID NO：7，其C端融合了含有6个组氨酸(6x His)的纯化标签以利于蛋白纯化。
Figure PCTCN2022102827-appb-000003
Figure PCTCN2022102827-appb-000004
1、获取插入片段
将全基因合成的含有信号肽的野生型剑乳点水蚤荧光素酶(pET28a Pxluc WT)用去离子水溶解，并稀释至10ng/μl，作为模板DNA。使用KOD FXD Neo酶，按照其说明书进行PCR反应体系的配制及PCR反应，进行插入片段的制备。
含有信号肽的野生型剑乳点水蚤荧光素酶(WT-Pxluc)和不含有信号肽的野生型剑乳点水蚤荧光素酶(WT-NS Pxluc)的PCR反应所使用的引物序列如表1所示，PCR反应体系如表2所示，反应条件如表3所示，PCR循环数30个。
表1
Figure PCTCN2022102827-appb-000005
表2
组分 体积(μL)
2x KOD FX Neo PCR缓冲液 25
2mM dNTPs 10
10μM Primer insert-F 1.5
10μM Primer insert-R 1.5
模板DNA(10ng/μL) 2.5
KOD FX Neo 1
PCR级水 8.5
总体积 50
表3
Figure PCTCN2022102827-appb-000006
向反应体系中加入0.5μL DpnI酶，37℃孵育3小时消化模板，然后胶回收约586bp和531bp的产物 即为插入片段insert。其中586bp的胶回收片段为含有信号肽的插入片段，531bp的胶回收片段为不含有信号肽的插入片段。
2、获取线性化载体
以pCold载体为模板，通过PCR的方式将载体线性化以便于同插入片段重组。使用KOD FX neo酶，按照其说明书进行PCR反应体系的配制及PCR反应。
PCR反应所使用的引物序列如表4所示，PCR反应体系如表5所示，反应条件如表6所示，PCR循环数30个。
表4
Figure PCTCN2022102827-appb-000007
表5
组分 体积(μL)
2x KOD FX Neo PCR缓冲液 25
2mM dNTPs 10
10μM Primer vector-F 1.5
10μM Primer vector-R 1.5
pCold(10ng/μL) 2.5
KOD FX Neo酶 1
PCR级水 8.5
总体积 50
表6
Figure PCTCN2022102827-appb-000008
向反应体系中加入0.5μL DpnI酶，37℃孵育3小时消化模板，然后胶回收约4433bp的产物即为线性化载体vector。
用In-Fusion Cloning试剂盒(TAKARA)对insert与vector按照如表7所示反应体系进行重组，于50℃孵育15分钟。
表7
组分 体积(μL)
插入片段(10ng/μL) 5
线性载体(10ng/μL) 3
Premix 2
总体积 10
取上述反应产物2.5μL转化入DH5α感受态细胞中，涂布于含终浓度为100μg/mL的氨苄抗性平板，次日从平板上挑取单克隆，抽提质粒进行测序，确保目标片段正确插入载体，获得的质粒即为含有信号肽 的野生剑乳点水蚤荧光素酶pCold-WT Pxluc(图2)和不含信号肽的野生剑乳点水蚤荧光素酶pCold WT NS Pxluc(图3)，符合实验预期。
实施例2 剑乳点水蚤荧光素酶的原核表达、纯化及蛋白水平活性测试
1、剑乳点水蚤荧光素酶的原核表达
将表达质粒pET28a-Pxluc WT转化入BL21(DE3)感受态细胞，表达质粒pCold WT Pxluc和pCold WT-NS Pxluc分别转化入OrigamiB(DE3)Chemically Competent Cell感受态细胞(唯地生物，EC1020S)中，涂平板，从平板上挑取单菌落，37℃过夜培养，次日按1:100的比例进行稀释，分别转接于新鲜的300ml含卡那霉素(50μg/ml)或者氨苄(100μg/ml)的LB培养基中，37℃，200rpm振荡培养至OD600≈0.5-0.6，冰上冷却1小时。按照终浓度为1mM的量加入诱导剂IPTG，16℃过夜诱导。
