Luciferase variants and their use

Abstract

Provided are luciferase variants and their uses, specifically copepod luciferase variants and their uses. By directed evolution of the Pxluc protein, mutants with a substrate specificity for ZS26 / F-CTZ improved by more than 2-fold and mutants with a substrate specificity for ZS2 / F-CTZ improved by more than 4-fold are obtained. This luciferase can be expressed in prokaryotic cells, has a simple purification process, is advantageous for large-scale production, has a luminescence brightness similar to Gluc, is easy to detect, and this luciferase is expected to have wide application prospects in many fields such as basic scientific research, biological monitoring, and biochemical diagnosis.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology. Specifically, the present invention relates to luciferase mutants and their uses, and particularly to copepoda luciferase mutants and their uses.

Background Art

[0002] Luciferase is an enzyme that can catalyze the chemiluminescence of luciferin or aliphatic aldehyde, and is widely present in insects, bacteria, fungi, and marine organisms. It has already become an important tool in scientific research and is widely used in fields such as life science research, genomics sequencing and analysis technology, clinical medicine and forensic detection, drug screening, environmental monitoring, and enzyme-linked detection. Utilizing the property of self-luminescence of luciferase, luciferase is always 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 biological monitoring technology, luciferase can be used to detect the presence or absence of chemical pollutants. Also, there is a broad application prospect in fields such as immunoassay and biochemical diagnosis. Moreover, as a reporter gene for studying the exogenous gene expression intensity and transcriptional regulation under different promoters, the combined use of multiple luciferases with similar self-luminescence brightness and different catalytic substrates is required.

[0003] Currently, luciferases with excellent research and development mainly include luciferases extracted from firefly luciferase (FLuc), bacterial luciferase (Lux), Renilla luciferase (RLuc), deep-sea shrimp (Oplophorus luciferase, OLuc), marine animal Gaussia princeps (Gaussia luciferase, GLuc), etc. Fluc requires ATP, O2, and Mg 2+It requires cofactors such as etc., has non-secretory expression, and many of the fluorescence reactions of Lux require molecules such as flavin mononucleotide (FMN), long-chain aliphatic aldehyde, oxygen, and reduced nicotinamide adenine dinucleotide (NADH). Rluc luminescence does not require ATP, but its fluorescence intensity is weak and it cannot be secreted in the same way.

[0004] In nature, more than 40 types of bioluminescence systems have already been discovered, but those that can be developed and utilized are very limited.

Summary of the Invention

Problems to be Solved by the Invention

[0005] The present invention solves at least to some extent one of the technical problems in the related art. For this reason, one object of the present invention is to provide mutants of copepod luciferase Pxluc (Pleuromamma xiphia, Pleuromamma xiphia luciferase). The inventor obtained mutants with a substrate specificity for ZS26 / F-CTZ improved by more than 2 times and mutants with a substrate specificity for ZS2 / F-CTZ improved by more than 4 times by subjecting the Pxluc protein to directed evolution. This luciferase can be expressed in both prokaryotic and eukaryotic cells, has a simple purification process, is advantageous for large-scale production, has a luminescence brightness similar to Gluc, is easy to detect, and this luciferase is expected to have a wide range of application prospects in many fields such as basic scientific research, biological monitoring, and biochemical diagnosis.

Means for Solving the Problems

[0006] Therefore, the first aspect of the present invention provides a mutant luciferase. According to an implementation means of the present invention, compared with the amino acid sequence shown in SEQ ID NO: 2, the mutant luciferase has at least one of the mutation sites at positions 98, 99, 100, and 101, and may or may not contain a signal peptide amino acid sequence. The mutant luciferase according to an implementation means of the present invention has strong catalytic activity against substrates such as coelenterazine, fluorinated coelenterazine, and coelenterazine derivatives, has a wider substrate spectrum than the conventional Pleuromamma xiphias luciferase, has stronger substrate selection specificity, has significantly enhanced luminescence brightness, and can be used in basic scientific research, biodetection technology, immunoassay, biochemical detection, or diagnosis and other fields for luminescence detection, and has broad application prospects.

[0007] The second aspect of the present invention provides a nucleic acid molecule. According to an implementation means 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 an implementation means of the present invention has strong catalytic activity against substrates such as coelenterazine, fluorinated coelenterazine, and coelenterazine derivatives, has a wider substrate spectrum than the conventional Pleuromamma xiphias luciferase, has stronger substrate selection specificity, has significantly enhanced luminescence brightness, and can be used in basic scientific research, biodetection technology, immunoassay, biochemical detection, or diagnosis and other fields for luminescence detection using the protein, and has broad application prospects.

[0008] The third aspect of the present invention provides an expression vector. According to one embodiment of the present invention, it contains the nucleic acid molecule described in the second aspect. The expression vector can contain selectable control sequences, and the control sequences are operably connected to the nucleic acid molecule. The control sequences are one or more control sequences capable of directing the expression of the nucleic acid molecule in a host. The expression vector according to one embodiment of the present invention can efficiently express a protein in an appropriate host cell, and the obtained protein has strong catalytic activity against substrates such as coelenterazine, fluorinated coelenterazine, and coelenterazine derivatives, has a broader substrate spectrum than conventional Pleuromamma xiphias luciferase, has stronger substrate selection specificity, has a significantly enhanced luminescence intensity, and can be used in basic scientific research, biodetection technology, immunoassay, biochemical detection, or diagnosis, etc. for luminescence detection using the protein, and has a wide application prospect.

[0009] The fourth aspect of the present invention provides a recombinant cell. According to one embodiment of the present invention, the nucleic acid molecule described in the second aspect or the expression vector described in the third aspect is carried. 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 mutant under appropriate conditions, and the mutant has strong catalytic activity against substrates such as coelenterazine, fluorinated coelenterazine, and coelenterazine derivatives, has a broader substrate spectrum than conventional Pleuromamma xiphias luciferase, has stronger substrate selection specificity, has a significantly enhanced luminescence intensity, and can be used in basic scientific research, biodetection technology, immunoassay, biochemical detection, or diagnosis, etc. for luminescence detection using the protein, and has a wide application prospect.

[0010] The fifth aspect of the present invention provides a method for producing a mutant luciferase. According to one embodiment of the present invention, the expression vector described in the third aspect is introduced into a recombinant cell, the recombinant cell is cultured and propagated, the culture propagation product is collected, and the mutant luciferase is extracted or purified.

[0011] The 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 is A) The mutant luciferase described in the first aspect forms a first mutant luciferase complex with the first specific recognition protein and the mutant luciferase in the form of a chemical bond, a biological bond, or a protein fusion. A second luciferase is used as a signal protein, and a second luciferase complex is formed with the second specific recognition protein and the second luciferase in the form of a chemical bond, a biological bond, or a protein fusion; B) The first mutant luciferase complex can react with a first substrate to generate a first luminescence signal, and the second luciferase complex can react with a second substrate to generate a second luminescence signal. The first mutant luciferase complex and the second substrate do not have a significant cross-substrate reaction, 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; C) By detecting the fluorescence signals and signal combinations of the self-luminescence systems of the mutant luciferase and the second luciferase, differentiating the four bases A, T, G, and C, and performing target nucleic acid sequencing.

[0012] 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. When the mutant luciferase and the second luciferase are different, the first substrate and the second substrate are different.

[0013] According to another embodiment of the present invention, the method for detecting a nucleic acid sequence is 1) Reacting different bases with reversible blocking modifications labeled with affinity labels under the action of a polymerase with a test template; 2) adding a plurality of luciferase complexes in step A, and specifically recognizing different affinity labels, so that the plurality of luciferase complexes are bound to different bases; 3) adding different substrates and determining the base type of polymerization by detecting the optical signal of the substrate or a combination thereof; 4) adding a cleavage reagent to cleave the blocking group and the linking group to prepare for the next polymerization reaction.

[0014] The seventh aspect of the present invention provides a nucleic acid sequencing reagent kit. According to an embodiment of the present invention, the reagent kit includes the mutant luciferase or luciferase complex described in the first aspect.

