High-fidelity DNA polymerase as well as preparation method and application thereof

Abstract

The invention discloses a high-fidelity DNA polymerase as well as a preparation method and application thereof, and relates to the technical field of biology. The invention provides a high-fidelity DNA polymerase, the high-fidelity DNA polymerase is formed by fusing a DNA binding protein and a Pfu DNA polymerase, the amino acid sequence of the DNA binding protein is as shown in SEQ ID NO: 3, the amino acid sequence of the Pfu DNA polymerase is as shown in SEQ ID NO: 5, and the amino acid sequence of the high-fidelity DNA polymerase is as shown in SEQ ID NO: 10. The high-fidelity DNA polymerase disclosed by the invention can splice and assemble oligonucleotides into long-fragment DNA, can reduce the number of mismatched bases, has a stable effect in splicing and assembling of multiple long-fragment DNA with different sequences, and has an important practical value for a genome assembling technology.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a high-fidelity DNA polymerase and a preparation method and application thereof. BACKGROUND

[0002] Synthetic biology realizes biological modification or empowerment by designing genetic elements or genomes, and has great application potential in biology, medicine, agriculture, energy and environmental protection. The length of DNA synthesized by existing methods is limited, and DNA assembly is the basis for realizing synthetic genetic elements, long fragment DNA or genomes, and is also a key technology of synthetic biology.

[0003] DNA polymerase is a class of enzymes that can catalyze the polymerization of deoxyribonucleotides to form DNA chains, and plays an irreplaceable core role in DNA replication, repair and molecular biology experimental operations. Its performance directly affects the accuracy and efficiency of gene amplification, sequencing, cloning and genome assembly technologies, and therefore has important application value in the fields of genetic engineering, biomedical research and development, agricultural breeding and clinical diagnosis. DNA polymerase can efficiently assemble oligonucleotide fragments together, and the commonly used one is Taq DNA polymerase. However, Taq DNA polymerase is not suitable for long fragment DNA assembly due to the lack of 3'→5' exonuclease proofreading activity. For long fragment DNA assembly, DNA polymerase needs to have three points: first, it has high temperature stability and maintains activity in multiple thermal cycles; second, it has high continuous synthesis ability and can amplify and enrich the assembled DNA long fragments; third, it has proofreading activity to correct the incorrectly incorporated bases in the polymerization reaction and reduce the error rate of the assembled fragments.

[0004] Pfu DNA polymerase is derived from Pyrococcus furiosus and has excellent thermal stability: the half-life is more than 18 hours at 95℃; it has 3'→5' exonuclease (proofreading) activity, and the error rate is about 10 times lower than that of Taq DNA polymerase (error rate about 1×10 -6 However, Pfu DNA polymerase has the problem of insufficient continuous synthesis ability, and its extension efficiency is nearly 6 times lower than that of Taq DNA polymerase.

[0005] The fusion of DNA binding protein and DNA polymerase to form high-fidelity DNA polymerase can significantly enhance the binding stability of the enzyme to DNA template and the continuous synthesis ability, however, the existing high-fidelity enzyme still needs to be improved in aspects of amplification rate, continuous synthesis ability, tolerance to complex templates and inhibitors, etc. The present application adopts the metagenomic mining strategy to directly find a new enzyme gene skeleton with natural high thermal stability and high efficiency from unculturable extreme environment microorganisms; then through protein fusion engineering, the catalytic core and the DNA binding domain are combined to design and screen a new high-fidelity DNA polymerase, which has high fidelity and high robustness, especially in the splicing assembly of long fragment DNA, and has high practical value. SUMMARY

[0006] The present application aims to provide a high-fidelity DNA polymerase and a preparation method and application thereof.

[0007] In order to achieve the above-mentioned application purposes, the technical solutions of the present application are as follows: In one aspect, the present application provides a high-fidelity DNA polymerase, wherein the high-fidelity DNA polymerase is a fusion of DNA binding protein and Pfu DNA polymerase, the amino acid sequence of the DNA binding protein is shown in SEQ ID NO: 3, the amino acid sequence of the Pfu DNA polymerase is shown in SEQ ID NO: 5, and the amino acid sequence of the DNA polymerase is shown in SEQ ID NO: 10.

[0008] In another aspect, the present application provides a DNA molecule encoding the above-mentioned high-fidelity DNA polymerase.

[0009] Specifically, the nucleotide sequence of the DNA molecule is shown in SEQ ID NO: 8.

[0010] In another aspect, the present application provides a recombinant expression vector, which is obtained by cloning the above-mentioned DNA molecule into an expression vector.