2、剑乳点水蚤荧光素酶的纯化
按8000rpm/min，10min的条件离心收集诱导后的菌液沉淀，加入30ml结合缓冲液(50mM Tris，250mM NaCl,pH 8.0)，300μL溶菌酶，冰上裂解30min，超声(2s on 3s off，60％功率)破碎30min，4℃12000rpm离心30min分离上清(细胞裂解液)和沉淀。
在手工柱(购自生工，型号为F506607-0001#亲和层析柱空柱)中加入2mL HisTrap FF填料，用30ml结合缓冲液冲洗平衡填料。然后加入约30ml过滤后的细胞裂解液。用漂洗液(50mM Tris，pH 8.0，250mM NaCl,10mM咪唑)冲洗10次(10ml/次)，然后用洗脱液(50mM Tris，pH 8.0，250mM NaCl,300mM咪唑)500ul洗脱蛋白4-5次，收集洗脱后的蛋白。4℃过夜透析。蛋白浓度和纯度分布通过BCA定量试剂盒法和SDS-PAGE法测定。
SDS-PAGE法测定洗脱得到的蛋白，纯化结果如图4所示，表明转化pET28a-Pxluc WT质粒的BL21(DE3)细胞成功表达出目的蛋白；转化表达质粒pCold WT Pxluc和pCold WT-NS Pxluc的OrigamiB(DE3)也分别成功表达出目的蛋白。
3、剑乳点水蚤荧光素酶蛋白活性测定
用BCA定量试剂盒(Thermo Scientific TM Pierce TM BCA Protein Assay Kit)精确测定蛋白的浓度，将上述获得的经纯化的荧光素酶用稀释液(50mM Tris-HCl pH 8.0，100mM NaCl，0.1％(v/v)Tween-20)稀释，将pET28a-Pxluc WT质粒表达的蛋白稀释至12μg/ml，将pCold WT Pxluc和pCold WT-NS Pxluc质粒表达的蛋白稀释至1μg/ml，各取10μL加入黑色96孔板。再加入用相同溶液稀释至100μM的底物腔肠素(购自百奥莱博)、氟代腔肠素、腔肠素的衍生物ZS2、ZS26各90μL(各化合物结构式如图5所示)，用酶标仪自发光模块读取发光强度。剑乳点水蚤荧光素酶与CTZ(腔肠素)的活性测试结果如图6，已知OrigamiB(DE3)感受态细胞中表达的Gluc发光活性较好，其作为对照组。图6结果表明，含有质粒pCold WT NS Pxluc的OrigamiB(DE3)感受态细胞的发光值最高，与含有质粒pCold Gluc的OrigamiB(DE3)感受态细胞的发光值相当。因此，不含信号肽的剑乳点水蚤荧光素酶对腔肠素的发光活性与Gluc对腔肠素的发光活性相近。相对含有信号肽的剑乳点水蚤荧光素酶，不含信号肽的剑乳点水蚤荧光素酶活性较高，后续实验中采用不含信号肽的剑乳点水蚤荧光素酶进行。
实施例3 剑乳点水蚤荧光素酶突变体库的设计与构建
通过PCR技术，以pCold WT-NS Pxluc质粒序列为模板，合成了在SEQ ID NO：2的G98、Q99、G100、G101位点的定点饱和突变体库(将特定氨基酸突变成其余任一氨基酸)，其中突变体库1为在氨基酸位点G98的定点饱和突变体库(L1)，突变体库2为在Q99位点的定点饱和突变体库(L2)，其中突变体库3为在氨基酸位点G100的定点饱和突变体库(L3)，突变体库4为在G101位点的定点饱和突变体库(L4)，突变体库5为在氨基酸位点G98和Q99的组合突变体库(L5)，突变体库6为在氨基酸位点G100和G101的组合突变体库(L6),突变体库7为在氨基酸位点G98、Q99、G100和G101位点的组合突变体库(L7)。
针对突变体库L1设计的一对引物：
上游引物Primer F1:
5’-GATAAATCTATTNNKCAGGGAGGCATAGGTGGCCCTATTG-3’(SEQ ID NO：14)
下游引物Primer R1:
5’-TAGGGCCACCTATGCCTCCCTGMNNAATAGATTTATCTCC-3’(SEQ ID NO：15)
针对突变体库L2设计的一对引物：
上游引物Primer F2:
5’-GATAAATCTATTGGANNKGGAGGCATAGGTGGCCCTATTG-3’(SEQ ID NO：16)
