WO2020047695A1 - 重组型kod聚合酶 - Google Patents

重组型kod聚合酶 Download PDF

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WO2020047695A1
WO2020047695A1 PCT/CN2018/103764 CN2018103764W WO2020047695A1 WO 2020047695 A1 WO2020047695 A1 WO 2020047695A1 CN 2018103764 W CN2018103764 W CN 2018103764W WO 2020047695 A1 WO2020047695 A1 WO 2020047695A1
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dna polymerase
protein
amino acid
replace
kod
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PCT/CN2018/103764
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English (en)
French (fr)
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翟莉莉
王林
章文蔚
董宇亮
郑越
刘芬
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深圳华大生命科学研究院
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Priority to US17/270,489 priority Critical patent/US20210324353A1/en
Priority to PCT/CN2018/103764 priority patent/WO2020047695A1/zh
Priority to CN201880096312.3A priority patent/CN112639089B/zh
Priority to EP18932681.2A priority patent/EP3862427A4/en
Priority to CN202311853075.XA priority patent/CN117802065A/zh
Priority to PCT/CN2019/102493 priority patent/WO2020048329A1/zh
Priority to CN201980051755.5A priority patent/CN112585264B/zh
Publication of WO2020047695A1 publication Critical patent/WO2020047695A1/zh

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    • C—CHEMISTRY; METALLURGY
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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
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • 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)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1252—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
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    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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    • 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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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/07—Nucleotidyltransferases (2.7.7)
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag

Definitions

  • the invention relates to the field of biotechnology, in particular to a recombinant KOD polymerase.
  • DNA polymerase The role of DNA polymerase is to quickly and accurately replicate DNA. It is important for living organisms to accurately transmit genetic information and maintain the stability of genetic material.
  • DNA polymerases There are five types of DNA polymerases in E. coli, which are divided into A, B, C and other types according to the sequence homology of the DNA polymerase.
  • the overall folding structure of DNA polymerase is similar to a human hand, and is divided into three parts: palm, thumb and finger. Although the structure of the finger and palm domains of different types of DNA polymerases will vary greatly, the differences in the catalyzed palm area are relatively different. small.
  • the main amino acid sequence of the active site of the DNA polymerase is conserved, but these conserved active sites of the enzyme are also relatively easy to change, and it is possible to add a non-natural specific dNTP while keeping the activity of the DNA polymerase not significantly reduced (see Patel Et al., Patent No. 6602695).
  • KOD DNA polymerase belongs to B-family DNA polymerase, which can quickly and accurately replicate DNA. It is a heat-resistant DNA polymerase with a wide range of applications.
  • One of the most important applications is in genome sequencing, such as SBS sequencing.
  • the SBS sequencing method uses a nucleotide having a modification at the 3 'sugar hydroxyl group, thereby blocking the addition of other nucleotides.
  • the use of a nucleotide with a 3 ' blocking group allows the nucleotide to be incorporated into the polynucleotide chain in a controlled manner. After each nucleotide is added, the presence of a 3 ' blocking group prevents other nucleotides from being added to the strand.
  • An object of the present invention is to provide a recombinant KOD polymerase.
  • the recombinant KOD polymerase provided by the present invention is a protein and is A) or B) as follows:
  • the protein shown is the 675, 385, 710, 674, 735, 736, 606, 709, 347, 349, 590, 676, 389, 589, 680, 384 in the amino acid sequence of the wild-type KOD DNA polymerase. Modification of at least one of the 18, 496 and 383 amino acid residues to obtain a protein having DNA polymerase activity;
  • the protein shown in B) is a protein derived from A) in which a tag sequence is added to the end of the amino acid sequence of the protein shown in A) and has DNA polymerase activity.
  • the protein shown in A) is the 675, 385, 710, 674, 735, 736, 606, 709, 347, 349, 590, 676, 389, 589
  • the amino acid residues of at least two of the 18th, 680, 384, 496, and 383 positions were modified to obtain a protein having DNA polymerase activity.
  • the protein shown in A) is the 675, 385, 710, 674, 735, 736, 606, 709, 347, 349, 590, 676, 389, 589
  • the amino acid residues at least 3 of the 18th, 680, 384, 496 and 383 positions were modified to obtain a protein having DNA polymerase activity.
  • the protein shown in A) is the 675, 385, 710, 674, 735, 736, 606, 709, 347, 349, 590, 676, 389, 589
  • the amino acid residues at least 4 of 18, 680, 384, 496, and 383 were modified to obtain a protein having DNA polymerase activity.
  • the protein shown in A) is the 675, 385, 710, 674, 735, 736, 606, 709, 347, 349, 590, 676, 389, 589
  • At least five of the 18th, 680, 384, 496, and 383 amino acid residues are modified to obtain a protein having DNA polymerase activity.
  • the protein shown in A) is the 675, 385, 710, 674, 735, 736, 606, 709, 347, 349, 590, 676, 389, 589
  • the amino acid residues of at least 6 of 18, 680, 384, 496, and 383 were modified to obtain a protein having DNA polymerase activity.
  • the modification is an amino acid substitution.
  • amino acid substitution is at least one of the following:
  • valine at position 389 with isoleucine or methionine
  • valine at position 589 with histidine or glutamine;
  • the serine at position 383 was replaced with threonine.
  • amino acid sequence of the wild-type KOD-type DNA polymerase is SEQ ID NO: 1.
  • the protein shown in A) above is a KOD-type DNA polymerase point mutant, specifically a single-point mutant in Table 1 in the Examples or a multi-point mutant in Table 2 in the Examples.
  • DNA molecules encoding the aforementioned proteins are also within the scope of the present invention.
  • An expression cassette, a recombinant vector, a recombinant bacterium, or a transgenic cell line containing the aforementioned DNA molecule is also within the scope of the present invention.
  • the product is a kit.
  • the invention Based on the kinetic simulation and statistical inference of KOD polymerase, the invention obtains mutation sites that can be used for experimental screening. And design experimental schemes, through semi-rational design, enzyme variant library construction and high-throughput screening to improve the catalytic and physical and chemical properties of polymerases, and develop DNA polymerases suitable for DNB sequencing work attached to the chip surface. Finally, the recombinant DNA polymerase was isolated and purified.
  • Figure 1 is a schematic diagram of the structure of a recombinant KOD DNA polymerase.
