WO2023109981A2 - 新型桡足类荧光素酶突变体及其应用 - Google Patents

新型桡足类荧光素酶突变体及其应用 Download PDF

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WO2023109981A2
WO2023109981A2 PCT/CN2023/087445 CN2023087445W WO2023109981A2 WO 2023109981 A2 WO2023109981 A2 WO 2023109981A2 CN 2023087445 W CN2023087445 W CN 2023087445W WO 2023109981 A2 WO2023109981 A2 WO 2023109981A2
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mutated
mutation
luciferase
mutant
nucleic acid
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WO2023109981A9 (zh
WO2023109981A3 (zh
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杨梦�
潘璐璐
刘文静
王艺
王若冰
杨理想
倪鸣
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深圳华大智造科技股份有限公司
青岛华大智造普惠科技有限公司
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Priority to PCT/CN2023/087445 priority Critical patent/WO2023109981A2/zh
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  • the present invention relates to the field of biotechnology. Specifically, the present invention relates to luciferase mutants and applications thereof, in particular to copepod luciferase mutants and applications thereof.
  • Bioluminescence is a phenomenon that exists widely in nature, mainly in organisms such as insects, bacteria, fungi and marine organisms. To date, more than 40 bioluminescent systems have been discovered in nature, and only a dozen luciferases and related substrates such as luciferin have been elucidated. In general, the bioluminescence reaction requires the presence of luciferase, a substrate (luciferin and its analogs, etc.) and an oxygen atom (molecular oxygen, etc.).
  • Luciferase is a general term for a class of enzymes that can catalyze the oxidation and luminescence of luciferin or aliphatic aldehydes. It is widely found in insects, bacteria, fungi and marine organisms. Sequencing and analysis technology, clinical medicine and forensic detection, drug screening, environmental monitoring and enzyme-linked detection and other fields. Taking advantage of the self-luminescence characteristics of luciferase, luciferase is often used in the fields of 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.
  • the luciferases with better research and development mainly include: firefly luciferase (firefly luciferase, FLuc), bacterial luciferase (bacterial luciferase, Lux), and Renilla luciferase (RLuc), Luciferase extracted from deep-sea shrimp (Oplophorus luciferase, OLuc), marine animal Gaussia princeps (Gaussia luciferase, GLuc), etc.
  • firefly luciferase firefly luciferase
  • FLuc firefly luciferase
  • bacterial luciferase bacterial luciferase
  • RLuc Renilla luciferase
  • Luciferase extracted from deep-sea shrimp Oplophorus luciferase, OLuc
  • marine animal Gaussia princeps Gaussia luciferase, GLuc
  • Fluc needs cofactors such as ATP, O 2 and Mg 2+ , and is non-secreted expression; most of the fluorescence reactions of Lux need flavin mononucleotide (FMN), long-chain fatty aldehyde, oxygen and reduced nicotinamide adenine dinuclear Nucleic acid (NADH) and other molecules; Rluc does not require ATP for luminescence, but its fluorescence intensity is weak, and it cannot be secreted.
  • FMN flavin mononucleotide
  • NADH nicotinamide adenine dinuclear Nucleic acid
  • Gaussia luciferase well compensates for the shortcomings of firefly luciferase and Renilla luciferase, and does not require cofactors such as ATP and Mg 2+ for luminescence.
  • Gaussian luciferase is a luciferase secreted by a marine copepod. It is one of the luciferases with the smallest molecular weight found so far, and it has a signal peptide that can be secreted extracellularly for easy activity detection. In the presence of oxygen, Gaussian luciferase can spontaneously catalyze the oxidation and luminescence of the substrate coelenterazine. However, it still faces the bottleneck of high-brightness and large-scale production.
  • the present invention aims to solve at least one of the technical problems existing in the prior art at least to a certain extent.
  • Gaussian luciferase mutant In order to increase the activity of Gaussian luciferase to the substrate coelenterazine (CTZ), and improve its expression yield.
  • the inventors obtained the Gaussian luciferase mutant with the highest catalytic activity to the substrate coelenterazine (CTZ) through protein-directed evolution of Gaussian luciferase Gluc, which was more than 9 times the catalytic activity of wild-type Gaussian luciferase.
  • the obtained luciferase mutant has small molecular weight, high stability, can be expressed by prokaryotic or eukaryotic, and the purification method and detection method are simple, which is beneficial to large-scale production. It has broad application prospects in immunoassay, biochemical diagnosis and sequencer application.
  • the present invention proposes a luciferase mutant.
  • the luciferase mutant refers to the amino acid sequence of wild-type Gaussian luciferase or the amino acid sequence of luciferase having at least 70% homology with the Gaussian luciferase as a reference , the luciferase mutant has a mutation in at least one of the following positions:
  • luciferase having at least 70% homology with the Gaussian luciferase refers to a highly conserved and homologous luciferase with wild-type Gaussian luciferase during evolution.
  • the amino acid positions in the amino acid sequence of the luciferase are positioned with reference to the amino acid positions in the amino acid sequence of the wild-type luciferase.
  • the Gaussian luciferase mutant according to the embodiment of the present invention has higher catalytic activity for substrates (coelenterazine and its analogues), and can be expressed in prokaryotic or eukaryotic soluble form to obtain large-scale production.
  • the above luciferase mutant may further include at least one of the following additional technical features:
  • the wild-type Gaussian luciferase without signal peptide has the amino acid sequence shown in SEQ ID NO: 3:
  • the luciferase having at least 70% homology to Gaussian luciferase is copepod luciferase.
  • the copepod luciferase comprises an enzyme selected from Mluc, Maluc, Pxluc, Mpluc at least one.
  • copepod luciferase is a class of luciferase with higher conservation, and at the active mutation-related site on Gluc, the remaining copepod luciferase such as Mluc, Maluc, Pxluc, Mpluc, etc. It has a similar activity-enhancing effect.
  • the mutant luciferase mutant has any one or more combinations of the following (1)-(22) mutations:
  • V at position 12 is mutated to A or S;
  • T mutation at position 20 is V or A
  • the L mutation at position 23 is A or T or I;
  • the H at position 62 is mutated to K or Q or N;
  • the P mutation at position 67 is L or K or A;
  • the T mutation at position 79 is H or K or P;
  • E at position 85 is mutated to S or D;
  • the S mutation at position 86 is T or I;
  • E mutation at position 93 is P or A or S or T;
  • V at position 121 is mutated to D or E.
  • the luciferase mutant compared with the amino acid sequence shown in SEQ ID NO:3, the luciferase mutant has the following mutations:
  • H at position 62 is mutated to K
  • P at 67 is mutated to L
  • H at 78 is mutated to A
  • E at 85 is mutated to S
  • S at 86 is mutated to T
  • S at 87 is mutated A mutation at position A to G, E at position 93 to S, L at position 107 to M, V at position 121 to E; or
  • V at position 12 is mutated to A, and the T at position 79 is mutated to H; or
  • V at position 12 is mutated to A, and the T at position 79 is mutated to K; or
  • the V at the 12th position is mutated to A
  • the H at the 62nd position is mutated to K
  • the P at the 67th position is mutated to L
  • the E at the 85th position is mutated to S
  • the S at the 86th position is mutated to T
  • the 87th position is mutated to T.
  • the A at position 93 is mutated to G
  • the E at position 93 is mutated to P
  • the L at position 107 is mutated to M
  • the V at position 121 is mutated to E; or
  • the T mutation at position 20 is A, and the I mutation at position 114 is M; or
  • L at position 23 is mutated to T
  • H at position 62 is mutated to K
  • P at position 67 is mutated to L
  • E at position 85 is mutated to S
  • S at position 86 is mutated to T
  • S at position 87 is mutated to T.
  • the A at position 93 is mutated to G
  • the E at position 93 is mutated to T
  • the L at position 107 is mutated to M
  • the V at position 121 is mutated to E; or
  • the H at position 62 is mutated to K
  • the P at position 67 is mutated to A
  • the E at position 85 is mutated to D
  • the S at position 86 is mutated to T
  • the A at position 87 is mutated to G
  • the 107th position is mutated mutation of L to M at position; or
  • the H at position 62 is mutated to K
  • the P at position 67 is mutated to K
  • the E at position 85 is mutated to D
  • the S at position 86 is mutated to T
  • the A at position 87 is mutated to G
  • the 107th position is mutated
  • the L at position 1 is mutated to M
  • the V at position 121 is mutated to E; or
  • the H at the 62nd position is mutated to K
  • the P at the 67th position is mutated to A
  • the E at the 85th position is mutated to D
  • the S at the 86th position is mutated to T
  • the A at the 87th position is mutated to G
  • the 93rd position is mutated
  • the E at position 1 is mutated to A
  • the L at position 107 is mutated to M
  • the V at position 121 is mutated to E; or
  • the H at the 62nd position is mutated to K
  • the P at the 67th position is mutated to A
  • the E at the 85th position is mutated to D
  • the S at the 86th position is mutated to I
  • the A at the 87th position is mutated to G
  • the 107th position is mutated
  • the L at the position is mutated to M
  • the V at the 121st position is mutated to E; or
  • the P mutation at the 67th position is A
  • the E mutation at the 85th position is D
  • the S mutation at the 86th position is T
  • the A mutation at the 87th position is G
  • the L mutation at the 107th position is M
  • the 121st position V in position is mutated to E; or
  • the H at the 62nd position is mutated to Q
  • the E at the 85th position is mutated to S
  • the S at the 86th position is mutated to T
  • the A at the 87th position is mutated to G
  • the E at the 93rd position is mutated to A
  • the 107th position is mutated
  • the L at position 1 is mutated to M
  • the V at position 121 is mutated to E; or
  • L at position 23 is mutated to A
  • H at position 62 is mutated to K
  • P at position 67 is mutated to L
  • E at position 85 is mutated to S
  • S at position 86 is mutated to T
  • S at position 87 is mutated to T.
  • the H at the 62nd position is mutated to K
  • the P at the 67th position is mutated to A
  • the E at the 85th position is mutated to D
  • the S at the 86th position is mutated to T
  • the A at the 87th position is mutated to G
  • the 107th position is mutated
  • the L at position 1 is mutated to M
  • the V at position 121 is mutated to D; or
  • the H at the 62nd position is mutated to K
  • the P at the 67th position is mutated to A
  • the E at the 85th position is mutated to D
  • the S at the 86th position is mutated to T
  • the A at the 87th position is mutated to G
  • the 107th position is mutated mutation of L to M at position; or
  • the T at the 20th position is mutated to A
  • the H at the 62nd position is mutated to K
  • the P at the 67th position is mutated to A
  • the E at the 85th position is mutated to D
  • the S at the 86th position is mutated to T
  • the 87th position is mutated to T.
