WO2024244010A1 - 用于测序的dna聚合酶突变体 - Google Patents

用于测序的dna聚合酶突变体 Download PDF

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WO2024244010A1
WO2024244010A1 PCT/CN2023/098118 CN2023098118W WO2024244010A1 WO 2024244010 A1 WO2024244010 A1 WO 2024244010A1 CN 2023098118 W CN2023098118 W CN 2023098118W WO 2024244010 A1 WO2024244010 A1 WO 2024244010A1
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mutated
mutates
amino acid
dna polymerase
mutant
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French (fr)
Inventor
刘芬
黄思谦
杨丽媛
高静
白佳琨
肖阳
杨涓
苏香
刘月鹏
宋承威
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Wuhan Mgi Tech Co Ltd
MGI Tech Co Ltd
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Wuhan Mgi Tech Co Ltd
MGI Tech Co Ltd
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Priority to CN202380098380.4A priority Critical patent/CN121241132A/zh
Priority to PCT/CN2023/098118 priority patent/WO2024244010A1/zh
Priority to EP23938994.3A priority patent/EP4722356A1/en
Publication of WO2024244010A1 publication Critical patent/WO2024244010A1/zh
Priority to US19/401,896 priority patent/US20260078358A1/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1252—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
    • C—CHEMISTRY; METALLURGY
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    • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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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
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    • 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
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    • 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
    • C12Q1/6869—Methods for sequencing
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    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli
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    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/07—Nucleotidyltransferases (2.7.7)
    • C12Y207/07007—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase

Definitions

  • the present invention relates to the field of biotechnology, and specifically, the present invention relates to a DNA polymerase mutant for sequencing, and in particular to a DNA polymerase mutant, a nucleic acid molecule, an expression vector, a recombinant cell, a recombinant strain, a method of DNA polymerase mutant, a complex, a method of nucleic acid sequencing, a nucleic acid sequencing kit and its use, and its use in preparing products related to catalyzing DNA amplification or nucleic acid sequencing.
  • the second-generation high-throughput DNA sequencing method based on sequencing by synthesis is the most widely used sequencing technology. Compared with the first-generation sequencing, it has the following advantages: fast sequencing speed and high throughput, which can complete large-scale genome sequencing in a short time; low sequencing cost, and the sequencing price of each base is much lower than that of the first-generation sequencing; high sequencing accuracy, which can detect low-frequency variants and heterozygous sites; high sequencing flexibility, which can be applied to different target areas and sample types.
  • the second-generation sequencing has a wide range of applications in the biomedical field, such as transcriptome sequencing, epigenetic sequencing, whole-genome association analysis, drug resistance detection and other applications (such as genetic identification, forensic analysis, genetic counseling, medical diagnosis), etc.
  • sequencing polymerase plays an important role in the sequencing process.
  • the principle of the second-generation sequencing is sequencing by synthesis, that is, the sequence of DNA is determined by capturing the special labels (generally fluorescent molecular labels) carried by the newly added bases during DNA replication. Sequencing polymerase is responsible for pairing and connecting the labeled bases with the template chain to form a complementary DNA chain.
  • the addition and detection of artificially modified nucleotides or nucleotide analogs is crucial.
  • DNA polymerization the biggest limiting factor is that the existing natural polymerases have a low ability to polymerize artificially modified nucleotides or nucleotide analogs.
  • enhancing the ability of polymerases to polymerize artificially modified nucleotides or nucleotides is a key step in this type of sequencing method.
  • DNA polymerases are divided into three families, called A, B, and C. Although the nucleotide binding sites of A and B polymerases are structurally similar, the motifs of polymerases from different families are significantly different, correspondingly representing the recognition mechanisms of nucleotides and their analogs. Among them, the thermostable B family polymerases from thermophilic archaea have the best incorporation performance for various nucleotide analogs, and their mutants are widely used in various second-generation sequencing methods.
  • thermostable B family polymerases of thermophilic archaea include KOD (Thermococcus kodakaraensis), 9°N (Thermococcus sp.9°N), TGO (Thermococcus gorgonarius), TOK (Desulfurococcus sp.Tok), Vent DNA polymerase (Thermococcus litoralis), JDF-3 and pfu DNA polymerase (Pyrococcus furiosis).
  • KOD Thermococcus kodakaraensis
  • 9°N Thermococcus sp.9°N
  • TGO Thermococcus gorgonarius
  • TOK Desulfurococcus sp.Tok
  • Vent DNA polymerase Thermococcus litoralis
  • JDF-3 and pfu DNA polymerase Pyrococcus furiosis
  • the present invention aims to solve at least one of the technical problems existing in the prior art to at least a certain extent.
  • the inventors have obtained new DNA polymerase mutants by enzyme engineering modification of the active sites related to the thermostable B family polymerase of thermophilic archaea, which can greatly improve the polymerase's incorporation efficiency of specific non-natural dNTPs, thereby improving the sequencing speed and sequencing quality of the sequencing by synthesis (SBS) method.
  • SBS sequencing by synthesis
  • the present invention proposes a DNA polymerase mutant.
  • the mutant includes: compared with the Pyrococcus abyssi DNA polymerase exo- (removal of 3'-5' exonuclease proofreading activity) mutant, at least three amino acid mutations in the following four sites or functionally equivalent sites: 409th, 410th, 411th, 486th; the Pyrococcus abyssi DNA polymerase Exo- mutant has an amino acid sequence as shown in SEQ ID NO: 1.
  • the DNA polymerase mutant described in the embodiment of the present invention can effectively improve the incorporation efficiency of the polymerase for specific non-natural dNTPs, thereby improving the sequencing speed and sequencing quality of the SBS sequencing method, which is of great significance.
  • amino acid positions in the amino acid sequence of the DNA polymerase mutant described in the present application are located with reference to the amino acid positions in the amino acid sequence of the wild-type Pyrococcus abyssi DNA polymerase or the wild-type Pyrococcus abyssi DNA polymerase exo-mutant.
  • the above-mentioned DNA enzyme mutant may further include at least one of the following additional technical features:
  • the DNA polymerase mutant has at least 90% identity with the Pyrococcus abyssi DNA polymerase exo-mutant.
  • the mutant has the following mutations:
  • amino acid L at position 409 is mutated to A, V, Y, H, F, or Q;
  • the mutant has any one of the following combined mutations (1)-(2):
  • amino acid Y at position 410 is mutated to A
  • amino acid L at position 409 is mutated to A
  • amino acid P at position 411 is mutated to V, G, C, S, or no mutation
  • amino acid A at position 486 is mutated to I, K, L, M, W, or F; or
  • the mutant has any one of the following combined mutations (1)-(7):
  • amino acid Y at position 410 is mutated to A
  • amino acid L at position 409 is mutated to A
  • amino acid P at position 411 is mutated to G, C, S or no mutation
  • amino acid A at position 486 is mutated to I, K, L, M, W or F; or
  • the sequencing recombinases obtained from the DNA polymerase mutants of the present invention
  • the sequencing recombinases produced by different mutation sites have different relative activities, which can provide more options for actual production needs.