  • Figure 2 shows the purity detection of wild-type KOD DNA polymerase fusion protein.
  • Figure 3 shows the Mie curve of the wild-type and recombinant KOD DNA polymerase fusion protein.
  • the KOD DNA polymerase and its mutants were constructed using the DNA 2.0 Electra TM Cloning Reagents Kit kit, and the His tag was used for Ni column affinity purification.
  • amino acid sequence of the wild-type KOD DNA polymerase is sequence 1 in the following sequence listing, and its coding gene sequence is sequence 2 in the following sequence listing.
  • the wild-type recombinant expression vector pD441-WT is a gene encoding a wild-type KOD-type DNA polymerase fused to a his-tag and recombined into the vector pD441-pelB according to the instructions of the Electra TM Cloning Reagents Kit (DNA2.0, EKT-02) kit.
  • DNA2.0, pD441-pelB the obtained vector is fused with a His-tagged wild-type KOD DNA polymerase-encoding gene, and expression is guided by a signal peptide on the vector pD441-pelB.
  • the nucleotide sequence of the His-tagged wild-type KOD-type DNA polymerase-encoding gene is a sequence obtained by ligating 6 His-tag codons to the 3 'end of Sequence 2.
  • the amino acid sequence of the wild-type KOD-type DNA polymerase fusion protein is obtained by linking the C-terminus of the amino acid shown in Sequence 1 with 6 His tags, as shown in Figure 1, where 1 is the signal peptide pelB, 2 is the fusion protein purification tag His, and 3 is Wild-type KOD DNA polymerase.
  • the wild-type recombinant expression vector pD441-WT was introduced into E. coli BL21 competent cells (purchased from Quanshijin Biotechnology Co., Ltd.), and smeared on a plate containing kanamycin 50 ⁇ g / ml to screen positive colonies. Three to five positive colonies were selected, and primers Cloning-F (sequence 3 in the sequence listing) and Cloning-R (sequence 4 in the sequence listing) were used to identify the selected positive colonies by PCR. A 2800 bp fragment was obtained that was basically consistent with the expected theoretical value. After sequencing, it was determined to be a positive clone and named BL21 / pD441-WT.
  • a single BL21 / pD441-WT colony was picked and cultured overnight in a 50 ml LB liquid medium (containing Kan 50 ⁇ g / ml) at 37 ° C, 220 rpm / min. The next day at a dilution of 1: 100, transfer to 1LLB liquid medium (containing Kan50 ⁇ g / ml), incubate at 37 ° C, 220rpm / min in a shaker to an OD600 of 0.5-0.8, and add IPTG to a final concentration of 0.5mM. The fusion protein was induced to express at 25 ° C overnight, and the BL21 / pD441-WT bacterial solution was obtained after induction. The blank control group did not add IPTG.
  • the crude cell extract was loaded at an appropriate flow rate for Ni column affinity chromatography (HiTrap FF prepacked column, 5ml, 17-5255-01, GE healthcare). After loading, 5CV was equilibrated with buffer1; 3% Buffer 2 (50 mM KPO 4 , 1M NaCl, 5% Glycerol, pH 7.0) elutes 5 CV; 50% buffer 2 elutes 5 CV, and collects Ni column affinity chromatography eluent corresponding to a peak of 100 mAU or more.
  • Buffer 2 50 mM KPO 4 , 1M NaCl, 5% Glycerol, pH 7.0
  • the ion exchange chromatography eluate corresponding to a peak of 100 mAU or more was subjected to gel chromatography (gel chromatography pre-packed column HiPrepSephacryl S-100HR, 26mm, 17-1194-01, GE healthcare), and then washed with 20% ethanol 3CV, wash 3CV with water, use 100% buffer 3 (20mM Tris, 200mM KCl, 0.2mM EDTA, 10% Glycerol, PH7.4) to equilibrate 3CV and load, and then use buffer 3 to elute 1.5CV.
  • the eluate is collected after purification Wild-type KOD-type DNA polymerase fusion protein.
  • the purified wild-type KOD DNA polymerase fusion protein was subjected to SDS-PAGE (concentrated gel was 5% and separated gel was 12%).
  • the protein sample was mixed with SDS-PAGE protein loading buffer (5X), and treated at 95 ° C for 5 min. To load.
  • 1 is a protein marker (PageRulerPrestainedProladin, Adder, 26616, Thermo Scientific)
  • 2 is 10 ⁇ l purified wild-type KOD DNA polymerase fusion protein at 1 mg / ml
  • 3 is 0.05 mg diluted 10-fold 20 times / ml purified wild-type KOD DNA polymerase fusion protein. It can be seen from the results of protein electrophoresis that the protein size in lanes 2 and 3 is about 91.5 KDa, which is consistent with the molecular weight reported in the literature.
  • the protein gel after electrophoresis was analyzed for protein purity using Quantity software. After purification, the purity of the wild-type KOD DNA polymerase fusion protein can reach 95% or more.
  • BL21 / pD441-WT did not induce the target protein with a size of about 91.5 KDa.
  • the control group introduced the empty vector pD441-pelB into E. coli BL21 to obtain BL21 / pD441-pelB.
  • the protein was expressed and purified by the above method, and the target protein having a size of about 91.5 KDa was not obtained.
  • KOD DNA polymerase mutant (amino acid sequence is sequence A) is the amino acid sequence (sequence 1) of the wild-type KOD DNA polymerase (sequence 1) Nos. 675, 385, 710, 674, 735, 736, 606, 709, 347, 349, 590 , 676, 389, 589, 680, 384, 496, and 383 have at least one amino acid substituted for the amino acid to obtain the protein; if there is only one amino acid substitution, the KOD DNA polymerase single point mutant is obtained, if there are two amino acids The two-point combination mutant of KOD DNA polymerase is obtained by substitution. If three amino acid substitutions are obtained, the three-point combination mutant of KOD DNA polymerase is obtained, and so on.
  • the KOD DNA polymerase mutant encoding gene (nucleotide sequence is sequence B) is the nucleotide sequence (sequence 2) of the wild-type KOD DNA polymerase encoding gene in accordance with paragraphs 675, 385, 710, 674, and 735 of Sequence 1.
  • a nucleic acid obtained by mutating at least one amino acid codon of 736, 606, 709, 347, 349, 590, 676, 389, 589, 680, 384, 496, and 383.
