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WO2025118178A1 - Dna聚合酶突变体及其应用 - Google Patents

Dna聚合酶突变体及其应用 Download PDF

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Publication number
WO2025118178A1
WO2025118178A1 PCT/CN2023/136740 CN2023136740W WO2025118178A1 WO 2025118178 A1 WO2025118178 A1 WO 2025118178A1 CN 2023136740 W CN2023136740 W CN 2023136740W WO 2025118178 A1 WO2025118178 A1 WO 2025118178A1
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mutated
dna polymerase
amino acid
mutant
mutation
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French (fr)
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杨梦�
杨理想
张慧君
王艺
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MGI Tech Co Ltd
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MGI Tech Co Ltd
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    • C—CHEMISTRY; METALLURGY
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]

Definitions

  • the present application relates to the field of biotechnology, and in particular, to DNA polymerase mutants and applications thereof.
  • DNA polymerase is widely used in PCR technology and plays an important role in life science research and related fields.
  • One of its main functions is to use single-stranded DNA as a template and deoxynucleotides (dNTPs) as substrates to efficiently and accurately synthesize a DNA sequence complementary to the template from the 5' end to the 3' end.
  • dNTPs deoxynucleotides
  • DNA polymerases can be divided into seven categories based on family classification, including A, B, C, D, X, Y and RT. There are some differences in the structure and function of DNA polymerases in different families. Among them, DNA polymerases in the B family usually have higher replication and elongation, which means that they can continuously polymerize a large number of nucleotides before dissociating from the DNA template.
  • the DNA polymerases of the B family also have DNA repair functions, including 3' ⁇ 5' exonuclease activity, which can detect mismatched bases during PCR amplification and remove them, and then reinsert the correct bases and continue DNA replication.
  • KOD is a representative of the B family DNA polymerase, derived from the thermophilic archaeon Thermococcuskodakarensis. Due to its high temperature resistance, KOD DNA polymerase has been widely used in PCR amplification, gene sequencing and other fields in recent years. However, the existing KOD DNA polymerase has a low polymerization ability in sequencing, which greatly limits the efficiency of sequencing.
  • the inventors obtained a new DNA polymerase mutant by enzyme engineering modification of the active sites related to the thermostable B family polymerase of thermophilic archaea, and improved the polymerization ability of KOD DNA polymerase in sequencing, thereby improving the sequencing speed and sequencing quality of the sequencing by synthesis (SBS) method.
  • a DNA polymerase mutant which, compared with the wild-type KOD DNA polymerase, has an amino acid mutation at position 408, position 409 or a functionally equivalent position, and an amino acid mutation at at least one position selected from the following 24 positions and functionally equivalent positions: position 141, position 143, position 147, position 382, position 383, position 384, position 389, position 485, position 584, position 589, position 390, position 491, position 592, position 603, position 604, position 605, position 606, position 607, position 610, position 611, position 612, position 613, position 614, position 615 97, 424, 432, 445, 523, 553, 561, 564, 461, 481, 605, 663, 711, 725; the amino acid sequence of the mutant other than the amino acid mutation site has at least 90% identity with the corresponding amino acid sequence of the wild-type KOD DNA polymerase; the wild-type KOD DNA polymerase has the
  • the DNA polymerase mutant compared with the wild-type KOD DNA polymerase, has an amino acid mutation at position 408, position 409 or a functionally equivalent position, and an amino acid mutation at at least one position selected from the following 23 positions and functionally equivalent positions: position 141, position 143, position 147, position 383, position 384, position 389, position 485, position 584, position 589, position 397, position 424, position 432, position 445, position 523, position 553, position 561, position 564, position 461, position 481, position 605, position 663, position 711, position 725; the amino acid sequence of the mutant other than the amino acid mutation site has at least 90% identity with the corresponding amino acid sequence of the wild-type KOD DNA polymerase; the wild-type KOD DNA polymerase has the amino acid sequence of SEQ ID The amino acid sequence shown in NO:2.
  • amino acid positions in the amino acid sequence of the DNA polymerase mutant described in the present application are referenced to the amino acid positions in SEQ ID NO: 2.
  • the DNA polymerase mutant has a 408th (point) mutation, it means that the 408th amino acid in SEQ ID NO: 2 is mutated.
  • the "functionally equivalent sites" used in the present invention include sites where, under specific circumstances, amino acids or nucleotides mutate but the function or properties of the DNA polymerase do not change.
  • identity includes 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.
  • amino acid sequence other than the amino acid mutation site of the mutant has at least 90% identity with the corresponding amino acid sequence of the wild-type KOD DNA polymerase" described in the present application refers to the amino acid sequence in the wild-type KOD DNA polymerase corresponding to the amino acid sequence other than the amino acid mutation site on the mutant.
  • the above-mentioned DNA polymerase mutant may further include at least one of the following technical features:
  • the amino acid sequence of the mutant other than the amino acid mutation site is the same as the corresponding amino acid sequence of the wild-type KOD DNA polymerase.
  • the DNA polymerase mutant compared with the wild-type KOD DNA polymerase, has an amino acid mutation at position 408, position 409 or a functionally equivalent position, and an amino acid mutation at at least one position selected from the following 9 positions and functionally equivalent positions: position 141, position 143, position 147, position 383, position 384, position 389, position 485, position 584, position 589.
  • the relative activity of the sequencing enzymes produced by different mutation sites is different, which can provide more options for actual production needs.
  • the DNA polymerase mutant compared with the wild-type KOD DNA polymerase, has an amino acid mutation at position 408, position 409 or a functionally equivalent position, and an amino acid mutation at at least one of the following two positions and functionally equivalent positions: position 147, position 584.
  • the inventors first discovered after a large number of screenings that mutations to amino acids at positions 147 and 584 can increase the activity of DNA polymerase.
  • the mutation type of the mutation site is:
  • the inventors found that a DNA polymerase having one of the above mutation types (or any combination) has higher enzyme activity.
  • the DNA polymerase mutant compared with the wild-type KOD DNA polymerase, has: positions 408, 409, 141, 143, 147, 383, 384, 389, 485, 584, 589 amino acid mutation at the position or functionally equivalent position; and amino acid mutation at at least one position selected from the following 15 positions and functionally equivalent positions: position 382, position 397, position 424, position 432, position 445, position 523, position 553, position 561, position 564, position 461, position 481, position 605, position 663, position 711, and position 725.
  • the DNA polymerase mutant compared with the wild-type KOD DNA polymerase, has: amino acid mutations at position 408, 409, 141, 143, 147, 383, 384, 389, 485, 584, 589 or functionally equivalent positions; and amino acid mutations at at least one of the following 14 positions and functionally equivalent positions: 397, 424, 432, 445, 523, 553, 561, 564, 461, 481, 605, 663, 711, 725.
  • a polymerase mutant (SEQ ID NO: 1) having an amino acid mutation at position 408, 409, 141, 143, 147, 383, 384, 389, 485, 584, 589 or a functionally equivalent position has a higher enzyme activity.
  • the inventors found that based on the amino acid sequence shown in SEQ ID NO: 1, mutations at one or more positions selected from position 397, 424, 432, 445, 523, 553, 561, 564, 461, 481, 605, 663, 711 and 725 can obtain mutants with different enzyme activities.
  • the DNA polymerase mutant compared with the wild-type KOD DNA polymerase, has: amino acid mutations at position 408, position 409, position 141, position 143, position 147, position 383, position 384, position 389, position 485, position 584, position 589 or functionally equivalent positions; and amino acid mutations at at least one of the following 4 positions and functionally equivalent positions: position 397, position 424, position 481, position 553.
  • mutations at one or more positions selected from positions 397, 424, 481 and 553 can obtain mutants with higher enzyme activity.
  • the mutation types of the mutation sites are: (1) D at position 141 mutates to A, (2) E at position 143 mutates to A, (3) H at position 147 mutates to E, (4) S at position 383 mutates to T, (5) Y at position 384 mutates to F, (6) V at position 389 mutates to I, (7) A at position 485 mutates to E, (8) K at position 584 mutates to E, (9) V at position 589 mutated to H, (10) L at position 408 mutated to I, (11) Y at position 409 mutated to A, (12) W at position 397 mutated to Y or H, (13) N at position 424 mutated to R, Q, H, I, L, M, F, W, K, Y, V, C or S, (14) D at position 432 mutated to R, E, H or M, (15) F mutates to R, (16) M at position 523 mutates to R
  • the mutation types of the mutation sites are: (1) D at position 141 mutates to A, (2) E at position 143 mutates to A, (3) H at position 147 mutates to E, (4) S at position 383 mutates to T, (5) Y at position 384 mutates to F, (6) V at position 389 mutates to I, (7) A at position 485 mutates to E, (8) K at position 584 mutates to E, (9) V at position 589 mutates to H, (10) L at position 408 mutates to I, (11) Y at position 409 mutates to A, (12) W at position 397 mutates to Y or H, (13) 3) N at position 424 mutated to R, Q, H, I, L, M, F, W, K, Y, V, C or S (14) D at position 432 mutated to R, E, H or M, (15) F at position 445 mutated to R, (16) M at position 523 mutates
  • DNA polymerase mutants with the above mutation types still have different degrees of enzyme activity.
  • the DNA polymerase mutant compared with the wild-type KOD DNA polymerase, has: amino acid mutations at positions 408, 409, 141, 143, 147, 383, 384, 389, 485, 584, 589 or functionally equivalent positions; and amino acid mutations at one of the following 15 positions and functionally equivalent positions: positions 382, Position 397, position 424, position 432, position 445, position 523, position 553, position 561, position 564, position 461, position 481, position 605, position 663, position 711, position 725; provided that the Q at position 382 cannot mutate to R, H, L and Y; or the N at position 424 cannot mutate to R and L; or the D at position 432 cannot mutate to M; or the H at position 663 cannot mutate to T, V, P and E.
  • Q at position 382 cannot mutate to R, H, L and Y means that Q at position 382 cannot mutate to R, or Q at position 382 cannot mutate to H, or Q at position 382 cannot mutate to L, or Q at position 382 cannot mutate to Y.
  • N at position 424 cannot mutate to R and L means that N at position 424 cannot mutate to R, or N at position 424 cannot mutate to L.
  • this expression method is also applicable to any embodiment. It will not be repeated in the following embodiments or implementation modes.
  • the DNA polymerase mutant compared with the wild-type KOD DNA polymerase, has: amino acid mutations at positions 408, 409, 141, 143, 147, 383, 384, 389, 485, 584, 589 or functionally equivalent positions; and one position selected from the following 14 positions and functionally equivalent positions Amino acid mutations: 397, 424, 432, 445, 523, 553, 561, 564, 461, 481, 605, 663, 711, 725; provided that the N at position 424 cannot mutate to R and L; or the D at position 432 cannot mutate to M; or the H at position 663 cannot mutate to T, V, P and E.
  • the DNA polymerase mutant compared with the wild-type KOD DNA polymerase, has: amino acid mutations at positions 408, 409, 141, 143, 147, 383, 384, 389, 485, 584, 589 or functionally equivalent positions; and amino acid mutations at two positions selected from the following 14 positions and functionally equivalent positions.
  • Acid mutations position 397, position 424, position 432, position 445, position 523, position 553, position 561, position 564, position 461, position 481, position 605, position 663, position 711, position 725; provided that, when the H at position 663 mutates to R, the A at position 553 cannot mutate to P or E; or when the H at position 663 mutates to Q, the Q at position 461 cannot mutate to Y.
  • the enzyme activity when the H at position 663 of the mutant mutates to R, and the A at position 553 mutates to P or E; or, when the H at position 663 mutates to Q, and the Q at position 461 mutates to Y, the enzyme activity is low.
  • the DNA polymerase mutant compared with the wild-type KOD DNA polymerase, has mutations (1)-(11), and any one mutation selected from (12)-(25).
  • amino acid sequence of a DNA polymerase mutant having mutations (1)-(11) is shown as SEQ ID NO:1.
  • a polymerase mutant with the amino acid sequence shown in SEQ ID NO:1 is used as a control group for polymerization activity detection.
  • a DNA polymerase mutant having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 1 has higher enzymatic activity. In some preferred examples, it has at least 91% identity, or at least 92% identity, or at least 93% identity, or at least 94% identity, or at least 95% identity, or at least 96% identity, or at least 97% identity, or at least 98% identity, or at least 99% identity, and in some most preferred examples, it has at least 100% identity.