  • the mutant has any one of the combined mutations (1)-(36):
  • L at position 409 is mutated to V
  • Y at position 410 is mutated to G
  • P at position 411 is not mutated
  • a at position 486 is mutated to I;
  • L at position 409 is mutated to V
  • Y at position 410 is mutated to G
  • P at position 411 is not mutated
  • a at position 486 is mutated to F
  • L at position 409 is mutated to Y, Y at position 410 is mutated to A, P at position 411 is not mutated, and A at position 486 is mutated to L; or
  • L at position 409 mutates to Y
  • Y at position 410 mutates to A
  • P at position 411 mutates to V
  • a at position 486 mutates to L
  • L at position 409 is mutated to H, Y at position 410 is mutated to A, P at position 411 is not mutated, and A at position 486 is mutated to L; or
  • L at position 409 is mutated to F
  • Y at position 410 is mutated to A
  • P at position 411 is not mutated
  • a at position 486 is mutated to L;
  • L at position 409 is mutated to A
  • Y at position 410 is mutated to A
  • P at position 411 is not mutated
  • a at position 486 is mutated to L;
  • L at position 409 is mutated to V
  • Y at position 410 is mutated to G
  • P at position 411 is not mutated
  • a at position 486 is mutated to M
  • L at position 409 is mutated to V
  • Y at position 410 is mutated to A
  • P at position 411 is not mutated
  • a at position 486 is mutated to W;
  • L at position 409 is mutated to V
  • Y at position 410 is mutated to A
  • P at position 411 is not mutated
  • a at position 486 is mutated to I;
  • L at position 409 is mutated to V
  • Y at position 410 is mutated to A
  • P at position 411 is not mutated
  • a at position 486 is mutated to F
  • the mutant has the following mutations:
  • L at position 409 is mutated to V
  • Y at position 410 is mutated to G
  • P at position 411 is not mutated
  • a at position 486 is mutated to I;
  • the sequencing recombinases obtained from the DNA polymerase mutants of the present invention
  • the sequencing recombinases produced by different mutation sites have different relative activities, which can provide more options for actual production needs.
  • mutant amino acid sequences were obtained by mutation based on the SEQ ID NO:1 sequence.
  • the relative activity of the recombinant polymerase obtained compared to the existing mutation is stronger, and the non-natural dNTP containing a fluorescently labeled 3'O-reversible terminator can be effectively added to the DNA chain, thereby improving the sequencing speed and sequencing quality.
  • the wild-type Pyrococcus abyssi DNA polymerase cannot add specific non-natural dNTPs (e.g., non-natural dNTPs containing 3’O-reversible terminators) to the DNA chain. Therefore, the inventors designed Pyrococcus abyssi DNA polymerase mutants to add specific non-natural dNTPs to the DNA chain.
  • the present invention provides a nucleic acid molecule.
  • the nucleic acid molecule encodes the DNA polymerase mutant described in the first aspect of the present invention.
  • the DNA polymerase mutant encoded by the nucleic acid molecule can be carried out in vivo or in vitro. Obtained in large quantities.
  • the present invention provides an expression vector.
  • the expression vector comprises the nucleic acid molecule described in the second aspect of the present invention.
  • 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 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.
  • the recombinant cell is used to express or secrete the DNA polymerase mutant described in the first aspect of the present invention.
  • the recombinant cell is selected from Escherichia coli, yeast or mammalian cells.
  • the present invention provides a recombinant strain.
  • the recombinant strain expresses the DNA polymerase mutant described in the first aspect of the present invention.
  • the DNA polymerase mutant can be obtained quickly and in large quantities by culturing the recombinant strain.
  • the present invention provides a method for obtaining a DNA polymerase mutant.
  • the method comprises culturing the recombinant cell described in the fourth aspect of the present invention or the recombinant strain described in the fifth aspect under conditions suitable for protein expression to obtain the DNA polymerase mutant.
  • the present invention provides a complex.
  • the complex comprises the DNA polymerase mutant and a small molecule compound or a macromolecule according to the first aspect of the present invention, wherein the DNA polymerase mutant and the small molecule compound or the macromolecule are coupled via a chemical bond.
  • the small molecule compound or macromolecule includes a fluorescent marker, fluorescein or an antibody.
  • the present invention provides a method for nucleic acid synthesis.
  • the method comprises: 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 invention 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 invention provides a method for nucleic acid sequencing.
  • the method comprises: subjecting a nucleic acid template to be tested and a mixed product of the DNA polymerase mutant described in the first aspect of the present invention and a non-natural dNTP with a fluorescently labeled 3'O-reversible terminator to amplification treatment and fluorescence 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 a nucleic acid template to be tested with the DNA polymerase mutant and a fluorescently labeled 3'O-reversible terminator non-natural dNTP, wherein the DNA polymerase mutant is responsible for matching the fluorescently labeled 3'O-reversible terminator non-natural dNTP with the nucleic acid template, and finally detecting multiple fluorescent labeling signals to obtain a sequence based on the obtained fluorescent labeling signals.
  • the nucleic acid sequence of the nucleic acid to be tested is then obtained.
  • the above polymerization reaction and fluorescent labeling reaction can be performed for multiple cycles according to the length of the sequencing template.
  • the present invention provides a nucleic acid sequencing kit.
  • the nucleic acid sequencing kit comprises the DNA polymerase mutant described in the first aspect or the complex described in the seventh aspect.
  • the kit of the present invention is used for efficient, accurate and rapid nucleic acid sequencing.
  • the present invention provides a use of the nucleic acid sequencing kit of the ninth aspect in sequencing.
  • the kit can be used for sequencing, including but not limited to sequencing by synthesis (SBS).
  • the present invention 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 shows the electrophoresis diagram of the Pyrococcus abyssi exo-type DNA polymerase fusion protein described in Example 1 of the present invention.
  • 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 the indicated technical features. Therefore, the features defined as “first” and “second” may explicitly or implicitly include at least one of the features. In the description of the present invention, the meaning of “plurality” is at least two, such as two, three, etc., unless otherwise clearly and specifically defined.
  • the amino acid sequence shown in SEQ ID NO: 1 is obtained by mutating the 141st amino acid D of the wild-type Pyrococcus abyssi DNA polymerase to A, and the 143rd amino acid E to A.
  • the wild-type Pyrococcus abyssi DNA polymerase has no exonuclease activity.
  • the present invention aims to provide a B family recombinant polymerase that can improve the quality of sequencing.
  • the enzyme is based on the DNA polymerase of the archaeon Pyrococcus abyssi (Erauso, et al. "Pyrococcus abyssi sp.nov.,a new hyperthermophilic archaeon isolated from a deep-sea hydrothermal vent.” Archives of microbiology 160(1993):338-349), has the characteristics of high activity and high heat resistance, but the wild-type polymerase cannot effectively incorporate non-natural dNTPs with fluorescent labels and 3'O-reversible terminators. It is necessary to improve the enzyme's incorporation efficiency of specific non-natural dNTPs through enzyme engineering and screening of related active sites for use in SBS sequencing methods.
  • the present invention provides a DNA polymerase mutant.
  • the DNA polymerase mutant provided in the embodiment of the present invention has an amino acid sequence with at least 90% homology to the wild-type Pyrococcus abyssi DNA polymerase exo-mutant, and has mutations in at least three of the following positions: position 409, position 410, position 411, and position 486.
  • the DNA polymerase mutant described in the present application has the advantages of high activity and high heat resistance, and has the ability to improve the performance of polymerizing 3'O-reversible terminator non-natural dNTPs, thereby improving sequencing speed and quality.