  • the KOD DNA polymerase mutant can be obtained by site-directed mutagenesis using wild-type KOD DNA polymerase, or it can be obtained by other existing methods.
  • Recombinant vectors expressing different KOD-type DNA polymerase mutants were obtained by recombining His-tagged different KOD-type DNA polymerase mutant protein-encoding genes to the vector pD441-pelB, and the resulting vectors were fused with His-tag different point mutants.
  • the protein-coding gene is guided by a signal peptide on the vector pD441-pelB for expression.
  • each KOD DNA polymerase mutant fusion protein is obtained by ligating 6 His tags to the C-terminus of the KOD DNA polymerase mutant shown in Sequence A; Sequence A is the wild-type KOD shown in Sequence 1. DNA polymerase amino acid sequence mutation sequence.
  • sequence B is the sequence obtained by mutating the corresponding amino acid codon in the nucleotide sequence of the wild-type KOD DNA polymerase shown in Sequence 2.
  • Table 1 shows the mutation positions and mutation information of KOD DNA polymerase single point mutants.
  • a KOD type DNA polymerase mutant using a multipoint mutant as an example is shown in Table 2 below.
  • the recombinant vector expressing different KOD type DNA polymerase mutants prepared in the above 1 was introduced into BL21 to obtain recombinant bacteria expressing different KOD type DNA polymerase mutant fusion proteins.
  • Different KOD type DNA polymerase mutant fusion proteins were subjected to SDS-PAGE (concentrated gel was 5% and separated gel was 12%) to obtain the target protein. For protein gels after electrophoresis. For the analysis of protein purity using Quantity software, the purity of different KOD DNA polymerase mutant fusion proteins can reach 95% or more.
  • the reaction solution was: 20 mM Tris-HCl (pH 7.5), 8 mM MgCl 2 , 50 ⁇ g / ml BSA, 0.15 mM each dNTPs (dATP, dTTP, dCTP, dGTP), [methyl-3H] -TTP (0.13mCi / nmol, final concentration, company MPBIO-FINE, catalog number CB2272108), 150mg / ml activated calf thymus DNA (company SIGMA-ALDRICH, product number GE27-4575-01), 7.5mM DTT, 1 ⁇ L wild-type KOD DNA polymerase (1mg / ml), the total reaction volume is 50 ⁇ l.
  • the above reaction solution was reacted at 75 ° for 30 minutes, and then the polymerization activity of the wild-type KOD-type DNA polymerase was calculated according to the definition of the enzyme activity to be 2 U / ⁇ l.
  • the KOD DNA polymerase mutants are the KOD DNA polymerase multipoint mutants in Table 2: AB60, AB70, AB71, CD72, CD73, EF74, EF75, EF76, GH77, GH78;
  • Table 2 is a KOD type DNA polymerase multipoint mutant
  • AB60 has an amino acid sequence in which arginine at position 709 of sequence 1 is mutated to serine, and valine at position 589 is mutated to histidine;
  • the amino acid sequence of AB70 is to mutate threonine at position 676 to lysine, valine at position 589 to histidine; valine at position 680 to methionine;
  • AB71 has an amino acid sequence in which the lysine at position 674 of sequence 1 is mutated to leucine, the valine at position 589 is mutated to histidine; the valine at position 680 is mutated to methionine;
  • the amino acid sequence of CD72 is to mutate threonine at position 676 to lysine, valine at position 589 to histidine, valine at position 680 to methionine, and 384 at Mutated to phenylalanine;
  • the amino acid sequence of CD73 is that the valine at position 389 of sequence 1 is mutated to isoleucine, and the lysine at position 674 is mutated to leucine; the valine at position 589 is mutated to histidine; Mutation to methionine;
  • the amino acid sequence of EF74 is to mutate lysine at position 674 to leucine, valine at position 589 to histidine, valine at position 680 to methionine, and serine at position 383. Mutation to threonine; tyrosine at position 384 to phenylalanine;
  • the amino acid sequence of EF75 is that the threonine at position 349 in sequence 1 is mutated to phenylalanine, the valine at position 589 is mutated to histidine; the threonine at position 676 is mutated to lysine; Mutation to methionine; tyrosine at position 496 to leucine;
  • the amino acid sequence of EF76 is to mutate valine position 589 of sequence 1 to histidine, mutine 676 position to lysine, valine position 680 to methionine, and serine position 383. Mutation to threonine; tyrosine at position 384 to phenylalanine;
  • the amino acid sequence of GH77 is to mutate valine at position 589 of sequence 1 to histidine, mutate hr at position 676 to lysine, mutate valine at position 680 to methionine, and serine at position 383. Mutation to threonine; tyrosine at position 384 to phenylalanine; arginine at position 709 to serine;
  • the amino acid sequence of GH78 is to mutate valine position 389 of sequence 1 to isoleucine, valine position 589 to histidine; 676 threonine to lysine; 680 position Valine is mutated to methionine; serine at position 383 is mutated to threonine; tyrosine at position 384 is mutated to phenylalanine.
  • the KOD-type DNA polymerase mutant fusion protein added 6 his tags to the C-terminus of each KOD-type DNA polymerase mutant.
  • This example uses Cy3 fluorescent dye-labeled dATP (dATP-Cy3) and Cy5 fluorescent dye-labeled substrate (P / T-2Cy5) to detect the relative reaction rate of recombinant KOD DNA polymerase mutants using a microplate reader.
  • dATP-Cy3 Cy3 fluorescent dye-labeled dATP
  • P / T-2Cy5 Cy5 fluorescent dye-labeled substrate
  • the 5'Cy5 fluorescently labeled single-stranded primers P1A (sequence 5 in the sequence listing) and P2A (sequence 6 in the sequence listing) were mixed in a 1: 1 mixture, and then kept at 65 ° C for 10 minutes, and then naturally cooled down to room temperature. The annealed product was stored at -20 ° C in the dark to obtain Cy5 fluorescent dye-labeled substrate P / T-2Cy5.
  • BioTek microplate reader was used for enzyme activity detection.
  • the reaction was performed in 384 plates (Corning black, clear bottom 384 plates).
  • the total volume of the reaction solution was 50 ⁇ l.