  • the DNA polymerase mutant has (1)-(11) mutation; and any one mutation selected from the following mutations: W at position 397 mutates to H, N at position 424 mutates to Q, A at position 553 mutates to R, A at position 553 mutates to N, A at position 553 mutates to E, A at position 553 mutates to G, A at position 553 mutates to H, A at position 553 mutates to P, A at position 553 mutates to S, Q at position 461 mutates to W, Q at position 461 mutates to Y, Y at position 481 mutates to M, Y at position 481 mutates to F, G at position 711 mutates to R, and G at position 711 mutates to S.
  • the inventors have experimentally verified that further amino acid single-point mutation based on the amino acid sequence shown in SEQ ID NO:1 can further increase the enzyme polymerization activity.
  • the DNA polymerase mutant compared with the wild-type KOD DNA polymerase, has mutations (1)-(11); and any one mutation combination selected from the following mutation combinations:
  • some mutants may have base bias in sequencing applications and can be adaptively selected based on actual experimental needs.
  • the DNA polymerase mutant has mutations (1)-(11), and any one of the following mutation combinations: Y at position 481 mutates to F, and A at position 553 mutates to P; Y at position 481 mutates to F, and A at position 553 mutates to S; W at position 397 mutates to H, and A at position 553 mutates to P; Q at position 461 mutates to Y, and A at position 553 mutates to P; N at position 424 mutates to Q, and A at position 553 mutates to P; W at position 397 mutates to H, and Y at position 481 mutates to F; A at position 553 mutates to P, and G at position 711 mutates to R; W at position 397 mutates to H, and A at position 553 mutates to P; A mutated to S; Y mutated to F, and G mutated to
  • the inventors have experimentally verified that double-site amino acid mutations based on the amino acid sequence shown in SEQ ID NO: 1 can further increase the enzyme polymerization activity.
  • the present application provides a nucleic acid molecule, which encodes the DNA polymerase mutant described in the first aspect of the present application.
  • the DNA polymerase mutant encoded by the nucleic acid molecule can be obtained in large quantities in vivo or in vitro.
  • nucleic acids mentioned in the specification and claims of the present invention those skilled in the art should understand that they actually include any one or both of the complementary double strands.
  • the 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 application proposes an expression vector, which includes or carries the nucleic acid molecule described in the second aspect of the present application.
  • the type of expression vector is not particularly limited, as long as it can replicate and express the corresponding mutant in the host cell.
  • the expression vector may further include a promoter, and the promoter is operably connected to the nucleic acid molecule.
  • the expression vector is a non-pathogenic viral vector
  • the non-pathogenic viral vector includes an adenoviral vector or a retroviral vector.
  • the expression vector is a non-viral vector
  • the non-viral vector includes but is not limited to a plasmid vector.
  • the present application provides a recombinant cell, the recombinant cell carrying the nucleic acid molecule described in the second aspect of the present application and the expression vector described in the third aspect of the present application.
  • the recombinant cell is used to express or secrete the DNA polymerase mutant described in the first aspect of the present application.
  • the recombinant cell is selected from Escherichia coli, yeast and mammalian cells.
  • 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 DNA polymerase mutant under appropriate conditions.
  • the present application provides a recombinant strain, wherein the recombinant strain expresses the DNA polymerase mutant described in the first aspect of the present application.
  • the DNA polymerase mutant can be obtained quickly and in large quantities by culturing the recombinant strain.
  • the present application proposes a method for obtaining a DNA polymerase mutant, the method comprising: The recombinant cell described in the first aspect or the recombinant strain described in the fifth aspect is cultured under conditions suitable for protein expression to obtain the DNA polymerase mutant.
  • the present application proposes a complex, which includes the DNA polymerase mutant described in the first aspect of the present application and a small molecule compound or a macromolecule, and the DNA polymerase mutant and the small molecule compound or the macromolecule are coupled by a chemical bond.
  • the small molecule compound or macromolecule includes a fluorescent marker, fluorescein or an antibody, etc.
  • DNA polymerase mutants and complexes proposed in this application include but are not limited to nucleic acid synthesis and nucleic acid sequencing, etc. They can also be used to screen drugs against viruses or cell division, and have great development prospects in forensic medicine and criminology.
  • the present application proposes a method for nucleic acid synthesis, the method comprising: subjecting a nucleic acid template, an amplification primer, dNTPs and a mixed product of the DNA polymerase mutant described in the first aspect of the present application to an amplification treatment under conditions suitable for nucleic acid amplification, so as to obtain the nucleic acid.
  • the aforementioned nucleic acid synthesis method can be used to efficiently and quickly amplify the nucleic acid template.
  • DNA polymerase mutant described in the present application has polymerization activity for all dNTPs (including dNTPs with or without fluorescent labels).
  • the present application proposes a method for nucleic acid sequencing, the method comprising: subjecting a nucleic acid template to be tested and a mixed product of the DNA polymerase mutant and modified dNTP described in the first aspect of the present application to an amplification treatment and a fluorescent signal detection treatment under conditions suitable for nucleic acid amplification; and determining the nucleic acid sequence of the nucleic acid to be tested based on the fluorescent signal obtained by detection.
  • the nucleic acid sequencing method includes mixing the nucleic acid template to be tested with the DNA polymerase mutant and the non-natural dNTP with a fluorescently labeled 3'O-reversible terminator, the DNA polymerase mutant is responsible for matching the non-natural dNTP with the fluorescently labeled 3'O-reversible terminator with the nucleic acid template, and finally detecting multiple fluorescent labeling signals, and obtaining the nucleic acid sequence based on the obtained fluorescent labeling signals to obtain the nucleic acid sequence of the nucleic acid to be tested.
  • the above polymerization reaction and fluorescent labeling reaction can be performed for multiple cycles according to the length of the sequencing template.
  • the present application proposes a nucleic acid sequencing kit, which comprises the DNA polymerase mutant described in the first aspect or the complex described in the seventh aspect.
  • the kit described in the present application is used for efficient, accurate and rapid nucleic acid sequencing.
  • the present application proposes a use of the nucleic acid sequencing kit of the tenth aspect in sequencing.
  • the kit can be used for sequencing, including but not limited to sequencing by synthesis (SBS).
  • the present application proposes a use of the DNA polymerase mutant described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, the recombinant cell described in the fourth aspect, the recombinant strain described in the fifth aspect, or the complex described in the seventh aspect in the preparation of products related to catalytic DNA amplification or nucleic acid sequencing.
  • the DNA polymerase mutant, nucleic acid molecule, expression vector, recombinant cell, recombinant strain or complex can be prepared alone or in combination for catalytic DNA amplification or nucleic acid sequencing related products.
  • Figure 1 is a schematic diagram of the recombinant KOD DNA polymerase expression plasmid pET22b-KOD plasmid map described in one embodiment of the present application;
  • Figure 2 is a schematic diagram of the expression of wild-type and some mutant KOD DNA polymerases and bacterial lysis results described in an embodiment of the present application;
  • Figure 3 is a schematic diagram of the results of detecting the activity of wild-type and some mutant KOD DNA polymerases using the FRET method described in an embodiment of the present application.
  • first and second are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Therefore, the features defined as “first” and “second” may explicitly or implicitly include up to In the description of the present invention, “plurality” means at least two, such as two, three, etc., unless otherwise clearly and specifically defined.
  • amino acid is represented by a single-letter or three-letter code and has the following meaning: A: Ala (alanine); R: Arg (arginine); N: Asn (asparagine); D: Asp (aspartic acid); C: Cys (cysteine); Q: Gln (glutamine); E: Glu (glutamate); G: Gly (glycine); H: His (histidine); I: Ile (isoleucine); L: Leu (leucine); K: Lys (lysine); M: Met (methionine); F: Phe (phenylalanine); P: Pro (proline); S: Ser (serine); T: Thr (threonine); W: Trp (tryptophan); Y: Tyr (tyrosine); V: Val (valine).
  • the term “identity” has the conventional meaning in the art and refers to the "homology” between two nucleic acid or amino acid sequences, and its percentage represents the statistically significant percentage of identical nucleotides or amino acid residues between the two sequences to be compared after the best alignment, and the differences between the two sequences are randomly distributed over their entire length.
  • the mutants are described according to their mutations at specific residues, and their positions are located with reference to the positions of amino acids in the amino acid sequence shown in SEQ ID NO:2 of the wild-type KOD polymerase.
  • the KOD DNA polymerase mutant (SEQ ID NO: 1) was obtained by mutation of the wild-type KOD DNA polymerase (SEQ ID NO: 2).
  • the mutation sites include: D at position 141 mutated to A, E at position 143 mutated to A, H at position 147 mutated to E, S at position 383 mutated to T, Y at position 384 mutated to F, V at position 389 mutated to I, L at position 408 mutated to I, Y at position 409 mutated to A, A at position 485 mutated to E, K at position 584 mutated to E, and V at position 589 mutated to H.
  • the term "relative polymerization activity" refers to the polymerization activity of each experimental group relative to the reference group calculated when the polymerization activity of the reference group is assumed to be 100%.
  • the reference group DNA polymerase referred to in this application is DP01, and its amino acid sequence is shown in SEQ ID NO: 1.
  • transformation refers to the introduction of DNA into a host cell so that the DNA can be replicated as an extrachromosomal element or by chromosomal integration. That is, transformation refers to the synthetic change of genes caused by the introduction of foreign DNA into cells.
  • amino acid sequences are displayed from 5' end to 3' end.
  • non-natural dNTPs or dNTPs with modifications have the same meaning, including but not limited to dNTPs with labels (e.g., fluorescent labels) and/or dNTPs with O-reversible terminators at the 3' end.
  • polymerase mutants used in the examples of the present application are all polymerase mutant fusion proteins with 6 His tags connected to the C-terminus. As known to those skilled in the art, they can all be replaced by untagged polymerase mutants.
  • thermostable B-family polymerases of thermophilic archaea perform well in incorporating natural nucleotides or their analogs in high-throughput sequencing.
  • thermostable B-family polymerases include KOD (Thermococcuskodakaraensis), 9°N (Thermococcussp.9°N), TGO (Thermococcusgorgonarius), TOK (Desulfurococcussp.Tok), VentDNA polymerase (Thermococcuslitoralis), JDF-3, and pfuDNA polymerase (Pyrococcusfuriosis).
  • KOD Thermococcuskodakaraensis
  • 9°N Thermococcussp.9°N
  • TGO Thermococcusgorgonarius
  • TOK Desulfurococcussp.Tok
  • VentDNA polymerase Thermococcuslitoralis
  • the inventors protected the functional domain sites of the wild-type polymerase to ensure that it can still perform its original basic functions. Through dynamic simulation and statistical inference of the palm, finger and thumb regions of KOD DNA polymerase, mutation sites that can be used for experimental screening were obtained. And through a large number of experimental verifications, DNA polymerases suitable for attachment to DNBs (DNA nanoballs) on the surface of the chip were finally obtained.
  • amino acid sequence of KOD DNA polymerase (DP01) is shown in SEQ ID NO:1.
  • the recombinant expression vector pET22b-WT is a vector in which the KOD DNA polymerase encoding gene fused with a His tag is recombined into the vector pET22b according to the instructions of the Seamless Cloning Kit (manufacturer: Novozymes; catalog number: C112-01).
  • the resulting vector is a KOD DNA polymerase encoding gene fused with a His tag ( Figure 1), and expression is induced by IPTG.
  • the nucleotide sequence of the KOD DNA polymerase encoding gene fused with a His tag is a sequence obtained by connecting six His tag codons to the 3' end of SEQ ID NO: 3;
  • the amino acid sequence of KOD DNA polymerase fusion protein is obtained by connecting 6 His tags to the C-terminus of the amino acid shown in SEQ ID NO:1.
  • the recombinant expression vector pET22b-WT was introduced into Escherichia coli BL21 competent cells (Beijing Solebold Technology Co., Ltd.), and the positive colonies were screened by smearing resistance plates (containing 50 ⁇ g/ml of ampicillin). 3-5 positive monoclonal colonies were selected, and the positive colonies were identified by bacterial liquid PCR using primers SQF (Table 1) and primers SQR (Table 1). The fragment of 2500 bp in size that was basically consistent with the expected theoretical value was obtained as a positive clone, and the positive clone was named BL21/pET22b-WT.