  • the present invention provides a nucleic acid molecule encoding the aforementioned DNA polymerase mutant.
  • 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 invention provides an expression vector comprising the aforementioned nucleic acid molecule.
  • the type of the expression vector is not particularly limited, as long as it can replicate and express the corresponding mutant in a host cell.
  • the present invention provides a recombinant cell carrying the aforementioned nucleic acid molecule, expression vector or expressing DNA polymerase mutant.
  • the recombinant cell is obtained by transfecting or transforming the expression vector.
  • the recombinant cell can efficiently express the aforementioned DNA polymerase mutant under appropriate conditions.
  • the present invention provides a recombinant strain that expresses the aforementioned DNA polymerase mutant.
  • the recombinant strain can be propagated in large quantities in a short time and the DNA polymerase mutant can be prepared efficiently.
  • the method for obtaining a DNA polymerase mutant provided by the present invention comprises the steps of: subjecting the aforementioned recombinant cell to a condition suitable for protein expression; The culture treatment is performed to obtain the DNA polymerase mutant.
  • the present invention provides a complex, wherein the complex comprises the aforementioned DNA polymerase mutant and a small molecule compound or a macromolecule, wherein the DNA polymerase mutant and the small molecule compound or the macromolecule are coupled via a chemical bond.
  • the nucleic acid sequence to be detected is determined by detecting the small molecule compound (fluorescent marker) or macromolecule (antibody, fluorescein) in the complex.
  • the preparation and crude enzyme activity screening of DNA polymerase and its mutants are performed based on Pyrococcus abyssi exo-type (deep-sea fireball exo-mutant).
  • the specific steps are as follows:
  • the primers were designed as follows: 5'-reverse complementary region (15-21 bp)-non-complementary region (at least 15 bp). Phanta Max Super-Fidelity DNA Polymerase was used to amplify the target plasmid.
  • L409 mutated to A, F, H, L, Q, S, V, Y;
  • P411 mutated to A, C, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y;
  • mutant site combination currently known to have better polymerization 3'O-reversible terminator non-natural dNTP activity in other B family polymerases (9°N, Pfu, Vent, etc.) is L409A-Y410A-P411I-A486L of 9°N polymerase (patent EP1664287B1, US8460910B2).
  • This mutant combination will be used as a control mutation site combination for the Pyrococcus abyssi polymerase screening experiment (SEQ ID NO: 2);
  • the amplified product contains the original template plasmid, in order to prevent it from forming false positive transformants after transformation, Dpn I digestion must be performed before recombination circularization to remove the methylated template plasmid.
  • step 1.1 In the 96-well plate of step 1.1, add IPTG (isopropylthiogalactoside) at a final concentration of 0.5mM, culture at 25°C, 220rpm/min for 12-16h to induce expression. Then add 1 ⁇ L 100mM PMSF (protease inhibitor) and 5 ⁇ L 10mg/ml lysozyme, gently blow with a pipette tip, mix, react at 37°C for 10min, and obtain the crude enzyme solution of bacterial lysis.
  • IPTG isopropylthiogalactoside
  • PMSF prote inhibitor
  • the modified nucleotides were introduced into the simulation high-throughput sequencing process, and the relative reaction rate of the polymerase mutants was detected by using an ELISA instrument to calculate the crude enzyme activity.
  • the specific experimental method is as follows:
  • Reaction buffer 20 mM Tris-HCl, 10 mM (NH 4 ) 2 SO 4 , 10 mM KCl, 2 mM MgSO 4 pH 8.5@25°C;
  • the reaction system is as follows: 1 ⁇ g of different polymerase proteins obtained in step 1.2 are mixed with reaction solutions containing 2 ⁇ M modified dATP and 1 ⁇ M DNA-Cy5;
  • Reaction solution formula 20mM Tris-HCl, 10mM (NH 4 ) 2 SO 4 , 10mM KCl, 2mM MgSO 4 , 2uM 3'-blocked modified dATP and 1 ⁇ M DNA-Cy5, the balance is water, pH 8.5;
  • DNA polymerase mutant fusion protein is performed based on Pyrococcus abyssi exo-type (deep-sea fireball exo-mutant). The specific steps are as follows:
  • step 2) The BL21/pD441 bacterial solution induced in step 1) was centrifuged at 8000 rpm/min for 10 min, the supernatant was discarded, the precipitated bacterial cells were collected, and the bacterial cells were resuspended in buffer 1 (50 mM KPO 4 , 500 mM NaCl, 10 mM imidazole, 5% Glycerol, pH 7.0), and PMSF (final concentration 0.5 mM), Triton X-100 (final concentration 0.5%), Lysozyme (lysozyme, final concentration 0.25%), then incubate at room temperature for 30 minutes, centrifuge at 12000 rpm/min, 4°C for 30 minutes, put in an ice bath, and disrupt the cells by ultrasound; centrifuge at 12000 rpm/min for 30 minutes, and place the supernatant obtained by centrifugation in a 75°C water bath for 20 minutes. During this period, pay attention to regular mixing to ensure uniform heating; centrifuge
  • step 3 The crude extract of the fusion protein obtained in step 2) was loaded onto Ni column affinity chromatography (affinity chromatography pre-packed column HisTrap FF, 5 ml, 17-5255-01, GE healthcare) at an appropriate flow rate. After loading, 5CV (column volume) of the column was equilibrated with buffer 1; 5CV of elution was performed with 3% buffer 2 (50 mM KPO 4 , 1 M NaCl, 5% Glycerol, pH 7.0); and 5CV of elution was performed with 50% buffer 2. The Ni column affinity chromatography eluate corresponding to the peak value greater than or equal to 100 mAU was collected.
  • Ni column affinity chromatography affinity chromatography pre-packed column HisTrap FF, 5 ml, 17-5255-01, GE healthcare
  • the eluate corresponding to the peak value greater than or equal to 100mAU was loaded at a certain flow rate for ion exchange chromatography (ion exchange prepacked column HiTrap Q HP, 5ml, 17-1154-01, GE healthcare), and after loading, it was equilibrated with buffer 2 for 5CV, and linearly eluted from 0% buffer 2 to 60% buffer 2, and the ion exchange chromatography eluate corresponding to the peak value greater than or equal to 100mAU was collected.
  • ion exchange chromatography ion exchange prepacked column HiTrap Q HP, 5ml, 17-1154-01, GE healthcare
  • the protein size in lanes 2 and 3 is about 90KDa, which is consistent with the molecular weight reported in the literature (Dietrich, J., Schmitt, P., Zieger, M., Preve, B., Rolland, J.L., Chaabihi, H., & Gueguen, Y. (2002). PCR performance of the highly thermosable proof-reading B-type DNA polymerase from Pyrococcus abyssi. FEMS microbiology letters, 217 (1), 89-94.). The protein purity was analyzed by Quantity one software on the protein gel after electrophoresis. The purity of the purified DNA polymerase mutant fusion protein can reach 85% or more. The target protein of about 90KDa was not obtained in the uninduced BL21/pD441 bacterial solution.
  • the actual sequencing performance test of the purified enzyme of the DNA polymerase mutant in the sequencer was carried out based on Pyrococcus abyssi exo-type (deep-sea fireball exo-mutant).
  • the purified enzyme of the dominant mutant was sequenced on the MGISEQ-2000 sequencer of BGI, and the sequencing results were analyzed.