[0015] The eighth aspect of the present invention provides a method for detecting the content of a test substance. According to an embodiment of the present invention, the method includes a) forming a complex of the specific recognition molecule of the test substance and the mutant luciferase in the form of a chemical bond, a biological bond, or a protein fusion, using the mutant luciferase described in the first aspect as a signal protein; b) contacting the test substance with the complex; c) adding a substrate or an analog of the substrate of preulomammar kifias luciferase to the reaction system; d) after adding the substrate or the analog of the substrate of preulomammar kifias luciferase, determining the content of the test substance based on the detected fluorescence intensity of the reaction system.

[0016] Utilizing the property that the mutant binds to and reacts with the substrate, the mutant is used as a signal protein to detect a test substance. For example, the mutant is bound to or fused with a protein that can specifically recognize the test substance through chemical bonding or formation of a fusion protein. The protein that can specifically recognize the test substance binds to the test substance, and the mutant catalyzes its substrate, emits self-luminescence, and the intensity of the bioluminescence emitted by the mutant during the process of catalyzing 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 test substance can be determined from the level of its activity. Therefore, the reagent kit containing the mutant can be used to accurately detect the content of the test substance.

[0017] According to the specific implementation means of the present invention, the test substance may be nucleic acid. The mutant according to the present invention has strong catalytic activity for substrates such as coelenterazine, fluorinated coelenterazine, and coelenterazine derivatives, has a wider substrate spectrum than conventional Pleuromamma-XK luciferase, has stronger substrate selection specificity, and has significantly enhanced luminescence brightness. Therefore, the method can be used to detect the expression status of RNA or protein by nucleic acid such as the expression level of RNA or protein, the localization of protein, or a tracer. The accuracy and sensitivity of the nucleic acid detection method are much higher than those of conventional Gaussian luciferase, and the obtained results are more accurate.

[0018] In a ninth aspect of the present invention, the present invention provides a method for screening a substrate for Pleuromamma-XK luciferase, I) contacting the mutant luciferase described in the first aspect with a substrate to be screened to obtain a reaction mixture; II) A step of determining whether the screening target substrate is a target substrate based on whether the reaction mixture obtained in step I) emits a chemiluminescent signal. The method utilizes the property that the mutant binds to the target substrate to generate bioluminescence. The screening target substrate of interest is contacted with the mutant. The mutant emits bioluminescence during the process of catalyzing the screening target substrate, and a chemiluminescence microplate reader can measure whether the mutant catalyzes the screening target substrate to emit chemiluminescence, thereby determining whether the screening target substrate is a target substrate. Therefore, the method according to an embodiment of the present invention can accurately screen for the target substrate.

[0019] It should be understood that within the scope of the present invention, each of the above technical features of the present invention can be combined with each of the technical features specifically described below (such as embodiments), constituting new or preferred technical solutions. Due to space constraints, it will not be repeated here one by one.

Brief Description of the Drawings

[0020] The above and / or additional aspects and advantages of the present invention will become apparent and easier to understand from the description of the embodiments in combination with the following drawings.

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Modes for Carrying Out the Invention

[0021] Hereinafter, embodiments of the present invention will be described in detail. The embodiments described below are exemplary and are used only for interpreting the present invention and not for limiting the present invention.

[0022] Note that the terms "first" and "second" are used only for the purpose of explanation and cannot be understood as indicating or suggesting the relative importance or the number of technical features shown. Therefore, the features limited by "first" and "second" can explicitly or implicitly include at least one such feature. In the description of the present invention, "a plurality" means at least two, for example, two, three, etc., unless otherwise specifically limited.

[0023] In the field of basic scientific research, the luciferase gene is widely used as a reporter gene for studying the expression intensity and transcriptional regulation of exogenous genes under various promoters. In the field of biological monitoring technology, luciferase can be used to detect the presence or absence of chemical contaminants. It also has broad application prospects in fields such as immunoassay and biochemical diagnosis.

[0024] Variant In one aspect of the present invention, the present invention proposes a luciferase variant. Compared with the amino acid sequence shown in SEQ ID NO: 2, the variant has any one or a combination of a plurality of mutation sites at positions 98, 99, 100, and 101, and may or may not include a signal peptide amino acid sequence.

[0025] Based on the mutation sites according to one embodiment of the present invention, by modifying the above amino acid sequence, the obtained mutant has strong catalytic activity against substrates such as coelenterazine, fluorinated coelenterazine, and coelenterazine derivatives ZS2 and ZS26. It has a broader spectrum, stronger specificity, and significantly enhanced luminescence intensity compared to conventional substrates of Pleuromamma xiphias luciferase. It can be used in basic scientific research, biological detection technology, immunoassay, biochemical detection, or diagnosis fields for luminescence detection, has broad application prospects. For example, as a reporter gene, it can quantitatively detect DNA, RNA, transcription factors, proteins, or cells, etc. As a luminescence signal protein in a fusion protein, it can quantitatively detect target small molecules, proteins, etc.

[0026] In this specification, a "reporter gene" is a concept in molecular biology, which refers to a gene that is expressed in cells, tissues / organs, or individuals under specific circumstances, generates traits that are easy to detect and not originally produced by experimental materials, that is, a gene encoding a detectable protein or enzyme. As a reporter gene, the following conditions are required in genetic selection and screening detection: 1. It is cloned or its entire sequence is measured; 2. The expression product does not originally exist in recipient cells, that is, there is no background, and there is no similar endogenous expression product in transfected cells; 3. Its expression product can be quantitatively measured. When used, it includes the usage mode of forming a chimeric gene by fusing a reporter gene and a gene expression regulatory sequence, or fusing with other target genes to perform nucleic acid expression under the control of a control sequence, and thereby detecting the expression control of the target gene using its expression product and studying nucleic acids, but is not limited thereto.

[0027] In this specification, "nucleic acid expression" refers to DNA being expressed as RNA, or DNA being expressed as RNA and RNA further being expressed as protein, or RNA being expressed as protein, that is, the product after nucleic acid expression in this specification may be either RNA or protein.

[0028] In this specification, "Chemiluminescence" is also called "Cold Light" and is the emission of light caused by a chemical reaction without excitation by light, heat, an electric field, etc. There is also chemiluminescence in the biological system, which is called "Bioluminescence" and is the light emitted by fireflies, certain bacteria or fungi, protozoa, worms, and crustaceans. In the present application, the mutant can perform self-luminescence, i.e., chemiluminescence.

[0029] According to some specific embodiments of the present invention, the mutant may further include at least one of the following additional technical features.

[0030] According to some specific embodiments of the present invention, the gene sequence of wild-type pleuromamma xiphias luciferase (WT Pxluc) containing a signal peptide is shown in SEQ ID NO:1, the encoded amino acid is shown in SEQ ID NO:2, and the amino acids at positions 1-17 are the signal peptide (in bold). The gene sequence of wild-type pleuromamma xiphias luciferase without a signal peptide (WT no signal peptide Pxluc:WT-NS Pxluc) is shown in SEQ ID NO:3, and the encoded amino acid is shown in SEQ ID NO:4. The sequences of SEQ ID NO:1-4 are specifically as follows. ATGTATATAAAAGTTTGGTTTGGTCTGGCTTGTCTTTCATTGGTTCTGGCCCAACCAACTGAAAACAAGCAGGAGTCTCATATTGTAGATTCAGATCTTGATGGCGACCGTGGTAGGAAGTTGCCCGGAAAAAAGCTTCCTATAGAAGTACTCAAAATCATGGAAGCCAATGCCAGGAGAGCTGGTTGCACTAGAGGATGTCTCATATGTCTTTCAAAAATCAAGTGTACAGCCAAAATGAAGCGATACATTCCAGGGAGATGTCATACTTATGAAGGAGATAAATCTATTGGACAGGGAGGCATAGGTGGCCCTATTGTTGATATTCCTGAAATTATTGGATTCAAGAACATGGAACCCATGGATCAGTTCATCGCACAAGTTGATCTGTGCGCCGACTGTACAACTGGGTGCCTGAAAGGCCTTGCTAATGTTAGGTGCAATGACTTGCTGAAGAAATGGCTGCCTGACAGATGTGCTGGTTTTGCCGACAAAATTCAAAATGAAGTGGATAGTATCAAGGGCATGGCTGGGGATCGC(SEQ ID NO:1) MYIKVWFGLACLSLVLAQPTENKQESHIVDSDLDGDRGRKLPGKKLPIEVLKIMEANARRAGCTRGCLICLSKIKCTAKMKRYIPGRCHTYEGDKSIGQGGIGGPIVDIPEIIGFKNMEPMDQFIAQVDLCADCTTGCLKGLANVRCNDLLKKWLPDRCAGFADKIQNEVDSIKGMAGDR(SEQ ID NO:2) CAACCAACTGAAAACAAGCAGGAGTCTCATATTGTAGATTCAGATCTTGATGGCGACCGTGGTAGGAAGTTGCCCGGAAAAAAGCTTCCTATAGAAGTACTCAAAATCATGGAAGCCAATGCCAGGAGAGCTGGTTGCACTAGAGGATGTCTCATATGTCTTTCAAAAATCAAGTGTACAGCCAAAATGAAGCGATACATTCCAGGGAGATGTCATACTTATGAAGGAGATAAATCTATTGGACAGGGAGGCATAGGTGGCCCTATTGTTGATATTCCTGAAATTATTGGATTCAAGAACATGGAACCCATGGATCAGTTCATCGCACAAGTTGATCTGTGCGCCGACTGTACAACTGGGTGCCTGAAAGGCCTTGCTAATGTTAGGTGCAATGACTTGCTGAAGAAATGGCTGCCTGACAGATGTGCTGGTTTTGCCGACAAAATTCAAAATGAAGTGGATAGTATCAAGGGCATGGCTGGGGATCGC(SEQ ID NO:3) QPTENKQESHIVDSDLDGDRGRKLPGKKLPIEVLKIMEANARRAGCTRGCLICLSKIKCTAKMKRYIPGRCHTYEGDKSIGQGGIGGPIVDIPEIIGFKNMEPMDQFIAQVDLCADCTTGCLKGLANVRCNDLLKKWLPDRCAGFADKIQNEVDSIKGMAGDR(SEQ ID NO:4)