  • Lane 1 is protein Marker (Page Ruler Prestained Protein Ladder, 26616, Thermo Fisher Scientific)
  • lane 12 is 5 ⁇ l KOD DNA polymerase fusion protein (DP01) + 5 ⁇ l 2X loading buffer
  • lanes 2-11 are 5 ⁇ l KOD DNA polymerase mutant fusion protein (DP001, DP002, DP004, DP005, DP007, DP008, DP009, DP024, DP011, DP012) + 5 ⁇ l 2X loading buffer; it can be seen that the protein size in lanes 2-12 is about 91.5kDa, which is consistent with the molecular weight reported in the literature.
  • the target protein of about 91.5 kDa was not obtained from the uninduced BL21/pET22b-WT bacterial solution.
  • the empty vector pET22b was introduced into E. coli BL21 to obtain BL21/pET22b.
  • the target protein of about 91.5 kDa was not obtained by expression and lysis using the above method.
  • the KOD DNA polymerase mutant fusion protein is a protein obtained by performing amino acid mutation on at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen or all fourteen amino acids in the 14 positions 397, 424, 432, 445, 523, 553, 561, 564, 461, 481, 605, 663, 711 and 725 of the amino acid sequence shown in DP01 (SEQ ID NO: 1); if only one amino acid is mutated, a single-point mutant of KOD DNA polymerase is obtained; if two amino acid mutations are obtained, a two-point combination mutation of KOD DNA polymerase is obtained, and so on.
  • the KOD DNA polymerase mutant encoding gene is a nucleic acid obtained by mutating the nucleotide sequence of the KOD DNA polymerase mutant DP01 encoding gene (SEQ ID NO:3) according to the corresponding amino acid sequence (SEQ ID NO:1) of at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen or all fourteen amino acid codons at positions 397, 424, 432, 445, 523, 553, 561, 564, 461, 481, 605, 663, 711, and 725.
  • the N at position 424 is mutated to R, Q, H, I, L, M, F, W, K, Y, V, C or S;
  • the D at position 432 mutated to R, E, H, or M;
  • the A at position 553 is mutated to T, R, N, E, G, H, L, M, F, Y, P, S, D, V, I or C;
  • the T at position 605 mutated to I or V;
  • H at position 663 is mutated to R, Q, L, M, F, T, Y, N, V, G, A, P, S, C, E, or K;
  • the H at position 725 was mutated to R.
  • amino acids at two positions selected from the 14 positions 397, 424, 432, 445, 523, 553, 561, 564, 461, 481, 605, 663, 711, and 725 in the amino acid sequence of the KOD DNA polymerase SEQ ID NO: 1 are mutated, and the other amino acid sequences remain unchanged to obtain a protein having DNA polymerase activity:
  • the amino acid sequence of DP80 is that the 481st Y in SEQ ID NO:1 is mutated to F, and the 553rd A is mutated to P;
  • the amino acid sequence of DP81 is that the 481st Y in SEQ ID NO:1 is mutated to F, and the 553rd A is mutated to S;
  • the amino acid sequence of DP82 is that the 397th W of SEQ ID NO:1 is mutated to H, and the 553rd A is mutated to P;
  • the amino acid sequence of DP83 is that the Q at position 461 of SEQ ID NO: 1 is mutated to Y, and the A at position 553 is mutated to P;
  • the amino acid sequence of DP84 is that the 481st Y in SEQ ID NO:1 is mutated to F, and the 553rd A is mutated to G;
  • the amino acid sequence of DP85 is that the 424th N of SEQ ID NO:1 is mutated to Q, and the 553rd A is mutated to P;
  • the amino acid sequence of DP86 is that the 397th W of SEQ ID NO:1 is mutated to H, and the 481st Y is mutated to F;
  • the amino acid sequence of DP87 is that the 553rd position A of SEQ ID NO:1 is mutated to P, and the 711th position G is mutated to R;
  • the amino acid sequence of DP88 is that the 397th W of SEQ ID NO:1 is mutated to H, and the 553rd A is mutated to S;
  • the amino acid sequence of DP89 is that the 481st Y in SEQ ID NO:1 is mutated to M, and the 553rd A is mutated to P;
  • the amino acid sequence of DP90 is that the Q at position 461 of SEQ ID NO: 1 is mutated to W, and the A at position 553 is mutated to P;
  • the amino acid sequence of DP91 is that the 553rd position A of SEQ ID NO:1 is mutated to P, and the 711th position G is mutated to S;
  • the amino acid sequence of DP92 is that the Q at position 461 of SEQ ID NO: 1 is mutated to Y, and the A at position 553 is mutated to S;
  • the amino acid sequence of DP93 is that the 553rd position A of SEQ ID NO:1 is mutated to P, and the 561st position M is mutated to W;
  • the amino acid sequence of DP94 is that the 553rd position A of SEQ ID NO:1 is mutated to P, and the 561st position M is mutated to R;
  • the amino acid sequence of DP96 is that the 481st Y in SEQ ID NO:1 is mutated to F, and the 711th G is mutated to R;
  • the amino acid sequence of DP97 is that the 461st Q in SEQ ID NO:1 is mutated to F, and the 553rd A is mutated to P;
  • the amino acid sequence of DP98 is that the 424th N of SEQ ID NO:1 is mutated to Q, and the 553rd A is mutated to S;
  • the amino acid sequence of DP99 is that the 432nd position D of SEQ ID NO:1 is mutated to H, and the 553rd position A is mutated to P;
  • the amino acid sequence of DP100 is that the 553rd position A of SEQ ID NO:1 is mutated to P, and the 605th position T is mutated to V;
  • the amino acid sequence of DP101 is that the 481st Y in SEQ ID NO:1 is mutated to F, and the 561st M is mutated to W;
  • the amino acid sequence of DP102 is that the 424th N of SEQ ID NO:1 is mutated to F, and the 553rd A is mutated to P;
  • the amino acid sequence of DP103 is that the 481st Y in SEQ ID NO:1 is mutated to F, and the 605th T is mutated to I;
  • the amino acid sequence of DP104 is that the 461st Q of SEQ ID NO:1 is mutated to F, and the 481st Y is mutated to F;
  • the amino acid sequence of DP105 is that the 553rd position A of SEQ ID NO:1 is mutated to P, and the 663rd position H is mutated to R;
  • the amino acid sequence of DP106 is that the 481st Y in SEQ ID NO:1 is mutated to M, and the 553rd A is mutated to S;
  • the amino acid sequence of DP107 is that the 553rd position A of SEQ ID NO:1 is mutated to P, and the 564th position L is mutated to M;
  • the amino acid sequence of DP108 is that the 432nd position D of SEQ ID NO:1 is mutated to E, and the 553rd position A is mutated to P;
  • the amino acid sequence of DP109 is that the 461st Q of SEQ ID NO:1 is mutated to F, and the 553rd A is mutated to S;
  • the amino acid sequence of DP110 is that the 481st Y in SEQ ID NO:1 is mutated to M, and the 553rd A is mutated to G;
  • the amino acid sequence of DP111 is that the 481st Y in SEQ ID NO:1 is mutated to F, and the 564th L is mutated to M;
  • the amino acid sequence of DP112 is that the 553rd position A of SEQ ID NO:1 is mutated to P, and the 663rd position H is mutated to N;
  • the amino acid sequence of DP113 is that the 432nd position D of SEQ ID NO:1 is mutated to H, and the 553rd position A is mutated to S;
  • the amino acid sequence of DP114 is that the 553rd position A of SEQ ID NO:1 is mutated to P, and the 663rd position H is mutated to F;
  • the amino acid sequence of DP115 is that the 553rd position A of SEQ ID NO:1 is mutated to P, and the 564th position L is mutated to R;
  • the amino acid sequence of DP116 is that the 424th N of SEQ ID NO:1 is mutated to S, and the 553rd A is mutated to P;
  • the amino acid sequence of DP117 is that the 481st Y in SEQ ID NO:1 is mutated to F, and the 553rd A is mutated to F;
  • the amino acid sequence of DP118 is that the 553rd position A of SEQ ID NO:1 is mutated to E, and the 663rd position H is mutated to R;
  • the amino acid sequence of DP119 is that the 481st Y in SEQ ID NO:1 is mutated to L, and the 711th G is mutated to R;
  • the amino acid sequence of DP120 is that the 424th N of SEQ ID NO:1 is mutated to Q, and the 725th H is mutated to R;
  • the amino acid sequence of DP121 is that the 424th N of SEQ ID NO:1 is mutated to Q, and the 663rd H is mutated to R;
  • the amino acid sequence of DP122 is that the 461st Q in SEQ ID NO:1 is mutated to W, and the 561st M is mutated to R;
  • the amino acid sequence of DP123 is that the D at position 432 of SEQ ID NO: 1 is mutated to R, and the A at position 553 is mutated to S;
  • the amino acid sequence of DP124 is that the 397th W of SEQ ID NO:1 is mutated to H, and the 663rd H is mutated to Y;
  • the amino acid sequence of DP126 is that the Q at position 461 of SEQ ID NO: 1 is mutated to Y, and the H at position 663 is mutated to Q;
  • the amino acid sequence of DP127 is that the 461st Q of SEQ ID NO:1 is mutated to I, and the 481st Y is mutated to M;
  • the amino acid sequence of DP128 is that the 553rd position A of SEQ ID NO:1 is mutated to Y, and the 605th position T is mutated to V;
  • the amino acid sequence of DP130 is that the 424th N of SEQ ID NO:1 is mutated to K, and the 663rd H is mutated to L;
  • the amino acid sequence of DP131 is that the 461st Q in SEQ ID NO:1 is mutated to F, and the 481st Y is mutated to R.
  • the recombinant vectors expressing different KOD DNA polymerase point mutants are taken as templates using the KOD DNA polymerase vector pET22b-WT and the operating steps in the instruction manual of the Mut Express II Fast Mutagenesis Kit V2 (manufacturer: Novazonics; item number: C214-02) to perform at least one site-directed mutagenesis to obtain each mutant, and the protein encoding genes of different KOD DNA polymerase point mutants fused with the His tag are recombined into the vector pET22b.
  • the obtained vectors, the protein encoding genes of different point mutants fused with the His tag are expressed by inducing IPTG.
  • each KOD DNA polymerase point mutant fusion protein is a point mutation based on SEQ ID NO: 1 and 6 His tags are connected to its C-terminus.
  • His tags are connected to its C-terminus.
  • a tag can be added to the N-terminus or C-terminus of the amino acid sequence to facilitate purification.
  • the tag is generally a sequence shown in Table 3; adding or not adding a tag has no effect on the performance of the enzyme.
  • the construction steps are the same as those in Example 1.
  • the recombinant vector expressing different KOD DNA polymerase point mutants prepared in step 3 is introduced into BL21 to obtain recombinant bacteria expressing different KOD DNA polymerase mutant fusion proteins.
  • the preparation steps are the same as those in Example 1, and the recombinant bacteria expressing different KOD DNA polymerase point mutant fusion proteins prepared in step 4 are expressed and lysed to obtain different KOD DNA polymerase point mutant fusion protein crude extracts.
  • the point mutant fusion proteins in this example were expressed and cleaved according to the method in Example 1.
  • dATP dATP-AF532
  • dTTP dTTP-AF532
  • DNA template template DNA-AF647 labeled with AF532 fluorescent dye
  • the single-stranded primers S1A (Table 1) and S2A (Table 1) (synthesized by Sangon Biotech Co., Ltd.) with 5'AF647 fluorescent label were mixed at 1:1 equimolar concentration, annealed at 80°C for 10 min, and the annealed product was stored at -20°C in the dark to obtain the template DNA-AF647 labeled with AF647 fluorescent dye.
  • Enzyme activity was detected using a TECAN microplate reader and the reaction was carried out in 96 plates (Corning black, clear bottom 96 plates). The total volume of the solution was 50 ⁇ l.