下游引物Primer R2:
5’-TAGGGCCACCTATGCCTCCMNNTCCAATAGATTTATCTCC-3’(SEQ ID NO：17)
针对突变体库L3设计的一对引物：
上游引物Primer F3:
5’-GATAAATCTATTGGACAGNNKGGCATAGGTGGCCCTATTG-3’(SEQ ID NO：18)
下游引物Primer R3:
5’-TAGGGCCACCTATGCCMNNCTGTCCAATAGATTTATCTCC-3’(SEQ ID NO：19)
针对突变体库L4设计的一对引物：
上游引物Primer F4：
5’-GATAAATCTATTGGACAGGGANNKATAGGTGGCCCTATTG-3’(SEQ ID NO：20)
下游引物Primer R4：
5’-TAGGGCCACCTATMNNTCCCTGTCCAATAGATTTATCTCC-3’(SEQ ID NO：21)
针对突变体库L5设计的一对引物：
上游引物Primer F5:
5’-GATAAATCTATTNNKNNKGGAGGCATAGGTGGCCCTATTG-3’(SEQ ID NO：22)
下游引物Primer R5:
5’-TAGGGCCACCTATGCCTCCMNNMNNAATAGATTTATCTCC-3’(SEQ ID NO：23)
针对突变体库L6设计的一对引物：
上游引物Primer F6：
5’-GATAAATCTATTGGACAGNNKNNKATAGGTGGCCCTATTG-3’(SEQ ID NO：24)
下游引物Primer R6：
5’-TAGGGCCACCTATMNNMNNCTGTCCAATAGATTTATCTCC-3’(SEQ ID NO：25)
针对突变体库L7设计的一对引物：
上游引物Primer F7：
5’-GAAGGAGATAAATCTATTNNKNNKNNKNNKATAGGTGGCCCTATTGTTGATA-3’(SEQ ID NO：26)
下游引物Primer R7：
5’-CAATAGGGCCACCTATMNNMNNMNNMNNAATAGATTTATCTCCTTCATAAGT-3’(SEQ ID NO：27)
在核酸序列中“N”为A/C/G/T，“K”为G/T，“M”为A/C。
表8显示了进行易错PCR的反应体系，表9显示了反应条件，PCR循环数30个。
表8
Figure PCTCN2022102827-appb-000009
表9
Figure PCTCN2022102827-appb-000010
待反应结束后，向反应体系中加入0.5μL DpnI酶，37℃孵育3小时消化模板。然后将酶切产物转化至DH5a感受态细胞，10ml过夜培养后提质粒。将提取的质粒转化至OrigamiB(DE3)Chemically Competent Cell感受态细胞(唯地生物，EC1020S)后，涂平板，从平板上挑取单菌落，37℃过夜培养，次日按1:100的比例进行稀释，转接于新鲜的0.4ml含氨苄抗性(100μg/ml)的LB培养基中，37℃，200rpm振荡培养至OD600≈0.5-0.6，冰上冷却1小时。按照终浓度为1mM的量加入诱导剂IPTG，16℃过夜诱导。
取50μL诱导后的菌液加入黑色96孔板，再分别加入10μL终浓度为100μM的底物腔肠素(CTZ)(购自百奥莱博)、氟代腔肠素、ZS2、ZS26，用酶标仪自发光模块读取发光强度，进行突变体的菌液筛选(筛选标准：以发光强度的比值进行筛选，将与底物ZS2或ZS26的发光强度与底物F-CTZ的发光强度比值，这个比值如果大于野生型对两个底物发光强度的比值，则认为优势突变体)。
实施例4 剑乳点水蚤荧光素酶突变体库的表达与纯化
1、剑乳点水蚤荧光素酶突变体的原核表达
将突变体库pCold-no sp Pxluc L1、pCold-no sp Pxluc L2、pCold-no sp Pxluc L3、pCold-no sp Pxluc L4、pCold-no sp Pxluc L5筛选出的突变体菌液37℃过夜培养，次日按1:100的比例进行稀释，转接于新鲜的15ml含氨苄(100μg/ml)的LB培养基中，37℃，200rpm振荡培养至OD600≈0.5-0.6，冰上冷却1小时。按照终浓度为1mM的量加入诱导剂IPTG，16℃过夜诱导。