[0031] According to some specific embodiments of the present invention, compared with the amino acid sequence shown in SEQ ID NO: 2, the variant has any one or a combination of the following mutations (1)-(4): (1) The G mutation at position 98 is L or P or Q or S or T; (2) The Q mutation at position 99 is R or W or I or Y or A or L or F or V or P or E or M; (3) The G mutation at position 100 is S or Q or R or W or T or A or L; (4) The G mutation at position 101 is F or R or S or C or Y or L or I or K or V or P.

[0032] According to some other specific embodiments of the present invention, compared with the amino acid sequence shown in SEQ ID NO:2, the mutant luciferase contains any one or two of the mutation sites at positions 98, 99, 100 and 101, and may or may not contain a signal peptide amino acid sequence.

[0033] According to some other specific embodiments of the present invention, compared with the amino acid sequence shown in SEQ ID NO:2, the mutant contains any one or two of the following mutations (1)-(4): (1) The G mutation at position 98 is L or P or Q or S or T. (2) The Q mutation at position 99 is R or W or I or Y or A or L or F or V or P or E or M. (3) The G mutation at position 100 is S or Q or R or W or T or A or L. (4) The G mutation at position 101 is 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 present invention, compared with the amino acid sequence shown in SEQ ID NO:2, the mutant has the following mutations: 1) The G mutation at position 98 is L and the Q mutation at position 99 is R, or 2) The G mutation at position 98 is P, or 3) The G mutation at position 98 is Q, or 4) The G mutation at position 98 is S and the Q mutation at position 99 is W, or 5) The Q mutation at position 98 is I, or 6) The Q mutation at position 99 is Y, or 7) The Q mutation at position 99 is A, or 8) The Q mutation at position 99 is L, or 9) The Q mutation at position 99 is F, or 10) The G mutation at position 98 is L and the Q mutation at position 99 is V, or 11) The G mutation at position 98 is T, the Q mutation at position 99 is P, or 12) The G mutation at position 98 is L, the Q mutation at position 99 is E, or 13) The Q mutation at position 99 is M, the G mutation at position 100 is S, or 14) The G mutation at position 100 is Q, the G mutation at position 101 is F, or 15) The G mutation at position 100 is R, the G mutation at position 101 is R, or 16) The G mutation at position 100 is W, the G mutation at position 101 is F, or 17) The G mutation at position 100 is S, or 18) The G mutation at position 100 is T, the G mutation at position 101 is S, or 19) The G mutation at position 100 is R, the G mutation at position 101 is C, or 20) The G mutation at position 100 is A, the G mutation at position 101 is R, or 21) The G mutation at position 100 is L, the G mutation at position 101 is Y, or 22) The G mutation at position 100 is S, the G mutation at position 101 is L, or 23) The G mutation at position 101 is I, or 24) The G mutation at position 100 is L, the G mutation at position 101 is K, or 25) The G mutation at position 100 is T, or 26) The G mutation at position 100 is A, the G mutation at position 101 is V, or 27) The G mutation at position 100 is A, the G mutation at position 101 is P.

[0034] According to some specific embodiments of the present invention, when the amino acid sequence shown in SEQ ID NO: 2 has the above mutations, the obtained protein has strong catalytic activity against substrates such as coelenterazine, fluorinated coelenterazine, and coelenterazine derivatives, has a wider spectrum than the conventional pleuromamma·kifianus luciferase substrate, has a significantly enhanced luminescence brightness, has a greatly improved detection accuracy in actual use, and can be used in basic scientific research, biological detection technology, immunoassay, biochemical detection, or diagnosis and other fields where luminescence detection is performed using the said protein, and has broad application prospects.

[0035] According to some specific embodiments of the present invention, the mutant luciferase does not contain a signal peptide sequence.

[0036] According to some specific embodiments of the present invention, the mutant luciferase may be a luciferase having a mutation at any one or a combination of sites at positions 98, 99, 100, and 101 (for example, any one of the four mutation sites, or any combination of two sites, or any combination of three sites, or all four sites are mutated) as compared with the amino acid sequence shown in SEQ ID NO:2.

[0037] According to some specific embodiments of the present invention, the mutant luciferase may have any one or a combination of sites at positions 98, 99, 100, and 101 as compared with the amino acid sequence shown in SEQ ID NO:2, and the mutant luciferase does not contain a signal peptide amino acid sequence.

[0038] The amino acids at positions 1-17 in the amino acid sequence shown in SEQ ID NO:2 are signal peptides. The inventors 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 mutates at any one or a combination of sites at positions 98, 99, 100, and 101 (these site numbers are numbered according to the SEQ ID NO:2 sequence), a mutant luciferase having this amino acid sequence can be obtained, which has strong catalytic activity against substrates such as coelenterazine, fluorinated coelenterazine, and coelenterazine derivative ZS2, and has a broader spectrum, stronger specificity, and significantly enhanced luminescence intensity than the conventional Pleuromamma xiphias luciferase substrate. According to some specific embodiments of the present invention, the mutant is a non-secretory protein or a secretory protein.

[0039] According to some specific embodiments of the present invention, the present invention provides a nucleic acid molecule encoding the aforementioned variant.

[0040] It should be understood by those skilled in the art that for the nucleic acids mentioned in the specification and claims of the present invention, they actually include either one or both of the complementary double strands. For the sake of convenience, in this specification and the claims, in most cases, only one strand is shown, but in fact, another complementary strand is disclosed. Also, the nucleic acid sequences in the present application include either DNA type or RNA type. Disclosing one means that the other is also disclosed.

[0041] Another aspect of the present invention is that the present invention proposes an expression vector containing the nucleic acid molecule as described above. The type of this expression vector is not particularly limited, and it only needs to copy and express the corresponding variant in the host cell. The expression vector may include a selectable control sequence, and the control sequence is operably connected 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 some specific embodiments of the present invention can efficiently express proteins in appropriate host cells. The obtained proteins have strong catalytic activity against substrates such as coelenterazine, fluorinated coelenterazine, and coelenterazine derivatives. They have a wider spectrum, stronger specificity, and significantly enhanced luminescence intensity compared to conventional pleuromamma luciferase substrates. They can be used in basic scientific research, biodetection technology, immunoassay, biochemical detection, or diagnosis and other fields for luminescence detection, and have broad application prospects.