  • the specific reaction system is as follows:
  • the reaction system is: 1UKOD polymerase mutant fusion protein, 1 ⁇ M dATP-Cy3, 10 ⁇ M dTTP, 10 ⁇ M dCTP, 10 ⁇ M dGTP, substrate P / T-2Cy5 according to 0.1, 0.2, 0.5, 1, 2, 3, 4, 6 ⁇ M, 8 Experiments were carried out at various concentration gradients.
  • the enzymatic reaction buffer was 20 mM Tris-HCl, 10 mM (NH4) 2SO4, 10 mM KCl, 2 mM MgSO4, pH 8.5@25°C.
  • the enzymatic reaction is in kinetic detection mode. Data are recorded every 30s.
  • the detection conditions are:
  • the data table and enzyme activity curve can be directly derived, and the reaction rate of its relative fluorescence value can be approximated.
  • the response rate to the fluorescence value depends on the concentration of the substrate P / T-2Cy5. Therefore, the activity of the recombinant KOD DNA polymerase mutant can be approximately determined by detecting the activity of the recombinant KOD DNA polymerase mutant under different concentrations of the substrate P / T-2Cy5. Its Km value is the corresponding substrate when the reaction rate reaches half of the maximum reaction rate.
  • KOD-WT wild-type KOD DNA polymerase fusion protein
  • the Michaelis-Menten curve of the KOD DNA polymerase mutant fusion protein is shown in Figure 3 and Table 3. It can be seen that the KOD DNA polymerase mutant mutant has higher polymerization activity than the wild type.
  • the specific experimental results can refer to Table 3 below.
  • the kinetic test results of KOD DNA polymerase mutants show greater potential than wild type. In the sequencing process, the faster the catalytic efficiency of the enzyme reaction, the larger the value of Vmax / Km, which can speed up the reaction to a certain extent and shorten the sequencing reaction time. From the experimental results, it can be seen that in terms of enzyme catalytic efficiency, the KOD DNA polymerase mutant has a better improvement than the wild-type KOD DNA polymerase.
  • the experiment of the present invention proves that this patent aims at improving the sequencing read length, shortening the reaction time and other improvement goals.
  • the KOD DNA polymerase is further modified, and the DNA polymerase B is involved in this area.
  • the original function site is protected to ensure that it can still complete the original basic function.
  • recombinant DNA polymerase has the advantages of stronger reaction rate and better affinity.

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Abstract

提供一种重组型KOD聚合酶，其是如下A）或B）：A）所示的聚合酶为将野生型KOD型DNA聚合酶氨基酸序列中第675、385、710、674、735、736、606、709、347、349、590、676、389、589、680、384、496和383位这18位中至少一位的氨基酸残基进行修饰，得到具有DNA聚合酶活性的蛋白；B）所述的聚合酶为将A）所示蛋白的氨基酸序列末端添加标签序列且具有DNA聚合酶活性的由A）衍生的蛋白质。

Description

重组型KOD聚合酶 技术领域
本发明涉及生物技术领域，尤其涉及一种重组型KOD聚合酶。
背景技术