  • the T mutation at the 21st position is A
  • the H mutation at the 62nd position is K
  • the P mutation at the 67th position is K
  • the E mutation at the 85th position is D
  • the S mutation at the 86th position is T
  • the 87th position A mutation at position A to G, L at position 107 to M, V at position 121 to E; or
  • the H at position 62 is mutated to K
  • the P at position 67 is mutated to K
  • the E at position 85 is mutated to D
  • the S at position 86 is mutated to T
  • the A at position 87 is mutated to G
  • the 107th position is mutated
  • the L at position 1 is mutated to M
  • the V at position 121 is mutated to D; or
  • the T at the 20th position is mutated to V
  • the T at the 21st position is mutated to A
  • the D at the 22nd position is mutated to E
  • the H at the 62nd position is mutated to K
  • the P at the 67th position is mutated to A
  • the 85th position is mutated
  • the E at position 86 is mutated to T
  • the A at position 87 is mutated to G
  • the L at position 107 is mutated to M
  • the V at position 121 is mutated to D; or
  • T at position 21 is mutated to A
  • H at position 62 is mutated to K
  • P at position 67 is mutated to A
  • E at position 85 is mutated to D
  • S at position 86 is mutated to T
  • S at position 87 is mutated to T.
  • the T at the 20th position is mutated to V
  • the T at the 21st position is mutated to A
  • the D at the 22nd position is mutated to G
  • the H at the 62nd position is mutated to K
  • the P at the 67th position is mutated to A
  • the 79th position is mutated
  • the T at position is mutated to P
  • the E at position 85 is mutated to D
  • the S mutation at position 86 is I
  • the A mutation at position 87 is G
  • the L mutation at position 107 is M
  • the V mutation at position 121 is E; or
  • the H at the 62nd position is mutated to K
  • the P at the 67th position is mutated to A
  • the E at the 85th position is mutated to D
  • the S at the 86th position is mutated to I
  • the A at the 87th position is mutated to G
  • the 107th position is mutated
  • the L at position 1 is mutated to M
  • the V at position 121 is mutated to D; or
  • the E at the 44th position is mutated to G
  • the H at the 62nd position is mutated to N
  • the P at the 67th position is mutated to A
  • the E at the 85th position is mutated to D
  • the S at the 86th position is mutated to T
  • the 87th position is mutated to T.
  • the T at the 21st position is mutated to A
  • the P at the 67th position is mutated to A
  • the E at the 85th position is mutated to D
  • the S at the 86th position is mutated to T
  • the A at the 87th position is mutated to G
  • the 107th position is mutated
  • the L at position 1 is mutated to M
  • the V at position 121 is mutated to D; or
  • the P mutation at the 67th position is A
  • the E mutation at the 85th position is D
  • the S mutation at the 86th position is T
  • the A mutation at the 87th position is G
  • the L mutation at the 107th position is M
  • the 121st position V in position is mutated to D; or
  • the V at the 12th position is mutated to A
  • the T at the 21st position is mutated to A
  • the D at the 22nd position is mutated to G
  • the L at the 35th position is mutated to F
  • the A at the 45th position is mutated to V
  • the 59th position is mutated
  • the C at the 85th position is mutated to S
  • the H at the 62nd position is mutated to K
  • the P at the 67th position is mutated to L
  • the E at the 85th position is mutated to D
  • the S at the 86th position is mutated to T
  • the A at the 87th position is mutated to G
  • mutation of E at position 93 to S mutation of L at position 107 to M
  • the V at the 12th position is mutated to A
  • the T at the 20th position is mutated to A
  • the T at the 21st position is mutated to A
  • the D at the 22nd position is mutated to G
  • the L at the 35th position is mutated to F
  • the 45th position is mutated to F.
  • the A at position 59 is mutated to S
  • the H at position 62 is mutated to K
  • the P at position 67 is mutated to L
  • the E at position 85 is mutated to D
  • the S at position 86 is mutated to T
  • a mutation at position 87 to G E at position 93 to S
  • L at position 107 to M and V at position 121 to E.
  • the V at the 12th position is mutated to A
  • the A at the 19th position is mutated to V
  • the T at the 20th position is mutated to A
  • the T at the 21st position is mutated to A
  • the D at the 22nd position is mutated to G
  • the 23rd position is mutated to G.
  • the L at position is mutated to I
  • the L at position 35 is mutated to F
  • the C at position 59 is mutated to S
  • the H at position 62 is mutated to K
  • the P at position 67 is mutated to L
  • the E at position 85 is mutated to D
  • the S at the 86th position is mutated to T
  • the A at the 87th position is mutated to G
  • the E at the 93rd position is mutated to S
  • the L at the 107th position is mutated to M
  • the V at the 121st position is mutated to E.
  • T at position 21 is mutated to A
  • H at position 62 is mutated to K
  • P at position 67 is mutated to L
  • E at position 85 is mutated to D
  • S at position 86 is mutated to T
  • S at position 87 is mutated to T.
  • the A at position is mutated to G
  • the E at position 93 is mutated to P
  • the L at position 107 is mutated to M
  • the V at position 121 is mutated to E.
  • the obtained protein when the amino acid sequence shown in SEQ ID NO: 3 has the above-mentioned mutation, the obtained protein has stronger catalytic activity on substrates such as coelenterazine. Compared with the existing Gaussian luciferase Luminous brightness is significantly enhanced.
  • the luciferase mutant further includes a signal peptide, and the signal peptide is arranged at the N-terminal of the luciferase mutant. It is used to guide the secretion of the above-mentioned luciferase mutants from the expressing cells.
  • the mutated luciferase mutant further includes a polyhistidine sequence, and the polyhistidine sequence is arranged at the C-terminus of the luciferase mutant. Used for affinity purification of the mutant luciferase mutants described above.
  • the invention proposes a nucleic acid molecule.
  • the nucleic acid molecule encodes the luciferase mutant described in the first aspect of the present invention.
  • the present invention provides an expression vector.
  • the expression vector includes the nucleic acid molecule described in the second aspect of the present invention.
  • the expression vector further includes a promoter.
  • the promoter is operably linked to the nucleic acid molecule.
  • the present invention provides a recombinant cell.
  • the recombinant cell carries the nucleic acid molecule described in the second aspect of the present invention and the expression vector described in the third aspect of the present invention. Then express or secrete the luciferase mutant described in the first aspect of the present invention.
  • the recombinant cells are selected from Escherichia coli, yeast or mammalian cells.
  • the present invention proposes a method for obtaining luciferase.
  • the recombinant cells described in the fourth aspect of the present invention are cultured under conditions suitable for protein expression, so as to obtain the luciferase.
  • the present invention proposes a conjugate.
  • the conjugate includes the luciferase mutant described in the first aspect of the present invention and a small molecule compound or a macromolecule, and the luciferase mutant and the small molecule compound or a macromolecule are coupled through a chemical bond. couplet.
  • the small molecular compound or macromolecule includes streptavidin (streptavidin, SA), digoxin, antibody, dNTP or dNTP analogue and the like.
  • the present invention provides a nucleic acid sequencing method.
  • the sequencing method includes: using the nucleic acid to be tested as a template, sequentially adding the conjugate and/or dNTP that can bind to the luciferase mutant described in the first aspect and/or the sixth aspect Or dNTP analogs or modified dNTPs are polymerized; according to the fluorescent signal emitted by the luciferase mutant and the Gaussian luciferase substrate or the analog of the substrate, the nucleic acid sequence of the nucleic acid to be tested is determined .
  • the method includes subsequently using the nucleic acid to be detected as a template
  • a variety of differently labeled dNTPs are added to the plate for polymerization, then luciferase mutants or conjugates that have differential activity with the substrate are added to recognize differently labeled dNTPs, and finally the substrate is added, according to the luciferase mutant
  • the various fluorescent signals emitted by reacting with the substrate of Gaussian luciferase or the analogue of the substrate finally obtain the nucleic acid sequence of the nucleic acid to be tested.
  • the above polymerization reaction and enzymatic fluorescence reaction can be carried out in multiple cycle.
  • the present invention provides a nucleic acid sequencing kit.
  • the nucleic acid sequencing kit includes the luciferase mutant described in the first aspect or the conjugate described in the sixth aspect.
  • the kit further includes dNTPs or dNTP analogs, universal primers, PCR polymerase or coelenterazine or coelenterazine analogs.
  • the present invention provides a method for detecting the content of the analyte.
  • the method for detecting the content of the analyte includes: combining the luciferase mutant described in the first aspect of the present invention or the conjugate described in the sixth aspect of the present invention Contact with the substrate of Gaussian luciferase or the analogue of the substrate, and finally determine the content of the analyte based on the strength of the fluorescent signal after the contact treatment.
  • the Gaussian luciferase substrate or substrate analogue includes at least one selected from coelenterazine and coelenterazine derivatives.
  • the coelenterazine derivative is represented by formula (I) or formula (II), or a salt or isomer thereof,
  • Figure 1 shows the plasmid map of the wild-type Gaussian luciferase prokaryotic expression plasmid pCold-Gluc-WT-NS without signal peptide;
  • Figure 2 shows the plasmid map of wild-type Gaussian luciferase eukaryotic expression plasmid pEE12.4-Gluc WT containing signal peptide;
  • FIG. 3 shows the structural diagram of the substrate coelenterazine (CTZ), and coelenterazine derivatives F-CTZ and ZS26;
  • Fig. 4 shows the protein electrophoresis picture of prokaryotic expression and purification of Gaussian luciferase mutant
  • Figure 5 shows the detection results of the activity of the prokaryotic expressed Gaussian luciferase mutant on the level of the substrate coelenterazine;
  • Figure 6 shows the electrophoresis of part of the Gaussian luciferase mutant eukaryotically expressed purified protein
  • Figure 7 shows the results of detection of the activity of the partially eukaryotic expressed Gaussian luciferase mutant on the level of the substrate coelenterazine protein
  • Figure 8 shows the electrophoresis of some Gaussian luciferase mutant biotin-coupled proteins
  • Figure 9 shows the protein-level activity detection results of some Gaussian luciferase mutant biotin-coupled proteins to substrates CTZ, F-CTZ and ZS26;
  • Figure 10 shows the signal values of some Gaussian luciferase mutant biotin-coupled proteins on the sequencer DNBSEQ E5.
  • first and second are used for descriptive purposes only, and cannot be interpreted as indicating or implying relative importance or implicitly specifying the quantity of indicated technical features.
  • the 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 specifically defined otherwise.
  • luciferase gene has been widely used as a reporter gene for the study of the expression intensity and transcriptional regulation of exogenous genes under different promoters.
  • luciferase can be used to detect the presence or absence of chemical pollutants.