2、剑乳点水蚤荧光素酶突变体的纯化
按8000rpm/min，10min的条件离心收集诱导后的菌液沉淀，加入600μL结合缓冲液(50mM Tris，pH 8.0，250mM NaCl)、6μL溶菌酶，冰上裂解30min，超声(2s on 3s off，60％功率)破碎10min，4℃12000rpm离心30min分离上清(细胞裂解液)和沉淀。
在手工柱(购自生工，型号为F506607-0001#亲和层析柱空柱)中加入50μL HisTrap FF填料，用3ml结合缓冲液冲洗平衡填料。然后加入600μL过滤后的细胞裂解液。用漂洗液(50mM Tris，pH 8.0，250mM NaCl,10mM咪唑)冲洗10次(3ml/次)，然后用洗脱液(50mM Tris，pH 8.0，250mM NaCl,300mM咪唑)100ul洗脱蛋白，收集洗脱后的蛋白。
将Ni柱洗脱后的蛋白用透析缓冲液(25mM Tris，pH 8.0，250mM NaCl)4℃过夜透析。蛋白浓度和纯度分布通过BCA定量试剂盒法(Thermo Scientific TM Pierce TM BCA Protein Assay Kit)和SDS-PAGE法测定。
实施例5 剑乳点水蚤荧光素酶优势突变体的底物特异性测试
用BCA定量试剂盒(Thermo Scientific TM Pierce TM BCA Protein Assay Kit)精确测定蛋白的浓度，将荧光素酶用稀释液(50mM Tris-HCl pH 8.0，100mM NaCl，0.1％(v/v)Tween-20)稀释至1μg/ml，取10μL加入黑色96孔板。再加入用相同溶液稀释至100μM的底物腔肠素(购自百奥莱博)、氟代腔肠素、腔肠素的衍生物ZS2、ZS26各90μL，用酶标仪自发光模块读取发光强度，并以ZS2/F-CTZ和ZS26/F-CTZ的活性比值来比较突变体的底物特异性。优势特异性突变体的活性测试结果如图7、图8。以下表10显示了剑乳点水蚤荧光素酶优势突变体的突变位点及ZS26/F-CTZ、ZS2/F-CTZ底物特异性的结果。
表10
Figure PCTCN2022102827-appb-000011
Figure PCTCN2022102827-appb-000012
表10以及图7、图8结果表明，不含信号肽的野生型剑乳点水蚤荧光素酶对ZS26/F-CTZ的底物特异性，约为1.77倍，对ZS2/F-CTZ的底物特异性，约为3.56倍；对该酶进行改造获得的优势突变体，其对底物ZS26/F-CTZ的特异性升高，最高约为74.857倍；其对底物ZS2/F-CTZ特异性升高，最高为约为24.750倍。
实施例6 真核表达剑乳点水蚤荧光素酶质粒的设计与构建
通过PCR技术，合成了用于pEE12.4载体的含信号肽的剑乳点水蚤荧光素酶，基因序列如SEQ ID NO：28所示，其N端信号肽后有用于纯化的组氨酸标签(6x His)，C端还有用于生物素化的Avitag：
Figure PCTCN2022102827-appb-000013
用于真核表达的剑乳点水蚤荧光素酶质粒以含有信号肽的原核表达质粒pET28a Pxluc WT为模板 DNA，构建表达质粒插入片段的PCR反应所用引物序列如表11所示，反应体系如表12所示，反应条件如表13所示。
表11
Figure PCTCN2022102827-appb-000014
表12
组分 体积(μL)
2x KOD FX Neo PCR缓冲液 25
2mM dNTPs 10
10μM Primer insert-F 1.5
10μM Primer insert-R 1.5
模板DNA pET28a Pxluc WT(10ng/μL) 2.5
KOD FX Neo酶 1
PCR级水 8.5
总体积 50
表13
Figure PCTCN2022102827-appb-000015
向反应体系中加入0.5μL DpnI酶，37℃孵育3小时消化模板，然后胶回收约528bp的产物即为插入片段insert。