[0042] In a further aspect of the present invention, the present invention proposes a recombinant cell carrying the nucleic acid molecule, expression vector or variant as described above. 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 variant under appropriate conditions, and the variant has strong catalytic activity against substrates such as coelenterazine, fluorinated coelenterazine and coelenterazine derivatives, and has a broader spectrum, stronger specificity and significantly enhanced luminescence intensity than the conventional pleuromamma kifianus luciferase substrate. It can be used in basic scientific research, biological detection technology, immunoassay, biochemical detection or diagnosis and other fields for luminescence detection using the protein, and has broad application prospects.

[0043] 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.

[0044] According to some specific embodiments of the present invention, the recombinant cell is Escherichia coli, yeast or mammalian cell. According to some specific embodiments of the present invention, the recombinant cell is not particularly limited, and cells of any of the above-mentioned variants that can be expressed in nucleic acids or vectors (such as plasmids), such as yeast cells, bacteria or mammalian cells such as human embryonic kidney cells, can be used.

[0045] According to some specific embodiments of the present invention, the recombinant cell does not include animal germ cells, fertilized eggs or embryonic stem cells.

[0046] According to some specific embodiments of the present invention, the present invention provides a method for producing mutant luciferase, which includes introducing the expression vector as described above into a recombinant cell, culturing and propagating the recombinant cell, and collecting the culture 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 mutant luciferase as described above in the detection of the nucleic acid sequence of the corresponding substrate.

[0047] In the present invention, "mutant luciferase", "luciferase mutant", and "mutant" can be equivalently substituted.

[0048] Reagent kit and use in the preparation of a reagent kit

[0049] In one aspect of the present invention, the present invention proposes a reagent kit for detecting the content of a test substance, the reagent kit includes a luciferase mutant as described above, and the specific recognition protein of the test substance is suitable for forming a complex with the mutant.

[0050] According to some specific embodiments of the present invention, the reagent kit further includes a substrate of Pleuromamma xiphias luciferase or an analogue of the substrate.

[0051] According to some specific embodiments of the present invention, the substrate is selected from at least one of coelenterazine, fluorinated coelenterazine or a coelenterazine derivative.

[0052] According to some specific embodiments of the present invention, the coelenterazine derivative is selected from coelenterazine derivative ZS2 or coelenterazine derivative ZS26. JPEG2025522599000001.jpg56170

[0053] According to some specific embodiments of the present invention, the reagent kit further includes the specific recognition protein of the test substance.

[0054] The present invention provides a nucleic acid sequencing reagent kit containing a mutant luciferase as described above.

[0055] In another aspect of the present invention, the present invention proposes the use in the manufacture of a reagent kit of a variant as described above, the reagent kit being used for detecting the content of a test substance, and the specific recognition protein of the test substance being suitable for forming a complex with the variant. Utilizing the property that the variant binds to a substrate and reacts, the variant can be used as a signal protein to detect the test substance. The variant is bound or fused to a protein that can specifically recognize the test substance by chemical bonding or formation of a fusion protein. When the test substance, the substrate of the variant, is in the same system, the protein that specifically recognizes the test substance binds to the test substance, and the variant catalyzes its substrate to emit self-luminescence. The intensity of the bioluminescence emitted during the process in which the variant catalyzes its substrate is measured by a chemiluminescence microplate reader. The intensity of the bioluminescence can reflect the activity of the variant, and the content of the test substance is judged according to the level of its activity. It is necessary to elute non-specific binding proteins in the system before the substrate binds to the variant to eliminate other factors that interfere with the detection result. Therefore, the variant can be used to manufacture a reagent kit to accurately detect the content of a test substance.

[0056] According to some specific embodiments of the present invention, the above use may further include at least one of the following additional technical features.

[0057] According to some specific embodiments of the present invention, the reagent kit further includes a substrate of pleuromamma kifianus luciferase or an analogue of the substrate.

[0058] According to some specific embodiments of the present invention, the substrate is selected from at least one of coelenterazine, fluorinated coelenterazine or a coelenterazine derivative.

[0059] According to some specific embodiments of the present invention, the substrate is not particularly limited, and any substance that can cause a chemical reaction with the variant is included within this range, not limited to chemiluminescence reactions. Those skilled in the art may substitute different substrates according to experimental needs.

[0060] According to some specific embodiments of the present invention, the said selenotriazine derivative is selected from selenotriazine derivative ZS2 or selenotriazine derivative ZS26.

[0061] According to some specific embodiments of the present invention, the said reagent kit further comprises a specific recognition protein of the said analyte.

[0062] Method

[0063] In one aspect of the present invention, the present invention proposes a method for detecting the content of a substance, i) forming a complex of a specific recognition protein of the analyte and a mutant as described above; ii) contacting the analyte with the said complex; iii) adding a substrate or an analogue of a substrate of prelumaman luciferase to the reaction system; iv) determining the content of the said analyte based on the fluorescence intensity of the said reaction system to which the substrate or the analogue of the substrate of prelumaman luciferase has been added.

[0064] According to some specific embodiments of the present invention, the present invention provides a method for detecting the content of an analyte, a) using a mutant luciferase as described above as a signal protein, and forming a complex of the specific recognition protein of the analyte and the mutant luciferase in the form of chemical bond, biological bond or protein fusion; b) contacting the analyte with the said complex; c) adding a substrate or an analogue of a substrate of prelumaman luciferase to the reaction system; d) determining the content of the test substance based on the fluorescence intensity of the reaction system detected after adding a substrate or a substrate analog of the prelumamanma kifias luciferase; This method includes the above steps. According to some specific embodiments of the present invention, the substrate is selected from at least one of coelenterazine, fluorinated coelenterazine, or a coelenterazine derivative.

[0065] According to some specific embodiments of the present invention, the coelenterazine derivative is selected from coelenterazine derivative ZS2 or coelenterazine derivative ZS26.

[0066] In a further aspect of the present invention, the present invention provides a method for screening a substrate of prelumamanma kifias luciferase, I) contacting the mutant as described above with the substrate to be screened to obtain a reaction mixture; II) determining whether the substrate to be screened is a target substrate based on whether the reaction mixture obtained in step I) emits a chemiluminescence signal.

[0067] According to some specific embodiments of the present invention, the fact that the reaction mixture obtained in step I) emits a chemiluminescence signal indicates that the substrate to be screened is a target substrate.

[0068] Hereinafter, the present invention will be described with reference to specific examples. These examples are illustrative and do not limit the present invention in any way.

[0069] Example 1 Design and construction of prokaryotic expression of wild-type prelumamanma kifias luciferase plasmid

[0070] In this example, by constructing wild-type pleuromona kieliense luciferase containing a signal peptide and wild-type pleuromona kieliense luciferase without a signal peptide, the influence of the signal peptide on wild-type pleuromona kieliense luciferase is compared.

[0071] The gene sequence of wild-type pleuromona kieliense luciferase containing a signal peptide (WT Pxluc) is shown in SEQ ID NO:1, the encoded amino acid is shown in SEQ ID NO:2, and the amino acids at positions 1-17 are the signal peptide (bold). The gene sequence of wild-type pleuromona kieliense luciferase without a signal peptide (WT no signal peptide Pxluc:WT-NS Pxluc) is shown in SEQ ID NO:3, and the encoded amino acid is shown in SEQ ID NO:4.