  • it also has broad application prospects in the fields of immune detection and biochemical diagnosis.
  • the luciferase mutant proposed by the present invention compared with the amino acid sequence shown in SEQ ID NO: 1 or the luciferase having at least 70% homology with the Gaussian luciferase (i.e. copepod luciferase ) amino acid sequence.
  • the mutant Compared with the amino acid sequence shown in SEQ ID NO: 3, the mutant has any one or more combinations of the following mutation sites: 10th, 12th, 19th, 20th, 21st bit, 22nd, 23rd, 35th, 44th, 45th, 59th, 62nd, 67th, 78th, 79th, 85th, 86th, Position 87, position 93, position 107, position 114, position 121, and may or may not include a signal peptide amino acid sequence.
  • the mutation site is modified to the amino acid sequence of wild-type Gaussian luciferase without signal peptide, and the obtained mutant is resistant to coelenterazine, fluorinated coelenterazine, and coelenterazine derivative ZS26.
  • the substrate has a wider spectrum, stronger specificity, and significantly enhanced luminescence brightness.
  • the mutant can be used in basic scientific research of luminescence detection, biological detection technology, immune detection, biochemical detection or diagnosis, and has broad application prospects.
  • the quantitative detection of DNA, RNA, Transcription factors, proteins or cells play an important role. It can also be used as a luminescent signal protein in a fusion protein to quantitatively detect small molecules of interest.
  • the present invention provides a nucleic acid molecule encoding the aforementioned mutant.
  • nucleic acid in the specification and claims of the present invention, those skilled in the art should understand that it actually includes any one or both of the complementary double strands.
  • nucleic acid sequence in the present application includes a DNA form or an RNA form, and disclosing one of them means that the other is also disclosed.
  • the present invention proposes an expression vector comprising the aforementioned nucleic acid molecule.
  • the type of the 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 on substrates such as coelenterazine, fluorocoelenterazine and coelenterazine derivatives , compared with the existing Gaussian luciferase substrate spectrum wider, more specific, significantly enhanced luminescence brightness, applied to the protein for luminescent detection of basic scientific research, biological detection technology, immunological detection, biochemical detection Or diagnosis and other fields can be used, has broad application prospects.
  • substrates such as coelenterazine, fluorocoelenterazine and coelenterazine derivatives
  • the present invention proposes a recombinant cell carrying the aforementioned nucleic acid molecule, expression vector or mutant.
  • the recombinant cells are obtained by transfecting or transforming the expression vector.
  • the recombinant cells can efficiently express the above-mentioned mutants under suitable conditions, and the catalytic activity of the mutants to substrates such as coelenterazine, fluorinated coelenterazine, and coelenterazine derivatives
  • substrates such as coelenterazine, fluorinated coelenterazine, and coelenterazine derivatives
  • the substrate spectrum is wider, the specificity is stronger, and the luminous brightness is significantly enhanced. It is applied to the protein for basic scientific research, biological detection technology, immune detection, It can be used in fields such as biochemical detection or diagnosis, and has broad application prospects.
  • a nucleic acid sequencing method proposed by the present invention refers to using the nucleic acid to be tested as a template, sequentially adding the dNTP polymerization reaction combined with the aforementioned luciferase mutant or the aforementioned conjugate; according to the fluorescent signal and signal combination, to distinguish A, The four bases of T, G, and C are subjected to target nucleic acid sequencing, and finally the nucleic acid sequence to be tested is obtained.
  • a method of detecting a nucleic acid sequence comprises the steps of:
  • the first mutant luciferase complex can react with the first substrate to generate a first luminescent signal; the second fluorescent The luciferase complex can react with the second substrate to generate a second luminescent signal; the first mutant luciferase complex does not have a significant cross-substrate reaction with the second substrate, and the second luciferin The enzyme complex does not have significant cross-substrate reactivity with said first substrate.
  • the first substrate and the second substrate are luciferase luminescence reaction substrates.
  • the first mutant luciferase complex and the second luciferase complex can be respectively combined with different modified bases.
  • the method for detecting nucleic acid sequence comprises the following steps:
  • step A 2) adding various luciferase complexes in step A, and coupling various luciferase complexes to different bases by specifically recognizing different affinity tags;
  • Another aspect of the present invention proposes a method for detecting the content of the analyte, comprising the following steps:
  • the analyte is suitable for forming a complex with the mutant.
  • the substrate of Gaussian luciferase includes coelenterazine, fluorocoelenterazine or coelenterazine derivatives.
  • the coelenterazine derivatives include coelenterazine derivative F-CTZ or coelenterazine derivative ZS26.
  • reporter gene is a molecular biology concept, which refers to a class of genes that are expressed in cells, tissues/organs or individuals under specific conditions and make them easy to detect, and the experimental materials do not originally produce traits, That is, a gene that encodes a protein or enzyme that can be detected.
  • reporter gene the following conditions must be met in terms of genetic selection and screening detection: 1. It has been cloned and the entire sequence has been determined; 2. The expression product does not exist in the recipient cells, that is, there is no background, and it is transfected. There is no similar endogenous expression product in the cells; 3.
  • the expression product can be quantitatively determined; when used, it includes but is not limited to the following methods of use: the reporter gene and the gene expression regulatory sequence are fused to form a chimeric gene, Or it can be fused with other target genes, and the nucleic acid can be expressed under the control of the regulatory sequence, so that its expression product can be used to detect the expression regulation of the target gene and study the nucleic acid.
  • “Luciferase” is a class of enzymes that catalyze a luminescent chemical reaction.
  • the substrate for this enzyme is called luciferin.
  • Light emission occurs when luciferin undergoes a chemical reaction due to the catalytic activity of luciferase in the presence of ATP.
  • fluorescence is also called chemical Chemiluminescence is light radiation produced by a chemical reaction without any excitation such as light, heat or electric field.
  • bioluminescence bioluminescence
  • the mutant catalyzes the oxidation of the substrate coelenterazine to emit light, that is, fluorescence.
  • the present invention relates to a nucleic acid comprising a base sequence encoding a luciferase mutant according to an embodiment of the present invention. That is, the nucleic acid contains a luciferase gene derived from a marine copepod organism, Daphnia longina. Nucleic acid refers to DNA or RNA.
  • the "gene" of luciferase mainly refers to the region into which the mRNA is transcribed, ie it refers to the structural gene.
  • the isomers of the structures shown in formula (I) or formula (II) described in the present application include all isomers (such as enantiomers, diastereomeric atropisomers (atropisomers) and geometric (or conformational)) forms; for example, the R and S configurations of each asymmetric center, (Z) and (E) double bond isomers, and (Z) and (E) conformational isomers. Accordingly, individual stereochemical isomers as well as mixtures of enantiomers, diastereomers and geometric isomers (or conformers) of the compounds of the present application are within the scope of the present application.
  • the salt of the structure represented by formula (I) or formula (II) used in this application refers to the organic salt and inorganic salt of the compound.
  • Pharmaceutically acceptable non-toxic acid salts include, but are not limited to, inorganic acid salts formed by reaction with amino groups such as hydrochloride, hydrobromide, phosphate, sulfate, perchlorate, And organic acid salts such as acetate, oxalate, maleate, tartrate, citrate, succinate, malonate, or other methods such as ion exchange methods recorded in books and literature these salts.
  • salts include adipate, alginate, ascorbate, aspartate, besylate, benzoate, bisulfate, borate, butyrate, camphorate Salt, camphorsulfonate, cyclopentylpropionate, digluconate, lauryl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate Salt, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, Malate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, palmitate, pamoate, pectate, persulfate, 3 - Phenylpropionate,
  • Salts obtained with appropriate bases include alkali metal, alkaline earth metal, ammonium and N+(C1-4alkyl) 4 salts. This application also contemplates the quaternary ammonium salts formed by any compound containing an N group. Water-soluble or oil-soluble or dispersed products can be obtained by quaternization.
  • Alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like.
  • Pharmaceutically acceptable salts further include suitable, non-toxic ammonium, quaternary ammonium salts and amine cations formed as counterions, such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, C1- 8 sulfonates and aromatic sulfonates.
  • luciferase luminescence When detecting luciferase luminescence using an imaging device, known detection methods can be applied. For example, by adding fluorescent Luciferin, ATP, Mg 2+ ions, etc. are appropriately added to cells expressing a luciferase-containing fusion protein to cause a luminescent reaction of luciferase, and the emitted light can be detected by an imaging device.
  • the imaging device is, for example, a microscope equipped with filters for capturing luminescence. Microscopy can be used to specify the localization of proteins based on the information obtained by identifying the location of the luminescence in the cell.
  • a microscope having a function capable of taking time-course images can be used, and time-course observation can be realized with the microscope.
  • wild-type and mutant Gaussian luciferases without signal peptide were constructed to compare the activity difference between the mutant and wild-type Gaussian luciferases.
  • the nucleotide sequence of wild-type Gaussian luciferase is SEQ ID NO:2, and the encoded amino acid sequence is SEQ ID NO:1.
  • the nucleotide sequence of wild-type Gaussian luciferase without signal peptide is SEQ ID NO: 4, and the amino acid sequence encoded by it is SEQ ID NO: 3.
  • the amino acid sequence is SEQ ID NO:5, and the nucleotide sequence is SEQ ID NO: 6, its C-terminus is fused with a purification tag containing 6 histidines (6x His) to facilitate protein purification, and the enzyme cleavage sites at both ends are Nde I and EcoR I.
  • Gaussian luciferase activity mutants B6 H62K, P67L, E85S, S86T, A87G, L107M, V121E
  • D6 H62K, P67A, E85D, S86T , A87G, L107M, V121E
  • 4-C12 H62K, E85S, S86T, A87G, E93P, L107M, V121E).
  • Gluc WT-NS B6, D6 and 4-C12 as templates, using multiplex PCR method, construct including N10, V12, A19, T20, T21, D22, L23, L35, E44, A45, C59, H62, P67, K71 , H78, T79, E85, S86, A87, G89, E93, I95, L107, I114, V121 and other loci combined random mutant library, compared with Gluc WT-NS, the mutant loci are mutated as shown in Table 3 Show.
  • the PCR reaction system is shown in Table 1, and the PCR reaction conditions are shown in Table 2.
  • prokaryotic expression and protein purification were carried out on the constructed pCold Gluc WT-NS, B6, D6, 4-C12 and the combined mutant library of each site.