以pEE12.4载体为模板，通过PCR的方式将载体线性化以便于同插入片段重组。使用KOD FX neo酶，按照其说明书进行PCR反应体系的配制及PCR反应；其中，所用引物序列如表14所示，PCR反应体系如表15所示，PCR反应条件如表16所示。
表14：
Figure PCTCN2022102827-appb-000016
表15：
组分 体积(μL)
2x KOD FX Neo PCR缓冲液 25
2mM dNTPs 10
10μM Primer vector-F 1.5
10μM Primer vector-R 1.5
pEE12.4 plasmid(10ng/μL) 2.5
KOD FX Neo 1
PCR级水 8.5
总体积 50
表16：
Figure PCTCN2022102827-appb-000017
向反应体系中加入0.5μL DpnI酶，于37℃孵育3小时消化模板，然后胶回收约7712bp的产物即为线性化载体vector。
用Takara In-Fusion Cloning试剂盒对本实施例中获得的insert与vector按照如表17中所示的反应体系进行重组，反应条件为50℃孵育15分钟。
表17：
组分 体积(μL)
insert(10ng/μL) 5
vector(10ng/μL) 3
Premix 2
总体积 10
取上述反应产物2.5μL转化入DH5α感受态细胞中，涂布于含终浓度为100μg/mL的氨苄抗性平板，次日从平板上挑取单克隆，抽提质粒。进行测序确保目标片段正确插入载体，获得的质粒即为用于真核表达的野生型荧光素酶pEE12.4-Pxluc WT，对获得的质粒进行检测，检测到的质粒图谱如图9所示，符合实验预期。
用于真核表达的突变体质粒pEE12.4 Pxluc P26-95，以构建好的pEE12.4-Pxluc WT为模板DNA，通过PCR方法合成；其中引物序列如表18所示，PCR反应体系如表19所示，PCR反应条件与表16相同。
表18：
Figure PCTCN2022102827-appb-000018
表19：
组分 体积(μL)
2x KOD FX Neo PCR缓冲液 25
2mM dNTPs 10
10μM Primer vector-F 1.5
10μM Primer vector-R 1.5
pEE12.4 Pxluc P26-95(10ng/μL) 2.5
KOD FX Neo 1
PCR级水 8.5
总体积 50
待反应结束后，向反应体系中加入0.5μL DpnI酶，37℃孵育3小时消化模板。取酶切反应产物2.5μL转化入DH5α感受态细胞中，涂布于含终浓度为100μg/mL的氨苄抗性平板，次日从平板上挑取单克隆，抽提质粒。进行测序确保突变体序列正确，获得的质粒即为用于真核表达的突变体荧光素酶pEE12.4  Pxluc P26-95(含有信号肽序列的荧光素酶突变体表达载体，突变位点G100A,G101P)。
实施例7 剑乳点水蚤荧光素酶的真核表达及蛋白纯化
将实施例6获得的pEE12.4 Pxluc WT和突变体pEE12.4 Pxluc P26-95质粒按照ExpiFectamine TM CHO转染试剂盒(Gibco，A29129)说明书分别转染入30mL Expi-CHO细胞中，转染7日后，测得细胞活力小于90％，4℃，8000rpm离心10min收集上清。
在手工柱(购自生工，型号为F506607-0001#亲和层析柱空柱)中分别加入2mL HisTrap FF填料，用30mL结合缓冲液冲洗平衡填料。然后分别加入约30mL过滤后的细胞上清液。用漂洗液(50mM Tris，pH 8.0，250mM NaCl,10mM咪唑)冲洗10次(10mL/次)，然后用洗脱液(50mM Tris，pH 8.0，250mM NaCl,300mM咪唑)500μL洗脱蛋白4-5次，分别收集洗脱后的蛋白。12％SDS-PAGE检测洗脱得到的蛋白，纯化结果如图10所示，可以得到较纯的Pxluc蛋白。
实施例8 剑乳点荧光素酶优势突变体的偶联及活性测试