[0011] Specifically, the expression vector is a prokaryotic expression vector. Further, the prokaryotic expression vector is a pET series vector.

[0012] Further, the expression vector is pET-28a.

[0013] In another aspect, the present application provides a recombinant engineering cell strain, which is obtained by transforming the above-mentioned recombinant expression vector into an engineering cell.

[0014] Specifically, the engineering cell is an Escherichia coli cell.

[0015] Further, the engineering cell is an Escherichia coli DE3 cell.

[0016] In another aspect, the present application provides the use of the high-fidelity DNA polymerase, the DNA molecule, the recombinant expression vector or the recombinant engineering cell strain in DNA amplification, DNA assembly, gene cloning or NGS sequencing library preparation.

[0017] In another aspect, the present application provides a method for preparing the high-fidelity DNA polymerase, comprising the following steps: S1, constructing a recombinant expression plasmid comprising the nucleotide sequence shown in SEQ ID NO: 8; S2, transforming the protein expression plasmid into a host cell, inducing expression, and obtaining a bacterial body; S3, destroying the bacterial body, collecting the supernatant after centrifugation, and obtaining the high-fidelity DNA polymerase after purification and dialysis.

[0018] Specifically, the expression plasmid in step S1 is pET-28a.

[0019] Specifically, the host cell in step S2 is a BL21 (DE3) competent cell.

[0020] According to some embodiments of the present application, the specific step of inducing expression is: spreading the transformed bacterial liquid on a solid LB culture plate containing kanamycin, culturing overnight at 37°C, picking a single colony to inoculate into LB medium containing kanamycin, and culturing under the condition of 37°C and 220 rpm until the OD600 value reaches 0.8-1.0, then adding IPTG solution to the culture medium to a final concentration of 1 mM. The bacterial liquid is further induced to culture at 37°C for 2 h.

[0021] Specifically, in step S3, the bacterial body is destroyed by adding a lysis solution, and then adding a lysozyme solution after resuspension. According to some embodiments of the present application, the lysis solution comprises 50 mM Tris HCl (pH 8.0), 500 mM NaCl, 0.1% NP-40 and 0.1% Triton X-100.

[0022] Further, the concentration of the lysozyme solution is 100 mg / ml.

[0023] Specifically, in step S3, before centrifugation, the step of placing the enzyme-degraded bacterial suspension into a 75°C water bath for heating for 1 h is further included.

[0024] Specifically, in step S3, the centrifugation condition is 4°C, 16904g centrifugation for 10 min.

[0025] Specifically, in step S3, after centrifugation, the supernatant is collected, filtered, and glycerol and benzalkonium chloride are added to the filtrate.

[0026] Specifically, the nickel column purification is used in step S3.

[0027] In another aspect, the present application provides a product of gene assembly, which comprises the high-fidelity DNA polymerase as described above.

[0028] The present application has the following advantages: The high-fidelity DNA polymerase provided by the present application has high enzyme activity (84.10±2.76) and long DNA fragment assembly capacity, and can reduce the number of mismatched bases and the assembly error rate to 0.29%. The engineering modification of the DNA polymerase to improve its performance has important research value for the gene assembly technology. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 The figure is a schematic diagram of the structure of four candidate fusion proteins.

[0030] Figure 2 The figure is the expression and purification identification results of four candidate fusion proteins. The positive control is bovine serum albumin (BSA) with a molecular weight of 66.5 kDa. The theoretical molecular weight of the four proteins calculated by Snapgene software (without His tag): hfDNAP-1 97,805.86 Da; hfDNAP-2 97,622.91 Da; hfDNAP-3 97,674.66 Da; hfDNAP-4 97,598.75 Da.

[0031] Figure 3 The figure is the DNA polymerase activity of four candidate fusion proteins.

[0032] Figure 4 The figure is the agarose gel electrophoresis diagram of four candidate fusion proteins assembling 4.9k DNA products.

[0033] Figure 5 The figure is the comparison of the enzyme hfDNAP-3 obtained by screening and wild type and commercial high-fidelity DNA polymerase in assembling other long DNA fragments. 1: DNA long fragment one, length 3804 bp; 2: DNA long fragment two, length 3877 bp; 3: DNA long fragment three, length 3836 bp. DETAILED DESCRIPTION

[0034] In order to make the technical means, creative features, purposes and effects of the present application easy to understand, the present application is further illustrated below in combination with specific examples, but the following examples are only preferred examples of the present application, not all. Based on the examples in the embodiments, other examples obtained by those skilled in the art without creative labor are within the protection scope of the present application. In the following examples, if not otherwise specified, the operation methods used are all conventional operation methods, the equipment used is all conventional equipment, and the equipment materials used in each example are all the same.