  • the reaction system was: 20 ⁇ l crude extract of KOD polymerase mutant fusion protein, 0.25 ⁇ M dATP-AF532, 0.25 ⁇ M dTTP, 0.25 ⁇ M dCTP, 0.25 ⁇ M dGTP, 0.1 ⁇ M template DNA-AF647 for the experiment, the enzyme reaction buffer was 20 mM Tris-HCl, 10 mM (NH 4 ) 2 SO4, 10 mM KCl, 2 mM MgSO 4 , 0.1% Triton, pH 8.8; the reaction temperature was 40°C.
  • the enzyme reaction was carried out in kinetic detection mode, and data were recorded once every minute.
  • the detection conditions were as shown in Table 4.
  • the data table or enzyme activity curve can be directly exported, and the reaction rate of its relative fluorescence value can be approximately calculated.
  • the polymerization reaction was carried out using KOD DNA polymerase fusion protein (DP01) and KOD DNA polymerase point mutant fusion protein as examples.
  • the C-terminus of the fusion protein contained 6 his tags.
  • KOD DNA polymerase point mutants For detailed information on KOD DNA polymerase point mutants, please refer to Table 2; for preparation methods, please refer to Examples 1 and 2.
  • DP01 fusion protein is a highly active KOD DNA polymerase, and its relative polymerization activity is 2-3 times higher than that of the polymerase with only mutations 408 and 409 (see patent number PCT/CN2019/102493). Based on the highly active variants, this application further optimizes and improves the polymerization activity of KOD DNA polymerase mutants.
  • the activity determination kinetic curves of some KOD DNA polymerase mutants are shown in Figure 3.
  • mutants DP001, DP002, DP004, DP005, DP006, DP007, DP008, DP009, DP010, DP015, DP016, DP017, DP180, etc. showed comparable polymerization activities.

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Abstract

提供了DNA聚合酶突变体及其应用。与野生型KOD DNA聚合酶相比，所述DNA聚合酶突变体具有第408位、第409位点或功能等同位点的氨基酸突变，以及选自以下23个位点和功能等同位点中的至少1个位点的氨基酸突变：第141位、第143位、第147位、第383位、第384位、第389位、第485位、第584位、第589位、第397位、第424位、第432位、第445位、第523位、第553位、第561位、第564位、第461位、第481位、第605位、第663位、第711位、第725位；所述突变体的所述氨基酸突变位点以外的氨基酸序列与所述野生型KOD DNA聚合酶的对应的氨基酸序列具有至少90％的同一性；所述野生型KOD DNA聚合酶具有如SEQ ID NO:2所示的氨基酸序列。

Description

DNA聚合酶突变体及其应用 技术领域
本申请涉及生物技术领域，具体的，本申请涉及DNA聚合酶突变体及其应用。
背景技术
DNA聚合酶在PCR技术中具有广泛应用，在生命科学研究及相关领域扮演着重要角色。其主要功能之一是以单链DNA为模板，利用脱氧核苷酸(dNTP)作为底物，沿5'端到3'端的方向高效、精确地合成互补于模板的DNA序列。其结构可以大致划分为手指、拇指和手掌区域，其中活性位点通常位于手掌区域。
DNA聚合酶根据家族分类可分为七类，包括A、B、C、D、X、Y和RT。不同家族的DNA聚合酶在结构和功能上存在一些差异。其中，B家族的DNA聚合酶通常具有较高的复制和延伸性，这意味其从DNA模板上解离前可以连续聚合数量较多的核苷酸。
此外，B家族的DNA聚合酶还具备DNA修复功能，包括3'→5'外切酶活性，能够检测PCR扩增过程中的错配碱基并将其切除，进而重新插入正确的碱基并继续DNA复制。KOD是B家族DNA聚合酶的一个代表，源自嗜热古细菌Thermococcuskodakarensis。由于其耐高温性质，KOD DNA聚合酶近年来在PCR扩增、基因测序等领域得到广泛应用。然而，现有的KOD DNA聚合酶在测序中聚合能力较低，极大限制了测序的效率。
为此，KOD DNA聚合酶仍有待改进。
发明内容
本申请是发明人基于对下列问题和事实的发现而完成的：
针对现有的KOD DNA聚合酶在边合成边测序(SBS)中反应读长较短，反应的速率较慢等。发明人通过对嗜热古细菌的热稳定B家族聚合酶相关活性位点进行酶工程改造，获得新的DNA聚合酶突变体，通过提高KOD DNA聚合酶在测序中的聚合能力，进而提升边合成边测序(SBS)方法的测序速度及测序质量。
为此，在本申请的第一方面，提出了一种DNA聚合酶突变体，与野生型KOD DNA聚合酶相比，所述DNA聚合酶突变体具有第408位、第409位点或功能等同位点的氨基酸突变，以及选自以下24个位点和功能等同位点中的至少1个位点的氨基酸突变：第141位、第143位、第147位、第382位、第383位、第384位、第389位、第485位、第584位、第589位、第397位、第424位、第432位、第445位、第523位、第553位、第561位、第564位、第461位、第481位、第605位、第663位、第711位、第725位；所述突变体的所述氨基酸突变位点以外的氨基酸序列与所述野生型KOD DNA聚合酶的对应的氨基酸序列具有至少90％的同一性；所述野生型KOD DNA聚合酶具有如SEQ ID NO:2所示的氨基酸序列。根据本申请的实施例，所述DNA聚合酶突变体，可以有效提高对dNTP的掺入能力，提升测序方法(如边合成边测序)的测序速度及测序质量。
在本申请的一些示例中，与野生型KOD DNA聚合酶相比，所述DNA聚合酶突变体具有第408位、第409位点或功能等同位点的氨基酸突变，以及选自以下23个位点和功能等同位点中的至少1个位点的氨基酸突变：第141位、第143位、第147位、第383位、第384位、第389位、第485位、第584位、第589位、第397位、第424位、第432位、第445位、第523位、第553位、第561位、第564位、第461位、第481位、第605位、第663位、第711位、第725位；所述突变体的所述氨基酸突变位点以外的氨基酸序列与所述野生型KOD DNA聚合酶的对应的氨基酸序列具有至少90％的同一性；所述野生型KOD DNA聚合酶具有如SEQ ID NO:2所示的氨基酸序列。

需要说明的是，本申请所述DNA聚合酶突变体的氨基酸序列中的氨基酸位置以SEQ ID NO:2中的氨基酸位置为参照。例如，DNA聚合酶突变体具有第408位(点)突变，则表示在SEQ ID NO:2中第408位氨基酸发生突变。
需要说明的是，本发明所使用的“功能等同位点”包括在特定情况下，氨基酸或核苷酸发生突变，但DNA聚合酶功能或性质不发生改变的位点。
需要说明的是，至少90％的同一性包括91％、92％、93％、94％、95％、96％、97％、98％或99％的同一性。
需要说明的是，本申请所述的“所述突变体的所述氨基酸突变位点以外的氨基酸序列与所述野生型KOD DNA聚合酶的对应的氨基酸序列具有至少90％的同一性”中“野生型KOD DNA聚合酶的对应的氨基酸序列”是指野生型KOD DNA聚合酶中，与所述突变体上氨基酸突变位点以外的氨基酸序列对应的氨基酸序列。
根据本申请的实施例，上述DNA聚合酶突变体可以进一步包括下列技术特征至少之一：
根据本申请的实施例，所述突变体的所述氨基酸突变位点以外的氨基酸序列与所述野生型KOD DNA聚合酶的对应的氨基酸序列相同。
根据本申请的实施例，与所述野生型KOD DNA聚合酶相比，所述DNA聚合酶突变体具有第408位、第409位点或功能等同位点的氨基酸突变，以及选自以下9个位点和功能等同位点中的至少1个位点的氨基酸突变：第141位、第143位、第147位、第383位、第384位、第389位、第485位、第584位、第589位。根据本申请的一些示例，不同的突变位点产生的测序酶相对活性不同，可以为实际生产需要提供更多的选择。
根据本申请的实施例，与所述野生型KOD DNA聚合酶相比，所述DNA聚合酶突变体具有第408位、第409位点或功能等同位点的氨基酸突变，以及选自以下2个位点和功能等同位点中的至少1个位点的氨基酸突变：第147位、第584位。根据本申请的实施例，发明人在经过大量筛选后，首次发现对第147位、第584位氨基酸进行突变可以增加DNA聚合酶的活性。
根据本申请的实施例，突变位点的突变类型为：
(1)第141位的D突变为A，(2)第143位的E突变为A，(3)第147位的H突变为E，(4)第383位的S突变为T，(5)第384位的Y突变为F，(6)第389位的V突变为I，(7)第485位的A突变为E，(8)第584位的K突变为E，(9)第589位的V突变为H，(10)第408位的L突变为I，(11)第409位的Y突变为A。
根据本申请的一些示例，发明人发现具有上述突变类型之一(或任意组合)的DNA聚合酶具有较高的酶活性。
根据本申请的实施例，与所述野生型KOD DNA聚合酶相比，所述DNA聚合酶突变体具有：第408位、第409位、第141位、第143位、第147位、第383位、第384位、第389位、第485位、第584位、第589 位点或功能等同位点的氨基酸突变；以及选自以下15个位点和功能等同位点中的至少1个位点的氨基酸突变：第382位、第397位、第424位、第432位、第445位、第523位、第553位、第561位、第564位、第461位、第481位、第605位、第663位、第711位、第725位。
在本申请的一些示例中，与所述野生型KOD DNA聚合酶相比，所述DNA聚合酶突变体具有：第408位、第409位、第141位、第143位、第147位、第383位、第384位、第389位、第485位、第584位、第589位点或功能等同位点的氨基酸突变；以及选自以下14个位点和功能等同位点中的至少1个位点的氨基酸突变：第397位、第424位、第432位、第445位、第523位、第553位、第561位、第564位、第461位、第481位、第605位、第663位、第711位、第725位。
在本申请的一些示例中，发明人发现具有第408位、第409位、第141位、第143位、第147位、第383位、第384位、第389位、第485位、第584位、第589位点或功能等同位点的氨基酸突变的聚合酶突变体(SEQ ID NO:1)具有较高的酶活性。发明人发现，在SEQ ID NO:1所示的氨基酸序列基础上再进行了选自第397位、第424位、第432位、第445位、第523位、第553位、第561位、第564位、第461位、第481位、第605位、第663位、第711位和第725位中的一个或多个位点的突变可以获得不同酶活性的突变体。
根据本申请的实施例，与所述野生型KOD DNA聚合酶相比，所述DNA聚合酶突变体具有：第408位、第409位、第141位、第143位、第147位、第383位、第384位、第389位、第485位、第584位、第589位点或功能等同位点的氨基酸突变；以及选自以下4个位点和功能等同位点中的至少1个位点的氨基酸突变：第397位、第424位、第481位、第553位。