[0072] By the total gene synthesis technique, the gene sequence SEQ ID NO:5 (Sangon Biotech) of pleuromona kieliense luciferase containing a signal peptide used in the pET28a vector (pET28a Pxluc WT) was synthesized. The plasmid map is shown in Figure 1, and a purification label containing six histidines (6xHis) is fused to its C-terminus to facilitate protein purification. The enzyme cleavage sites at both ends are BamHI and NotI. By the PCR technique, the gene sequence SEQ ID NO:6 of the pleuromona kieliense luciferase protein containing a signal peptide used in the pCold vector was synthesized, and a purification label containing six histidines (6xHis) is fused to its C-terminus to facilitate protein purification. By the PCR technique, the gene sequence SEQ ID NO:7 of the pleuromona kieliense luciferase protein with the signal peptide removed used in the pCold vector was synthesized, and a purification label containing six histidines (6xHis) is fused to its C-terminus to facilitate protein purification. ATGTATATAAAAGTTTGGTTTGGTCTGGCTTGTCTTTCATTGGTTCTGGCCCAACCAACTGAAAACAAGCAGGAGTCTCATATTGTAGATTCAGATCTTGATGGCGACCGTGGTAGGAAGTTGCCCGGAAAAAAGCTTCCTATAGAAGTACTCAAAATCATGGAAGCCAATGCCAGGAGAGCTGGTTGCACTAGAGGATGTCTCATATGTCTTTCAAAAATCAAGTGTACAGCCAAAATGAAGCGATACATTCCAGGGAGATGTCATACTTATGAAGGAGATAAATCTATTGGACAGGGAGGCATAGGTGGCCCTATTGTTGATATTCCTGAAATTATTGGATTCAAGAACATGGAACCCATGGATCAGTTCATCGCACAAGTTGATCTGTGCGCCGACTGTACAACTGGGTGCCTGAAAGGCCTTGCTAATGTTAGGTGCAATGACTTGCTGAAGAAATGGCTGCCTGACAGATGTGCTGGTTTTGCCGACAAAATTCAAAATGAAGTGGATAGTATCAAGGGCATGGCTGGGGATCGC(SEQ ID NO:1) MYIKVWFGLACLSLVLAQPTENKQESHIVDSDLDGDRGRKLPGKKLPIEVLKIMEANARRAGCTRGCLICLSKIKCTAKMKRYIPGRCHTYEGDKSIGQGGIGGPIVDIPEIIGFKNMEPMDQFIAQVDLCADCTTGCLKGLANVRCNDLLKKWLPDRCAGFADKIQNEVDSIKGMAGDR(SEQ ID NO:2) CAACCAACTGAAAACAAGCAGGAGTCTCATATTGTAGATTCAGATCTTGATGGCGACCGTGGTAGGAAGTTGCCCGGAAAAAAGCTTCCTATAGAAGTACTCAAAATCATGGAAGCCAATGCCAGGAGAGCTGGTTGCACTAGAGGATGTCTCATATGTCTTTCAAAAATCAAGTGTACAGCCAAAATGAAGCGATACATTCCAGGGAGATGTCATACTTATGAAGGAGATAAATCTATTGGACAGGGAGGCATAGGTGGCCCTATTGTTGATATTCCTGAAATTATTGGATTCAAGAACATGGAACCCATGGATCAGTTCATCGCACAAGTTGATCTGTGCGCCGACTGTACAACTGGGTGCCTGAAAGGCCTTGCTAATGTTAGGTGCAATGACTTGCTGAAGAAATGGCTGCCTGACAGATGTGCTGGTTTTGCCGACAAAATTCAAAATGAAGTGGATAGTATCAAGGGCATGGCTGGGGATCGC(SEQ ID NO:3) QPTENKQESHIVDSDLDGDRGRKLPGKKLPIEVLKIMEANARRAGCTRGCLICLSKIKCTAKMKRYIPGRCHTYEGDKSIGQGGIGGPIVDIPEIIGFKNMEPMDQFIAQVDLCADCTTGCLKGLANVRCNDLLKKWLPDRCAGFADKIQNEVDSIKGMAGDR(SEQ ID NO:4) aagcttgccgccaccATGTATATAAAAGTTTGGTTTGGTCTGGCTTGTCTTTCATTGGTTCTGGCCCAACCAACTGAAAACAAGCAGGAGTCTCATATTGTAGATTCAGATCTTGATGGCGACCGTGGTAGGAAGTTGCCCGGAAAAAAGCTTCCTATAGAAGTACTCAAAATCATGGAAGCCAATGCCAGGAGAGCTGGTTGCACTAGAGGATGTCTCATATGTCTTTCAAAAATCAAGTGTACAGCCAAAATGAAGCGATACATTCCAGGGAGATGTCATACTTATGAAGGAGATAAATCTATTGGACAGGGAGGCATAGGTGGCCCTATTGTTGATATTCCTGAAATTATTGGATTCAAGAACATGGAACCCATGGATCAGTTCATCGCACAAGTTGATCTGTGCGCCGACTGTACAACTGGGTGCCTGAAAGGCCTTGCTAATGTTAGGTGCAATGACTTGCTGAAGAAATGGCTGCCTGACAGATGTGCTGGTTTTGCCGACAAAATTCAAAATGAAGTGGATAGTATCAAGGGCATGGCTGGGGATCGCggtggcagtgaaaatctgtattttcagagcggcggccatcatcatcatcatcatggcagctgatgagaattc(SEQ ID NO:5) caccatgaatcacaaagtgcatatgATGTATATAAAAGTTTGGTTTGGTCTGGCTTGTCTTTCATTGGTTCTGGCCCAACCAACTGAAAACAAGCAGGAGTCTCATATTGTAGATTCAGATCTTGATGGCGACCGTGGTAGGAAGTTGCCCGGAAAAAAGCTTCCTATAGAAGTACTCAAAATCATGGAAGCCAATGCCAGGAGAGCTGGTTGCACTAGAGGATGTCTCATATGTCTTTCAAAAATCAAGTGTACAGCCAAAATGAAGCGATACATTCCAGGGAGATGTCATACTTATGAAGGAGATAAATCTATTGGACAGGGAGGCATAGGTGGCCCTATTGTTGATATTCCTGAAATTATTGGATTCAAGAACATGGAACCCATGGATCAGTTCATCGCACAAGTTGATCTGTGCGCCGACTGTACAACTGGGTGCCTGAAAGGCCTTGCTAATGTTAGGTGCAATGACTTGCTGAAGAAATGGCTGCCTGACAGATGTGCTGGTTTTGCCGACAAAATTCAAAATGAAGTGGATAGTATCAAGGGCATGGCTGGGGATCGCggtggcagtgagaacctgtac(SEQ ID NO:6) catgaatcacaaagtgcatatgCAACCAACTGAAAACAAGCAGGAGTCTCATATTGTAGATTCAGATCTTGATGGCGACCGTGGTAGGAAGTTGCCCGGAAAAAAGCTTCCTATAGAAGTACTCAAAATCATGGAAGCCAATGCCAGGAGAGCTGGTTGCACTAGAGGATGTCTCATATGTCTTTCAAAAATCAAGTGTACAGCCAAAATGAAGCGATACATTCCAGGGAGATGTCATACTTATGAAGGAGATAAATCTATTGGACAGGGAGGCATAGGTGGCCCTATTGTTGATATTCCTGAAATTATTGGATTCAAGAACATGGAACCCATGGATCAGTTCATCGCACAAGTTGATCTGTGCGCCGACTGTACAACTGGGTGCCTGAAAGGCCTTGCTAATGTTAGGTGCAATGACTTGCTGAAGAAATGGCTGCCTGACAGATGTGCTGGTTTTGCCGACAAAATTCAAAATGAAGTGGATAGTATCAAGGGCATGGCTGGGGATCGCggtggcagtgagaacctgta(SEQ ID NO:7)

[0073] 1. Acquisition of Insert Fragment Wild-type prefuroma mammal Kifia luciferase (pET28a Pxluc WT) containing a signal peptide synthesized from the full gene is dissolved in deionized water and diluted to 10 ng / μl to serve as template DNA. Using the KOD FXD Neo enzyme, prepare the PCR reaction system and perform the PCR reaction according to its specification to produce the insert fragment.

[0074] The primer sequences used in the PCR reactions of wild-type prefuroma mammal Kifia luciferase containing a signal peptide (WT-Pxluc) and wild-type prefuroma mammal Kifia 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 was 30.

[0075]

Table 1

[0076]

Table 2

[0077]

Table 3

[0078] Add 0.5 μL of DpnI enzyme to the reaction system and incubate at 37 °C for 3 hours to digest the template. Then, the gel-extracted products of approximately 586 bp and 531 bp are the insertion fragments insert. The 586-bp gel-extracted fragment is the insertion fragment containing the signal peptide, and the 531-bp gel-extracted fragment is the insertion fragment without the signal peptide.

[0079] 2. Obtaining the linearized vector Using the pCold vector as a template, linearize the vector by PCR to facilitate recombination with the insertion fragment. Use the KOD FX neo enzyme and prepare the PCR reaction system and perform the PCR reaction according to its manual.

[0080] 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.