  • the specific experimental procedure is as follows:
  • the expression plasmid pCold Gluc WT-NS and the mutant obtained in Example 1 were respectively transformed into OrigamiB(DE3) Chemically Competent Cell Competent Cells (Vidibiology, EC1020S) were applied to a plate containing ampicillin resistance (100 ⁇ g/mL), and a single colony was picked from the plate, cultured overnight at 37°C, and the next day at a ratio of 1:100 Proportionally diluted, transferred to fresh 3mL LB medium containing ampicillin resistance (100 ⁇ g/mL), cultured at 37°C and 200rpm with shaking until OD600 ⁇ 0.5-0.6, and cooled on ice for 1 hour after the culture.
  • the inducer IPTG was added according to the final concentration of 1 mM, and induced overnight at 16°C.
  • the induced bacterial liquid precipitate was collected by centrifugation, and 600 ⁇ L of binding buffer (50mM Tris-HCl, pH 8.0, 250mM NaCl,) was added, lysed on ice for 30min, and ultrasonic (2s on 3s off, 60% power) for 30 min, 4°C, 12000 rpm centrifugation for 30 min to separate the supernatant (cell lysate) and the precipitate.
  • binding buffer 50mM Tris-HCl, pH 8.0, 250mM NaCl,
  • BCA quantitative kit (Thermo Scientific TM Pierce TM BCA Protein Assay Kit) was used to accurately measure the concentration of protein, and the purified luciferase obtained in Example 2 was diluted with diluent (50mM Tris-HCl pH 8.0, 100mM NaCl, 0.1% (v/v) Tween-20) was diluted to 1 ⁇ g/mL, and 10 ⁇ L was added to a black 96-well plate. Then add 90 ⁇ L of the substrate coelenterazine (MGI, FIG. 3 ) diluted to 100 ⁇ M with the same solution, and read the luminescence intensity from the luminescence module with a microplate reader ( FIG. 5 ).
  • diluent 50mM Tris-HCl pH 8.0, 100mM NaCl, 0.1% (v/v) Tween-20
  • MMI substrate coelenterazine
  • the activity test results of the dominant mutant relative to the wild-type Gaussian luciferase catalytic substrate coelenterazine that does not contain the signal peptide of the sequence shown in SEQ ID NO: 3 are shown in Table 3, wherein the following 33 mutant combinations
  • the catalytic activity of coelenterazine was increased by more than 2.5 times, respectively 17-1 (H62K, P67L, H78A, E85S, S86T, A87G, E93S, L107M, V121E), 18-2 (H62K, P67L, T79P, E85S, S86T , A87G, E93S, L107M, V121E), 20-3 (N10S, V12A), 21-1 (V12A, T79H), 23-1 (V12A, T79K), 24-2 (N10S, V12S), 27-2 ( V12A, H62K, P67L, E85S, S86T, A87G, E93P, L107M, V121E), 30-3 (T
  • the wild-type Gaussian luciferase containing the signal peptide (pEE12.4-Gluc WT) of the pEE12.4 vector was synthesized (Fig. 2), which contains the amino acid of the wild-type Gaussian luciferase containing the signal peptide
  • the sequence is SEQ ID NO: 8
  • the gene sequence is SEQ ID NO: 7
  • its N-terminus is fused with a purification tag containing 6 histidines (6x His) after the signal peptide to facilitate protein purification
  • its C-terminus is fused with a in biotinylated Avi-tag.
  • the Gaussian luciferase plasmid for eukaryotic expression uses mutant prokaryotic pCold plasmids 4-1, 6-1, A2-2, A2-3, A8-2, and A12-5 without signal peptides as templates to construct expression
  • the primer sequences used in the PCR reaction are shown in Table 4
  • the reaction system is shown in Table 5
  • the reaction conditions are shown in Table 6.
  • the pEE12.4 vector is used as a template, and the N-terminal of the template has a histidine tag for purification, and the C-terminal has Avitag for biotinylation.
  • the vector is linearized by PCR to facilitate recombination with the insert.
  • Use KOD FX neo enzyme carry out the preparation of PCR reaction system and PCR reaction according to its specification; Wherein, the primer sequence used is as shown in Table 7, PCR reaction system is as shown in Table 8, and PCR reaction condition is as shown in Table 9.
  • the Gluc mutant plasmid of the pEE12.4 vector obtained in Example 4 and the pEE12.4-Gluc WT synthesized by the whole gene were subjected to large-scale extraction of the plasmids, and then transfected into 30 mL HEK293E cells with PEI (cell density: 4 ⁇ 10 6 cells 5 days after transfection, the cell viability was measured to be less than 90%, and the supernatant was collected by centrifugation at 8000rpm for 10min at 4°C.
  • washing solution 50mM Tris-HCl, (pH 8.0, 250mM NaCl, 10mM imidazole) was washed 10 times (10mL/time), and 500 ⁇ L of the eluent (50mM Tris-HCl, pH 8.0, 250mM NaCl, 300mM imidazole) was used to elute the protein 4-5 times. off protein. The eluted protein was detected by 12% SDS-PAGE, and the purification results are shown in FIG. 6 .
  • Gluc WT and Gluc mutants were tested for activity according to the method described in Example 3, and the results are shown in FIG. 7 .
  • the Gluc protein obtained in Example 5 contains an AviTag tag and can be biotinylated by BirA enzyme (MGI) to Biotin-Avi-tag-Gluc.
  • MMI BirA enzyme
  • the biotinylation reaction system is shown in Table 11.
  • SA-Gluc After standing at room temperature for 30 minutes, SA-Gluc can be prepared by adding SA to the system.
  • the histidine tag on the mutant can be used for further purification to obtain a relatively pure SA-Gluc (mutant).
  • the purification result is shown in Figure 8 .
  • SA-Gluc Gluc wt
  • SA-Gluc mutants were tested for on-machine activity using the sequencer DNBSEQ E5 (MGI), and the on-machine results are shown in Figure 10.

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Abstract