[0035] Example 1 Design and preparation of high-fidelity DNA polymerase 1. Design The structure (PDB ID: 1BNZ) of Sso7d protein was taken as a query structure, and Foldseek protein structure search tool was used to perform structure homology search in multiple protein structure databases (including AF50, AFDB-proteome, AFDB-swissport, Cath50, GMGCL, MGnify_ESM30 and PDB100), and no more than 1000 hit records were reserved in each database, and a total of 3748 candidate protein structures were obtained. From the Foldseek alignment results, according to the starting position of the structure homology region, the corresponding protein amino acid sequence fragments were extracted by extending not more than 20 amino acid residues to both ends.

[0036] The obtained candidate proteins were subjected to the following filtering treatment: removing hit records with bitscore value less than 50 in Foldseek alignment results; removing hit records with probability value (prob) less than 0.5; removing hit records with query coverage (qcov) not more than 50%. The homologous protein sequences after preliminary screening were aligned with the Sso7d prototype protein sequence, and the protein records with sequence consistency more than 75% were removed, and the homologous proteins with sequence novelty were reserved.

[0037] Based on the key residue characteristics of DNA recognition and binding, the screened homologous proteins were further evaluated and screened, and finally 4 candidate DNA binding proteins were determined, and the sequences were as follows: The amino acid sequence of DNA binding protein 1 is shown as SEQ ID NO: 1: MAEFIVVHENGEERIVNLAWVEEIRPDDGRAVFYYAFQGAGYMEQDSIKTDEPYNAVKRMIWR.

[0038] The amino acid sequence of DNA binding protein 2 is shown as SEQ ID NO: 2: MKYLTLKYTDEKYKINLDKITMVQIREGYICITFDAHNISEIYENECSNFFEIKKILENL.

[0039] The amino acid sequence of DNA binding protein 3 is shown as SEQ ID NO: 3: MRLYRFTNEHGQDAALNIDKIGDMHQDRKDVHVRWGGAYQETTRIPNTTLEELINNLKLLGES.

[0040] The amino acid sequence of DNA binding protein 4 is shown as SEQ ID NO: 4: MAFIKVKDKKTKEDTIINTNMICRISRNKNGYTVFFSSGNVGAAYYEYDEDNAKKIFDAIGVSL.

[0041] Nucleotide sequence design and synthesis: Sequence design: the amino acid sequence of wild-type Pfu DNA polymerase is shown as SEQ ID NO: 5, and four candidate DNA binding proteins are fused to the C-terminal of Pfu DNA polymerase through Linker sequences (as shown in SEQ ID NO: 6-9). Figure 1 The four fusion proteins are candidate high-fidelity DNA polymerases (denoted as hfDNAP-1, hfDNAP-2, hfDNAP-3, and hfDNAP-4), and the nucleotide sequences generated and optimized according to the amino acid sequences are shown as SEQ ID NO: 6-9.

[0042] SEQ ID NO: 5: MILDVDYITEEGKPVIRLFKKENGKFKIEHDRTFRPYIYALLRDDSKIEEVKKITGERHGKIVRIVDVEKVEKKFLGKPITVWKLYLEHPQDVPTIREKVREHPAVVDIFEYDIPFAKRYLIDKGLIPMEGEEELKILAFDIETLYHEGEEFGKGPIIMISYADENEAKVITWKNIDLPYVEVVSSEREMIKRFLRIIREKDPDIIVTYNGDSFDFPYLAKRAEKLGIKLTIGRDGSEPKMQRIGDMTAVEVKGRIHFDLYHVITRTINLPTYTLEAVYEAIFGKPKEKVYADEIAKAWESGENLERVAKYSMEDAKATYELGKEFLPMEIQLSRLVGQPLWDVSRSSTGNLVEWFLLRKAYERNEVAPNKPSEEEYQRRLRESYTGGFVKEPEKGLWENIVYLDFRALYPSIIITHNVSPDTLNLEGCKNYDIAPQVGHKFCKDIPGFIPSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQKAIKLLANSFYGYYGYAKARWYCKECAESVTAWGRKYIELVWKELEEKFGFKVLYIDTDGLYATIPGGESEEIKKKALEFVKYINSKLPGLLELEYEGFYKRGFFVTKKRYAVIDEEGKVITRGLEIVRRDWSEIAKETQARVLETILKHGDVEEAVRIVKEVIQKLANYEIPPEKLAIYEQITRPLHEYKAIGPHVAVAKKLAAKGVKIKPGMVIGYIVLRGDGPISNRAILAEEYDPKKHKYDAEYYIENQVLPAVLRILEGFGYRKEDLRYQKTRQVGLTSWLNIKKS.