[0081]

Table 4

[0082]

Table 5

[0083]

Table 6

[0084] Add 0.5 μL of DpnI enzyme to the reaction system and incubate at 37 °C for 3 hours to digest the template. Then, the gel-purified product of about 4433 bp is the linearized vector.

[0085] Recombine the insert and vector using the In-Fusion Cloning reagent kit (TAKARA) according to the reaction system shown in Table 7 and incubate at 50 °C for 15 minutes.

[0086]

Table 7

[0087] Transform 2.5 μL of the above reaction product into DH5α competent cells, spread them on an ampicillin-resistant plate with a final concentration of 100 μg / mL. The next day, pick out monoclonal colonies from the plate, extract the plasmid and sequence it to confirm that the target fragment is accurately inserted into the vector. The obtained plasmids are the wild-type prefuroma mammal luciferase pCold-WT Pxluc containing the signal peptide (Figure 2) and the wild-type prefuroma mammal luciferase pCold WT NS Pxluc without the signal peptide (Figure 3), which is in line with the expectation of the experiment.

[0088] Example 2 Prokaryotic expression, purification and protein-level activity measurement of prefuroma mammal luciferase

[0089] 1. Prokaryotic expression of prefuroma mammal luciferase 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 respectively transformed into OrigamiB (DE3) Chemically Competent Cell (Vidi Biotechnology, EC1020S). They were spread on plates, single colonies were taken out from the plates, cultured overnight at 37°C, diluted at a ratio of 1:100 the next day, and transferred into fresh 300 ml of LB medium containing kanamycin (50 μg / ml) or ampicillin (100 μg / ml) respectively. They were cultured with shaking at 37°C and 200 rpm until OD600 ≈ 0.5 - 0.6, and then cooled on ice for 1 hour. The inducer IPTG was added in an amount with a final concentration of 1 mM, and induction was carried out overnight at 16°C.

[0090] 2. Purification of Pleuromamma kaffiensis luciferase After induction, the bacterial liquid 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, dissolved on ice for 30 min, disrupted by ultrasound (2 s on 3 s off, 60% power) for 30 min, and centrifuged at 12000 rpm at 4°C for 30 min to separate the supernatant (cell lysate) and the precipitate.

[0091] 2 mL of HisTrap FF filler was added to a manual column (purchased from Sangon Biotech, model number: F506607-0001# affinity chromatography column empty column), and the filler was equilibrated by washing with 30 ml of binding buffer. Then, about 30 ml of filtered cell lysate was added. It was washed 10 times (10 ml each time) with a rinse solution (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 solution (50 mM Tris, pH 8.0, 250 mM NaCl, 300 mM imidazole), and the eluted protein was collected. Dialysis was carried out overnight at 4°C. The protein concentration and purity distribution were measured by the BCA quantitative reagent kit method and the SDS-PAGE method.

[0092] The proteins eluted by SDS-PAGE method were measured, and the purification results are shown in Figure 4, indicating that the BL21(DE3) cells transformed with the pET28a-Pxluc WT plasmid successfully expressed the target protein, and OrigamiB (DE3) transformed with the expression plasmids pCold WT Pxluc and pCold WT-NS Pxluc also successfully expressed the target protein respectively.

[0093] 3. Measurement of the protein activity of pleuromamma kifferi luciferase Accurately measure the protein concentration using a BCA quantitative reagent kit (Thermo ScientificTM PierceTM BCA Protein Assay Kit), dilute the purified luciferase obtained above with a diluent (50 mM Tris-HCl pH 8.0, 100 mM NaCl, 0.1% (v / v) Tween-20), dilute the protein expressed by the pET28a-Pxluc WT plasmid to 12 μg / ml, dilute the proteins expressed by the pCold WT Pxluc and pCold WT-NS Pxluc plasmids to 1 μg / ml, and take 10 μL of each and add them to a black 96-well plate. Add 90 μL each of the substrates coelenterazine (purchased from CapitalBio), fluorinated coelenterazine, coelenterazine derivatives ZS2 and ZS26 diluted to 100 μM with the same solution (the structural formula of each compound is shown in Figure 5), and read the luminescence intensity with the self-luminescence module of the enzyme marker. The measurement results of the activities of prefuroma mammas luciferase and CTZ (coelenterazine) are shown in Figure 6. It is known that the Gluc luminescence activity expressed in OrigamiB (DE3) competent cells is good, and it is used as a control group. From the results in Figure 6, it is shown that the luminescence value of OrigamiB (DE3) competent cells containing the plasmid pCold WT NS Pxluc is the highest and is equivalent to the luminescence value of OrigamiB (DE3) competent cells containing the plasmid pCold Gluc. Therefore, the luminescence activity of coelenterazine by prefuroma mammas luciferase without a signal peptide is close to the luminescence activity of coelenterazine by Gluc. The activity of prefuroma mammas luciferase without a signal peptide is higher than that of prefuroma mammas luciferase with a signal peptide, and subsequent experiments will be carried out with prefuroma mammas luciferase without a signal peptide.

[0094] Example 3 Design and construction of a prefuroma mammas luciferase mutant library

[0095] Using the PCR technique, with the pCold WT-NS Pxluc plasmid sequence as a template, a site-directed saturation mutant library at sites G98, Q99, G100, and G101 of SEQ ID NO:2 (mutating a specific amino acid to any of the other amino acids) was synthesized. Mutant library 1 is the site-directed saturation mutant library (L1) at amino acid site G98, mutant library 2 is the site-directed saturation mutant library (L2) at site Q99, mutant library 3 is the site-directed saturation mutant library (L3) at amino acid site G100, mutant library 4 is the site-directed saturation mutant library (L4) at site G101, mutant library 5 is the combined mutant library (L5) at amino acid sites G98 and Q99, mutant library 6 is the combined mutant library (L6) at amino acid sites G100 and G101, and mutant library 7 is the combined mutant library (L7) at amino acid sites G98, Q99, G100, and G101 sites.

[0096] A pair of primers designed for mutant library L1: Forward primer Primer F1: 5’-GATAAATCTATTNNKCAGGGAGGCATAGGTGGCCCTATTG-3’ (SEQ ID NO:14) Reverse primer Primer R1: 5’-TAGGGCCACCTATGCCTCCCTGMNNAATAGATTTATCTCC-3’ (SEQ ID NO:15) A pair of primers designed for mutant library L2: Forward primer Primer F2: 5’-GATAAATCTATTGGANNKGGAGGCATAGGTGGCCCTATTG-3’ (SEQ ID NO:16) Reverse primer Primer R2: 5’-TAGGGCCACCTATGCCTCCMNNTCCAATAGATTTATCTCC-3’ (SEQ ID NO:17) A pair of primers designed for mutant library L3: Forward primer Primer F3: 5’-GATAAATCTATTGGACAGNNKGGCATAGGTGGCCCTATTG-3’(SEQ ID NO:18) Reverse primer Primer R3: 5’-TAGGGCCACCTATGCCMNNCTGTCCAATAGATTTATCTCC-3’ (SEQ ID NO:19) A pair of primers designed for mutant library L4: Forward primer Primer F4: 5’-GATAAATCTATTGGACAGGGANNKATAGGTGGCCCTATTG-3’(SEQ ID NO:20) Reverse primer Primer R4: 5’-TAGGGCCACCTATMNNTCCCTGTCCAATAGATTTATCTCC-3’(SEQ ID NO:21) A pair of primers designed for mutant library L5: Forward primer Primer F5: 5’-GATAAATCTATTNNKNNKGGAGGCATAGGTGGCCCTATTG-3’ (SEQ ID NO:22) Reverse primer Primer R5: 5’-TAGGGCCACCTATGCCTCCMNNMNNAATAGATTTATCTCC-3’ (SEQ ID NO:23) A pair of primers designed for mutant library L6: Forward primer Primer F6: 5’- GATAAATCTATTGGACAGNNKNNKATAGGTGGCCCTATTG-3’(SEQ ID NO:24) Reverse primer Primer R6: 5’- TAGGGCCACCTATMNNMNNCTGTCCAATAGATTTATCTCC-3’(SEQ ID NO:25) A pair of primers designed for variant library L7: Upstream primer Primer F7: 5’-GAAGGAGATAAATCTATTNNKNNKNNKNNKATAGGTGGCCCTATTGTTGATA-3’(SEQ ID NO:26) Downstream primer Primer R7: 5’-CAATAGGGCCACCTATMNNMNNMNNMNNAATAGATTTATCTCCTTCATAAGT-3’(SEQ ID NO:27)

[0097] In the nucleic acid sequence, "N" is A / C / G / T, "K" is G / T, and "M" is A / C.