本申请涉及新型桡足类荧光素酶突变体及其应用。所述荧光素酶突变体，以野生型高斯荧光素酶的氨基酸序列或与所述高斯荧光素酶SEQ ID NO:3具有至少70％同源性的荧光素酶的氨基酸序列为参考，相较于SEQ ID NO:3所示的氨基酸序列，具有以下至少之一位置的突变：第10位、第12位、第19位、第20位、第21位、第22位、第23位、第35位、第44位、第45位、第59位、第62位、第67位、第78位、第79位、第85位、第86位、第87位、第93位、第107位、第114位、第121位。

Description

新型桡足类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
生物发光是自然界广泛存在的一种现象，主要存在于昆虫、细菌、真菌和海洋生物等生物中。迄今为止，在自然界中发现了超过40种生物发光系统，只有十余种荧光素酶和相关底物如荧光素被阐明。一般来说，生物发光反应需要荧光素酶、底物(荧光素及类似物等)和氧原子(分子氧等)的存在。
荧光素酶是能够催化荧光素或脂肪醛氧化发光的一类酶的总称，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，可以与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、O2和Mg2+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。
高斯荧光素酶(Gaussia luciferase,Gluc)很好地弥补了萤火虫荧光素酶和海肾荧光素酶的缺点，发光时不需要ATP和Mg2+等辅助因子。高斯荧光素酶是由一种海洋桡足类动物分泌的荧光素酶，是目前发现的分子量最小的荧光素酶之一，并且具有信号肽，可以分泌到胞外，便于活性检测。在氧气存在的条件下，高斯荧光素酶可自发催化底物腔肠素氧化发光。但目前仍面临高亮度、规模化生产的瓶颈。
发明内容
本发明旨在至少在一定程度上解决现有技术中存在的技术问题至少之一。
为了能够增加高斯荧光素酶对底物腔肠素(CTZ)的活性，并提高其表达产量。发明人通过对高斯荧光素酶Gluc进行蛋白定向进化，获得对底物腔肠素(CTZ)催化活性最高的高斯荧光素酶突变体，且为野生型高斯荧光素酶催化活性的9倍以上。并且获得此类荧光素酶突变体分子量小、稳定高、可通过原核或真核表达，且纯化方法和检测方法简单，有利于规模化生产。在免疫检测、生化诊断及测序仪应用等方面具有广阔的应用前景。
为此，在本发明的一个方面，本发明提出了一种荧光素酶突变体。根据本发明的实施例，所述荧光素酶突变体是指以野生型高斯荧光素酶的氨基酸序列或与所述高斯荧光素酶具有至少70％同源性的荧光素酶的氨基酸序列为参考，所述荧光素酶突变体具有以下至少之一位置的突变：
第10位、第12位、第19位、第20位、第21位、第22位、第23位、第35位、第44位、第45位、第59位、第62位、第67位、第78位、第79位、第85位、第86位、第87位、第93位、第107位、第114位、第121位。需要说明的是，本申请所述的“与所述高斯荧光素酶具有至少70％同源性的荧光素酶”是指在进化过程中，与野生型高斯荧光素酶高度保守和同源的荧光素酶，该荧光素酶的氨基酸序列中的氨基酸的位置参照野生型荧光素酶的氨基酸序列中氨基酸的位置进行定位。根据本发明实施例的高斯荧光素酶突变体对于底物(腔肠素及其类似物)具有更高的催化活性，并且可以进行原核或真核可溶性表达，获得规模化生产。
根据本发明的实施例，上述荧光素酶突变体可以进一步包括下列附加技术特征至少之一：
根据本发明的实施例，所述不含信号肽的野生型高斯荧光素酶具有SEQ ID NO:3所示的氨基酸序列：
Figure PCTCN2023087445-ftappb-I100001
根据本发明的实施例，所述高斯荧光素酶具有至少70％同源性的荧光素酶为桡足类荧光素酶。
根据本发明的实施例，所述桡足类荧光素酶包括选自Mluc、Maluc、Pxluc、Mpluc的 至少之一。发明人发现，桡足类荧光素酶是保守性较高的一类荧光素酶，在Gluc上的活性突变相关位点，在其余的桡足类荧光素酶如Mluc、Maluc、Pxluc、Mpluc等上有类似的活性提升作用。
根据本发明的实施例，相较于SEQ ID NO:3所示的氨基酸序列，所述突变荧光素酶突变体具有以下(1)-(22)突变中的任意一个或者多个组合：
(1)第10位的N突变为S；
(2)第12位的V突变为A或S；
(3)第19位的A突变为V；
(4)第20位的T突变为V或A；
(5)第21位的T突变为A；
(6)第22位的D突变为E或G；
(7)第23位的L突变为A或T或I；
(8)第35位的L突变为F；
(9)第44位的E突变为G；
(10)第45位的A突变为V；
(11)第59位的C突变为S；
(12)第62位的H突变为K或Q或N；
(13)第67位的P突变为L或K或A；
(14)第78位的H突变为A；
(15)第79位的T突变为H或K或P；
(16)第85位的E突变为S或D；
(17)第86位的S突变为T或I；
(18)第87位的A突变为G；
(19)第93位的E突变为P或A或S或T；
(20)第107位的L突变为M；
(21)第114位的I突变为M；
(22)第121位的V突变为D或E。
不同的突变位点对于底物(腔肠素及其类似物)有不同的活性，可以为实际生产需要提供更多的选择。
根据本发明的实施例，相较于SEQ ID NO:3所示的氨基酸序列，所述荧光素酶突变体具有以下突变：
(1)第62位的H突变为K，第67位的P突变为L，第78位的H突变为A，第85位的E突变为S，第86位的S突变为T，第87位的A突变为G，第93位的E突变为S，第107位的L突变为M，第121位的V突变为E；或
(2)第62位的H突变为K，第67位的P突变为L，第79位的T突变为P，第85位的E突变为S，第86位的S突变为T，第87位的A突变为G，第93位的E突变为S，第107位的L突变为M，第121位的V突变为E；或
(3)第10位的N突变为S，第12位的V突变为A；或
(4)第12位的V突变为A，第79位的T突变为H；或
(5)第12位的V突变为A，第79位的T突变为K；或
(6)第10位的N突变为S，第12位的V突变为S；或
(7)第12位的V突变为A,第62位的H突变为K,第67位的P突变为L,第85位的E突变为S，第86位的S突变为T,第87位的A突变为G，第93位的E突变为P,第107位的L突变为M,第121位的V突变为E；或
(8)第20位的T突变为A，第114位的I突变为M；或
(9)第23位的L突变为T,第62位的H突变为K,第67位的P突变为L,第85位的E突变为S，第86位的S突变为T,第87位的A突变为G，第93位的E突变为T,第107位的L突变为M,第121位的V突变为E；或
(10)第62位的H突变为K,第85位的E突变为D，第86位的S突变为T,第87位的A突变为G，第107位的L突变为M,第121位的V突变为E；或
(11)第62位的H突变为K,第67位的P突变为A,第85位的E突变为D，第86位的S突变为T,第87位的A突变为G，第107位的L突变为M；或
(12)第62位的H突变为K，第67位的P突变为A，第85位的E突变为D，第86位的S突变为T，第87位的A突变为G，第121位的V突变为E；或
(13)第62位的H突变为K,第67位的P突变为K,第85位的E突变为D，第86位的S突变为T,第87位的A突变为G，第107位的L突变为M,第121位的V突变为E；或
(14)第62位的H突变为K，第67位的P突变为A，第85位的E突变为D，第86位的S突变为T，第87位的A突变为G，第93位的E突变为A，第107位的L突变为M，第121位的V突变为E；或
(15)第62位的H突变为K，第67位的P突变为A，第85位的E突变为D，第86位的S突变为I，第87位的A突变为G，第107位的L突变为M，第121位的V突变为E； 或
(16)第67位的P突变为A，第85位的E突变为D，第86位的S突变为T，第87位的A突变为G，第107位的L突变为M，第121位的V突变为E；或
(17)第62位的H突变为Q，第85位的E突变为S，第86位的S突变为T，第87位的A突变为G，第93位的E突变为A，第107位的L突变为M，第121位的V突变为E；或
(18)第23位的L突变为A，第62位的H突变为K，第67位的P突变为L，第85位的E突变为S，第86位的S突变为T，第87位的A突变为G，第93位的E突变为S，第107位的L突变为M，第121位的V突变为E；或
(19)第62位的H突变为K，第67位的P突变为A，第85位的E突变为D，第86位的S突变为T，第87位的A突变为G，第107位的L突变为M，第121位的V突变为D；或
(20)第62位的H突变为K，第67位的P突变为A，第85位的E突变为D，第86位的S突变为T，第87位的A突变为G，第107位的L突变为M；或
(21)第20位的T突变为A,第62位的H突变为K，第67位的P突变为A，第85位的E突变为D，第86位的S突变为T，第87位的A突变为G，第107位的L突变为M，第121位的V突变为E；或
(22)第21位的T突变为A，第62位的H突变为K，第67位的P突变为K,第85位的E突变为D，第86位的S突变为T，第87位的A突变为G，第107位的L突变为M，第121位的V突变为E；或
(23)第62位的H突变为K，第67位的P突变为K,第85位的E突变为D，第86位的S突变为T，第87位的A突变为G，第107位的L突变为M，第121位的V突变为D；或
(24)第20位的T突变为V，第21位的T突变为A，第22位的D突变为E，第62位的H突变为K，第67位的P突变为A，第85位的E突变为D，第86位的S突变为T，第87位的A突变为G，第107位的L突变为M，第121位的V突变为D；或
(25)第21位的T突变为A，第62位的H突变为K，第67位的P突变为A,第85位的E突变为D，第86位的S突变为T，第87位的A突变为G，第107位的L突变为M，第121位的V突变为E；或
(26)第20位的T突变为V，第21位的T突变为A，第22位的D突变为G，第62位的H突变为K，第67位的P突变为A，第79位的T突变为P，第85位的E突变为D， 第86位的S突变为I，第87位的A突变为G，第107位的L突变为M，第121位的V突变为E；或
(27)第62位的H突变为K，第67位的P突变为A，第85位的E突变为D，第86位的S突变为I，第87位的A突变为G，第107位的L突变为M，第121位的V突变为D；或
(28)第44位的E突变为G，第62位的H突变为N，第67位的P突变为A，第85位的E突变为D，第86位的S突变为T，第87位的A突变为G，第107位的L突变为M，第121位的V突变为D；或
(29)第21位的T突变为A，第67位的P突变为A，第85位的E突变为D，第86位的S突变为T，第87位的A突变为G，第107位的L突变为M，第121位的V突变为D；或
(30)第67位的P突变为A,第85位的E突变为D，第86位的S突变为T，第87位的A突变为G，第107位的L突变为M，第121位的V突变为D；或
(31)第62位的H突变为Q，第85位的E突变为D，第86位的S突变为T，第87位的A突变为G，第107位的L突变为M，第121位的V突变为E；或
(32)第12位的V突变为A,第21位的T突变为A，第22位的D突变为G,第35位的L突变为F，第45位的A突变为V，第59位的C突变为S,第62位的H突变为K，第67位的P突变为L，第85位的E突变为D，第86位的S突变为T，第87位的A突变为G，第93位的E突变为S，第107位的L突变为M，第121位的V突变为E；或
(33)第12位的V突变为A,第20位的T突变为A,第21位的T突变为A，第22位的D突变为G，第35位的L突变为F，第45位的A突变为V，第59位的C突变为S，第62位的H突变为K，第67位的P突变为L，第85位的E突变为D，第86位的S突变为T，第87位的A突变为G，第93位的E突变为S，第107位的L突变为M，第121位的V突变为E。
(34)第12位的V突变为A,第19位的A突变为V，第20位的T突变为A,第21位的T突变为A，第22位的D突变为G,第23位的L突变为I，第35位的L突变为F，第59位的C突变为S，第62位的H突变为K，第67位的P突变为L，第85位的E突变为D，第86位的S突变为T，第87位的A突变为G，第93位的E突变为S，第107位的L突变为M，第121位的V突变为E。
(35)第21位的T突变为A，第62位的H突变为K，第67位的P突变为L，第85位的E突变为D，第86位的S突变为T，第87位的A突变为G，第93位的E突变为P， 第107位的L突变为M，第121位的V突变为E。
根据本发明的一些具体实施例，当SEQ ID NO:3所示的氨基酸序列具有上述突变时，获得的蛋白对腔肠素等底物的催化活性较强，相对于现有的高斯荧光素酶发光亮度显著增强。
根据本发明的实施例，所述荧光素酶突变体进一步包括信号肽，所述信号肽设置于所述荧光素酶突变体的N端。用于引导上述荧光素酶突变体从表达细胞内向外分泌。