实施例7中得到的Pxluc蛋白含有AviTag标签，即AviTag-Pxluc-WT和突变体AviTag-Pxluc-P26-95，可被BirA酶(Avidity,BIRA500)生物素化为Biotin-Avitag-Pxluc-WT和Biotin-AviTag-Pxluc-P26-95，生物素化的反应体系如表18。
表20：
组分 体积(μL)
AviTag-Pxluc(1mg/mL) 12
BiomixA 10
BiomixB 10
BirA(1mg/mL) 3
H 2O 65
总体积 100
在表20所示的体系中加入SA即可制备SA-Pxluc，利用Pxluc上的组氨酸标签可以用来进一步纯化得到较纯的SA-Pxluc，纯化结果如图11所示。
按照实施例5中所述方法对pEE12.4 Pxluc WT和突变体pEE12.4 Pxluc P26-95，以及偶联后产物SA-Pxluc WT和SA-Pxluc P26-95进行活性测试，结果如图12所示，表明真核表达的Pxluc WT和突变体Pxluc P26-95均具有催化底物腔肠素衍生物ZS2、ZS26的活性，并且与Pxluc WT相比，突变体Pxluc P26-95对ZS26/F-CTZ的底物特异性显著提高，对ZS26/F-CTZ的底物特异性提高。偶联后产物SA-Pxluc P26-95相比SA-Pxluc WT对ZS26/F-CTZ的底物特异性显著提高。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。

Claims (18)

  1. 一种突变荧光素酶，其中，相较于SEQ ID NO:2所示的氨基酸序列，所述突变荧光素酶具有以下突变位点中的至少之一：第98位、99位、100位和101位，并且可以包括或者不包括信号肽氨基酸序列。
  2. 根据权利要求1所述的突变荧光素酶，其中，相较于SEQ ID NO:2所示的氨基酸序列，所述突变荧光素酶具有以下(1)-(4)突变中的任意一个或者多个组合：
    (1)第98位的G突变为L或P或Q或S或T；
    (2)第99位的Q突变为R或W或I或Y或A或L或F或V或P或E或M；
    (3)第100位的G突变为S或Q或R或W或T或A或L；
    (4)第101位的G突变为F或R或S或C或Y或L或I或K或V或P。
  3. 根据权利要求1所述的突变荧光素酶，其中，相较于SEQ ID NO:2所示的氨基酸序列，所述突变荧光素酶具有以下突变位点中的任意一个或者两个：第98位、99位、100位和101位，并且可以包括或者不包括信号肽氨基酸序列。
  4. 根据权利要求3所述的突变荧光素酶，其中，相较于SEQ ID NO:2所示的氨基酸序列，所述突变荧光素酶具有以下(1)-(4)突变中的任意一个或者两个：
    (1)第98位的G突变为L或P或Q或S或T；
    (2)第99位的Q突变为R或W或I或Y或A或L或F或V或P或E或M；
    (3)第100位的G突变为S或Q或R或W或T或A或L；
    (4)第101位的G突变为F或R或S或C或Y或L或I或K或V或P。
  5. 根据权利要求1-4中任一项所述的突变荧光素酶，其中，相较于SEQ ID NO:2所示的氨基酸序列，所述突变荧光素酶具有以下突变：
    1)第98位的G突变为L，第99位的Q突变为R；或
    2)第98位的G突变为P；或
    3)第98位的G突变为Q；或
    4)第98位的G突变为S，第99位的Q突变为W；或
    5)第99位的Q突变为I；或
    6)第99位的Q突变为Y；或
    7)第99位的Q突变为A；或
    8)第99位的Q突变为L；或
    9)第99位的Q突变为F；或
    10)第98位的G突变为L，第99位的Q突变为V；或
    11)第98位的G突变为T，第99位的Q突变为P；或
    12)第98位的G突变为L，第99位的Q突变为E；或
    13)第99位的Q突变为M，第100位的G突变为S；或
    14)第100位的G突变为Q，第101位的G突变为F；或
    15)第100位的G突变为R，第101位的G突变为R；或
    16)第100位的G突变为W，第101位的G突变为F；或