[0098] Table 8 shows the PCR reaction system prone to errors, Table 9 shows the reaction conditions, and the number of PCR cycles is 30.

[0099]

Table 8

[0100]

Table 9

[0101] After the reaction is completed, 0.5 μL of DpnI enzyme is added to the reaction system and incubated at 37 °C for 3 hours to digest the template. Then, the enzyme cleavage product is transformed into DH5a competent cells, cultured overnight in 10 ml, and then the plasmid is extracted. After the extracted plasmid is transformed into OrigamiB(DE3) Chemically Competent Cell (Vidi Biotechnology, EC1020S) competent cells, it is spread on a plate, a single colony is taken out from the plate, cultured overnight at 37 °C, diluted at a ratio of 1:100 the next day, transferred to fresh 0.4 ml of LB medium containing ampicillin resistance (100 μg / ml), shaken and cultured at 37 °C and 200 rpm until OD600 ≈ 0.5 - 0.6, and cooled on ice for 1 hour. The inducer IPTG is added in an amount with a final concentration of 1 mM, and induced overnight at 16 °C.

[0102] Take 50 μL of the induced bacterial solution and add it to a black 96-well plate, and add 10 μL each of the substrates coelenterazine (CTZ) (purchased from CapitalBio), fluorinated coelenterazine, ZS2, and ZS26 with a final concentration of 100 μM. Read the luminescence intensity by the self-luminescence module of the enzyme marker, and perform mutant bacterial solution screening (screening criteria: screening is performed by the ratio of luminescence intensity, compare the luminescence intensity ratio of the substrates ZS2 or ZS26 to the luminescence intensity of the substrate F-CTZ, and if this ratio is greater than the ratio of the luminescence intensities of the two substrates by the wild type, it is regarded as a dominant mutant).

[0103] Example 4 Expression and Purification of Pleuromamma xiphias Luciferase Mutant Library 1. Prokaryotic Expression of Pleuromamma xiphias Luciferase Mutants The mutant bacterial solutions 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, diluted at a ratio of 1:100 the next day, transferred to fresh 15 ml of LB medium containing ampicillin (100 μg / ml), shaken and cultured at 37°C and 200 rpm until OD600 ≈ 0.5 - 0.6, and cooled on ice for 1 hour. An inducer IPTG was added in an amount with a final concentration of 1 mM, and induction was carried out overnight at 16°C.

[0104] 2. Purification of the pleuromamma kiffiensis luciferase mutant The bacterial solution precipitate after induction was collected by centrifugation at 8000 rpm / min for 10 min, 600 μL of binding buffer (50 mM Tris, pH 8.0, 250 mM NaCl) and 6 μL of lysozyme were added, dissolved on ice for 30 min, disrupted by ultrasonic waves (2 s on 3 s off, 60% power) for 10 min, and centrifuged at 4°C and 12000 rpm for 30 min to separate the supernatant (cell lysate) and precipitate.

[0105] 50 μL of HisTrap FF filler was added to a manual column (purchased from Sangon Biotech, model number: F506607-0001# affinity chromatography column empty column), and the filler was equilibrated by washing with 3 ml of binding buffer. Then, 600 μL of the filtered cell lysate was added. It was washed 10 times (3 ml each time) with a rinse solution (50 mM Tris, pH 8.0, 250 mM NaCl, 10 mM imidazole), and then the protein was eluted with 100 μl of an eluent (50 mM Tris, pH 8.0, 250 mM NaCl, 300 mM imidazole), and the protein after elution was collected.

[0106] The protein eluted from the Ni column is dialyzed overnight at 4°C against a dialysis buffer (25 mM Tris, pH 8.0, 250 mM NaCl). The protein concentration and purity distribution are measured by the BCA quantification reagent kit method (Thermo ScientificTM PierceTM BCA Protein Assay Kit) and SDS-PAGE method.

[0107] Example 5 Substrate Specificity Test of Pleuromamma kifui Luciferase Dominant Mutants

[0108] The protein concentration is accurately measured using a BCA quantification reagent kit (Thermo ScientificTM PierceTM BCA Protein Assay Kit). Luciferase is 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 is taken and added to a black 96-well plate. 90 μL each of the substrates coelenterazine (purchased from CapitalBio), fluorinated coelenterazine, and coelenterazine derivatives ZS2 and ZS26, diluted to 100 μM with the same solution, are added. The luminescence intensity is read using the chemiluminescence module of the enzyme marker, and the substrate specificity of the mutants is compared based on the activity ratios of ZS2 / F-CTZ and ZS26 / F-CTZ. The activity measurement results of the dominant specific mutants are shown in Figures 7 and 8. Table 10 shows the mutation sites of the Pleuromamma kifui luciferase dominant mutants and the results of the ZS26 / F-CTZ and ZS2 / F-CTZ substrate specificities.

[0109]

Table 10

[0110] From the results of Table 10 and Figures 7 and 8, the substrate specificity of wild-type pleuromamma kifianus luciferase without a signal peptide for ZS26 / F-CTZ was approximately 1.77-fold, and for ZS2 / F-CTZ was approximately 3.56-fold. The dominant mutants obtained by modifying the enzyme showed improved specificity for the substrate ZS26 / F-CTZ, up to approximately 74.857-fold at most, and improved specificity for the substrate ZS2 / F-CTZ, up to approximately 24.750-fold at most.

[0111] Example 6 Design and construction of eukaryotic expression of pleuromamma kifianus luciferase plasmid

[0112] By PCR technology, pleuromamma kifianus luciferase containing a signal peptide used in the pEE12.4 vector was synthesized. The gene sequence is shown in SEQ ID NO:28. There is a histidine label for purification (6xHis) after the N-terminal signal peptide and an Avitag for biotinylation at the C-terminal. ATGTATATAAAAGTTTGGTTTGGTCTGGCTTGTCTTTCATTGGTTCTGGCCcaccaccaccaccatcacggcagcCAACCAACTGAAAACAAGCAGGAGTCTCATATTGTAGATTCAGATCTTGATGGCGACCGTGGTAGGAAGTTGCCCGGAAAAAAGCTTCCTATAGAAGTACTCAAAATCATGGAAGCCAATGCCAGGAGAGCTGGTTGCACTAGAGGATGTCTCATATGTCTTTCAAAAATCAAGTGTACAGCCAAAATGAAGCGATACATTCCAGGGAGATGTCATACTTATGAAGGAGATAAATCTATTGGACAGGGAGGCATAGGTGGCCCTATTGTTGATATTCCTGAAATTATTGGATTCAAGAACATGGAACCCATGGATCAGTTCATCGCACAAGTTGATCTGTGCGCCGACTGTACAACTGGGTGCCTGAAAGGCCTTGCTAATGTTAGGTGCAATGACTTGCTGAAGAAATGGCTGCCTGACAGATGTGCTGGTTTTGCCGACAAAATTCAAAATGAAGTGGATAGTATCAAGGGcatggctggggatcgcggctccggactgaatgatatc(SEQ ID NO:28)

[0113] For the eukaryotic expression, the prokaryotic expression plasmid pET28a Pxluc WT containing a signal peptide is used as the template DNA for the pleuromamma xiphias luciferase plasmid. The primer sequences used in the PCR reaction for constructing the expression plasmid insertion fragment are shown in Table 11, the reaction system is shown in Table 12, and the reaction conditions are shown in Table 13.

[0114]

Table 11

[0115]

Table 12

[0116]

Table 13

[0117] Add 0.5 μL of DpnI enzyme to the reaction system, incubate at 37 °C for 3 hours to digest the template. Then, the gel-purified product of approximately 528 bp is the insert fragment insert.

[0118] Using the pEE12.4 vector as a template, linearize the vector by PCR to facilitate recombination with the insert fragment. Prepare the PCR reaction system and perform the PCR reaction with the KOD FX neo enzyme according to its manual. 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.