DNA聚合酶的作用是快速而准确地复制DNA，对于生命体能够精准的传递遗传信息并且维持遗传物质稳定性具有重要的作用。在大肠杆菌中有五种DNA聚合酶，根据DNA聚合酶的序列同源性的不同，又分为A,B,C等几种类型。DNA聚合酶的总折叠结构类似人手，分为手掌、拇指和手指三部分，虽然不同种类的DNA聚合酶的手指和手掌的结构域的结构会有变化会很大，但催化的手掌区域差异较小。DNA聚合酶的活性位点的主要的氨基酸序列是保守的，但是这些保守的酶活性位点也是相对容易改变的，能够加入非天然的特定dNTP而保持DNA聚合酶的活性不显著下降(参见Patel等的专利号6602695)。
KOD DNA聚合酶属于B-family DNA聚合酶，能够快速准确的复制DNA，是一种耐热的DNA聚合酶，应用范围广泛，其中最重要的一个应用就是应用于基因组测序中，例如：SBS测序。SBS测序方法中使用在3’糖羟基具有修饰的核苷酸，从而阻断其它核苷酸的加入。使用带有3’阻断基团的核苷酸允许以受控方式将核苷酸掺入多核苷酸链中。在加入每个核苷酸后，3’阻断基团的存在阻止其它核苷酸加入所述链中。在去除阻断基团后，恢复天然游离的3’羟基基团用于加入下一个核苷酸。但是目前SBS测序也存在很多技术问题，如：测序的读长较短，反应的速率太慢等。KOD聚合酶是针对这些技术问题进行专门改进的酶产品，其去除了野生型KOD聚合酶的3’-5’外切酶活性，并通过对该聚合酶的DNA结合区域、dNTP催化位点，蛋白其他结构域的计算机模拟和预测，筛选出一定量的优化突变位点，并对突变后的KOD聚合酶进行实验筛选，得到一批可用的突变位点信息，并发现可以添加部分非天然氨基酸对KOD聚合酶进行改造，目前此类产品已应用于测序中(参见刘芬等的专利号)。
发明公开
本发明的一个目的提供一种重组型KOD聚合酶。
本发明提供的重组型KOD聚合酶，为一种蛋白，是如下A)或B)：
A)所示的蛋白为将野生型KOD型DNA聚合酶氨基酸序列中第675、385、 710、674、735、736、606、709、347、349、590、676、389、589、680、384、496和383位这18位中至少一位的氨基酸残基进行修饰，得到具有DNA聚合酶活性的蛋白；
B)所示的蛋白为将A)所示蛋白的氨基酸序列末端添加标签序列且具有DNA聚合酶活性的由A)衍生的蛋白质。
上述蛋白中，A)所示的蛋白为将野生型KOD型DNA聚合酶氨基酸序列中第675、385、710、674、735、736、606、709、347、349、590、676、389、589、680、384、496和383位这18位中至少2位的氨基酸残基进行修饰，得到具有DNA聚合酶活性的蛋白。
上述蛋白中，A)所示的蛋白为将野生型KOD型DNA聚合酶氨基酸序列中第675、385、710、674、735、736、606、709、347、349、590、676、389、589、680、384、496和383位这18位中至少3位的氨基酸残基进行修饰，得到具有DNA聚合酶活性的蛋白。
上述蛋白中，A)所示的蛋白为将野生型KOD型DNA聚合酶氨基酸序列中第675、385、710、674、735、736、606、709、347、349、590、676、389、589、680、384、496和383位这18位中至少4位的氨基酸残基进行修饰，得到具有DNA聚合酶活性的蛋白。
上述蛋白中，A)所示的蛋白为将野生型KOD型DNA聚合酶氨基酸序列中第675、385、710、674、735、736、606、709、347、349、590、676、389、589、680、384、496和383位这18位中至少5位的氨基酸残基进行修饰，得到具有DNA聚合酶活性的蛋白。
上述蛋白中，A)所示的蛋白为将野生型KOD型DNA聚合酶氨基酸序列中第675、385、710、674、735、736、606、709、347、349、590、676、389、589、680、384、496和383位这18位中至少6位的氨基酸残基进行修饰，得到具有DNA聚合酶活性的蛋白。
上述蛋白中，所述修饰为氨基酸置换。
上述蛋白中，所述氨基酸置换为如下至少一种：
第675位的丙氨酸置换为苯丙氨酸或甲硫氨酸；
第385位的谷氨酸置换为甲硫氨酸或色氨酸；
第710位的异亮氨酸置换为丝氨酸或组氨酸；
第674位的赖氨酸置换为亮氨酸或半胱氨酸；
第735位的天冬酰胺置换为组氨酸；
第736位的谷氨酰胺置换为谷氨酸或丙氨酸；
第606位的精氨酸置换为组氨酸或甲硫氨酸；
第709位的精氨酸置换为丝氨酸或组氨酸；
第347位的丝氨酸置换为异亮氨酸或甲硫氨酸；
第349位的苏氨酸置换为苯丙氨酸或异亮氨酸；
第590位的苏氨酸置换为赖氨酸或亮氨酸；
第676位的苏氨酸置换为赖氨酸或酪氨酸；
第389位的缬氨酸置换为异亮氨酸或甲硫氨酸；
第589位的缬氨酸置换为组氨酸或谷氨酰胺；
第680位的缬氨酸置换为甲硫氨酸或谷氨酸；
第384位的酪氨酸置换为苯丙氨酸或色氨酸；
第496位的酪氨酸置换为亮氨酸或异亮氨酸；
第383位的丝氨酸置换为苏氨酸。
上述蛋白中，所述野生型KOD型DNA聚合酶的氨基酸序列为序列1。
上述A)所示的蛋白为KOD型DNA聚合酶点突变体，具体为实施例中表1的单点突变体或者实施例中表2的多点突变体。
编码上述蛋白的DNA分子也是本发明保护的范围。
含有上述DNA分子的表达盒、重组载体、重组菌或转基因细胞系也是本发明保护的范围。
上述蛋白在作为DNA聚合酶中的应用也是本发明保护的范围；
或上述DNA分子或上述表达盒、重组载体、重组菌或转基因细胞系在制备DNA聚合酶中的应用也是本发明保护的范围。
上述蛋白或上述DNA分子或上述的表达盒、重组载体、重组菌或转基因细胞在测序中的应用也是本发明保护的范围；上述测序具体为DNA测序。
或上述蛋白或上述DNA分子或上述的表达盒、重组载体、重组菌或转基因细胞在制备用于测序的产品中的应用也是本发明保护的范围。
上述应用中，所述产品为试剂盒。
本发明对KOD聚合酶行了动力学模拟与统计推断为基础，获得了可用于实 验筛选的突变位点。并设计实验方案，通过半理性设计、酶变体库构建和高通量筛选达到改善聚合酶的催化及理化特性，开发适合于在附着于芯片表面DNB测序工作的DNA聚合酶。最后，重组性的DNA聚合酶进行了分离和纯化。
附图说明
图1为重组型KOD DNA聚合酶结构示意图。
图2为野生型KOD DNA聚合酶融合蛋白纯度检测。
图3为野生型和重组型KOD DNA聚合酶融合蛋白米氏曲线。
实施发明的最佳方式
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1、KOD DNA聚合酶突变体的制备
在本发明中，将KOD DNA聚合酶及其突变体采用DNA 2.0的Electra TM Cloning Reagents Kit试剂盒进行表达载体的构建，并且利用His标签进行Ni柱亲和纯化。
一、野生型KOD DNA聚合酶的制备
野生型KOD DNA聚合酶的氨基酸序列为如下序列表中的序列1，其编码基因序列为如下序列表中序列2。