[0043] SEQ ID NO: 6:

[0044] SEQ ID NO:7:

[0045] SEQ ID NO:8:

[0046] SEQ ID NO: 9:

[0047] 2. Plasmid vector construction: Four genes were synthesized and assembled according to SEQ ID NO: 6-9. The construction was in the open reading frame region of pET-28a expression vector (P0023, MCLAB). The four plasmid DNAs were transformed into BL21 (DE3) competent cells, respectively. The transformed bacterial liquid was spread on solid LB plates containing kanamycin and incubated at 37°C overnight. Single colonies were picked and verified by Sanger sequencing method. The single colonies with correct sequences were selected and the corresponding plasmids and bacterial liquid were preserved.

[0048] 3. Preparation of solutions: 1) Isopropyl β-D-thiogalactoside (IPTG) solution (1M): 2.38g of IPTG was weighed and added to 10mL of enzyme-free water. After dissolving, it was filtered with a 0.22µm filter membrane and stored at -20°C for standby.

[0049] 2) Lysis solution: 50mM Tris HCl (pH8.0), 500mM NaCl, 0.1% NP-40 and 0.1% Triton X-100.

[0050] 3) Imidazole solution stock (2M): 1.36g of imidazole was weighed and added to 10mL of enzyme-free water. After dissolving, it was adjusted to pH=8 with hydrochloric acid and stored at 4°C for standby.

[0051] 4) Gradient elution buffer: 10mM eluent: 50mM Tris HCl (pH8.0), 500mM NaCl / 100mM NaCl, 0.05% NP-40, 5% glycerol, 5mM β-mercaptoethanol, 1mM benzamidine and 10mM imidazole.

[0052] 20mM eluent: 50mM Tris HCl (pH8.0), 100mM NaCl, 0.05% NP-40, 5% glycerol, 5mM β-mercaptoethanol, 1mM benzamidine and 20mM imidazole.

[0053] 50mM eluent: 50mM Tris HCl (pH8.0), 100mM NaCl, 0.05% NP-40, 5% glycerol, 5mM β-mercaptoethanol, 1mM benzamidine and 50mM imidazole.

[0054] 300 mM Elution Buffer: 50 mM Tris HC1 (pH 8.0), 100 mM NaCl, 0.05% NP-40, 5% glycerol, 5 mM β-mercaptoethanol, 1 mM benzamidine, and 300 mM imidazole.

[0055] 5) Storage Buffer: 20 mM Tris HC1 (pH 8.0), 500 mM NaCl.

[0056] 4. Protein Induction Expression: The four monoclonal bacteria solutions that were verified to be correct by sequencing were recovered and inoculated into 200 mL of LB medium containing 50 μg / mL of kanamycin and cultured at 37°C, 220 rpm, until the OD 600 value reached 0.8-1.0, IPTG solution was added to the culture medium to a final concentration of 1 mM. The bacteria solution was induced to continue to culture at 37°C for 2 h, and the bacteria were collected by centrifugation.

[0057] 5. Protein Purification: The bacteria were washed twice with PBS, the supernatant was discarded by centrifugation, 4 mL of lysis solution was added to the bacteria precipitate, 20 μL of lysozyme solution (100 mg / mL) was added after resuspension, and the mixture was mixed evenly. The mixed solution was placed in a four-dimensional rotary mixer and rotated at room temperature for 15 min. The enzyme-treated bacteria suspension was placed in a 75°C water bath for 1 h, and then centrifuged at 16000 x g at 4°C for 10 min. The supernatant was collected, filtered using a 0.22 μm filter, and 400 uL of glycerol, 1.4 μL of 100 mM benzamidine was added to the filtrate, mixed and used. Nickel column (Ni-NTA) purification was used. The nickel column was equilibrated with lysis solution, 20 μL of imidazole stock solution (2 M) was added to the cell lysate, mixed, and then combined with the nickel column. The target protein was eluted with gradient elution buffer from low to high concentration.