  • the specific experimental steps are as follows:
  • Sequencing library Ecoli.fa library (Part No.1000005038, MGI Tech co., Ltd);
  • Sequencing conditions polymerization time 90 seconds, excision 60 seconds.
  • control P1 mutant and 5 mutants with different performances in the enzyme activity test were selected for on-machine testing. These mutants were sequenced for 100 cycles (SE100) on the MGISEQ-2000 sequencer. The test results are shown in Table 2, indicating that the purified enzyme of the dominant mutant performs better in actual sequencing.

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Abstract

提供用于测序的DNA聚合酶突变体。所述DNA聚合酶突变体包括：与Pyrococcus abyssi DNA聚合酶exo-突变体相比，在以下四个位点或功能等同位点中具有至少之三个氨基酸突变：第409位、第410位、第411位、第486位；所述Pyrococcus abyssi DNA聚合酶exo-突变体具有如SEQ ID NO:1所示的氨基酸序列。

Description

用于测序的DNA聚合酶突变体 技术领域
本发明涉及生物技术领域，具体的，本发明涉及用于测序的DNA聚合酶突变体，特别涉及一种DNA聚合酶突变体、核酸分子、表达载体、重组细胞、重组菌株、DNA聚合酶突变体的方法、复合物、核酸测序的方法、核酸测序试剂盒及其用途和制备用于催化DNA扩增或核酸测序相关产品中的用途。
背景技术
核酸序列对于理解基因的功能至关重要，在基因分析中具有广泛应用，而DNA测序方法是基因分析中重要的工具。基于边合成边测序(Sequencing by Synthesis，SBS)的第二代高通量DNA测序方法是现在应用最广泛的测序技术。相比于一代测序，具有以下几个优势：测序速度快，通量高，可以在短时间内完成大规模的基因组测序；测序成本低，每个碱基的测序价格远低于一代测序；测序准确性高，可以检测出低频的变异和杂合位点；测序灵活性高，可以应用于不同的目标区域和样本类型。二代测序在生物医学领域有广泛的应用，例如，转录组测序、表观遗传学测序、全基因组关联分析、药物耐药检测及其他应用(如遗传鉴定、法医分析、遗传咨询、医学诊断)等。
测序聚合酶作为二代测序中最重要的组分之一，在测序过程中起着重要的作用。二代测序的原理是边合成边测序，即在DNA复制过程中通过捕捉新添加的碱基所携带的特殊标记(一般为荧光分子标记)来确定DNA的序列。测序聚合酶就是负责将带有标记的碱基与模板链配对并连接起来，形成互补的DNA链。在这种测序方法中，添加和检测人工修饰的核苷酸或核苷酸类似物(例如，带有荧光标记的可逆终止子的DNA聚合酶)至关重要。在DNA聚合中，最大的限制因素是现有的天然聚合酶对人工修饰的核苷酸或核苷酸类似物的聚合能力较低。有鉴于此，增强聚合酶对人工修饰的核苷酸或核苷酸的聚合能力是此类测序方法中的关键步骤。
根据大肠杆菌聚合酶I、II和III的氨基酸序列差异，DNA聚合酶分为三个家族，分别称为A、B和C。虽然A和B聚合酶的核苷酸结合位点在结构上相似，但不同家族聚合酶的基序明显不同，相应地代表了核苷酸及其类似物的识别机制。其中，来自嗜热古细菌的热稳定B家族聚合酶对各种核苷酸类似物的掺入性能最好，其突变体被广泛应用于各种二代测序方法中。嗜热古细菌的热稳定B家族聚合酶包括KOD(Thermococcus kodakaraensis)、9°N(Thermococcus sp.9°N)、TGO(Thermococcus gorgonarius),TOK(Desulfurococcus sp.Tok)、Vent DNA聚合酶(Thermococcus litoralis)、JDF-3和pfu DNA聚合酶(Pyrococcus furiosis)等，它们的DNA聚合酶活性位点的主要氨基酸序列是保守的，但这些保守的酶活性位点也容易改变，可以在保持DNA活性的同时加入非天然特异性dNTP聚合酶。
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。
针对现有测序酶的聚合速度及测序质量(如，Lag(影响测序信号和质量)，链偏等问题)存在的问题。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，可以很好的提高聚合酶对特定的非天然dNTP的掺入效率，进而提高边合成边测序(SBS)方法的测序速度及测序质量。
为此，在本发明的一个方面，本发明提出了一种DNA聚合酶突变体。根据本发明的实施例，所述突变体包括：与Pyrococcus abyssi DNA聚合酶exo-(去除3'–5'外切酶校正活性)突变体相比，在以下四个位点或功能等同位点中具有至少之三个氨基酸突变：第409位、第410位、第411位、第486位；所述Pyrococcus abyssi DNA聚合酶Exo-突变体具有如SEQ ID NO:1所示的氨基酸序列。本发明实施例所述的DNA聚合酶突变体，可以有效提高聚合酶对特定的非天然dNTP的掺入效率，进而改善SBS测序方法的测序速度及测序质量方面具有重要意义。
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参照野生型Pyrococcus abyssi DNA聚合酶或野生型Pyrococcus abyssi DNA聚合酶exo-突变体的氨基酸序列中氨基酸的位置进行定位。
根据本发明的实施例，上述DNA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DNA聚合酶突变体与所述Pyrococcus abyssi DNA聚合酶exo-突变体具有至少90％的同一性。
根据本发明的实施例，突变体具有以下突变：
(1)第409位氨基酸L突变为A或V或Y或H或F或Q；
(2)第410位氨基酸Y突变为A或G；
(3)第411位氨基酸P不突变或突变为V或G或C或S；
(4)第486位氨基酸A突变为I或K或L或M或W或F。
根据本发明的实施例，所述突变体具有以下(1)-(2)任意一种组合突变：
(1)第410位氨基酸Y突变为A，第409位氨基酸L突变为A或V或Y或H或F或Q，第411位氨基酸P突变为V或G或C或S或不突变，第486位氨基酸A突变为I或K或L或M或W或F；或
(2)第410位氨基酸Y突变为G，第409位氨基酸L突变为A或V或Y或H或F或Q，第411位氨基酸P突变为V或G或C或S或不突变，第486位氨基酸A突变为I或K或L或M或W或F。
根据本发明的实施例，所述突变体具有以下(1)-(7)任意一种组合突变：
(1)第410位氨基酸Y突变为A，第409位氨基酸L突变为A，第411位氨基酸P突变为G或C或S或不突变，第486位氨基酸A突变为I或K或L或M或W或F；或
(2)第410位氨基酸Y突变为A，第409位氨基酸L突变为V，第411位氨基酸P突变为V或G或C或不突变，第486位氨基酸A突变为I或L或W或F；或
(3)第410位氨基酸Y突变为A，第409位氨基酸L突变为Y，第411位氨基酸P突变为V或G或不突变，第486位氨基酸A突变为L；或
(4)第410位氨基酸Y突变为A，第409位氨基酸L突变为H，第411位氨基酸P突变为V或G或不突变，第486位氨基酸A突变为L；或
(5)第410位氨基酸Y突变为A，第409位氨基酸L突变为F，第411位氨基酸P突变为V或G或不突变，第486位氨基酸A突变为L；或
(6)第410位氨基酸Y突变为A，第409位氨基酸L突变为Q，第411位氨基酸P突变为G或不突变，第486位氨基酸A突变为L；或
(7)第410位氨基酸Y突变为G，第409位氨基酸L突变为V，第411位氨基酸P不突变，第486位氨基酸A突变为I或K或M或F。
根据本发明的一些具体实施例，不同的突变位点产生的测序重组酶(由本发明所述DNA聚合酶突变体获得)相对活性不同，可以为实际生产需要提供更多的选择。