1、野生型表达载体pD441-WT的构建
野生型重组表达载体pD441-WT为将融合his标签的野生型KOD型DNA聚合酶编码基因按Electra TM Cloning Reagents Kit(DNA2.0,EKT-02)试剂盒说明书操作步骤重组到载体pD441-pelB(DNA2.0,pD441-pelB)上，得到的载体，融合His标签的野生型KOD型DNA聚合酶编码基因，通过载体pD441-pelB上的信号肽引导表达。
融合His标签的野生型KOD型DNA聚合酶编码基因的核苷酸序列为在序列2的3’端连接有6个His标签密码子得到的序列。
野生型KOD型DNA聚合酶融合蛋白的氨基酸序列为将序列1所示的氨基酸的C端连接6个His标签得到，如下图1，1为信号肽pelB；2为融合蛋白纯化标签His；3为野生型KOD DNA聚合酶。
2、重组菌的构建
将野生型重组表达载体pD441-WT导入大肠杆菌BL21感受态细胞(购自全式金生物科技有限公司)中，涂抹在含有卡那霉素50μg/ml的平板上来筛选阳性菌落。挑选阳性菌落3-5个，利用引物Cloning-F(序列表中序列3)和Cloning-R(序列表中序列4)对所挑选的阳性菌落进行PCR鉴定。得到与预计的理论值基本一致的2800bp大小的片段，经测序结果比对判定为阳性克隆，命名为BL21/pD441-WT。
序列3:Cloning-F：5’GGTTTTTTTATGGGGGGAGTTTAGG 3’
序列4:Cloning-R：5’CATCTCATCTGTAACATCATTGGCA 3’
3、野生型KOD型DNA聚合酶融合蛋白的表达和纯化
挑取BL21/pD441-WT单菌落，于50ml LB液体培养基中(含Kan50μg/ml)中，37℃，220rpm/min，过夜培养。第二天按1:100稀释量，转接于1LLB液体培养基中(含Kan50μg/ml)，37℃，220rpm/min摇床培养至OD 600为0.5-0.8，加入IPTG终浓度为0.5mM，25℃过夜诱导表达融合蛋白，获得诱导后BL21/pD441-WT菌液。空白对照组菌液不加IPTG。
将上述BL21/pD441-WT菌液于转速为8000rpm/min离心10min，弃上清，收集沉淀菌体，重悬细胞于buffer(50mM KPO4，500mMNaCl，10mM imidazole，5％Glycerol，PH 7.0)中，另加入PMSF(终浓度0.5mM)。然后使用超声破碎仪进行菌体破碎，工作时间40min，功率200W，超声1S停2S,报警温度15℃。超声破碎完成后，将细胞破碎液于80℃水浴20min，期间注意定时混匀，使受热均匀；12000rpm/min，4℃离心30min，将上清用0.22um滤膜过滤，收集滤液即为细胞粗提物。
将细胞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buffer1平衡5CV；3％buffer 2(50mMKPO 4，1M NaCl，5％Glycerol，PH 7.0)洗脱5CV；50％buffer 2洗脱5CV，收集大于等于100mAU峰值对应的Ni柱亲和层析洗脱液。
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buffer2平衡5CV，0％buffer 2→60％buffer 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。
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HiPrepSephacryl S-100HR，26mm，17-1194-01，GE healthcare)，先用20％乙醇洗3CV，水洗3CV，利用100％buffer 3(20mMTris，200mMKCl，0.2mM EDTA，10％Glycerol，PH7.4)平衡3CV后上样，再用buffer 3洗脱1.5CV，收集洗脱液即为纯化后野生型KOD型DNA聚合酶融合蛋白。
将纯化后野生型KOD型DNA聚合酶融合蛋白进行SDS-PAGE(浓缩胶为5％分离胶为12％)，蛋白样品与SDS-PAGE蛋白上样缓冲液(5X)进行混合，95℃处理5min，进行上样。结果如图2所示，1为蛋白Marker(PageRulerPrestained Protein Ladder，26616，Thermo Scientific)，2为10μl 1mg/ml纯化后野生型KOD型DNA聚合酶融合蛋白，3为10μl稀释20倍后的0.05mg/ml纯化后野生型KOD型DNA聚合酶融合蛋白。从蛋白电泳结果可以看出，泳道2和泳道3中蛋白大小约91.5KDa，与文献报道分子量一致。
对电泳后的蛋白胶利用Quantity one软件分析蛋白纯度，纯化后野生型KOD型DNA聚合酶融合蛋白纯度能达到95％或以上。
未诱导BL21/pD441-WT菌液未得到约91.5KDa大小的目的蛋白。
对照组将空载体pD441-pelB导入大肠杆菌BL21中，得到BL21/pD441-pelB。采用上述方法进行表达和纯化蛋白，也未得到约91.5KDa大小的目的蛋白。
二、KOD DNA聚合酶突变体融合蛋白的制备
KOD DNA聚合酶突变体(氨基酸序列为序列A)为将野生型KOD DNA聚合酶的氨基酸序列(序列1)第675、385、710、674、735、736、606、709、347、349、590、676、389、589、680、384、496、383中至少有一个氨基酸进行氨基酸置换，得到的蛋白；若只有一个氨基酸置换所到的是KOD DNA聚合酶单点突变体，若有两个氨基酸置换所得到的是KOD DAN聚合酶两点组合突变体，若有三个氨基酸置换所得到的是KOD DNA聚合酶三点组合突变体，依次类推。
KOD DNA聚合酶突变体编码基因(核苷酸序列为序列B)为将野生型KOD DNA聚合酶编码基因的核苷酸序列(序列2)按照序列1中第675、385、710、674、735、736、606、709、347、349、590、676、389、589、680、 384、496、383中至少有一个氨基酸的密码子进行突变，得到的核酸。
可以将野生型KOD型DNA聚合酶用定点突变的方式获得KOD DNA聚合酶突变体，也可以用别的现有方法获得。
1、表达KOD型DNA聚合酶突变体的重组载体的制备
表达不同KOD型DNA聚合酶突变体的重组载体为将融合His标签的的不同KOD型DNA聚合酶突变体蛋白编码基因重组到载体pD441-pelB上，得到的载体，融合His标签的不同点突变体蛋白编码基因，通过载体pD441-pelB上的信号肽引导表达。