根据本发明的实施例，所述突变荧光素酶突变体进一步包括多聚组氨酸序列，所述多聚组氨酸序列设置于所述荧光素酶突变体的C端。用于亲和纯化上述突变荧光素酶突变体。
在本发明的第二方面，本发明提出了一种核酸分子。根据本发明的实施例，所述核酸分子编码本发明第一方面所述的荧光素酶突变体。
在本发明的第三方面，本发明提出了一种表达载体。根据本发明的实施例，所述表达载体包括本发明第二方面所述的核酸分子。
根据本发明的实施例，所述表达载体进一步包括启动子。
根据本发明的实施例，所述启动子与所述核酸分子可操作的连接。
在本发明的第四方面，本发明提出了一种重组细胞。根据本发明的实施例，所述重组细胞携带本发明第二方面所述的核酸分子以及本发明第三方面所述的表达载体。进而表达或分泌本发明第一方面所述的荧光素酶突变体。
根据本发明的实施例，所述重组细胞选自大肠杆菌、酵母或哺乳动物细胞。
在本发明的第五方面，本发明提出了一种获得荧光素酶的方法。根据本发明的实施例，将本发明第四方面所述的重组细胞在适于蛋白表达的条件下进行培养处理，以便获得所述荧光素酶。
在本发明的第六方面，本发明提出了一种缀合物。根据本发明的实施例，所述缀合物包括本发明第一方面所述荧光素酶突变体和小分子化合物或大分子，所述荧光素酶突变体和小分子化合物或大分子通过化学键偶联。
根据本发明的实施例，所述小分子化合物或大分子包括链霉亲和素(streptavidin，SA)、地高辛、抗体、dNTP或dNTP类似物等。
在本发明的第七方面，本发明提出了一种核酸测序的方法。根据本发明的实施例，所述测序方法包括：以待测核酸为模板，依次加入可以结合第一方面所述荧光素酶突变体和/或第六方面所述的缀合物和/或dNTP或dNTP类似物或修饰的dNTP进行聚合反应；依据所述荧光素酶突变体与高斯荧光素酶的底物或底物的类似物反应所发出的荧光信号，确定所述待测核酸的核酸序列。根据本发明的具体实施例，所述方法包括随后以待测核酸为模 板加入多种不同标记的dNTP进行聚合反应，然后加入与底物反应有差异活性的荧光素酶突变体或缀合物识别不同标记的dNTP，最后加入底物，依据所述荧光素酶突变体与高斯荧光素酶的底物或底物的类似物反应所发出的多种荧光信号，最终获得所述待测核酸的核酸序列，以上聚合反应和酶促荧光反应可以根据测序模板长度进行多个循环。
在本发明的第八方面，本发明提出了一种核酸测序试剂盒。根据本发明的实施例，所述核酸测序试剂盒包括第一方面所述的荧光素酶突变体或第六方面所述的缀合物。
根据本发明的实施例，所述试剂盒进一步包括dNTP或dNTP类似物、通用引物、PCR聚合酶或腔肠素或腔肠素类似物。
在本发明的第九方面，本发明提出了一种检测待测物含量的方法。根据本发明的实施例，所述检测待测物含量的方法包括：将结合有本发明第一方面所述的荧光素酶突变体或本发明第六方面所述的缀合物的待测物与高斯荧光素酶的底物或底物的类似物进行接触，最终基于接触处理后的荧光信号的强弱，确定所述待测物的含量。
根据本发明的实施例，所述高斯荧光素酶的底物或底物的类似物包括选自腔肠素、腔肠素衍生物中的至少之一。
根据本发明的实施例，所述腔肠素衍生物如式(I)或式(Ⅱ)所示，或其盐或异构体，
Figure PCTCN2023087445-ftappb-I100002
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显示了不含信号肽的野生型高斯荧光素酶原核表达质粒pCold-Gluc-WT-NS质粒图谱；
图2显示了含信号肽的野生型高斯荧光素酶真核表达质粒pEE12.4-Gluc WT质粒图谱；
图3显示了底物腔肠素(CTZ)，及腔肠素衍生物F-CTZ和ZS26的结构图；
图4显示了高斯荧光素酶突变体原核表达纯化的蛋白电泳图；
图5显示了原核表达的高斯荧光素酶突变体对底物腔肠素蛋白水平活性检测结果；
图6显示了部分高斯荧光素酶突变体真核表达纯化蛋白的电泳图；
图7显示了部分真核表达的高斯荧光素酶突变体对底物腔肠素蛋白水平活性检测结果；
图8显示了部分高斯荧光素酶突变体生物素偶联蛋白的电泳图；
图9显示了部分高斯荧光素酶突变体生物素偶联蛋白对底物CTZ、F-CTZ和ZS26的蛋白水平活性检测结果；
图10显示了部分高斯荧光素酶突变体生物素偶联蛋白在测序仪DNBSEQ E5上的信号值。
具体实施方式
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。
本发明提出的荧光素酶突变体，相较于SEQ ID NO:1所示的氨基酸序列或与所述高斯荧光素酶具有至少70％同源性的荧光素酶(即桡足类荧光素酶)的氨基酸序列。相较于SEQ ID NO:3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10位、第12位、第19位、第20位、第21位、第22位、第23位、第35位、第44位、第45位、第59位、第62位、第67位、第78位、第79位、第85位、第86位、第87位、第93位、第107位、第114位、第121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不含信号肽的野生型高斯荧光素酶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ZS26等底物具有较强的催化活性，相较于现有的高斯荧光素酶底物谱更广、特异性更强、发光亮度显著增强。在应用上，所述突变体在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。此外，在报告基因，定量检测DNA、RNA、 转录因子、蛋白或细胞等方面具有重要作用。也可作为融合蛋白中的发光信号蛋白，定量检测目标小分子等。
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。
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，相较于现有的高斯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。
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，相较于现有的高斯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。
本发明提出的一种核酸测序的方法是指以待测核酸为模板，依次加入结合有前述荧光素酶突变体或前述缀合物的dNTP聚合反应；依据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，最终获得所述待测核酸序列。
具体的，为了方便理解，下面对本申请的技术方案进行详细解释和说明。一种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；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。所述第一底物和第二底物为荧光素酶的发光反应底物。所述第一突变荧光素酶复合物与第二荧光素酶复合物，可分别结合在不同修饰过的碱基上。
C)通过检测所述第一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。
本发明的再一方面提出了一种检测待测物含量的方法，包括以下步骤：
1)将待测物、所述待测物的特异性识别蛋白、前述突变体以及高斯荧光素酶的底物进行接触，所述待测物适于与所述突变体形成复合物。
2)基于所述复合物形成前后引起所述突变体荧光强度的变化，确定所述待测物的含量；
根据本发明的实施例，所述高斯荧光素酶的底物包括腔肠素、氟代腔肠素或腔肠素衍生物。
根据本发明的实施例，所述腔肠素衍生物包括腔肠素衍生物F-CTZ或腔肠素衍生物ZS26。
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。
“荧光素酶”是催化发光化学反应的一类酶。该酶的底物称为荧光素。在ATP的存在下，在荧光素因荧光素酶的催化活性而发生化学反应时出现光发射。本文中，“荧光”又称为化 学发光，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。生命系统中也有荧光，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，所述突变体催化底物腔肠素氧化发光，即荧光。
本发明涉及一种核酸，所述核酸含有编码本发明实施方式的荧光素酶突变体的碱基序列。即，该核酸含有源自海洋桡足类生物长腹水蚤的荧光素酶基因。核酸是指DNA或RNA。荧光素酶的“基因”主要是指mRNA所转录的区域，即，其是指结构基因。
除非另外指出，本申请描述的式(I)或式(Ⅱ)所示结构的异构体包括所有异构体(如，对映体、非对映体阻转异构体(atropisomer)和几何(或构象))形式；例如，各不对称中心的R和S构型，(Z)和(E)双键异构体，以及(Z)和(E)构象异构体。因此，本申请化合物的单个立体化学异构体以及对映体混合物、非对映体混合物和几何异构体(或构象异构体)混合物均在本申请的范围之内。
本申请所使用的式(I)或式(Ⅱ)所示结构的盐是指该化合物的有机盐和无机盐。如文献(S.M.Berge et al.,describe pharmaceutically acceptable salts in detail in J.Pharmaceutical Sciences,1977,66:1-19.)所记载。药学上可接受的无毒的酸形成的盐包括，但并不限于，与氨基基团反应形成的无机酸盐有盐酸盐，氢溴酸盐，磷酸盐，硫酸盐，高氯酸盐，和有机酸盐如乙酸盐，草酸盐，马来酸盐，酒石酸盐，柠檬酸盐，琥珀酸盐，丙二酸盐，或通过书籍文献上所记载的其他方法如离子交换法来得到这些盐。其他药学上可接受的盐包括己二酸盐，藻酸盐，抗坏血酸盐，天冬氨酸盐，苯磺酸盐，苯甲酸盐，重硫酸盐，硼酸盐，丁酸盐，樟脑酸盐，樟脑磺酸盐，环戊基丙酸盐，二葡萄糖酸盐，十二烷基硫酸盐，乙磺酸盐，甲酸盐，反丁烯二酸盐，葡庚糖酸盐，甘油磷酸盐，葡萄糖酸盐，半硫酸盐，庚酸盐，己酸盐，氢碘酸盐，2-羟基-乙磺酸盐，乳糖醛酸盐，乳酸盐，月桂酸盐，月桂基硫酸盐，苹果酸盐，丙二酸盐，甲磺酸盐，2-萘磺酸盐，烟酸盐，硝酸盐，油酸盐，棕榈酸盐，扑酸盐，果胶酸盐，过硫酸盐，3-苯基丙酸盐，苦味酸盐，特戊酸盐，丙酸盐，硬脂酸盐，硫氰酸盐，对甲苯磺酸盐，十一酸盐，戊酸盐，等等。通过适当的碱得到的盐包括碱金属，碱土金属，铵和N+(C1-4烷基)4的盐。本申请也拟构思了任何所包含N的基团的化合物所形成的季铵盐。水溶性或油溶性或分散产物可以通过季铵化作用得到。碱金属或碱土金属盐包括钠，锂，钾，钙，镁，等等。药学上可接受的盐进一步包括适当的、无毒的铵，季铵盐和抗平衡离子形成的胺阳离子，如卤化物，氢氧化物，羧化物，硫酸化物，磷酸化物，硝酸化物，C1-8磺酸化物和芳香磺酸化物。
在使用成像设备检测荧光素酶的发光时，可以应用公知的检测方法。例如，通过将荧 光素、ATP和Mg2+离子等适当地添加到表达含荧光素酶的融合蛋白的细胞中来引起荧光素酶的发光反应，并且通过成像设备可以检测发出的光。所述成像设备是例如配备有用于捕获发光的滤光器的显微镜。基于通过鉴定细胞中的发光位置而获得的信息，可以使用显微镜来具体说明蛋白的定位。作为成像设备，可以使用具有能够进行时程图像拍摄功能的显微镜，并且可以用所述显微镜实现时程观察。
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原核表达高斯荧光素酶突变体的质粒设计与构建
本实施例对不含有信号肽的野生型和突变体高斯荧光素酶进行构建，用以比较突变体与野生型高斯荧光素酶的活性差异。
野生型高斯荧光素酶(Gluc WT)的核苷酸序列为SEQ ID NO:2，其编码的氨基酸序列为SEQ ID NO:1。不含信号肽的野生型高斯荧光素酶(Gluc WT-NS:Gluc WT no signal peptide)的核苷酸序列为SEQ ID NO:4，其编码的氨基酸序列为SEQ ID NO:3。通过全基因合成方法，合成了pCold-Gluc WT-NS质粒(图1)，包含了不含有信号肽的野生型高斯荧光素酶的氨基酸序列为SEQ ID NO:5，核苷酸序列为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，两端的酶切位点为Nde I和EcoR I。通过全基因合成方法，分别合成了pCold载体的不含信号肽的高斯荧光素酶活性突变体B6(H62K,P67L,E85S,S86T,A87G,L107M,V121E)，D6(H62K,P67A,E85D,S86T,A87G,L107M,V121E)，4-C12(H62K,E85S,S86T,A87G,E93P,L107M,V121E)。以Gluc WT-NS、B6、D6和4-C12为模板，利用多重PCR方法，构建包括N10，V12，A19，T20，T21，D22,L23，L35，E44，A45，C59，H62，P67，K71，H78,T79，E85，S86，A87，G89，E93，I95,L107，I114，V121等位点的组合随机突变体库，突变体相较于Gluc WT-NS进行突变的位点如表3所示。PCR反应体系如表1所示，PCR反应条件如表2所示。