    17)第100位的G突变为S；或
    18)第100位的G突变为T，第101位的G突变为S；或
    19)第100位的G突变为R，第101位的G突变为C；或
    20)第100位的G突变为A，第101位的G突变为R；或
    21)第100位的G突变为L，第101位的G突变为Y；或
    22)第100位的G突变为S，第101位的G突变为L；或
    23)第101位的G突变为I；或
    24)第100位的G突变为L，第101位的G突变为K；或
    25)第100位的G突变为T；或
    26)第100位的G突变为A，第101位的G突变为V；或
    27)第100位的G突变为A，第101位的G突变为P。
  6. 根据权利要求1-5中任一项所述的突变荧光素酶，其中，所述突变荧光素酶不含有信号肽氨基酸序列。
  7. 一种核酸分子，其中，所述核酸分子编码权利要求1-6中任一项所述的突变荧光素酶。
  8. 一种表达载体，其中，包含权利要求7所述的核酸分子。
  9. 一种重组细胞，其中，携带权利要求7所述的核酸分子或权利要求8所述的表达载体。
  10. 根据权利要求9所述的重组细胞，其中，所述重组细胞选自大肠杆菌、酵母或哺乳动物细胞。
  11. 一种产生突变荧光素酶的方法，包括：将权利要求8所述的表达载体导入重组细胞，培养繁殖重组细胞，收集培养繁殖产物提取或纯化突变荧光素酶。
  12. 一种利用权利要求1-6中任一项所述突变荧光素酶检测核酸序列的方法，所述方法包括以下步骤：
    A)以权利要求1-6中任一项所述突变荧光素酶通过化学偶联、生物偶联或融合蛋白的形式，将第一特异性识别蛋白与所述突变荧光素酶形成第一突变荧光素酶复合物；以第二荧光素酶为信号蛋白，通过化学偶联、生物偶联或融合蛋白的形式，将第二特异性识别蛋白与所述第二荧光素酶形成第二荧光素酶复合物；
    B)所述第一突变荧光素酶复合物能与第一底物反应生成第一发光信号；所述第二荧光素酶复合物能与第二底物反应生成第二发光信号；所述第一突变荧光素酶复合物与所述第二底物不具有显著的交叉底物反应，且所述第二荧光素酶复合物与所述第一底物不具有显著的交叉基底反应；
    C)通过检测所述突变荧光素酶和所述第二荧光素酶自发光体系的荧光信号及信号组合，来分辨A、T、G、C四种碱基进行目标核酸测序。
  13. 根据权利要求12所述的方法，其中，当所述突变荧光素酶和所述第二荧光素酶相同时，所述第一底物和所述第二底物相同；
    当所述突变荧光素酶和所述第二荧光素酶不同时，所述第一底物和所述第二底物不同。
  14. 一种核酸测序试剂盒，包括权利要求1-6中任一项所述的突变荧光素酶。
  15. 一种检测待测物含量的方法，包括以下步骤：
    a)以权利要求1-6中任一项所述的突变荧光素酶为信号蛋白，通过化学偶联、生物偶联或融合蛋白的形式，将所述待测物的特异性识别蛋白与所述突变荧光素酶形成复合物；
    b)将所述待测物与所述复合物接触；
    c)向反应体系中加入剑乳点水蚤荧光素酶的底物或底物的类似物；
    d)基于加入所述剑乳点水蚤荧光素酶的底物或底物的类似物后，检测的所述反应体系的荧光强度，确定所述待测物的含量。
  16. 根据权利要求15所述的方法，所述剑乳点水蚤荧光素酶的底物选自腔肠素、氟代腔肠素或腔肠素衍生物中的至少之一；
    任选地，所述腔肠素衍生物选自腔肠素衍生物ZS2或腔肠素衍生物ZS26，
    Figure PCTCN2022102827-appb-100001
  17. 一种筛选剑乳点水蚤荧光素酶的底物的方法，其中，包括：
    Ⅰ)将权利要求1-6中任一项所述的突变荧光素酶与待筛选底物进行接触，获得反应混合物；
    Ⅱ)基于步骤Ⅰ)获得的所述反应混合物是否发出化学光信号，判断所述待筛选底物是否为目标底物。
  18. 根据权利要求17所述的方法，其中，步骤Ⅰ)获得的所述反应混合物发出化学光信号为所述待筛选底物是目标底物的指示。
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