根据本发明的实施例，所述突变体具有(1)-(36)任意一种组合突变：
(1)第409位的L突变为A，第410位的Y突变为A，第411位的P不突变，第486位的A突变为I；或
(2)第409位的L突变为V，第410位的Y突变为A，第411位的P不突变，第486位的A突变 为L；或
(3)第409位的L突变为A，第410位的Y突变为A，第411位的P不突变，第486位的A突变为W；或
(4)第409位的L突变为V，第410位的Y突变为G，第411位的P不突变，第486位的A突变为K；或
(5)第409位的L突变为V，第410位的Y突变为G，第411位的P不突变，第486位的A突变为I；或
(6)第409位的L突变为A，第410位的Y突变为A，第411位的P不突变，第486位的A突变为M；或
(7)第409位的L突变为V，第410位的Y突变为G，第411位的P不突变，第486位的A突变为F；或
(8)第409位的L突变为V，第410位的Y突变为A，第411位的P突变为V，第486位的A突变为L；或
(9)第409位的L突变为A，第410位的Y突变为A，第411位的P不突变，第486位的A突变为F；或
(10)第409位的L突变为A，第410位的Y突变为A，第411位的P突变为G，第486位的A突变为I；或
(11)第409位的L突变为A，第410位的Y突变为A，第411位的P突变为C，第486位的A突变为I；或
(12)第409位的L突变为Y，第410位的Y突变为A，第411位的P不突变，第486位的A突变为L；或
(13)第409位的L突变为A，第410位的Y突变为A，第411位的P突变为S，第486位的A突变为K；或
(14)第409位的L突变为Y，第410位的Y突变为A，第411位的P突变为G，第486位的A突变为L；或
(15)第409位的L突变为Y，第410位的Y突变为A，第411位的P突变V，第486位的A突变为L；或
(16)第409位的L突变为H，第410位的Y突变为A，第411位的P不突变，第486位的A突变为L；或
(17)第409位的L突变为F，第410位的Y突变为A，第411位的P不突变，第486位的A突变为L；或
(18)第409位的L突变为A，第410位的Y突变为A，第411位的P不突变，第486位的A突变为L；或
(19)第409位的L突变为A，第410位的Y突变为A，第411位的P突变为C，第486位的A突变为M；或
(20)第409位的L突变为V，第410位的Y突变为G，第411位的P不突变，第486位的A突变为M；或
(21)第409位的L突变为A，第410位的Y突变为A，第411位的P突变为G，第486位的A突变为W；或
(22)第409位的L突变为A，第410位的Y突变为A，第411位的P突变为G，第486位的A突变为M；或
(23)第409位的L突变为V，第410位的Y突变为A，第411位的P不突变，第486位的A突变为W；或
(24)第409位的L突变为V，第410位的Y突变为A，第411位的P突变为C，第486位的A突变为L；或
(25)第409位的L突变为Q，第410位的Y突变为A，第411位的P突变为G，第486位的A突变为L；或
(26)第409位的L突变为H，第410位的Y突变为A，第411位的P突变为G，第486位的A突变为L；或
(27)第409位的L突变为A，第410位的Y突变为A，第411位的P突变为S，第486位的A突变为F；或
(28)第409位的L突变为V，第410位的Y突变为A，第411位的P不突变，第486位的A突变为I；或
(29)第409位的L突变为H，第410位的Y突变为A，第411位的P突变为V，第486位的A突变为L；或
(30)第409位的L突变为A，第410位的Y突变为A，第411位的P突变为S，第486位的A突变为I；或
(31)第409位的L突变为A，第410位的Y突变为A，第411位的P突变为S，第486位的A突变为M；或
(32)第409位的L突变为F，第410位的Y突变为A，第411位的P突变为G，第486位的A突变为L；或
(33)第409位的L突变为V，第410位的Y突变为A，第411位的P突变为G，第486位的A 突变为L；或
(34)第409位的L突变为V，第410位的Y突变为A，第411位的P不突变，第486位的A突变为F；或
(35)第409位的L突变为A，第410位的Y突变为A，第411位的P突变为G，第486位的A突变为F；或
(36)第409位的L突变为F，第410位的Y突变为A，第411位的P突变为V，第486位的A突变为L。
根据本发明的实施例，所述突变体具有以下突变：
(1)第409位的L突变为V，第410位的Y突变为G，第411位的P不突变，第486位的A突变为I；或
(2)第409位的L突变为A，第410位的Y突变为A，第411位的P不突变，第486位的A突变为I；或
(3)第409位的L突变为A，第410位的Y突变为A，第411位的P不突变，第486位的A突变为M；或
(4)第409位的L突变为A，第410位的Y突变为A，第411位的P不突变，第486位的A突变为L；或
(5)第409位的L突变为V，第410位的Y突变为A，第411位的P不突变，第486位的A突变为I；或
(6)第409位的L突变为A，第410位的Y突变为A，第411位的P突变为C，第486位的A突变为F。
根据本发明的一些具体实施例，不同的突变位点产生的测序重组酶(由本发明所述DNA聚合酶突变体获得)相对活性不同，可以为实际生产需要提供更多的选择。
需要说明的是，上述突变体氨基酸序列均在SEQ ID NO:1序列基础上突变获得。
根据本发明的一些具体实施例，当所述DNA聚合酶突变体的氨基酸序列具有上述突变时，相对于现有突变获得的重组聚合酶的相对活性更强，能够有效将含有荧光标记的3’O-可逆终止子非天然dNTP添加到DNA链中，提高测序速度及测序质量。
需要说明的是，基于野生型Pyrococcus abyssi DNA聚合酶无法将特定(如，非天然dNTP含有3’O-可逆终止子)的非天然dNTP加入到DNA链中，因此，发明人通过设计Pyrococcus abyssi DNA聚合酶突变体，进而将特定的非天然dNTP添加到DNA链中。
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。
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。
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。
根据本发明的实施例，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。
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。所述重组细胞用于表达或分泌本发明第一方面所述的DNA聚合酶突变体。
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，本发明提出了一种重组菌株。根据本发明的实施例，所述重组菌株表达本发明第一方面所述的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。
在本发明的第六方面，本发明提出了一种获得DNA聚合酶突变体的方法。根据本发明的实施例，所述方法包括将本发明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。
在本发明的第七方面，本发明提出了一种复合物。根据本发明的实施例，所述复合物包括本发明第一方面所述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。
根据本发明的实施例，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。
在本发明的第八方面，本发明提出了一种核酸合成方法。根据本发明的实施例，所述方法包括：将核酸模板、扩增引物、dNTP与本发明第一方面所述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。
在本发明的第九方面，本发明提出了一种核酸测序的方法。根据本发明的实施例，所述方法包括：将待测核酸模板与本发明第一方面所述的DNA聚合酶突变体和带有荧光标记的3’O-可逆终止子非天然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。