Figure PCTCN2023087445-ftappb-I100003

Figure PCTCN2023087445-ftappb-I100004

Figure PCTCN2023087445-ftappb-I100005
表1：PCR反应体系：
Figure PCTCN2023087445-ftappb-I100006
表2：PCR反应条件
Figure PCTCN2023087445-ftappb-I100007
在反应后的PCR体系中加入0.5μL DpnI酶，37℃孵育3小时消化模板，然后进行胶回收约4800bp的产物即各位点的组合突变体库。
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，37℃过夜培养，次日从平板上挑取单克隆，进行摇菌扩繁，抽提质粒。进行测序确保多重PCR的正确扩增，获得的质粒即为不含信号肽的野生高斯荧光素酶pCold Gluc WT-NS、B6、D6、4-C12和各位点的组合突变体库。
实施例2高斯荧光素酶突变体的原核表达与纯化
本实施例对构建的pCold Gluc WT-NS、B6、D6、4-C12和各位点的组合突变体库进行原核表达和蛋白纯化，具体的实验流程如下：
将实施例1中获得的表达质粒pCold Gluc WT-NS和突变体分别转化入OrigamiB(DE3) Chemically Competent Cell感受态细胞(唯地生物，EC1020S)中，涂至含有氨苄抗性的平板(100μg/mL)，从平板上挑取单菌落，于37℃过夜培养，次日按1:100的比例进行稀释，转接于新鲜的含氨苄抗性(100μg/mL)的3mL LB培养基中，于37℃、200rpm振荡培养至OD600≈0.5-0.6，培养结束后冰上冷却1小时。按照终浓度为1mM的量加入诱导剂IPTG，并于16℃条件下过夜诱导。
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-HCl，pH 8.0，250mM NaCl,),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96孔纯化板中(GE)中加入500μL去离子水清洗后，加入500μL漂洗液(50mM Tris-HCl，pH 8.0，250mM NaCl，10mM咪唑)平衡后，加入过滤后的细胞裂解液。用漂洗液(50mM Tris-HCl，pH 8.0，250mM NaCl，10mM咪唑)冲洗10次(200μL/次)，然后用洗脱液(50mM Tris-HCl，pH 8.0，250mM NaCl，300mM咪唑)100μL洗脱蛋白，收集洗脱后的蛋白，部分蛋白电泳图如图4所示。
实施例3高斯荧光素酶突变体的活性检测
本实施例采用BCA定量试剂盒(Thermo ScientificTM PierceTM BCA Protein Assay Kit)精确测定蛋白的浓度，将实施例2获得的纯化的所述荧光素酶用稀释液(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(MGI，图3)90μL，用酶标仪自发光模块读取发光强度(图5)。优势突变体相对于SEQ ID NO:3所示序列的野生型不含有信号肽的高斯荧光素酶催化底物腔肠素的活性测试结果如表3所示，其中，下述33个突变体组合在催化腔肠素活性方面提升2.5倍以上，分别是17-1(H62K,P67L,H78A,E85S,S86T,A87G,E93S,L107M,V121E)，18-2(H62K,P67L,T79P,E85S,S86T,A87G,E93S,L107M,V121E)，20-3(N10S,V12A)，21-1(V12A,T79H)，23-1(V12A,T79K)，24-2(N10S,V12S)，27-2(V12A,H62K,P67L,E85S,S86T,A87G,E93P,L107M,V121E)，30-3(T20A,I114M)，36-1(L23T,H62K,P67L,E85S,S86T,A87G,E93T,L107M,V121E)，1-1(H62K,E85D,S86T,A87G,L107M,V121E)，2-3(H62K,P67A,E85D,S86T,A87G,L107M)，4-1(H62K,P67A,E85D,S86T,A87G,V121E)，5-1(H62K,P67K,E85D,S86T,A87G,L107M,V121E)，6-1(H62K,P67A,E85D,S86T,A87G,E93A,L107M,V121E)，8-3(H62K,P67A,E85D,S86I,A87G,L107M,V121E)，12-2(P67A,E85D,S86T,A87G,L107M,V121E)，13-2(H62Q,E85S,S86T,A87G,E93A,L107M,V121E)，28-1(L23A,H62K,P67L,E85S,S86T, A87G,E93S,L107M,V121E)，A2-2(H62K,P67A,E85D,S86T,A87G,L107M,V121D)，A2-3(H62K,P67A,E85D,S86T,A87G,L107M)，A3-1(T20A,H62K,P67A,E85D,S86T,A87G,L107M,V121E)，A5-1(T21A,H62K,P67K,E85D,S86T,A87G,L107M,V121E)，A5-2(H62K,P67K,E85D,S86T,A87G,L107M,V121E)，A7-1(T20V,T21A,D22E,H62K,P67A,E85D,S86T,A87G,L107M,V121D),A7-2(T21A,H62K,P67A,E85D,S86T,A87G,L107M,V121E),A8-1(T20V,T21A,D22G,H62K,P67A,E85D,S86I,A87G,T79P,L107M,V121E)，A8-2(H62K,P67A,E85D,S86I,A87G,L107M,V121D),A12-1(E44G,H62N,P67A,E85D,S86T,A87G,L107M,V121D)，A12-4(T21A,P67A,E85D,S86T,A87G,L107M,V121D)，A12-5(P67A,E85D,S86T,A87G,L107M,V121D),A14-1(H62Q,E85D,S86T,A87G,L107M,V121E)，A28-1(V12A,T21A,D22G,L35F,A45V,C59S,H62K,P67L,E85D,S86T,A87G,E93S,L107M,V121E)，A28-2(V12A,T20A,T21A,D22G,L35F,A45V,C59S,H62K,P67L,E85D,S86T,A87G,E93S,L107M,V121E),A28-3(V12A，A19V，T20A，T21A，D22G，L23I，L35F，C59S，H62K，P67L，E85D，S86T，A87G，E93S，L107M，V121E)，A29-3(T21A，H62K，P67L，E85D，S86T，A87G，E93P，L107M，V121E)。
表3：高斯荧光素酶优势突变体的突变位点及活性结果汇总表
Figure PCTCN2023087445-ftappb-I100008

Figure PCTCN2023087445-ftappb-I100009

Figure PCTCN2023087445-ftappb-I100010
实施例4真核表达高斯荧光素酶质粒的设计与构建
通过全基因合成方法，合成了pEE12.4载体的包含信号肽的野生型高斯荧光素酶(pEE12.4-Gluc WT)(图2)，包含了含有信号肽的野生型高斯荧光素酶的氨基酸序列为SEQ ID NO:8，基因序列为SEQ ID NO:7，其N端在信号肽后融合了含有6个组氨酸(6x His)的纯化标签以利于蛋白纯化，其C端融合了用于生物素化的Avi-tag。用于真核表达的高斯荧光素酶质粒以不含信号肽的突变体原核pCold质粒4-1、6-1、A2-2、A2-3、A8-2、A12-5为模板，构建表达质粒插入片段，PCR反应所使用的引物序列如表4所示，反应体系如表5所示，反应条件如表6所示。
Figure PCTCN2023087445-ftappb-I100011
表4：PCR反应引物序列
Figure PCTCN2023087445-ftappb-I100012

Figure PCTCN2023087445-ftappb-I100013
表5：反应体系
Figure PCTCN2023087445-ftappb-I100014
表6：反应条件
Figure PCTCN2023087445-ftappb-I100015
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543bp的产物即为插入片段(insert)。
以pEE12.4载体为模板，其模板N端还有用于纯化的组氨酸标签，C端还有用于生物素化的Avitag。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7所示，PCR反应体系如表8所示，PCR反应条件如表9所示。
表7：PCR反应引物序列
Figure PCTCN2023087445-ftappb-I100016
表8：反应体系
Figure PCTCN2023087445-ftappb-I100017

Figure PCTCN2023087445-ftappb-I100018
表9：反应条件
Figure PCTCN2023087445-ftappb-I100019
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7600b p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10中所示的反应体系进行重组，反应条件为50℃孵育15分钟。
表10：重组反应体系
Figure PCTCN2023087445-ftappb-I100020
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Amp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Gluc突变体。
实施例5高斯荧光素酶突变体的真核表达、纯化与酶活检测
将实施例4获得的pEE12.4载体的Gluc突变体质粒和全基因合成的pEE12.4-Gluc WT经过质粒大提后，用PEI转染入30mL HEK293E细胞中(细胞密度：4×106细胞/ml，活力95％以上)，转染5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 Excel填料，依次用20mL的去离子水、用10mL洗脱液I(50mM Tris-HCl，pH 8.0，250mM NaCl,1M咪唑)、20ml结合缓冲液(50mM Tris-HCl，pH 8.0，250mM NaCl)冲洗平衡填料。将约30mL过滤后的细胞上清液加入平衡好的填料。用漂洗液(50mM Tris-HCl， pH 8.0，250mM NaCl,10mM咪唑)冲洗10次(10mL/次)后，用洗脱液(50mM Tris-HCl，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6所示。
按照实施例3中所述方法对Gluc WT及Gluc突变体进行活性测试，结果如图7所示。
实施例6高斯荧光素酶突变体的偶联及酶活检测
本实施例5中得到的Gluc蛋白含有AviTag标签，可被BirA酶(MGI)生物素化为Biotin-Avi-tag-Gluc，生物素化的反应体系如表11。
表11：反应体系
Figure PCTCN2023087445-ftappb-I100021
室温放置30min后，在体系中加入SA即可制备SA-Gluc，利用突变体上的组氨酸标签可以用来进一步纯化得到较纯的SA-Gluc(突变体)，纯化结果如图8所示。
按照实施例3中所述方法对SA-Gluc(Gluc wt)及SA-Gluc突变体进行底物腔肠素和腔肠素衍生物F-CTZ、ZS26的活性测试，结果如图9所示。
实施例7高斯荧光素酶突变体的上机验证
偶联后SA-Gluc(Gluc wt)及SA-Gluc突变体利用测序仪DNBSEQ E5(MGI)进行上机活性检测，上机结果如图10所示。
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 (22)