[0058] 6. Protein Analysis, Quantification and Storage: The eluted components were subjected to SDS-PAGE electrophoresis and Coomassie blue staining, and according to the staining, the components with high purity of the target protein were collected for overnight dialysis. After dialysis, 50 kDa ultrafiltration tube was used for ultrafiltration concentration, the concentrated solution was collected for BCA quantitative determination of concentration, SDS-PAGE electrophoresis and Coomassie blue staining to determine the purity (. Figure 2 ). The product was divided and stored at -80°C to avoid repeated freezing and thawing.

[0059] Example 2 Polymerase Activity Assay The purified proteins were uniformly diluted to 0.01 μg / μL, and the purified mutant enzyme activity was detected using a fluorescent polymerase activity assay kit (Biotium, USA, #29051). The detection steps were performed according to the kit instructions, and the imported commercial reagent Phusion® ultra-fidelity DNA polymerase (NEB, USA, #M0530S) was used to make a standard curve. The results are shown in Figure 3 FIG. 10, and the polymerase activities of the four candidate proteins were 62.18 ± 0.80, 55.47 ± 1.30, 84.10 ± 2.76, and 72.42 ± 5.58, respectively. The highest polymerase activity was hfDNAP-3, and the amino acid sequence is shown in SEQ ID NO: 10.

[0060] SEQ ID NO: 10 MILDVDYITEEGKPVIRLFKKENGKFKIEHDRTFRPYIYALLRDDSKIEEVKKITGERHGKIVRIVDVEKVEKKFLGKPITVWKLYLEHPQDVPTIREKVREHPAVVDIFEYDIPFAKRYLIDKGLIPMEGEEELKILAFDIETLYHEGEEFGKGPIIMISYADENEAKVITWKNIDLPYVEVVSSEREMIKRFLRIIREKDPDIIVTYNGDSFDFPYLAKRAEKLGIKLTIGRDGSEPKMQRIGDMTAVEVKGRIHFDLYHVITRTINLPTYTLEAVYEAIFGKPKEKVYADEIAKAWESGENLERVAKYSMEDAKATYELGKEFLPMEIQLSRLVGQPLWDVSRSSTGNLVEWFLLRKAYERNEVAPNKPSEEEYQRRLRESYTGGFVKEPEKGLWENIVYLDFRALYPSIIITHNVSPDTLNLEGCKNYDIAPQVGHKFCKDIPGFIPSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQKAIKLLANSFYGYYGYAKARWYCKECAESVTAWGRKYIELVWKELEEKFGFKVLYIDTDGLYATIPGGESEEIKKKALEFVKYINSKLPGLLELEYEGFYKRGFFVTKKRYAVIDEEGKVITRGLEIVRRDWSEIAKETQARVLETILKHGDVEEAVRIVKEVIQKLANYEIPPEKLAIYEQITRPLHEYKAIGPHVAVAKKLAAKGVKIKPGMVIGYIVLRGDGPISNRAILAEEYDPKKHKYDAEYYIENQVLPAVLRILEGFGYRKEDLRYQKTRQVGLTSWLNIKKSGTGGGGRLYRFTNEHGQDAALNIDKIGDMHQDRKDVHVRWGGAYQETTRIPNTTLEELINNLKLLGES.

[0061] Example 34.9k DNA fragment assembly error rate detection A DNA sequence of a gene segment was selected, with a length of 4914 bp, and the sequence is shown as SEQ ID NO: 11. DNAWorks software was used to design oligonucleotide (Oligo) sequences (SEQ ID NO: 12-SEQ ID NO: 137) for assembling the DNA fragment, each sequence was synthesized and mixed to prepare an Oligo Mix solution (10 µM). A total of 6 groups were set up for the experiment: the Phu-WT group was the Phusion high-fidelity DNA polymerase expressed and purified in the laboratory, the hfDNAP four groups were four candidate high-fidelity DNA polymerases, and the Positive Control group was an imported commercial reagent Phusion® Ultra High-Fidelity DNA Polymerase (NEB, USA, Cat. No.: M0530S).

[0062] SEQ ID NO: 11:

[0063] 10x PCR Buffer was prepared: 100 mM Tris-HCl (pH 8.9), 500 mM KCl, 15 mM MgCl2. The first round reaction system was: 2.0 μL 10x PCR Buffer, 1.6 μL dNTP (2.5 mM each), 2 μL hfDNAP protein, 4.0 μL Oligos Mix, ddH2O to 20 μL. The reaction program was: 95 °C for 3 min; 98 °C for 10 s, 60 °C for 20 s, 72 °C for 2 min / cycle, for a total of 30 cycles; 72 °C for 5 min; 4 °C for ∞. The second round reaction system was: 5.0 μL 10x PCR Buffer, 4.0 μL dNTP (2.5 mM each), 5 μL hfDNAP protein, 1 μL first round PCA product, 1.5 μL Primer F (10 μM), 1.5 μL Primer R (10 μM), enzyme-free ultrapure water to 50 μL. The reaction program was the same as the first round, the forward primer Primer F was the sequence shown in SEQ ID NO: 12, and the reverse primer Primer R was the sequence shown in SEQ ID NO: 137.