每个KOD型DNA聚合酶突变体融合蛋白的氨基酸序列为将序列A所示的KOD型DNA聚合酶突变体的C端连接6个His标签得到的；序列A为序列1所示的野生型KOD DNA聚合酶氨基酸序列突变得到的序列。
融合His标签的不同KOD型DNA聚合酶突变体蛋白编码基因的核苷酸序列为将序列B所示的不同KOD型DNA聚合酶突变体蛋白编码基因的3‘端连接6个His标签密码子得到的；序列B为序列2所示的野生型KOD DNA聚合酶编码核苷酸序列进行相应的氨基酸密码子突变得到的序列。
下面以单点突变体为例，如表1所示。
表1为KOD DNA聚合酶单点突变体的突变位置及突变信息
Figure PCTCN2018103764-appb-000001
Figure PCTCN2018103764-appb-000002
以多点突变体为例的KOD型DNA聚合酶突变体，如下表2所示。
2、重组菌的构建
与上述一中的2方法相同，将上述1制备的表达不同KOD型DNA聚合酶突变体重组载体导入BL21中，得到表达不同KOD型DNA聚合酶突变体融合蛋白的重组菌。
3、突变体的表达和纯化
与上述一野生型KOD型DNA聚合酶融合蛋白表达和纯化方法相同，将上述2制备的表达不同KOD型DNA聚合酶突变体融合蛋白的重组菌进行表达纯化，得到不同的KOD型DNA聚合酶突变体融合蛋白。
将不同的KOD型DNA聚合酶突变体融合蛋白行SDS-PAGE(浓缩胶为5％分离胶为12％)检测，得到目的蛋白。对电泳后的蛋白胶对于用Quantity one软件分析蛋白纯度，不同的KOD型DNA聚合酶突变体融合蛋白纯度均得到能达到95％或以上。
实施例2、重组型KOD DNA聚合酶突变体融合蛋白的性能检测
一、重组型KOD DNA聚合酶突变体融合蛋白的聚合活性检测
聚合酶活性检测参照Nishioka,M.,et al.(2001.J.Biotechnol.88)文献方法进行，一个酶活单位定义为:单位酶在50μl的反应体系中于75°反应30min聚合10nmol dNTP生成酸不溶性物质的量。
反应液为：20mMTris-HCl(pH7.5)，8mM MgCl 2，50μg/ml BSA，0.15 mM each dNTPs(dATP、dTTP、dCTP、dGTP)，[methyl-3H]-TTP(0.13mCi/nmol，final concentration,公司MPBIO-FINE,产品目录号CB2272108)，150mg/ml activated calf thymus DNA(公司SIGMA-ALDRICH，产品货号GE27-4575-01),7.5mM DTT，1μL野生型KOD型DNA聚合酶(1mg/ml),总反应体积为50μl。
上述反应液于75°反应30min，后按酶活定义计算野生型KOD型DNA聚合酶的聚合活性为2U/μl。
以野生型KOD型DNA聚合酶融合蛋白及KOD型DNA聚合酶突变体融合蛋白为例，进行上述聚合反应。
其中KOD型DNA聚合酶突变体为表2中的KOD型DNA聚合酶多点突变体：AB60、AB70、AB71、CD72、CD73、EF74、EF75、EF76、GH77、GH78；
表2为KOD型DNA聚合酶多点突变体
名称 聚合活性
WT(野生型KOD型DNA聚合酶) 2.0
AB60 2.5
AB70 3.1
AB71 3.3
CD72 2.8
CD73 2.4
EF74 2.7
EF75 3.5
EF76 3.2
GH77 3.8
GH78 4.5
表2所示的突变体具体突变位置及方式如下：
AB60其氨基酸序列为将序列1的第709位精氨酸突变为丝氨酸，且将第589位缬氨酸突变组氨酸；
AB70其氨基酸序列为将序列1的第676位苏氨酸突变为赖氨酸，第589位缬氨酸突变为组氨酸；第680位缬氨酸突变为甲硫氨酸；
AB71其氨基酸序列为将序列1的第674位赖氨酸突变为亮氨酸，第589位缬氨酸突变为组氨酸；第680位缬氨酸突变为甲硫氨酸；
CD72其氨基酸序列为将序列1的第676位苏氨酸突变为赖氨酸，第589位缬氨酸突变为组氨酸；第680位缬氨酸突变为甲硫氨酸；第384位酪氨酸突变为苯丙氨酸；
CD73其氨基酸序列为将序列1的第389位缬氨酸突变为异亮氨酸，第674位赖氨酸突变为亮氨酸；第589位缬氨酸突变为组氨酸；第680位缬氨酸突变为甲硫氨酸；
EF74其氨基酸序列为将序列1的第674位赖氨酸突变为亮氨酸，第589位缬氨酸突变为组氨酸；第680位缬氨酸突变为甲硫氨酸；第383位丝氨酸突变为苏氨酸；第384位酪氨酸突变为苯丙氨酸；
EF75其氨基酸序列为将序列1的第349位苏氨酸突变为苯丙氨酸，第589位缬氨酸突变为组氨酸；第676位苏氨酸突变为赖氨酸；第680位缬氨酸突变为甲硫氨酸；第496位酪氨酸突变为亮氨酸；
EF76其氨基酸序列为将序列1的第589位缬氨酸突变为组氨酸，第676位苏氨酸突变为赖氨酸；第680位缬氨酸突变为甲硫氨酸；第383位丝氨酸突变为苏氨酸；第384位酪氨酸突变为苯丙氨酸；
GH77其氨基酸序列为将序列1的第589位缬氨酸突变为组氨酸，第676位苏氨酸突变为赖氨酸；第680位缬氨酸突变为甲硫氨酸；第383位丝氨酸突变为苏氨酸；第384位酪氨酸突变为苯丙氨酸；第709位精氨酸突变为丝氨酸；
GH78其氨基酸序列为为将序列1的第389位缬氨酸突变为异亮氨酸，第589位缬氨酸突变为组氨酸；第676位苏氨酸突变为赖氨酸；第680位缬氨酸突变为甲硫氨酸；第383位丝氨酸突变为苏氨酸；第384位酪氨酸突变为苯丙氨酸。
KOD型DNA聚合酶突变体融合蛋白为在各自KOD型DNA聚合酶突变体的C端加入6个his标签。
KOD型DNA聚合酶突变体融合蛋白活性结果如表2所示，可以看出，KOD型DNA聚合酶突变体具有聚合酶活性。
其他突变体的聚合活性结果类似。
二、KOD型DNA聚合酶突变体融合蛋白的单碱基掺入动力学
本实施例利用Cy3荧光染料标记的的dATP(dATP-Cy3)和Cy5荧光染料标记的底物(P/T-2Cy5)，利用酶标仪来检测重组型KOD DNA聚合酶突变体的相对反应速率，近似描绘各突变体的米氏动力学曲线，具体实验方法如下：
将带有5’Cy5荧光标记的单链引物P1A(序列表中序列5)和P2A(序列表中序列6)按照1:1混合后，于65℃保持10min,自然降温至室温即可，得到的退火产物避光保存至-20℃，即得到Cy5荧光染料标记的底物P/T-2Cy5。
使用BioTek酶标仪进行酶活检测，反应在384板(Corning black,clear bottom 384plates)中进行，反应液总体积50μl，具体的反应体系如下：
反应体系为：1UKOD聚合酶突变体融合蛋白，1μM dATP-Cy3，10μM dTTP,10μM dCTP,10μM dGTP,底物P/T-2Cy5按0.1、0.2、0.5、1、2、3、4、6μM 8个浓度梯度进行实验，酶促反应缓冲液为20mMTris-HCl、10mM(NH4)2SO4、10mMKCl、2mM MgSO4 pH 8.5@25℃。