  1. 一种荧光素酶突变体，其特征在于，以野生型高斯荧光素酶的氨基酸序列或与所述高斯荧光素酶SEQ ID NO:3具有至少70％同源性的荧光素酶的氨基酸序列为参考，相较于SEQ ID NO:3所示的氨基酸序列，所述荧光素酶突变体具有以下至少之一位置的突变：
    第10位、第12位、第19位、第20位、第21位、第22位、第23位、第35位、第44位、第45位、第59位、第62位、第67位、第78位、第79位、第85位、第86位、第87位、第93位、第107位、第114位、第121位。
  2. 根据权利要求1所述的荧光素酶突变体，其特征在于，所述野生型高斯荧光素酶具有SEQ ID NO:1所示的氨基酸序列。
  3. 根据权利要求1所述的荧光素酶突变体，其特征在于，所述高斯荧光素酶具有至少70％同源性的荧光素酶为桡足类荧光素酶。
  4. 根据权利要求3所述的荧光素酶突变体，其特征在于，所述桡足类荧光素酶包括选自Mluc、Maluc、Pxluc、Mpluc的至少之一。
  5. 根据权利要求1～4任一项所述的荧光素酶突变体，其特征在于，所述突变荧光素酶突变体具有以下(1)-(24)突变中的任意一个或者多个组合：
    (1)第10位的N突变为S；
    (2)第12位的V突变为A或S；
    (3)第19位的A突变为V；
    (4)第20位的T突变为V或A；
    (5)第21位的T突变为A；
    (6)第22位的D突变为E或G；
    (7)第23位的L突变为A或T或I；
    (8)第35位的L突变为F；
    (9)第44位的E突变为G；
    (10)第45位的A突变为V；
    (11)第59位的C突变为S；
    (12)第62位的H突变为K或Q或N；
    (13)第67位的P突变为L或K或A；
    (14)第78位的H突变为A；
    (15)第79位的T突变为H或K或P；
    (16)第85位的E突变为S或D；
    (17)第86位的S突变为T或I；
    (18)第87位的A突变为G；
    (19)第93位的E突变为P或A或S或T；
    (20)第107位的L突变为M；
    (21)第114位的I突变为M；
    (22)第121位的V突变为D或E。
  6. 根据权利要求1所述的荧光素酶突变体，其特征在于，所述突变荧光素酶具有以下突变：
    (1)第62位的H突变为K，第67位的P突变为L，第78位的H突变为A，第85位的E突变为S，第86位的S突变为T，第87位的A突变为G，第93位的E突变为S，第107位的L突变为M，第121位的V突变为E；或
    (2)第62位的H突变为K，第67位的P突变为L，第79位的T突变为P，第85位的E突变为S，第86位的S突变为T，第87位的A突变为G，第93位的E突变为S，第107位的L突变为M，第121位的V突变为E；或
    (3)第10位的N突变为S，第12位的V突变为A；或
    (4)第12位的V突变为A，第79位的T突变为H；或
    (5)第12位的V突变为A，第79位的T突变为K；或
    (6)第10位的N突变为S，第12位的V突变为S；或
    (7)第12位的V突变为A,第62位的H突变为K,第67位的P突变为L,第85位的E突变为S，第86位的S突变为T,第87位的A突变为G，第93位的E突变为P,第107位的L突变为M,第121位的V突变为E；或
    (8)第20位的T突变为A，第114位的I突变为M；或
    (9)第23位的L突变为T,第62位的H突变为K,第67位的P突变为L,第85位的E突变为S，第86位的S突变为T,第87位的A突变为G，第93位的E突变为T,第107位的L突变为M,第121位的V突变为E；或
    (10)第62位的H突变为K,第85位的E突变为D，第86位的S突变为T,第87位的A突变为G，第107位的L突变为M,第121位的V突变为E；或
    (11)第62位的H突变为K,第67位的P突变为A,第85位的E突变为D，第86位的S突变为T,第87位的A突变为G，第107位的L突变为M；或
    (12)第62位的H突变为K，第67位的P突变为A，第85位的E突变为D，第86 位的S突变为T，第87位的A突变为G，第121位的V突变为E；或
    (13)第62位的H突变为K,第67位的P突变为K,第85位的E突变为D，第86位的S突变为T,第87位的A突变为G，第107位的L突变为M,第121位的V突变为E；或
    (14)第62位的H突变为K，第67位的P突变为A，第85位的E突变为D，第86位的S突变为T，第87位的A突变为G，第93位的E突变为A，第107位的L突变为M，第121位的V突变为E；或
    (15)第62位的H突变为K，第67位的P突变为A，第85位的E突变为D，第86位的S突变为I，第87位的A突变为G，第107位的L突变为M，第121位的V突变为E；或
    (16)第67位的P突变为A，第85位的E突变为D，第86位的S突变为T，第87位的A突变为G，第107位的L突变为M，第121位的V突变为E；或
    (17)第62位的H突变为Q，第85位的E突变为S，第86位的S突变为T，第87位的A突变为G，第93位的E突变为A，第107位的L突变为M，第121位的V突变为E；或
    (18)第23位的L突变为A，第62位的H突变为K，第67位的P突变为L，第85位的E突变为S，第86位的S突变为T，第87位的A突变为G，第93位的E突变为S，第107位的L突变为M，第121位的V突变为E；或(19)第62位的H突变为K，第67位的P突变为A，第85位的E突变为D，第86位的S突变为T，第87位的A突变为G，第107位的L突变为M，第121位的V突变为D；或
    (20)第62位的H突变为K，第67位的P突变为A，第85位的E突变为D，第86位的S突变为T，第87位的A突变为G，第107位的L突变为M；或
    (21)第20位的T突变为A,第62位的H突变为K，第67位的P突变为A,第85位的E突变为D，第86位的S突变为T，第87位的A突变为G，第107位的L突变为M，第121位的V突变为E；或
    (22)第21位的T突变为A，第62位的H突变为K，第67位的P突变为K,第85位的E突变为D，第86位的S突变为T，第87位的A突变为G，第107位的L突变为M，第121位的V突变为E；或
    (23)第62位的H突变为K，第67位的P突变为K,第85位的E突变为D，第86位的S突变为T，第87位的A突变为G，第107位的L突变为M，第121位的V突变为 D；或
    (24)第20位的T突变为V，第21位的T突变为A，第22位的D突变为E，第62位的H突变为K，第67位的P突变为A，第85位的E突变为D，第86位的S突变为T，第87位的A突变为G，第107位的L突变为M，第121位的V突变为D；或
    (25)第21位的T突变为A，第62位的H突变为K，第67位的P突变为A,第85位的E突变为D，第86位的S突变为T，第87位的A突变为G，第107位的L突变为M，第121位的V突变为E；或
    (26)第20位的T突变为V，第21位的T突变为A，第22位的D突变为G，第62位的H突变为K，第67位的P突变为A，第79位的T突变为P，第85位的E突变为D，第86位的S突变为I，第87位的A突变为G，第107位的L突变为M，第121位的V突变为E；或
    (27)第62位的H突变为K，第67位的P突变为A，第85位的E突变为D，第86位的S突变为I，第87位的A突变为G，第107位的L突变为M，第121位的V突变为D；或
    (28)第44位的E突变为G，第62位的H突变为N，第67位的P突变为A，第85位的E突变为D，第86位的S突变为T，第87位的A突变为G，第107位的L突变为M，第121位的V突变为D；或
    (29)第21位的T突变为A，第67位的P突变为A，第85位的E突变为D，第86位的S突变为T，第87位的A突变为G，第107位的L突变为M，第121位的V突变为D；或
    (30)第67位的P突变为A,第85位的E突变为D，第86位的S突变为T，第87位的A突变为G，第107位的L突变为M，第121位的V突变为D；或
    (31)第62位的H突变为Q，第85位的E突变为D，第86位的S突变为T，第87位的A突变为G，第107位的L突变为M，第121位的V突变为E；或
    (32)第12位的V突变为A,第21位的T突变为A，第22位的D突变为G,第35位的L突变为F，第45位的A突变为V，第59位的C突变为S,第62位的H突变为K，第67位的P突变为L，第85位的E突变为D，第86位的S突变为T，第87位的A突变为G，第93位的E突变为S，第107位的L突变为M，第121位的V突变为E；或
    (33)第12位的V突变为A,第20位的T突变为A,第21位的T突变为A，第22位的D突变为G,第35位的L突变为F，第45位的A突变为V，第59位的C突变为S 第62位的H突变为K，第67位的P突变为L，第85位的E突变为D，第86位的S突变为T，第87位的A突变为G，第93位的E突变为S，第107位的L突变为M，第121位的V突变为E；或
    (34)第12位的V突变为A,第19位的A突变为V，第20位的T突变为A,第21位的T突变为A，第22位的D突变为G,第23位的L突变为I，第35位的L突变为F，第59位的C突变为S，第62位的H突变为K，第67位的P突变为L，第85位的E突变为D，第86位的S突变为T，第87位的A突变为G，第93位的E突变为S，第107位的L突变为M，第121位的V突变为E；或
    (35)第21位的T突变为A，第62位的H突变为K，第67位的P突变为L，第85位的E突变为D，第86位的S突变为T，第87位的A突变为G，第93位的E突变为P，第107位的L突变为M，第121位的V突变为E。
  7. 根据权利要求1所述的荧光素酶突变体，其特征在于，所述突变荧光素酶突变体进一步包括信号肽，所述信号肽设置于所述荧光素酶突变体的N端。
  8. 根据权利要求1所述的荧光素酶突变体，其特征在于，所述突变荧光素酶突变体进一步包括多聚组氨酸序列，所述多聚组氨酸序列设置于所述荧光素酶突变体的C端。
  9. 一种核酸分子，其特征在于，所述核酸分子编码权利要求1-8中任一项所述的荧光素酶突变体。
  10. 一种表达载体，其特征在于，包含权利要求9所述的核酸分子。
  11. 根据权利要求10所述的表达载体，其特征在于，进一步包括启动子，所述启动子与所述核酸分子可操作的连接。
  12. 一种重组细胞，其特征在于，携带权利要求9所述的核酸分子或权利要求10或11所述的表达载体。
  13. 根据权利要求12所述的重组细胞，其特征在于，所述重组细胞选自大肠杆菌、酵母或哺乳动物细胞。
  14. 一种获得荧光素酶的方法，其特征在于，包括：将权利要求12所述的重组细胞在适于蛋白表达的条件下进行培养处理，以便获得所述荧光素酶。
  15. 一种缀合物，其特征在于，包括权利要求1～8任一项所述荧光素酶突变体和小分子化合物或大分子，所述荧光素酶突变体和小分子化合物或大分子通过化学键偶联。
  16. 根据权利要求15所述的缀合物，其特征在于，所述小分子化合物或大分子包括链霉亲和素、地高辛、抗体、dNTP或dNTP类似物。
  17. 一种核酸测序的方法，其特征在于，包括：
    以待测核酸为模板，将可以结合权利要求1～8任一项所述荧光素酶突变体和/或权利要求15～16任一项所述的缀合物和/或dNTP或dNTP类似物或修饰的dNTP进行聚合反应；
    依据所述荧光素酶与高斯荧光素酶的底物或底物的类似物反应所发出的荧光信号，确定所述待测核酸的核酸序列。
  18. 一种核酸测序试剂盒，其特征在于，包括权利要求1-8中任一项所述的荧光素酶突变体或权利要求15或16所述的缀合物。
  19. 根据权利要求18所述的试剂盒，其特征在于，进一步包括：dNTP、通用引物、PCR聚合酶或腔肠素或腔肠素类似物。
  20. 一种检测待测物含量的方法，其特征在于，包括：
    将结合有权利要求1-8中任一项所述的荧光素酶突变体或权利要求15或16所述的缀合物的待测物与高斯荧光素酶的底物或底物的类似物进行接触；以及基于接触处理后的荧光信号的强弱，确定所述待测物的含量。
  21. 根据权利要求17或20所述的方法，其特征在于，所述高斯荧光素酶的底物或底物的类似物包括选自腔肠素、腔肠素衍生物中的至少之一。
  22. 根据权利要求21所述的方法，所述腔肠素衍生物如式(I)或式(Ⅱ)所示，或其盐或异构体，
    Figure PCTCN2023087445-ftappb-I200001
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