在本申请的一些示例中，在具有第408位、第409位、第141位、第143位、第147位、第383位、第384位、第389位、第485位、第584位、第589位点或功能等同位点的氨基酸突变的基础上再进行选自第397位、第424位、第481位和第553位中的一个或多个位点的突变能够获得酶活性更高的突变体。
根据本申请的实施例，突变位点的突变类型为：(1)第141位的D突变为A，(2)第143位的E突变为A，(3)第147位的H突变为E，(4)第383位的S突变为T，(5)第384位的Y突变为F，(6)第389位的V突变为I，(7)第485位的A突变为E，(8)第584位的K突变为E，(9)第589位的V突变为H，(10)第408位的L突变为I，(11)第409位的Y突变为A，(12)第397位的W突变为Y或H，(13)第424位的N突变为R、Q、H、I、L、M、F、W、K、Y、V、C或S，(14)第432位的D突变为R、E、H或M，(15)第445位的F突变为R，(16)第523位的M突变为R或L，(17)第553位的A突变为T、R、N、E、G、H、L、M、F、Y、P、S、D、V、I或C，(18)第561位的M突变为R或W，(19)第564位的L突变为R或M，(20)第461位的Q突变为I、L、M、F、W或Y，(21)第481位的Y突变为R、H、L、M或F，(22)第605位的T突变为I或V，(23)第663位的H突变为R、Q、L、M、F、T、Y、N、V、G、A、P、S、C、E或K，(24)第711位的G突变为R、M或S，(25)第725位的H突变为R，(26)第382位的Q突变为R、H、L或Y。
在本申请的一些示例中，突变位点的突变类型为：(1)第141位的D突变为A，(2)第143位的E突变为A，(3)第147位的H突变为E，(4)第383位的S突变为T，(5)第384位的Y突变为F，(6)第389位的V突变为I，(7)第485位的A突变为E，(8)第584位的K突变为E，(9)第589位的V突变为H，(10)第408位的L突变为I，(11)第409位的Y突变为A，(12)第397位的W突变为Y或H，(13)第424位的N突变为R、Q、H、I、L、M、F、W、K、Y、V、C或S(14)第432位的D突变为R、E、H或M，(15)第445位的F突变为R，(16)第523位的M突变为R或L，(17)第553位的A突变为T、R、N、E、G、H、L、M、F、Y、P、S、D、V、I或C，(18)第561位的M突变为R或W，(19)第564位的L突变为R或M，(20)第461位的Q突变为I、L、M、F、W或Y，(21)第481 位的Y突变为R、H、L、M或F，(22)第605位的T突变为I或V，(23)第663位的H突变为R、Q、L、M、F、T、Y、N、V、G、A、P、S、C、E或K，(24)第711位的G突变为R、M或S，(25)第725位的H突变为R。
在本申请的一些示例中，发明人发现具有上述突变类型的DNA聚合酶突变体仍有不同程度的酶活性。
根据本申请的实施例，与所述野生型KOD DNA聚合酶相比，所述DNA聚合酶突变体具有：第408位、第409位、第141位、第143位、第147位、第383位、第384位、第389位、第485位、第584位、第589位点或功能等同位点的氨基酸突变；以及选自以下15个位点和功能等同位点中的一个位点的氨基酸突变：第382位、第397位、第424位、第432位、第445位、第523位、第553位、第561位、第564位、第461位、第481位、第605位、第663位、第711位、第725位；条件是，第382位的Q不能突变为R、H、L和Y；或第424位的N不能突变为R和L；或第432位的D不能突变为M；或第663位的H不能突变为T、V、P和E。
需要说明的是，所称的第382位的Q不能突变为R、H、L和Y是指第382位的Q不能突变为R，或第382位的Q不能突变为H，或第382位的Q不能突变为L，或第382位的Q不能突变为Y。同理，第424位的N不能突变为R和L是指第424位的N不能突变为R，或第424位的N不能突变为L。在本申请中，该表述方法同样适用于任一实施例。在以下实施例或实施方式中不再赘述。
根据本申请的实施例，与所述野生型KOD DNA聚合酶相比，所述DNA聚合酶突变体具有：第408位、第409位、第141位、第143位、第147位、第383位、第384位、第389位、第485位、第584位、第589位点或功能等同位点的氨基酸突变；以及选自以下14个位点和功能等同位点中的一个位点的氨基酸突变：第397位、第424位、第432位、第445位、第523位、第553位、第561位、第564位、第461位、第481位、第605位、第663位、第711位、第725位；条件是，第424位的N不能突变为R和L；或第432位的D不能突变为M；或第663位的H不能突变为T、V、P和E。
根据本申请的实施例，与所述野生型KOD DNA聚合酶相比，所述DNA聚合酶突变体具有：第408位、第409位、第141位、第143位、第147位、第383位、第384位、第389位、第485位、第584位、第589位点或功能等同位点的氨基酸突变；以及选自以下14个位点和功能等同位点中的两个位点的氨基酸突变：第397位、第424位、第432位、第445位、第523位、第553位、第561位、第564位、第461位、第481位、第605位、第663位、第711位、第725位；条件是，当第663位的H突变为R时，第553位的A不能突变为P或E；或当第663位的H突变为Q时，第461位的Q不能突变为Y。
在本申请的一些示例中，所述突变体的第663位的H突变为R，且第553位的A突变为P或E时；或者，第663位的H突变为Q，且第461位的Q突变为Y时，酶活性较低。
根据本申请的实施例，与所述野生型KOD DNA聚合酶相比，所述DNA聚合酶突变体具有(1)-(11)的突变，以及选自(12)-(25)中的任意一种突变。
在本申请的一些示例中，具有(1)-(11)的突变的DNA聚合酶突变体的氨基酸序列如SEQ ID NO:1所示。
在本申请的一些示例中，以SEQ ID NO:1所示氨基酸序列的聚合酶突变体作为聚合活性检测的对照组。
在本申请的一些示例中，与SEQ ID NO:1所示氨基酸序列具有至少90％的同一性的DNA聚合酶突变体有较高的酶活性。在一些优选示例中，具有至少91％的同一性或至少92％的同一性或至少93％的同一性或至少94％的同一性或至少95％的同一性或至少96％的同一性或至少97％的同一性或至少98％的同一性或至少99％的同一性，在一些最优选示例中，具有至少100％同一性。
根据本申请的实施例，与所述野生型KOD DNA聚合酶相比，所述DNA聚合酶突变体具有(1)-(11) 的突变；以及选自以下突变中的任意一种突变：第397位的W突变为H，第424位的N突变为Q，第553位的A突变为R，第553位的A突变为N，第553位的A突变为E，第553位的A突变为G，第553位的A突变为H，第553位的A突变为P，第553位的A突变为S，第461位的Q突变为W，第461位的Q突变为Y，第481位的Y突变为M，第481位的Y突变为F，第711位的G突变为R，第711位的G突变为S。
在本申请的一些示例中，发明人经过实验验证发现，在SEQ ID NO:1所示氨基酸序列的基础上再进行氨基酸单位点突变可以进一步增加酶聚合活性。
根据本申请的实施例，与所述野生型KOD DNA聚合酶相比，所述DNA聚合酶突变体具有(1)-(11)的突变；以及选自以下突变组合中的任意一种突变组合：
第481位Y突变为F，且第553位A突变为P；第481位Y突变为F，且第553位A突变为S；第397位W突变为H，且第553位A突变为P；第461位Q突变为Y，且第553位A突变为P；第481位Y突变为F，且第553位A突变为G；第424位N突变为Q，且第553位A突变为P；第397位W突变为H，且第481位Y突变为F；第553位A突变为P，且第711位G突变为R；第397位W突变为H，且第553位A突变为S；第481位Y突变为M，且第553位A突变为P；第461位Q突变为W，且第553位A突变为P；第553位A突变为P，且第711位G突变为S；第461位Q突变为Y，且第553位A突变为S；第553位A突变为P，且第561位M突变为W；第553位A突变为P，且第561位M突变为R；第481位Y突变为F，且第711位G突变为R；第461位Q突变为F，且第553位A突变为P；第424位N突变为Q，且第553位A突变为S；第432位D突变为H，且第553位A突变为P；第553位A突变为P，且第605位T突变为V；第481位Y突变为F，且第561位M突变为W；第424位N突变为F，且第553位A突变为P；第481位Y突变为F，且第605位T突变为I；第461位Q突变为F，且第481位Y突变为F；第553位A突变为P，且第663位H突变为R；第481位Y突变为M，且第553位A突变为S；第553位A突变为P，且第564位L突变为M；第432位D突变为E，且第553位A突变为P；第461位Q突变为F，且第553位A突变为S；第481位Y突变为M，且第553位A突变为G；第481位Y突变为F，且第564位L突变为M；第553位A突变为P，且第663位H突变为N；第432位D突变为H，且第553位A突变为S；第553位A突变为P，且第663位H突变为F；第553位A突变为P，且第564位L突变为R；第424位N突变为S，且第553位A突变为P；第481位Y突变为F，且第553位A突变为F；第553位A突变为E，且第663位H突变为R；第481位Y突变为L，且第711位G突变为R；第424位N突变为Q，且第725位H突变为R；第424位N突变为Q，且第663位H突变为R；第461位Q突变为W，且第561位M突变为R；第432位D突变为R，且第553位A突变为S；第397位W突变为H，且第663位H突变为Y；第461位Q突变为Y，且第663位H突变为Q；第461位Q突变为I，且第481位Y突变为M；第553位A突变为Y，且第605位T突变为V；第424位N突变为K，且第663位H突变为L；第461位Q突变为F，且第481位Y突变为R。
根据本申请的示例，部分突变体在测序应用中可能具有碱基偏向性，可基于实际实验需要进行适应性选择。
根据本申请的实施例，与所述野生型KOD DNA聚合酶相比，所述DNA聚合酶突变体具有(1)-(11)的突变，以及选自以下突变组合中的任意一种突变组合：第481位Y突变为F，且第553位A突变为P；第481位Y突变为F，且第553位A突变为S；第397位W突变为H，且第553位A突变为P；第461位Q突变为Y，且第553位A突变为P；第424位N突变为Q，且第553位A突变为P；第397位W突变为H，且第481位Y突变为F；第553位A突变为P，且第711位G突变为R；第397位W突变为H，且第553 位A突变为S；第481位Y突变为F，且第711位G突变为R；第461位Q突变为F，且第481位Y突变为F；第553位A突变为P，且第564位L突变为M；第481位Y突变为F，且第564位L突变为M；第424位N突变为S，且第553位A突变为P；第481位Y突变为L，且第711位G突变为R；第424位N突变为Q，且第725位H突变为R；第397位W突变为H，且第663位H突变为Y；第461位Q突变为I，且第481位Y突变为M。
在本申请的一些示例中，发明人经过实验验证发现，在SEQ ID NO:1所示氨基酸序列的基础上再进行氨基酸双位点突变可以进一步增加酶聚合活性。
在本申请的第二方面，本申请提出了一种核酸分子，所述核酸分子编码本申请第一方面所述的DNA聚合酶突变体。所述核酸分子编码的DNA聚合酶突变体可在体内或体外进行大量获得。
需要说明的是，对于本发明说明书和权利要求书中所提及的核酸，本领域技术人员应当理解，实际包括互补双链的任意一条，或者两条。为了方便，在本说明书和权利要求书中，虽然多数情况下只给出了一条链，但实际上也公开了与之互补的另一条链。另外，本申请中的核酸序列包括DNA形式或RNA形式，公开其中一种，意味着另一种也被公开。
在本申请的第三方面，本申请提出了一种表达载体，所述表达载体包括或携带本申请第二方面所述的核酸分子。在本申请中，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即可。
需要说明的是，所述表达载体也可进一步包括启动子，所述启动子与所述核酸分子可操作的连接。
根据本申请的实施例，所述表达载体为非致病性病毒载体，所述非致病性病毒载体包括腺病毒载体或逆转录病毒载体。
根据本申请的实施例，所述表达载体为非病毒载体，所述非病毒载体包括但不限于质粒载体。
在本申请的第四方面，本申请提出了一种重组细胞，所述重组细胞携带本申请第二方面所述的核酸分子以及本申请第三方面所述的表达载体。所述重组细胞用于表达或分泌本申请第一方面所述的DNA聚合酶突变体。
根据本申请的实施例，所述重组细胞选自大肠杆菌、酵母和哺乳动物细胞。
在本申请的一些示例中，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DNA聚合酶突变体。
在本申请的第五方面，本申请提出了一种重组菌株，所述重组菌株表达本申请第一方面所述的DNA聚合酶突变体。通过培养所述重组菌株可以快速大量获得DNA聚合酶突变体。