[0119]

Table 14

[0120]

Table 15

[0121]

Table 16

[0122] Add 0.5 μL of DpnI enzyme to the reaction system, incubate at 37 °C for 3 hours to digest the template. Then, the gel-purified product of approximately 7712 bp is the linearized vector vector.

[0123] Recombine the insert and vector obtained in this example using the Takara In-Fusion Cloning reagent kit according to the reaction system shown in Table 17, and the reaction conditions are to incubate at 50 °C for 15 minutes.

[0124]

Table 17

[0125] 2.5 μL of the above reaction product was transformed into DH5α competent cells and plated on an ampicillin-resistant plate with a final concentration of 100 μg / mL. The next day, monoclonal colonies were picked from the plate, plasmids were extracted and sequenced to confirm that the target fragment was accurately inserted into the vector. The obtained plasmid was the wild-type luciferase pEE12.4-Pxluc WT used for eukaryotic expression. The obtained plasmid was detected, and the detected plasmid map is shown in Figure 9, which is as expected for the experiment.

[0126] The mutant plasmid pEE12.4 Pxluc P26-95 used for eukaryotic expression was synthesized by the PCR method using the constructed pEE12.4-Pxluc WT as the template DNA. The primer sequences are shown in Table 18, and the PCR reaction system is shown in Table 19. The PCR reaction conditions are the same as those in Table 16.

[0127]

Table 18

[0128]

Table 19

[0129] After the reaction was completed, 0.5 μL of DpnI enzyme was added to the reaction system and incubated at 37 °C for 3 hours to digest the template. Then, 2.5 μL of the enzyme cleavage product was taken and transformed into DH5α competent cells, and plated on an ampicillin-resistant plate with a final concentration of 100 μg / mL. The next day, monoclonal colonies were picked from the plate and plasmids were extracted. Sequencing was performed to confirm that the mutant sequence was correct. The obtained plasmid was the mutant luciferase pEE12.4 Pxluc P26-95 (luciferase mutant expression vector containing the signal peptide sequence, mutation sites G100A, G101P) used for eukaryotic expression.

[0130] Example 7 Eukaryotic Expression of Pleuromamma kifui Luciferase and Protein Purification

[0131] The pEE12.4 Pxluc WT and mutant pEE12.4 Pxluc P26-95 plasmids obtained in Example 6 were transfected into 30 mL of Expi-CHO cells respectively according to the instructions of the ExpiFectamineTM CHO Transfection Reagent Kit (Gibco, A29129). After transfection for 7 days, when the measured cell viability was less than 90%, the cells were centrifuged at 8000 rpm for 10 min at 4 °C, and the supernatant was collected.

[0132] Add 2 mL of HisTrap FF filler to a manual column (purchased from Sangon Biotech, model number: F506607-0001# affinity chromatography column empty column) respectively, and wash it with 30 mL of binding buffer to equilibrate the filler. Then add about 30 mL of filtered cell supernatant respectively. Wash it 10 times (10 mL each time) with the rinse solution (50 mM Tris, pH 8.0, 250 mM NaCl, 10 mM imidazole), and then elute the protein 4-5 times with 500 μL of the elution solution (50 mM Tris, pH 8.0, 250 mM NaCl, 300 mM imidazole), and collect the protein after each elution. Detect the protein eluted by 12% SDS-PAGE, and the purification results are shown in Figure 10, and relatively pure Pxluc protein can be obtained.

[0133] Example 8 Binding and Activity Tests of Dominant Mutants of Sordomilk Dot Luciferase

[0134] The Pxluc protein obtained in Example 7 contains an AviTag label. That is, AviTag-Pxluc-WT and the mutant AviTag-Pxluc-P26-95 can be biotinylated to Biotin-Avitag-Pxluc-WT and Biotin-AviTag-Pxluc-P26-95 by the BirA enzyme (Avidity, BIRA500). The reaction system for biotinylation is shown in Table 18.

[0135]

Table 20

[0136] If SA is added to the system shown in Table 20, SA-Pxluc can be produced. By further purification using the histidine label on Pxluc, relatively pure SA-Pxluc can be obtained. The purification results are shown in Figure 11.

[0137] The activities of pEE12.4 Pxluc WT, the mutant pEE12.4 Pxluc P26-95, and the post-binding products SA-Pxluc WT and SA-Pxluc P26-95 were measured by the method described in Example 5. The results are shown in Figure 12. Both the eukaryotic-expressed Pxluc WT and the mutant Pxluc P26-95 have the activity to catalyze the substrates coelenterazine derivatives ZS2 and ZS26. 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 substrate specificity of the post-binding product SA-Pxluc P26-95 for ZS26 / F-CTZ is significantly improved compared with SA-Pxluc WT.

[0138] In the description of this specification, descriptions referring to terms such as "one embodiment", "several embodiments", "example", "specific example", or "several examples" mean that the specific features, structures, materials, or characteristics described with reference to the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic expressions of the above terms do not necessarily target the same embodiment or example. In addition, the specific features, structures, materials, or characteristics described may be incorporated in any one or more embodiments or examples in an appropriate manner. Furthermore, those skilled in the art can combine and combine the various embodiments or examples described in this specification and the features related to the various embodiments or examples without contradiction.

[0139] Embodiments of the present invention have been presented and described, but the above embodiments are illustrative and cannot be understood as limiting the present invention. Those skilled in the art can understand that various changes, modifications, substitutions, and variations can be made to the above embodiments within the scope of the present invention.

Claims

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

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

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

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

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

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

7. A nucleic acid molecule encoding the mutant luciferase according to any one of claims 1 to 6.

8. An expression vector containing the nucleic acid molecule according to claim 7.

9. A recombinant cell carrying the nucleic acid molecule according to claim 7 or the expression vector according to claim 8.

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

11. A method for producing a mutant luciferase, comprising introducing the expression vector according to claim 8 into a recombinant cell, culturing and propagating the recombinant cell, and collecting the culture propagation product to extract or purify the mutant luciferase, a method for producing a mutant luciferase.

12. A method for detecting a nucleic acid sequence using the mutant luciferase according to any one of claims 1 to 6, wherein the method comprises: A) forming a first mutant luciferase complex by chemically bonding, biologically bonding or fusing proteins the mutant luciferase according to any one of claims 1 to 6 and a first specific recognition protein; and forming a second luciferase complex by chemically bonding, biologically bonding or fusing proteins a second luciferase as a signal protein and a second specific recognition protein; B) the first mutant luciferase complex can react with a first substrate to generate a first luminescence signal, the second luciferase complex can react with a second substrate to generate a second luminescence signal, the first mutant luciferase complex has no significant cross-substrate reaction with the second substrate, and the second luciferase complex has no significant cross-substrate reaction with the first substrate; C) performing sequencing of the target nucleic acid by recognizing the four bases A, T, G, and C by detecting the fluorescence signals and signal combinations of the self-luminescence systems of the mutant luciferase and the second luciferase. A method comprising the steps of.

13. 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. The method according to claim 12.

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

15. A method for detecting the content of a test substance, comprising: a) forming a complex by chemically bonding, biologically bonding or fusing proteins the mutant luciferase according to any one of claims 1 to 6 as a signal protein and a specific recognition protein of the test substance; b) contacting the test substance with the complex; c) adding a substrate or a substrate analog of Pleuromamma xiphias luciferase to the reaction system. d) determining the content of the test substance based on the fluorescence intensity of the reaction system detected after adding a substrate or an analog of the substrate of Pleuromamma xiphias luciferase; a method comprising the steps of.

16. The substrate of Pleuromamma xiphias luciferase is selected from at least one of coelenterazine, fluorinated coelenterazine or a coelenterazine derivative, Optionally, the coelenterazine derivative is selected from coelenterazine derivative ZS2 or coelenterazine derivative ZS26, The method according to claim 15.

17. A method for screening a substrate of Pleuromamma xiphias luciferase, comprising: I) contacting the mutant luciferase according to any one of claims 1 to 6 with the substrate to be screened to obtain a reaction mixture; II) determining whether the substrate to be screened is a target substrate based on whether the reaction mixture obtained in step I) emits a chemiluminescence signal; a method comprising the steps of.

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

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