酶促反应以动力学检测模式，每30s记录一次数据，检测条件为:
Figure PCTCN2018103764-appb-000003
反应完成后可直接导出数据表格和酶活性曲线，可近似计算其相对荧光值的反应速率。
对于荧光值的反应速率的大小取决于底物P/T-2Cy5的浓度，因此通过在不同浓度的底物P/T-2Cy5条件下，检测重组型KOD DNA聚合酶突变体的活性可以近似确定其Km值即反应速率达到最大反应速率的一半时所对应的底物。
以野生型KOD DNA聚合酶融合蛋白(KOD-WT)为对照。
KOD DNA聚合酶突变体融合蛋白的Michaelis-Menten曲线如图3和表3所示，可以看出KOD DNA聚合酶突变体突变体相对野生型具有较高的 聚合活性。具体的实验结果可参照下表3,KOD DNA聚合酶突变体的动力学测试结果显示较野生型有较大的潜力。而在测序过程中，酶反应的催化效率越快，Vmax/Km的值就越大，从而可以在一定程度上加快反应完成，缩短测序反应时间。从实验结果中，可以看出，在酶催化效率方面，KOD DNA聚合酶突变体较野生型KOD DNA聚合酶都有一个较好的提升。
表3 KOD DNA聚合酶突变体的动力学测试结果
Figure PCTCN2018103764-appb-000004
工业应用
本发明的实验证明，本专利针对进一步延长测序读长，缩短反应时间等改进目标，在此前工作的基础上对KOD DNA聚合酶进行了更加深入的改造,对DNA聚合酶B family在此区域涉及到的原有功能位点进行了保护，确保其仍然能够完成原有的基本功能。提高在测序中DNA聚合酶的反应速率，增加反应读长。重组性的DNA聚合酶相比野生型的DNA聚合酶在催化方面，展示了较强的反应速率，更好的亲和力等优点。

Claims (13)

  1. 一种蛋白，是如下A)或B)：
    A)所示的蛋白为将野生型KOD型DNA聚合酶氨基酸序列中第675、385、710、674、735、736、606、709、347、349、590、676、389、589、680、384、496和383位这18位中至少一位的氨基酸残基进行修饰，得到具有DNA聚合酶活性的蛋白；
    B)所示的蛋白为将A)所示蛋白的氨基酸序列末端添加标签序列且具有DNA聚合酶活性的由A)衍生的蛋白质。
  2. 根据权利要求1所述的蛋白，其特征在于：
    A)所示的蛋白为将野生型KOD型DNA聚合酶氨基酸序列中第675、385、710、674、735、736、606、709、347、349、590、676、389、589、680、384、496和383位这18位中至少2位的氨基酸残基进行修饰，得到具有DNA聚合酶活性的蛋白。
  3. 根据权利要求1或2所述的蛋白，其特征在于：
    A)所示的蛋白为将野生型KOD型DNA聚合酶氨基酸序列中第675、385、710、674、735、736、606、709、347、349、590、676、389、589、680、384、496和383位这18位中至少3位的氨基酸残基进行修饰，得到具有DNA聚合酶活性的蛋白。
  4. 根据权利要求1-3中任一所述的蛋白，其特征在于：
    A)所示的蛋白为将野生型KOD型DNA聚合酶氨基酸序列中第675、385、710、674、735、736、606、709、347、349、590、676、389、589、680、384、496和383位这18位中至少4位的氨基酸残基进行修饰，得到具有DNA聚合酶活性的蛋白。
  5. 根据权利要求1-4中任一所述的蛋白，其特征在于：
    A)所示的蛋白为将野生型KOD型DNA聚合酶氨基酸序列中第675、385、710、674、735、736、606、709、347、349、590、676、389、589、680、384、496和383位这18位中至少5位的氨基酸残基进行修饰，得到具有DNA聚合酶活性的蛋白。
  6. 根据权利要求1-5中任一所述的蛋白，其特征在于：
    A)所示的蛋白为将野生型KOD型DNA聚合酶氨基酸序列中第675、385、710、674、735、736、606、709、347、349、590、676、389、589、680、384、496和383位这18位中至少6位的氨基酸残基进行修饰，得到具有DNA聚合酶活性的蛋白。
  7. 根据权利要求1-6中任一所述的蛋白，其特征在于：所述修饰为氨基酸置换。
  8. 根据权利要求7所述的蛋白，其特征在于：
    所述氨基酸置换为如下至少一种：
    第675位的丙氨酸置换为苯丙氨酸或甲硫氨酸；
    第385位的谷氨酸置换为甲硫氨酸或色氨酸；
    第710位的异亮氨酸置换为丝氨酸或组氨酸；
    第674位的赖氨酸置换为亮氨酸或半胱氨酸；
    第735位的天冬酰胺置换为组氨酸；
    第736位的谷氨酰胺置换为谷氨酸或丙氨酸；
    第606位的精氨酸置换为组氨酸或甲硫氨酸；
    第709位的精氨酸置换为丝氨酸或组氨酸；
    第347位的丝氨酸置换为异亮氨酸或甲硫氨酸；
    第349位的苏氨酸置换为苯丙氨酸或异亮氨酸；
    第590位的苏氨酸置换为赖氨酸或亮氨酸；
    第676位的苏氨酸置换为赖氨酸或酪氨酸；
    第389位的缬氨酸置换为异亮氨酸或甲硫氨酸；
    第589位的缬氨酸置换为组氨酸或谷氨酰胺；
    第680位的缬氨酸置换为甲硫氨酸或谷氨酸；
    第384位的酪氨酸置换为苯丙氨酸或色氨酸；
    第496位的酪氨酸置换为亮氨酸或异亮氨酸；
    第383位的丝氨酸置换为苏氨酸。
  9. 根据权利要求1-8中任一所述蛋白，其特征在于：所述野生型KOD型DNA聚合酶的氨基酸序列为序列1。
  10. 编码权利要求1-9中任一所述蛋白的DNA分子。
  11. 含有权利要求10所述DNA分子的表达盒、重组载体、重组菌或 转基因细胞系。
  12. 权利要求1-9中任一所述蛋白在作为DNA聚合酶中的应用；
    或权利要求10所述DNA分子或权利要求11所述表达盒、重组载体、重组菌或转基因细胞系在制备DNA聚合酶中的应用。
  13. 权利要求1-9中任一所述蛋白或权利要求10所述DNA分子或权利要求11所述的表达盒、重组载体、重组菌或转基因细胞在测序中的应用；
    或权利要求1-9中任一所述蛋白或权利要求10所述DNA分子或所述的表达盒、重组载体、重组菌或转基因细胞在制备用于测序的产品中的应用。
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