在本申请的第六方面，本申请提出了一种获得DNA聚合酶突变体的方法，所述方法包括将本申请第四 方面所述的重组细胞或第五方面所述的重组菌株在适于蛋白表达的条件下进行培养处理，以便获得所述DNA聚合酶突变体。
在本申请的第七方面，本申请提出了一种复合物，所述复合物包括本申请第一方面所述DNA聚合酶突变体和小分子化合物或大分子，所述DNA聚合酶突变体和小分子化合物或大分子通过化学键偶联。
根据本申请的实施例，所述小分子化合物或大分子包括荧光标记、荧光素或抗体等。
需要说明的是，本申请提出的DNA聚合酶突变体、复合物的应用场景包括但不限于核酸合成和核酸测序等，还可以用于筛选针对病毒或者细胞分裂的药物，在法医学和犯罪学方面也具有较大的发展前景。
在本申请的第八方面，本申请提出了一种核酸合成方法，所述方法包括：将核酸模板、扩增引物、dNTP与本申请第一方面所述DNA聚合酶突变体的混合产物在适于核酸扩增的条件下进行扩增处理，以便获得所述核酸。利用前述核酸合成方法可以对核酸模板进行高效快速扩增。
需要说明的是，本申请所述DNA聚合酶突变体对所有的dNTP(包括有荧光标记或无荧光标记的dNTP)均有聚合活性。
在本申请的第九方面，本申请提出了一种核酸测序的方法，所述方法包括：将待测核酸模板与本申请第一方面所述的DNA聚合酶突变体和带有修饰的dNTP的混合产物在适于核酸扩增的条件下进行扩增处理和荧光信号检测处理；以及基于检测获得的荧光信号，确定所述待测核酸的核酸序列。
示例性的，所述核酸测序方法包括将待测核酸模板与所述DNA聚合酶突变体、带有荧光标记的3’O-可逆终止子非天然dNTP进行混合处理，DNA聚合酶突变体负责将所述带有荧光标记的3’O-可逆终止子非天然dNTP与核酸模板进行匹配，最后通过检测多种荧光标记信号，基于获得的荧光标记信号获得的核酸序列进而得到待测核酸的核酸序列，以上聚合反应和荧光标记反应可以根据测序模板长度进行多个循环。
在本申请的第十方面，本申请提出了一种核酸测序试剂盒，所述核酸测序试剂盒包括第一方面所述的DNA聚合酶突变体或第七方面所述的复合物。本申请所述的试剂盒用于高效、准确、快速进行核酸测序。
在本申请的第十一方面，本申请提出了一种第十方面所述核酸测序试剂盒在测序中的用途。根据本申请的实施例，所述试剂盒可用于测序，包括但不限于边合成边测序(SBS)。
在本申请的第十二方面，本申请提出了一种第一方面所述DNA聚合酶突变体、第二方面所述核酸分子、第三方面所述表达载体、第四方面所述重组细胞、第五方面所述重组菌株或第七方面所述复合物在制备用于催化DNA扩增或核酸测序相关产品中的用途。根据本申请的实施例，所述DNA聚合酶突变体、核酸分子、表达载体、重组细胞、重组菌株或复合物可以单独或者组合制备用于催化DNA扩增或核酸测序相关产品。
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为本申请一个实施例所述重组型KOD DNA聚合酶表达质粒pET22b-KOD质粒图谱示意图；
图2为本申请一个实施例所述野生型以及部分突变体KOD DNA聚合酶表达和菌裂解液结果示意图；
图3为本申请一个实施例所述FRET方法检测野生型以及部分突变体KOD DNA聚合酶活性结果示意图。
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。
在本申请中，除非另有说明，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。
在本申请中，除非另有说明，术语“氨基酸”由单字母或三字母代码表示，具有如下含义：A：Ala(丙氨酸)；R：Arg(精氨酸)；N：Asn(天冬酰胺)；D：Asp(天冬氨酸)；C：Cys(半胱氨酸)；Q：Gln(谷氨酰胺)；E：Glu(谷氨酸)；G：Gly(甘氨酸)；H：His(组氨酸)；I：Ile(异亮氨酸)；L：Leu(亮氨酸)；K：Lys(赖氨酸)；M：Met(甲硫氨酸)；F：Phe(苯丙氨酸)；P：Pro(脯氨酸)；S：Ser(丝氨酸)；T：Thr(苏氨酸)；W：Trp(色氨酸)；Y：Tyr(酪氨酸)；V：Val(缬氨酸)。
本申请中，除非另有说明，术语“同一性”具有本领域常规的含义，是指两个核酸或氨基酸序列之间的“同源性”，其百分比表示在最佳比对(best alignment)后获得的待比较的两个序列之间的相同核苷酸或氨基酸残基的统计学意义的百分比，两个序列之间的差异随机地分布在其整个长度上。本申请中，所述突变体根据它们在特定残基上的突变来描述，其位置参照野生型KOD聚合酶SEQ ID NO:2所示氨基酸序列中氨基酸的位置进行定位。
其中，KOD DNA聚合酶突变体(SEQ ID NO:1)是由野生型KOD DNA聚合酶(SEQ ID NO:2)突变获得的，以SEQ ID NO:2序列位置为参考，突变位点包括：第141位D突变为A，第143位E突变为A，第147位H突变为E，第383位S突变为T，第384位Y突变为F，第389位V突变为I，第408位L突变为I，第409位Y突变为A，第485位A突变为E，第584位K突变为E，第589位V突变为H。
在本申请中，除非另有说明，术语“相对聚合活性”是认定参照组聚合活力为100％时，计算的各实验组相对于参照组的聚合活力。如根据本申请的实施例，本申请所称的参照组DNA聚合酶为DP01，其氨基酸序列如SEQ ID NO:1所示。
在本申请中，除非另有说明，术语“转化”是指将DNA导入宿主细胞以便DNA可作为染色体外元件或通过染色体整合被复制。即，转化指通过将外源DNA导入细胞引起的基因的合成改变。
在本申请中，除非另有说明，氨基酸序列展示方式均为5’端→3’端。
在本申请中，除非另有说明，非天然dNTP或带有修饰的dNTP表示相同含义，包括但不限于带有标记(例如荧光标记)的dNTP和/或3’端含有O-可逆终止子的dNTP等。
在本申请中，除非另有说明，本申请实施例所用聚合酶突变体均为C端连接6个His标签的聚合酶突变体融合蛋白，如本领域人员所知，均可以替换为不加标签的聚合酶突变体。
已有研究报道，嗜热古细菌的热稳定B家族聚合酶在高通量测序中掺入天然核苷酸或其类似物表现出色。这些热稳定B家族聚合酶包括KOD(Thermococcuskodakaraensis)、9°N(Thermococcussp.9°N)、TGO(Thermococcusgorgonarius)、TOK(Desulfurococcussp.Tok)、VentDNA聚合酶(Thermococcuslitoralis)、JDF-3和pfuDNA聚合酶(Pyrococcusfuriosis)等。
为了能够在高通量测序过程(如边合成边测序(SequencingbySynthesis，SBS))中提升现有的DNA聚合酶的聚合速度，并增加反应读长。发明人经过深入研究发现，可以通过对KOD DNA聚合酶进行酶工程改造来增强其对带有修饰dNTP的掺入效率。
发明人通过对野生型聚合酶功能结构域位点进行了保护，确保其仍然能够完成原有的基本功能。通过对KOD DNA聚合酶的手掌区、手指区以及拇指区进行了动力学模拟与统计推断，获得了可用于实验筛选的突变位点。并通过大量实验验证，最终获得适合附着于芯片表面的DNB(DNA纳米球)上的DNA聚合酶。
下面参考示例，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。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1：KOD DNA聚合酶突变体的制备
在本实施例中，KOD DNA聚合酶(DP01)的氨基酸序列如SEQ ID NO:1所示。
1、DP01表达载体的构建
重组表达载体pET22b-WT为将融合His标签的KOD DNA聚合酶编码基因按SeamlessCloningKit试剂盒(厂家：诺唯赞；货号：C112-01)说明书操作步骤重组到载体pET22b上，得到的载体为融合His标签的KOD DNA聚合酶编码基因(图1)，通过IPTG诱导表达。
融合His标签的KOD DNA聚合酶编码基因的核苷酸序列为在SEQ ID NO:3的3’端连接6个His标签密码子得到的序列；

KOD DNA聚合酶融合蛋白的氨基酸序列为将SEQ ID NO:1所示的氨基酸的C端连接6个His标签得到。
2、重组菌的构建
重组表达载体pET22b-WT导入大肠杆菌BL21感受态细胞(北京索莱宝科技有限公司)中，涂抹抗性平板(含氨苄50μg/ml)筛选阳性菌落。挑选3-5个阳性单克隆菌落，利用引物SQF(表1)和引物SQR(表1)对阳性菌落进行菌液PCR鉴定。得到与预计的理论值基本一致的2500bp大小的片段为阳性克隆，将该阳性克隆命名为BL21/pET22b-WT。
表1：引物序列信息
3、DP01的表达及粗提物制备
挑取BL21/pET22b-WT单菌落，于150μlLB液体培养基(含氨苄50μg/ml)中，37℃，200rpm，过夜培养。次日按1:100稀释量，转接于150μl LB液体培养基(含氨苄50μg/ml)，37℃，220rpm振荡培养至OD600为0.6-0.8，加入终浓度为0.5mM的IPTG，37℃过夜诱导培养，收集诱导后BL21/pET22b-WT菌液。同时设不加IPTG作为空白对照，收集未诱导的BL21/pET22b-WT菌液。
在上述诱导后BL21/pET22b-WT菌液中加入1μM PMSF(蛋白酶抑制剂)和50μg溶菌酶，混匀，于37℃孵育10min进行菌裂解反应，即为融合蛋白粗提物。此蛋白粗提物可用于后续蛋白活性测定。
将裂解后的KOD DNA聚合酶融合蛋白粗提物进行SDS-PAGE(浓缩胶为5％、分离胶为12％)，结果如图2所示，泳道1为蛋白Marker(Page Ruler Prestained Protein Ladder，26616，Thermo Fisher Scientific)，泳道12为5μl KOD DNA聚合酶融合蛋白(DP01)+5μl 2X上样缓冲液，泳道2-11为5μl KOD DNA聚合酶突变体融合蛋白(DP001、DP002、DP004、DP005、DP007、DP008、DP009、DP024、DP011、DP012)+5μl 2X上样缓冲液；可以看出，泳道2-12中蛋白大小约91.5kDa，与文献报道分子量一致(Huber C,Marx A.Variants of sequence family B Thermococcus kodakaraensis DNA polymerase with increased mismatch extension selectivity.PLoS One.2017 Aug 23；12(8):e0183623；Chim N,Shi C,Sau SP,Nikoomanzar A,Chaput JC.Structural basis for TNA synthesis by an engineered TNA polymerase.Nat Commun.2017Nov 27；8(1):1810.)。
未诱导BL21/pET22b-WT菌液未得到约91.5kDa大小的目的蛋白。将空载体pET22b导入大肠杆菌BL21中，得到BL21/pET22b。采用上述方法进行表达和裂解，也未得到约91.5kDa大小的目的蛋白。
实施例2：KOD DNA聚合酶突变体融合蛋白的制备
在本实施例中，KOD DNA聚合酶突变体融合蛋白为将DP01所示的氨基酸序列(SEQ ID NO:1)的第397、424、432、445、523、553、561、564、461、481、605、663、711、725这14位中至少1个、至少2个、至少3个、至少4个、至少5个、至少6个、至少7个、至少8个、至少9个、至少10个、至少11个、至少12个、至少13个或全部14个氨基酸进行氨基酸突变后获得的蛋白；若只有一个氨基酸突变所得到的是KOD DNA聚合酶单点突变体，若有两个氨基酸突变所得到的是KOD DNA聚合酶两点组合突变，以此类推。
KOD DNA聚合酶突变体编码基因为将KOD DNA聚合酶突变体DP01编码基因的核苷酸序列(SEQ ID NO:3)按照其对应的氨基酸序列中(SEQ ID NO:1)第397、424、432、445、523、553、561、564、461、481、605、663、711、725这14位中至少1个、至少2个、至少3个、至少4个、至少5个、至少6个、至少7个、至少8个、至少9个、至少10个、至少11个、至少12个、至少13个或全部14个氨基酸密码子进行氨基酸密码子突变，得到的核酸。
KOD DNA聚合酶突变体蛋白中，氨基酸位置突变的方式如下：
第397位的W突变为Y或H；
第424位的N突变为R、Q、H、I、L、M、F、W、K、Y、V、C或S；
第432位的D突变为R、E、H或M；
第445位的F突变为R；
第523位的M突变为R或L；
第553位的A突变为T、R、N、E、G、H、L、M、F、Y、P、S、D、V、I或C；
第561位的M突变为R或W；
第564位的L突变为R或M；
第461位的Q突变为I、L、M、F、W或Y；
第481位的Y突变为R、H、L、M或F；
第605位的T突变为I或V；
第663位的H突变为R、Q、L、M、F、T、Y、N、V、G、A、P、S、C、E或K；
第711位的G突变为R、M或S；
第725位的H突变为R。
1、单点突变KOD DNA聚合酶突变体蛋白