[0064] After the reaction, the assembly products were subjected to agarose gel electrophoresis, and the results are shown in Figure 4 After the reaction, the assembly products were subjected to agarose gel electrophoresis, and the results are shown in

[0065] Table 1. Statistical analysis of error types in hf-DNAP-1 assembly

[0066] Table 2. Statistical analysis of error types in hfDNAP-3 assembly

[0067] Table 3. Statistical analysis of error types in NEB Phusion® Ultra-Pfu DNA Polymerase assembly

[0068] Example 4 DNA assembly effect stability test To test the stability or universality of hfDNAP-3 in DNA assembly, different sequences were selected for assembly according to the method of Example 3, and whether there was a band of the desired length was identified by agarose gel electrophoresis. The CDS sequence (NM_004006.3) of the human DMD gene (NCBI Gene ID: 1756) was selected, and the sequence was divided into three segments: DNA long fragment one, length 3804 bp; DNA long fragment two, length 3877 bp; DNA long fragment three, length 3836 bp. The experiment was divided into four groups: Phu-WT group, Phusion high-fidelity DNA polymerase expressed and purified in the laboratory; hfDNAP-3 group, high-fidelity DNA polymerase hfDNAP-3 screened; NEB Phu group, imported commercial reagent Phusion® Ultra High-Fidelity DNA Polymerase (NEB, USA, Cat No: M0530S); NEB Q5 group, imported commercial reagent Q5® Ultra High-Fidelity DNA Polymerase (NEB, USA, Cat No: M0491S). Each group was assembled with the corresponding high-fidelity enzyme. The assembly reaction and procedure were the same as in Example 3. The gel electrophoresis results of the assembly products are shown in Figure 5 As shown, hfDNAP-3 can successfully assemble the three target fragments, the content of non-specific assembly products is low, and the assembly effect stability is better than that of existing high-fidelity DNA polymerases and commercial ultra-high-fidelity DNA polymerases.

[0069] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A high-fidelity DNA polymerase, characterized in that, The high-fidelity DNA polymerase is fused by a DNA binding protein and Pfu DNA polymerase, the amino acid sequence of the DNA binding protein is shown as SEQ ID NO: 3, and the amino acid sequence of the Pfu DNA polymerase is shown as SEQ ID NO:

5.

2. The high-fidelity DNA polymerase of claim 1, wherein, The amino acid sequence of the high-fidelity DNA polymerase is shown as SEQ ID NO:

10.

3. A DNA molecule encoding the high-fidelity DNA polymerase according to any one of claims 1-2.

4. The DNA molecule of claim 3, wherein, The nucleotide sequence of the DNA molecule is shown as SEQ ID NO:

8.

5. A recombinant expression vector, characterized in that, The DNA molecule according to any one of claims 3-4 is cloned into an expression vector to obtain.

6. A recombinant engineered cell line, characterized in that, The recombinant expression vector according to claim 5 is transformed into an engineering cell to obtain.

7. The recombineering cell strain of claim 6, wherein, The engineering cell is an E. coli cell.

8. The high-fidelity DNA polymerase according to any one of claims 1-2, the DNA molecule according to any one of claims 3-4, the recombinant expression vector according to claim 5, or the recombinant engineering cell strain according to any one of claims 6-7 is applied to DNA amplification, DNA splicing assembly, gene cloning, and NGS sequencing library preparation.

9. A method of producing a high-fidelity DNA polymerase according to any one of claims 1 to 2, characterized in that, The method comprises the following steps: S1, constructing a recombinant expression plasmid comprising the nucleotide sequence shown as SEQ ID NO: 8; S2, transforming the protein expression plasmid into a host cell, inducing expression, and obtaining a bacterial body; S3, destroying the bacterial body, collecting the supernatant after centrifugation, and obtaining the high-fidelity DNA polymerase after purification and dialysis.

10. A product of a gene assembly, characterized in that, The product comprises the high-fidelity DNA polymerase according to any one of claims 1-2.

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