在本发明的第十方面，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DNA聚合酶突变体或第七方面所述的复合物。本发明所述的试剂盒用于高效、准确、快速进行核酸测序。
在本发明的第十一方面，本发明提出了一种第九方面所述核酸测序试剂盒在测序中的用途。根据本发明的实施例，所述试剂盒可用于测序，包括但不限于边合成边测序(SBS)。
在本发明的第十二方面，本发明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。根据本发明的实施例，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显示了本发明实施例1所述Pyrococcus abyssi exo-型DNA聚合酶融合蛋白电泳图。
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。
定义与说明
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。
本申请中，所述如SEQ ID NO:1所示的氨基酸序列为野生型Pyrococcus abyssi DNA聚合酶第141位氨基酸D突变为A，第143位氨基酸E突变为A获得。所述野生型Pyrococcus abyssi DNA聚合酶无核酸外切酶活性。
在本申请中，除非另有说明，术语“同一性”与“同源性”表示含义一致。
本发明的目的是提供一种可提高测序质量的B家族重组聚合酶。该酶是基于古菌Pyrococcus abyssi的DNA聚合酶(Erauso,et al."Pyrococcus abyssi sp.nov.,a new hyperthermophilic archaeon isolated from a deep-sea hydrothermal vent."Archives of microbiology 160(1993):338-349)，具有高活性及高耐热性的特点，但野生型的聚合酶不能有效掺入带有荧光标记及3‘O-可逆终止子(3‘O-reversible terminator)的非天然dNTP。需要通过对相关活性位点的酶工程改造及筛选，才能提高酶对特定的非天然dNTP的掺入效率，以用于SBS测序方法中。
DNA聚合酶突变体
本发明提出了一种DNA聚合酶突变体。本发明的实施例提出的DNA聚合酶突变体，与野生型Pyrococcus abyssi DNA聚合酶exo-突变体具有至少90％同源性的氨基酸序列，且具有以下至少之三位置的突变：第409位、第410位、第411位、第486位。
本申请所述的DNA聚合酶突变体具有高活性，高耐热性的优势，具有提高聚合3’O-可逆终止子非天然dNTP性能，进而提高测序速度及质量。
核酸分子
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DNA聚合酶突变体。
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。
表达载体
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即可。
重组细胞
本发明提出了一种重组细胞，携带前面所述的核酸分子、表达载体或表达DNA聚合酶突变体。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DNA聚合酶突变体。
重组菌株
本发明提出了一种重组菌株，表达前述DNA聚合酶突变体。所述重组菌株可以在短时间内进行大量繁殖，高效制备DNA聚合酶突变体。
获得DNA聚合酶突变体的方法
本发明提出的获得DNA聚合酶突变体的方法，通过将前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DNA聚合酶突变体。
复合物
本发明提出了一种复合物，所述复合物包括前述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。在基因测序领域中，通过检测复合物中的小分子化合物(荧光标记)或大分子(抗体、荧光素)进而确定待测核酸序列。
下面参考具体实施例，对本发明进行描述，需要说明的是，这些实施例仅是描述性的，而不以任何方式限制本发明。
实施例1
根据本发明实施例，基于Pyrococcus abyssi exo-型(深海火球菌exo-突变体)进行DNA聚合酶及其突变体的制备与粗酶活性筛选。具体步骤如下：
1.1重组表达载体的构建及转化
生工生物工程(上海)股份有限公司合成融合C端6x His标签的Pyrococcus abyssi exo-型DNA聚合酶基因(SEQ ID NO:1)的pD441-WT载体质粒及409，410，411，485位点的定点突变引物。
其中，引物设计方式为：5’-反向互补区域(15～21bp)-非互补区域(至少15bp)。使用Phanta Max Super-Fidelity DNA Polymerase(Phanta Max超保真DNA聚合酶)对目标质粒进行扩增。
409，410，411，485位点突变为如下氨基酸：
L409突变至A、F、H、L、Q、S、V、Y；
Y410突变至A、G；
P411突变至A、C、E、F、H、I、K、L、M、N、P、Q、R、S、T、V、W、Y；
A485突变至F、I、L、K、M、W；
对目前已知在其他B家族聚合酶中(9°N、Pfu、Vent等)具有较佳的聚合3’O-可逆终止子非天然dNTP活性的突变体位点组合为9°N聚合酶的L409A-Y410A-P411I-A486L等(专利EP1664287B1、US8460910B2)。此突变体组合将作为Pyrococcus abyssi聚合酶筛选实验的对照突变位点组合(SEQ ID NO:2)；

酶突变体中4个位点的一部分突变组合如1所示，其中对照突变体Mut1组合为L409A-Y410A-P411I-A486L。
表1：

由于扩增产物中包含原始模板质粒，为防止其在转化后形成假阳性转化子，必须在进行重组环化之前进行Dpn I消化，去除甲基化模板质粒。
将重组聚合酶突变体表达载体导入大肠杆菌BL21感受态细胞中，涂抹抗性平板(含卡那50μg/ml)筛选阳性菌落。过夜培养后挑取实验组的单菌落，于5ml LB(含卡那50μg/ml)培养基中，37℃，200rpm/min，过夜培养。次日按1:100稀释量，转接于含150μl新鲜LB(含卡那50μg/ml)培养基96孔细胞板中37℃，220rpm振荡培养至OD600＝0.6。
1.2重组蛋白诱导表达和菌体裂解
在步骤1.1的96孔板中，按终浓度0.5mM加入IPTG(异丙基硫代半乳糖苷)，25℃，220rpm/min，培养12-16h，诱导表达。之后加入1μL 100mM PMSF(蛋白酶抑制剂)，另加5μL 10mg/ml溶菌酶，用枪头轻轻吹打，混匀，于37℃反应10min，获得菌体裂解粗酶液。
1.3Fret重组蛋白和非天然dNTP掺入效率测试
在步骤1.2获得的粗酶液中加入AF532荧光染料标记的3’O-阻断修饰的dATP(华大智造US10988501B2专利)和Cy5荧光染料标记的DNA链(生工生物工程(上海)股份有限公司)，序列信息为：
F：Cy5-CGTGTATGCGTAATAGGATCCCGACTCACTATGGACG；
R：Cy5-CGTGTATCGTCCATAGTGAGTCGGGATCCTATTACGC。
模拟高通量测序过程中参入修饰核苷酸，利用酶标仪来检测聚合酶突变体的相对反应速率，计算粗酶活性，具体实验方法如下：
反应缓冲液：20mM Tris-HCl、10mM(NH4)2SO4、10mM KCl、2mM MgSO4pH 8.5@25℃；
反应体系为：步骤1.2中得到的不同的1μg聚合酶蛋白分别与含有2μM修饰dATP和1μM的DNA-Cy5的反应液混合；
反应液配方：20mM Tris-HCl、10mM(NH4)2SO4、10mM KCl、2mM MgSO4、2uM的3’阻断修饰的dATP和1μM的DNA-Cy5，余量为水，pH 8.5；
反应温度：60℃；
反应时间：1h。
反应完成后导出数据表格和酶活性曲线，计算粗酶对比对照酶突变体P1的相对活性Relative Vmax/Km。部分结果如表1所示，Pyrococcus abyssi exo-型DNA聚合酶野生型P0(WT-Exo-)不能掺入带AF532荧光染料标记的3’O-阻断修饰的dATP，而部分突变体蛋白可以掺入，产生了荧光信号值。从实验结果可以看出，不同的突变体对修饰的3’阻断的dNTP有不同聚合活性，表明在不同位点进行了有效突变和位点组合，列表中Ralative Vmax/Km值高于对照的突变位点组合均可视为能高效掺入3’阻断dNTP的突变体。表明这些突变体在高通量测序中具有很大应用前景。