将KOD DNA聚合酶SEQ ID NO:1氨基酸序列中选自397、424、432、445、523、553、561、564、461、481、605、663、711、725这14位中的一位的氨基酸进行突变，其他氨基酸序列不变，得到具有DNA聚合酶活性的蛋白(表2)。
表2：KOD DNA聚合酶单点突变体的突变及突变信息


2、双点突变KOD DNA聚合酶突变体蛋白
将所述KOD DNA聚合酶SEQ ID NO:1氨基酸序列中选自397、424、432、445、523、553、561、564、461、481、605、663、711、725这14位中的2位氨基酸进行突变，其他氨基酸序列不变，得到具有DNA聚合酶活性的蛋白：
DP80其氨基酸序列为将SEQ ID NO:1的第481位Y突变为F，且将第553位A突变为P；
DP81其氨基酸序列为将SEQ ID NO:1的第481位Y突变为F，且将第553位A突变为S；
DP82其氨基酸序列为将SEQ ID NO:1的第397位W突变为H，且将第553位A突变为P；
DP83其氨基酸序列为将SEQ ID NO:1的第461位Q突变为Y，且将第553位A突变为P；
DP84其氨基酸序列为将SEQ ID NO:1的第481位Y突变为F，且将第553位A突变为G；
DP85其氨基酸序列为将SEQ ID NO:1的第424位N突变为Q，且将第553位A突变为P；
DP86其氨基酸序列为将SEQ ID NO:1的第397位W突变为H，且将第481位Y突变为F；
DP87其氨基酸序列为将SEQ ID NO:1的第553位A突变为P，且将第711位G突变为R；
DP88其氨基酸序列为将SEQ ID NO:1的第397位W突变为H，且将第553位A突变为S；
DP89其氨基酸序列为将SEQ ID NO:1的第481位Y突变为M，且将第553位A突变为P；
DP90其氨基酸序列为将SEQ ID NO:1的第461位Q突变为W，且将第553位A突变为P；
DP91其氨基酸序列为将SEQ ID NO:1的第553位A突变为P，且将第711位G突变为S；
DP92其氨基酸序列为将SEQ ID NO:1的第461位Q突变为Y，且将第553位A突变为S；
DP93其氨基酸序列为将SEQ ID NO:1的第553位A突变为P，且将第561位M突变为W；
DP94其氨基酸序列为将SEQ ID NO:1的第553位A突变为P，且将第561位M突变为R；
DP96其氨基酸序列为将SEQ ID NO:1的第481位Y突变为F，且将第711位G突变为R；
DP97其氨基酸序列为将SEQ ID NO:1的第461位Q突变为F，且将第553位A突变为P；
DP98其氨基酸序列为将SEQ ID NO:1的第424位N突变为Q，且将第553位A突变为S；
DP99其氨基酸序列为将SEQ ID NO:1的第432位D突变为H，且将第553位A突变为P；
DP100其氨基酸序列为将SEQ ID NO:1的第553位A突变为P，且将第605位T突变为V；
DP101其氨基酸序列为将SEQ ID NO:1的第481位Y突变为F，且将第561位M突变为W；
DP102其氨基酸序列为将SEQ ID NO:1的第424位N突变为F，且将第553位A突变为P；
DP103其氨基酸序列为将SEQ ID NO:1的第481位Y突变为F，且将第605位T突变为I；
DP104其氨基酸序列为将SEQ ID NO:1的第461位Q突变为F，且将第481位Y突变为F；
DP105其氨基酸序列为将SEQ ID NO:1的第553位A突变为P，且将第663位H突变为R；
DP106其氨基酸序列为将SEQ ID NO:1的第481位Y突变为M，且将第553位A突变为S；
DP107其氨基酸序列为将SEQ ID NO:1的第553位A突变为P，且将第564位L突变为M；
DP108其氨基酸序列为将SEQ ID NO:1的第432位D突变为E，且将第553位A突变为P；
DP109其氨基酸序列为将SEQ ID NO:1的第461位Q突变为F，且将第553位A突变为S；
DP110其氨基酸序列为将SEQ ID NO:1的第481位Y突变为M，且将第553位A突变为G；
DP111其氨基酸序列为将SEQ ID NO:1的第481位Y突变为F，且将第564位L突变为M；
DP112其氨基酸序列为将SEQ ID NO:1的第553位A突变为P，且将第663位H突变为N；
DP113其氨基酸序列为将SEQ ID NO:1的第432位D突变为H，且将第553位A突变为S；
DP114其氨基酸序列为将SEQ ID NO:1的第553位A突变为P，且将第663位H突变为F；
DP115其氨基酸序列为将SEQ ID NO:1的第553位A突变为P，且将第564位L突变为R；
DP116其氨基酸序列为将SEQ ID NO:1的第424位N突变为S，且将第553位A突变为P；
DP117其氨基酸序列为将SEQ ID NO:1的第481位Y突变为F，且将第553位A突变为F；
DP118其氨基酸序列为将SEQ ID NO:1的第553位A突变为E，且将第663位H突变为R；
DP119其氨基酸序列为将SEQ ID NO:1的第481位Y突变为L，且将第711位G突变为R；
DP120其氨基酸序列为将SEQ ID NO:1的第424位N突变为Q，且将第725位H突变为R；
DP121其氨基酸序列为将SEQ ID NO:1的第424位N突变为Q，且将第663位H突变为R；
DP122其氨基酸序列为将SEQ ID NO:1的第461位Q突变为W，且将第561位M突变为R；
DP123其氨基酸序列为将SEQ ID NO:1的第432位D突变为R，且将第553位A突变为S；
DP124其氨基酸序列为将SEQ ID NO:1的第397位W突变为H，且将第663位H突变为Y；
DP126其氨基酸序列为将SEQ ID NO:1的第461位Q突变为Y，且将第663位H突变为Q；
DP127其氨基酸序列为将SEQ ID NO:1的第461位Q突变为I，且将第481位Y突变为M；
DP128其氨基酸序列为将SEQ ID NO:1的第553位A突变为Y，且将第605位T突变为V；
DP130其氨基酸序列为将SEQ ID NO:1的第424位N突变为K，且将第663位H突变为L；
DP131其氨基酸序列为将SEQ ID NO:1的第461位Q突变为F，且将第481位Y突变为R。
3、表达KOD DNA聚合酶突变体的重组载体的制备
表达不同KOD DNA聚合酶点突变体的重组载体，以KOD DNA聚合酶载体pET22b-WT为模板，采用Mut Express II Fast Mutagenesis Kit V2试剂盒(厂家：诺唯赞；货号：C214-02)说明书操作步骤，进行至少一次定点突变获得各突变体，将融合His标签的不同KOD DNA聚合酶点突变体蛋白编码基因重组到载体pET22b上，得到的载体，融合His标签的不同点突变体蛋白编码基因，通过IPTG诱导表达。
每个KOD DNA聚合酶点突变体融合蛋白的氨基酸序列为在SEQ ID NO:1基础上进行点突变并在其C端连接6个His标签。如本领域人员所知，对于不同的酶，可在氨基酸序列的N端或C端加标签，以便于纯化，标签一般为表3所示的序列；加或不加标签对于酶的性能没有影响。
表3：标签序列
4、重组菌的构建
构建步骤同实施例1，将步骤3制备的表达不同KOD DNA聚合酶点突变体重组载体导入BL21中，得到表达不同KOD DNA聚合酶突变体融合蛋白的重组菌。
5、突变体表达及粗提物制备
制备步骤同实施例1，将步骤4制备的表达不同KOD DNA聚合酶点突变体融合蛋白的重组菌进行表达和裂解，得到不同的KOD DNA聚合酶点突变体融合蛋白粗提物。
采用SDS-PAGE凝胶电泳检测不同的KOD DNA聚合酶点突变体融合蛋白粗提物，结果如图2所示，表明获得均一性较好的蛋白。
实施例3：重组型KOD DNA聚合酶突变体融合蛋白的性能检测
本实施例中的点突变体融合蛋白均按照实施例1中的方法进行表达裂解。
1、KOD DNA聚合酶点突变体融合蛋白的单碱基掺入动力学
本实施例利用AF532荧光染料标记的dATP(dATP-AF532)、dTTP(dTTP-AF532)和AF647荧光染料标记的DNA模板(模板DNA-AF647)，模拟SBS测序过程中掺入修饰核苷酸，利用酶标仪检测重组型KOD DNA聚合酶突变体相对反应速率，检测各突变体相对KOD聚合酶DP01活性比，具体实验方法如下：
将带有5’AF647荧光标记的单链引物S1A(表1)和S2A(表1)(由生工生物工程公司合成)按1:1等摩尔浓度进行混合，按80℃10min退火，退火产物避光保存至-20℃，即得到AF647荧光染料标记的模板DNA-AF647。
使用TECAN酶标仪进行酶活检测，反应在96板(Corning black，clear bottom 96plates)中进行，反应 液总体积50μl。
反应体系为：20μl KOD聚合酶突变体融合蛋白粗提物，0.25μM dATP-AF532，0.25μM dTTP，0.25μM dCTP，0.25μM dGTP，0.1μM模板DNA-AF647进行实验，酶反应缓冲液为20mM Tris-HCl、10mM(NH4)2SO4、10mM KCl、2mM MgSO4、0.1％Triton、pH 8.8；反应温度为40℃。
酶反应以动力学检测模式，每分钟记录一次数据，检测条件为如表4所示。
表4：酶动力学检测反应条件
反应完成后可直接导出数据表格或者酶活曲线，可近似计算其相对荧光值的反应速率。
以KOD DNA聚合酶融合蛋白(DP01)及KOD DNA聚合酶点突变体融合蛋白为例，进行聚合反应。其中，融合蛋白的C端包含6个his标签。
KOD DNA聚合酶点突变体具体信息参考表2，制备方法参考实施例1和2。
DP01融合蛋白是高活性KOD DNA聚合酶，其相对聚合活性相对于仅突变408和409的聚合酶(参见专利号PCT/CN2019/102493)提高2-3倍。本申请在高活性变体的基础上，进一步优化并提高KOD DNA聚合酶突变体的聚合活性。在以单链DNA为模板、dATP-AF532为底物时，部分KOD DNA聚合酶突变体的活性测定动力学曲线如图3所示。
野生型KOD DNA聚合酶、仅突变408和409的聚合酶、以及各KOD DNA聚合酶突变体的聚合活性相对KOD DNA聚合酶DP01的聚合活性检测结果如表5所示。与KOD DNA聚合酶(DP01)相比，突变体DP003、DP011、DP012、DP013、DP014、DP019、DP020、DP023、DP024、DP041、DP83、DP112、DP113、DP115、DP117、DP130等表现出相当的聚合活性；突变体DP001、DP002、DP004、DP005、DP006、DP007、DP008、DP009、DP010、DP015、DP016、DP017、DP018、DP021、DP022、DP80、DP81、DP82、DP83、DP84、DP85、DP86、DP87、DP88、DP89、DP91、DP92、DP93、DP96、DP101、DP104、DP106、DP107、DP108、DP109、DP110、DP111、DP116、DP119、DP120、DP121、DP122、DP123、DP124、DP127等表现出更好的聚合活性。
表5：KOD DNA聚合酶突变体的动力学测试结果




注：因WT(野生型KOD DNA聚合酶)无法掺入修饰dATP，所以相对聚合活性为0。
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 (28)

  1. 一种DNA聚合酶突变体，其特征在于，
    与野生型KOD DNA聚合酶相比，所述突变体具有第408位、第409位点或功能等同位点的氨基酸突变，以及选自以下23个位点和功能等同位点中的至少1个位点的氨基酸突变：
    第141位、第143位、第147位、第383位、第384位、第389位、第485位、第584位、第589位、第397位、第424位、第432位、第445位、第523位、第553位、第561位、第564位、第461位、第481位、第605位、第663位、第711位、第725位；
    所述突变体的所述氨基酸突变位点以外的氨基酸序列与所述野生型KOD DNA聚合酶的对应的氨基酸序列具有至少90％的同一性；
    所述野生型KOD DNA聚合酶具有如SEQ ID NO:2所示的氨基酸序列。
  2. 根据权利要求1所述的DNA聚合酶突变体，其特征在于，所述突变体的所述氨基酸突变位点以外的氨基酸序列与所述野生型KOD DNA聚合酶的对应的氨基酸序列相同。
  3. 根据权利要求1所述的DNA聚合酶突变体，其特征在于，与所述野生型KOD DNA聚合酶相比，所述突变体具有第408位、第409位点或功能等同位点的氨基酸突变，以及选自以下9个位点和功能等同位点中的至少1个位点的氨基酸突变：
    第141位、第143位、第147位、第383位、第384位、第389位、第485位、第584位、第589位。
  4. 根据权利要求3所述的DNA聚合酶突变体，其特征在于，与所述野生型KOD DNA聚合酶相比，所述突变体具有第408位、第409位点或功能等同位点的氨基酸突变，以及选自以下2个位点和功能等同位点中的至少1个位点的氨基酸突变：
    第147位、第584位。