实施例2
根据本发明的实施例，基于Pyrococcus abyssi exo-型(深海火球菌exo-突变体)进行DNA聚合酶突变体融合蛋白的表达和纯化。具体步骤如下：
1)挑取突变体的BL21/pD441单菌落，于50ml LB液体培养集中(含卡那50μg/ml)中，37℃，220rpm/min，过夜培养。次日按1:100稀释量，转接于1000ml LB液体培养基中(含卡那50μg/ml)，37℃，220rpm/min振荡培养至OD600为0.5-0.8，加入终浓度为0.5mM的IPTG，25℃过夜诱导培养，收集诱导后BL21/pD441菌液。同时设不加IPTG作为空白对照，收集未诱导BL21/pD441菌液。
2)将步骤1)诱导后的BL21/pD441菌液，在转速为8000rpm/min离心10min，弃上清，收集沉淀菌体，重悬菌体细胞于缓冲液1(50mM KPO4，500mM NaCl，10mM imidazole(咪唑)，5％Glycerol(甘油)，pH 7.0)中，另加入PMSF(终浓度0.5mM)、Triton X-100(终浓度0.5％)、Lysozyme(溶菌酶，终浓度0.25％)，后室温孵育30min，12000rpm/min、4℃离心30min，冰浴，超声破碎细胞；再以12000rpm/min的转速离心30min，将离心所得上清液于75℃水浴20min，期间注意定时混匀，使受热均匀；12000rpm/min，4℃离心30min，将上清液用0.22μm滤膜过滤，即得融合蛋白粗提物。
3)将步骤2)获得融合蛋白粗提物以适当流速上样进行Ni柱亲和层析(亲和层析预装柱HisTrap FF，5ml，17-5255-01，GE healthcare)，上样后利用缓冲液1平衡5CV(柱体积)；3％缓冲液2(50mM KPO4，1M NaCl，5％Glycerol，pH 7.0)洗脱5CV；50％缓冲液2洗脱5CV，收集大于等于100mAU峰值对应的Ni柱亲和层析洗液。
4)将大于等于100mAU峰值对应的洗脱液按一定的流速上样进行离子交换层析(离子交换预装柱HiTrap Q HP，5ml，17-1154-01，GE healthcare)，上样后利用缓冲液2平衡5CV，0％缓冲液2→60％缓冲液2线性洗脱，收集大于等于100mAU峰值对应的离子交换层析洗脱液。
5)将大于等于100mAU峰值对应的离子交换层析洗脱液进行凝胶层析(凝胶层析预装柱HiPrep Sephacryl S-100HR，26mm，17-1194-01，GE healthcare)，先用20％乙醇洗3CV，水洗3CV，利用100％缓冲液3(20mM Tris，200mM KCl，0.2mM EDTA，10％Glycerol，pH 7.4)平衡3CV后上样，再用缓冲液3洗脱1.5CV，收集洗脱液即为纯化后野生型DNA聚合酶融合蛋白。
6)将Pyrococcus abyssi exo-型DNA聚合酶融合蛋白进行SDS-PAGE(浓缩胶为5％分离胶为12％)，结果如图1所示，1泳道为蛋白Marker(PageRuler Prestained Protein Ladder，26616，Thermo Scientific)，2泳道为1ul融合蛋白粗提物上清液，3泳道为1μl 1mg/ml Ni柱亲和层析纯化后的Pyrococcus abyssi exo-型DNA聚合酶突变体融合蛋白。可以看出，泳道2和泳道3中蛋白大小约90KDa，与文献(Dietrich,J.,Schmitt,P.,Zieger,M.,Preve,B.,Rolland,J.L.,Chaabihi,H.,&Gueguen,Y.(2002).PCR performance of the highly thermostable proof-reading B-type DNA polymerase from Pyrococcus abyssi.FEMS microbiology letters,217(1),89-94.)报道分子量一致。对电泳后的蛋白胶利用Quantity one软件分析蛋白纯度，纯化后DNA聚合酶突变体融合蛋白纯度能达到85％或以上。未诱导BL21/pD441菌液未得到约90KDa大小的目的蛋白。
实施例3
根据本发明的实施例，基于Pyrococcus abyssi exo-型(深海火球菌exo-突变体)进行DNA聚合酶突变体纯化酶在测序仪中的实际测序性能测试。其中，优势突变体的纯化酶在华大的MGISEQ-2000测序仪上进行了测序尝试，并且对测序结果进行了分析，具体实验步骤如下：
测序文库：Ecoli.fa文库(Part No.1000005038,MGI Tech co.,Ltd)；
测序条件：聚合时间90秒，切除60秒。
使用相同的测序文库Ecoli.fa文库和测序条件，挑选了对照P1突变体以及其中5个在酶活测试中表现不一的突变体进行上机测试，这些突变体在MGISEQ-2000测序仪上进行了100个循环的测序测试(SE100)。测试结果如表2所示，表明优势突变体的纯化酶再实际测序中性能更佳。
表2：

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 (21)

  1. 一种DNA聚合酶突变体，其特征在于，
    与Pyrococcus abyssi DNA聚合酶exo-突变体相比，在以下四个位点或功能等同位点中具有至少三个氨基酸突变：
    第409位、第410位、第411位、第486位；
    所述Pyrococcus abyssi DNA聚合酶exo-突变体具有如SEQ ID NO:1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DNA聚合酶突变体与所述Pyrococcus abyssi DNA聚合酶exo-突变体具有至少90％的同一性。
  3. 根据权利要求1所述的DNA聚合酶突变体，其特征在于，所述突变体具有以下突变：
    (1)第409位氨基酸L突变为A或V或Y或H或F或Q；
    (2)第410位氨基酸Y突变为A或G；
    (3)第411位氨基酸P不突变或突变为V或G或C或S；
    (4)第486位氨基酸A突变为I或K或L或M或W或F。
  4. 根据权利要求3所述的DNA聚合酶突变体，其特征在于，所述突变体具有以下(1)-(2)任意一种组合突变：
    (1)第410位氨基酸Y突变为A，第409位氨基酸L突变为A或V或Y或H或F或Q，第411位氨基酸P突变为V或G或C或S或不突变，第486位氨基酸A突变为I或K或L或M或W或F；或
    (2)第410位氨基酸Y突变为G，第409位氨基酸L突变为A或V或Y或H或F或Q，第411位氨基酸P突变为V或G或C或S或不突变，第486位氨基酸A突变为I或K或L或M或W或F。
  5. 根据权利要求3所述的DNA聚合酶突变体，其特征在于，所述突变体具有以下(1)-(7)任意一种组合突变：
    (1)第410位氨基酸Y突变为A，第409位氨基酸L突变为A，第411位氨基酸P突变为G或C或S或不突变，第486位氨基酸A突变为I或K或L或M或W或F；或
    (2)第410位氨基酸Y突变为A，第409位氨基酸L突变为V，第411位氨基酸P突变为V或G或C或不突变，第486位氨基酸A突变为I或L或W或F；或
    (3)第410位氨基酸Y突变为A，第409位氨基酸L突变为Y，第411位氨基酸P突变为V或G或不突变，第486位氨基酸A突变为L；或
    (4)第410位氨基酸Y突变为A，第409位氨基酸L突变为H，第411位氨基酸P突变为V或G或不突变，第486位氨基酸A突变为L；或
    (5)第410位氨基酸Y突变为A，第409位氨基酸L突变为F，第411位氨基酸P突变为V或G 或不突变，第486位氨基酸A突变为L；或
    (6)第410位氨基酸Y突变为A，第409位氨基酸L突变为Q，第411位氨基酸P突变为G或不突变，第486位氨基酸A突变为L；或
    (7)第410位氨基酸Y突变为G，第409位氨基酸L突变为V，第411位氨基酸P不突变，第486位氨基酸A突变为I或K或M或F。
  6. 根据权利要求4所述的DNA聚合酶突变体，其特征在于，所述突变体具有(1)-(36)任意一种组合突变：