  5. 根据权利要求3或4所述的DNA聚合酶突变体，其特征在于，突变位点的突变类型为：
    (1)第141位的D突变为A，
    (2)第143位的E突变为A，
    (3)第147位的H突变为E，
    (4)第383位的S突变为T，
    (5)第384位的Y突变为F，
    (6)第389位的V突变为I，
    (7)第485位的A突变为E，
    (8)第584位的K突变为E，
    (9)第589位的V突变为H，
    (10)第408位的L突变为I，
    (11)第409位的Y突变为A。
  6. 根据权利要求1所述的DNA聚合酶突变体，其特征在于，与所述野生型KOD DNA聚合酶相比，所述突变体具有：
    第408位、第409位、第141位、第143位、第147位、第383位、第384位、第389位、第485位、第584位、第589位点或功能等同位点的氨基酸突变，以及
    选自以下14个位点和功能等同位点中的至少1个位点的氨基酸突变：
    第397位、第424位、第432位、第445位、第523位、第553位、第561位、第564位、第461位、 第481位、第605位、第663位、第711位、第725位。
  7. 根据权利要求6所述的DNA聚合酶突变体，其特征在于，与所述野生型KOD DNA聚合酶相比，所述突变体具有：
    第408位、第409位、第141位、第143位、第147位、第383位、第384位、第389位、第485位、第584位、第589位点或功能等同位点的氨基酸突变；以及
    选自以下4个位点和功能等同位点中的至少1个位点的氨基酸突变：
    第397位、第424位、第481位、第553位。
  8. 根据权利要求6所述的DNA聚合酶突变体，其特征在于，突变位点的突变类型为：
    (1)第141位的D突变为A，
    (2)第143位的E突变为A，
    (3)第147位的H突变为E，
    (4)第383位的S突变为T，
    (5)第384位的Y突变为F，
    (6)第389位的V突变为I，
    (7)第485位的A突变为E，
    (8)第584位的K突变为E，
    (9)第589位的V突变为H，
    (10)第408位的L突变为I，
    (11)第409位的Y突变为A，
    (12)第397位的W突变为Y或H，
    (13)第424位的N突变为R、Q、H、I、L、M、F、W、K、Y、V、C或S，
    (14)第432位的D突变为R、E、H或M，
    (15)第445位的F突变为R，
    (16)第523位的M突变为R或L，
    (17)第553位的A突变为T、R、N、E、G、H、L、M、F、Y、P、S、D、V、I或C，
    (18)第561位的M突变为R或W，
    (19)第564位的L突变为R或M，
    (20)第461位的Q突变为I、L、M、F、W或Y，
    (21)第481位的Y突变为R、H、L、M或F，
    (22)第605位的T突变为I或V，
    (23)第663位的H突变为R、Q、L、M、F、T、Y、N、V、G、A、P、S、C、E或K，
    (24)第711位的G突变为R、M或S，
    (25)第725位的H突变为R。
  9. 根据权利要求6所述的DNA聚合酶突变体，其特征在于，与所述野生型KOD DNA聚合酶相比，所述突变体具有：
    第408位、第409位、第141位、第143位、第147位、第383位、第384位、第389位、第485位、第584位、第589位点或功能等同位点的氨基酸突变；以及
    选自以下14个位点和功能等同位点中的一个位点的氨基酸突变：
    第397位、第424位、第432位、第445位、第523位、第553位、第561位、第564位、第461位、第481位、第605位、第663位、第711位、第725位；
    条件是，第424位的N不能突变为R和L；或
    第432位的D不能突变为M；或
    第663位的H不能突变为T、V、P和E。
  10. 根据权利要求6所述的DNA聚合酶突变体，其特征在于，与所述野生型KOD DNA聚合酶相比，所述突变体具有：
    第408位、第409位、第141位、第143位、第147位、第383位、第384位、第389位、第485位、第584位、第589位点或功能等同位点的氨基酸突变；以及
    选自以下14个位点和功能等同位点中的两个位点的氨基酸突变：
    第397位、第424位、第432位、第445位、第523位、第553位、第561位、第564位、第461位、第481位、第605位、第663位、第711位、第725位；
    条件是，当第663位的H突变为R时，第553位的A不能突变为P和E；或
    当第663位的H突变为Q时，第461位的Q不能突变为Y。
  11. 根据权利要求8所述的DNA聚合酶突变体，其特征在于，与所述野生型KOD DNA聚合酶相比，所述突变体具有(1)-(11)的突变，以及选自(12)-(25)中的任意一种突变。
  12. 根据权利要求11所述的DNA聚合酶突变体，其特征在于，与所述野生型KOD DNA聚合酶相比，所述突变体具有(1)-(11)的突变，以及选自以下突变中的任意一种突变：
    第397位的W突变为H，
    第424位的N突变为Q，
    第553位的A突变为R，
    第553位的A突变为N，
    第553位的A突变为E，
    第553位的A突变为G，
    第553位的A突变为H，
    第553位的A突变为P，
    第553位的A突变为S，
    第461位的Q突变为W，
    第461位的Q突变为Y，
    第481位的Y突变为M，
    第481位的Y突变为F，
    第711位的G突变为R，
    第711位的G突变为S。
  13. 根据权利要求8所述的DNA聚合酶突变体，其特征在于，与所述野生型KOD DNA聚合酶相比，所述突变体具有(1)-(11)的突变，以及选自以下突变组合中的任意一种突变组合：
    第481位Y突变为F，且第553位A突变为P；
    第481位Y突变为F，且第553位A突变为S；
    第397位W突变为H，且第553位A突变为P；
    第461位Q突变为Y，且第553位A突变为P；
    第481位Y突变为F，且第553位A突变为G；
    第424位N突变为Q，且第553位A突变为P；
    第397位W突变为H，且第481位Y突变为F；
    第553位A突变为P，且第711位G突变为R；
    第397位W突变为H，且第553位A突变为S；
    第481位Y突变为M，且第553位A突变为P；
    第461位Q突变为W，且第553位A突变为P；
    第553位A突变为P，且第711位G突变为S；
    第461位Q突变为Y，且第553位A突变为S；
    第553位A突变为P，且第561位M突变为W；
    第553位A突变为P，且第561位M突变为R；
    第481位Y突变为F，且第711位G突变为R；
    第461位Q突变为F，且第553位A突变为P；
    第424位N突变为Q，且第553位A突变为S；
    第432位D突变为H，且第553位A突变为P；
    第553位A突变为P，且第605位T突变为V；
    第481位Y突变为F，且第561位M突变为W；
    第424位N突变为F，且第553位A突变为P；
    第481位Y突变为F，且第605位T突变为I；
    第461位Q突变为F，且第481位Y突变为F；
    第553位A突变为P，且第663位H突变为R；
    第481位Y突变为M，且第553位A突变为S；
    第553位A突变为P，且第564位L突变为M；
    第432位D突变为E，且第553位A突变为P；
    第461位Q突变为F，且第553位A突变为S；
    第481位Y突变为M，且第553位A突变为G；
    第481位Y突变为F，且第564位L突变为M；
    第553位A突变为P，且第663位H突变为N；
    第432位D突变为H，且第553位A突变为S；
    第553位A突变为P，且第663位H突变为F；
    第553位A突变为P，且第564位L突变为R；
    第424位N突变为S，且第553位A突变为P；
    第481位Y突变为F，且第553位A突变为F；
    第553位A突变为E，且第663位H突变为R；
    第481位Y突变为L，且第711位G突变为R；
    第424位N突变为Q，且第725位H突变为R；
    第424位N突变为Q，且第663位H突变为R；
    第461位Q突变为W，且第561位M突变为R；
    第432位D突变为R，且第553位A突变为S；
    第397位W突变为H，且第663位H突变为Y；
    第461位Q突变为Y，且第663位H突变为Q；
    第461位Q突变为I，且第481位Y突变为M；
    第553位A突变为Y，且第605位T突变为V；
    第424位N突变为K，且第663位H突变为L；
    第461位Q突变为F，且第481位Y突变为R。
  14. 根据权利要求13所述的DNA聚合酶突变体，其特征在于，与所述野生型KOD DNA聚合酶相比，所述突变体具有(1)-(11)的突变，以及选自以下突变组合中的任意一种突变组合：
    第481位Y突变为F，且第553位A突变为P；
    第481位Y突变为F，且第553位A突变为S；
    第397位W突变为H，且第553位A突变为P；
    第461位Q突变为Y，且第553位A突变为P；
    第424位N突变为Q，且第553位A突变为P；
    第397位W突变为H，且第481位Y突变为F；
    第553位A突变为P，且第711位G突变为R；
    第397位W突变为H，且第553位A突变为S；
    第481位Y突变为F，且第711位G突变为R；
    第461位Q突变为F，且第481位Y突变为F；
    第553位A突变为P，且第564位L突变为M；
    第481位Y突变为F，且第564位L突变为M；
    第424位N突变为S，且第553位A突变为P；
    第481位Y突变为L，且第711位G突变为R；
    第424位N突变为Q，且第725位H突变为R；
    第397位W突变为H，且第663位H突变为Y；
    第461位Q突变为I，且第481位Y突变为M。
  15. 一种核酸分子，其特征在于，所述核酸分子编码权利要求1～14任一项所述的DNA聚合酶突变体。
  16. 一种表达载体，其特征在于，所述表达载体包含或携带权利要求15所述的核酸分子。
  17. 根据权利要求16所述的表达载体，其特征在于，所述表达载体为非致病性病毒载体或非病毒载体，所述非致病性病毒载体包括腺病毒载体或逆转录病毒载体；所述非病毒载体包括质粒载体。
  18. 一种重组细胞，其特征在于，所述重组细胞携带权利要求15所述的核酸分子或权利要求16～17任一项所述的表达载体。
  19. 根据权利要求18所述的重组细胞，其特征在于，所述重组细胞选自大肠杆菌、酵母和哺乳动物细胞。
  20. 一种重组菌株，其特征在于，所述重组菌株表达权利要求1～14任一项所述的DNA聚合酶突变体。
  21. 一种获得DNA聚合酶突变体的方法，其特征在于，所述方法包括：将权利要求18～19任一项所述的重组细胞或权利要求20所述的重组菌株在适于蛋白表达的条件下进行培养处理，以便获得所述DNA聚合酶突变体。
  22. 一种复合物，其特征在于，所述复合物包括权利要求1～14任一项所述DNA聚合酶突变体和小分子化合物或大分子，所述突变体和小分子化合物或大分子通过化学键偶联。
  23. 根据权利要求22所述的复合物，其特征在于，所述小分子化合物或大分子包括荧光标记、荧光素或抗体。
  24. 一种核酸合成方法，其特征在于，所述方法包括：
    将核酸模板、扩增引物、dNTP与权利要求1～14任一项所述DNA聚合酶突变体的混合产物在适于核酸扩增的条件下进行扩增处理，以便获得所述核酸。
  25. 一种核酸测序的方法，其特征在于，所述方法包括：
    将待测核酸与权利要求1～14任一项所述DNA聚合酶突变体和带有修饰的dNTP的混合产物在适于核酸扩增的条件下进行扩增处理和荧光信号检测处理；以及
    基于检测获得的荧光信号，确定所述待测核酸的核酸序列。
  26. 一种核酸测序试剂盒，其特征在于，所述试剂盒包括权利要求1～14任一项所述的DNA聚合酶突变体或权利要求22～23任一项所述的复合物。
  27. 权利要求26所述核酸测序试剂盒在测序中的用途。
  28. 权利要求1～14任一项所述的DNA聚合酶突变体、权利要求15所述的核酸分子、权利要求16～17任一项所述表达载体、权利要求18～19任一项所述的重组细胞、权利要求20所述的重组菌株或权利要求22～23任一项所述的复合物在制备用于催化DNA扩增或核酸测序相关产品中的用途。
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