    (1)第409位的L突变为A，第410位的Y突变为A，第411位的P不突变，第486位的A突变为I；或
    (2)第409位的L突变为V，第410位的Y突变为A，第411位的P不突变，第486位的A突变为L；或
    (3)第409位的L突变为A，第410位的Y突变为A，第411位的P不突变，第486位的A突变为W；或
    (4)第409位的L突变为V，第410位的Y突变为G，第411位的P不突变，第486位的A突变为K；或
    (5)第409位的L突变为V，第410位的Y突变为G，第411位的P不突变，第486位的A突变为I；或
    (6)第409位的L突变为A，第410位的Y突变为A，第411位的P不突变，第486位的A突变为M；或
    (7)第409位的L突变为V，第410位的Y突变为G，第411位的P不突变，第486位的A突变为F；或
    (8)第409位的L突变为V，第410位的Y突变为A，第411位的P突变为V，第486位的A突变为L；或
    (9)第409位的L突变为A，第410位的Y突变为A，第411位的P不突变，第486位的A突变为F；或
    (10)第409位的L突变为A，第410位的Y突变为A，第411位的P突变为G，第486位的A突变为I；或
    (11)第409位的L突变为A，第410位的Y突变为A，第411位的P突变为C，第486位的A突变为I；或
    (12)第409位的L突变为Y，第410位的Y突变为A，第411位的P不突变，第486位的A突 变为L；或
    (13)第409位的L突变为A，第410位的Y突变为A，第411位的P突变为S，第486位的A突变为K；或
    (14)第409位的L突变为Y，第410位的Y突变为A，第411位的P突变为G，第486位的A突变为L；或
    (15)第409位的L突变为Y，第410位的Y突变为A，第411位的P突变V，第486位的A突变为L；或
    (16)第409位的L突变为H，第410位的Y突变为A，第411位的P不突变，第486位的A突变为L；或
    (17)第409位的L突变为F，第410位的Y突变为A，第411位的P不突变，第486位的A突变为L；或
    (18)第409位的L突变为A，第410位的Y突变为A，第411位的P不突变，第486位的A突变为L；或
    (19)第409位的L突变为A，第410位的Y突变为A，第411位的P突变为C，第486位的A突变为M；或
    (20)第409位的L突变为V，第410位的Y突变为G，第411位的P不突变，第486位的A突变为M；或
    (21)第409位的L突变为A，第410位的Y突变为A，第411位的P突变为G，第486位的A突变为W；或
    (22)第409位的L突变为A，第410位的Y突变为A，第411位的P突变为G，第486位的A突变为M；或
    (23)第409位的L突变为V，第410位的Y突变为A，第411位的P不突变，第486位的A突变为W；或
    (24)第409位的L突变为V，第410位的Y突变为A，第411位的P突变为C，第486位的A突变为L；或
    (25)第409位的L突变为Q，第410位的Y突变为A，第411位的P突变为G，第486位的A突变为L；或
    (26)第409位的L突变为H，第410位的Y突变为A，第411位的P突变为G，第486位的A突变为L；或
    (27)第409位的L突变为A，第410位的Y突变为A，第411位的P突变为S，第486位的A 突变为F；或
    (28)第409位的L突变为V，第410位的Y突变为A，第411位的P不突变，第486位的A突变为I；或
    (29)第409位的L突变为H，第410位的Y突变为A，第411位的P突变为V，第486位的A突变为L；或
    (30)第409位的L突变为A，第410位的Y突变为A，第411位的P突变为S，第486位的A突变为I；或
    (31)第409位的L突变为A，第410位的Y突变为A，第411位的P突变为S，第486位的A突变为M；或
    (32)第409位的L突变为F，第410位的Y突变为A，第411位的P突变为G，第486位的A突变为L；或
    (33)第409位的L突变为V，第410位的Y突变为A，第411位的P突变为G，第486位的A突变为L；或
    (34)第409位的L突变为V，第410位的Y突变为A，第411位的P不突变，第486位的A突变为F；或
    (35)第409位的L突变为A，第410位的Y突变为A，第411位的P突变为G，第486位的A突变为F；或
    (36)第409位的L突变为F，第410位的Y突变为A，第411位的P突变为V，第486位的A突变为L。
  7. 根据权利要求1所述的DNA聚合酶突变体，其特征在于，所述突变体具有以下突变：
    (1)第409位的L突变为V，第410位的Y突变为G，第411位的P不突变，第486位的A突变为I；或
    (2)第409位的L突变为A，第410位的Y突变为A，第411位的P不突变，第486位的A突变为I；或
    (3)第409位的L突变为A，第410位的Y突变为A，第411位的P不突变，第486位的A突变为M；或
    (4)第409位的L突变为A，第410位的Y突变为A，第411位的P不突变，第486位的A突变为L；或
    (5)第409位的L突变为V，第410位的Y突变为A，第411位的P不突变，第486位的A突变为I；或
    (6)第409位的L突变为A，第410位的Y突变为A，第411位的P突变为C，第486位的A突变为F。
  8. 一种核酸分子，其特征在于，所述核酸分子编码权利要求1～7任一项所述的DNA聚合酶突变体。
  9. 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。
  10. 根据权利要求9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；
    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。
  11. 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所述的表达载体。
  12. 根据权利要求11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。
  13. 一种重组菌株，其特征在于，所述重组菌株表达权利要求1～7任一项所述的DNA聚合酶突变体。
  14. 一种获得DNA聚合酶突变体的方法，其特征在于，包括：将权利要求11～12任一项所述的重组细胞或权利要求13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。
  15. 一种复合物，其特征在于，包括权利要求1～7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。
  16. 根据权利要求15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。
  17. 一种核酸合成方法，其特征在于，包括：
    将核酸模板、扩增引物、dNTP与权利要求1～7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。
  18. 一种核酸测序的方法，其特征在于，包括：
    将待测核酸与权利要求1～7任一项所述DNA聚合酶突变体和带有荧光标记的3’O-可逆终止子非天然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。
  19. 一种核酸测序试剂盒，其特征在于，包括权利要求1～7任一项所述的DNA聚合酶突变体或权利要求15或16所述的复合物。
  20. 权利要求19所述核酸测序试剂盒在测序中的用途。
  21. 权利要求1～7任一项所述的DNA聚合酶突变体、权利要求8所述的核酸分子、权利要求9～10 任一项所述表达载体、权利要求11～12任一项所述的重组细胞、权利要求13所述的重组菌株或权利要求15～16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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