A high-fidelity DNA polymerase, its preparation method and application

Abstract

This invention discloses a high-fidelity DNA polymerase, its preparation method, and its applications, relating to the field of biotechnology. The invention provides a high-fidelity DNA polymerase, which is a fusion of a DNA-binding protein and a 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 high-fidelity DNA polymerase is shown in SEQ ID NO:10. The high-fidelity DNA polymerase of this invention can assemble oligonucleotides into long DNA fragments, reducing the number of mismatched bases, and exhibits stable performance in assembling various long DNA fragments with different sequences. This invention has significant practical value for genome assembly technology.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a high-fidelity DNA polymerase, its preparation method, and its applications. Background Technology

[0002] Synthetic biology, through the design of genetic elements or genomes, enables bioengineering or empowerment, and has enormous application potential in biology, medicine, agriculture, energy, and environmental protection. Current methods for directly synthesizing DNA have limitations in length; DNA assembly is fundamental to the synthesis of genetic elements, long DNA fragments, or genomes, and is a key technology in synthetic biology.

[0003] DNA polymerases are a class of enzymes that catalyze the polymerization of deoxyribonucleotides to form DNA chains, playing an irreplaceable core role in DNA replication, repair, and molecular biology experiments. Their performance directly affects the accuracy and efficiency of gene amplification, sequencing, cloning, and genome assembly technologies, thus having significant application value in genetic engineering, biomedical research and development, agricultural breeding, and clinical diagnostics. DNA polymerases can efficiently and rapidly assemble oligonucleotide fragments together; Taq DNA polymerase is a commonly used example. However, Taq DNA polymerase lacks 3'→5' exonuclease proofreading activity, making it unsuitable for assembling long DNA fragments. For the assembly of long DNA fragments, DNA polymerases need to possess three characteristics: first, high-temperature stability, maintaining activity during multiple thermal cycles; second, high continuous synthesis capacity, capable of amplifying and enriching assembled long DNA fragments; and third, proofreading activity, correcting incorrectly incorporated bases during the polymerization reaction and reducing the error rate of assembled fragments.

[0004] Pfu DNA polymerase, derived from *Pyrococcus furiosus*, exhibits excellent thermostability: a half-life greater than 18 hours at 95°C; it possesses 3'→5' exonuclease (proofreading) activity, with an error rate approximately 10 times lower than Taq DNA polymerase (error rate approximately 1×10⁻⁶). -6 / base). However, Pfu DNA polymerase suffers from insufficient sustained synthesis capacity, with its elongation efficiency being nearly 6 times lower than that of Taq DNA polymerase.

[0005] The fusion of DNA-binding proteins with DNA polymerase to form a high-fidelity DNA polymerase can significantly enhance the binding stability of the enzyme to the DNA template and its continuous synthesis capacity. However, existing high-fidelity enzymes still need improvement in terms of amplification rate, continuous synthesis capacity, and tolerance to complex templates and inhibitors. This invention employs a metagenomic mining strategy to directly discover novel enzyme gene backbones with naturally high thermal stability and high efficiency from unculturable microorganisms in extreme environments. Then, through protein fusion engineering, the catalytic core is combined with the DNA-binding domain to design and screen a new high-fidelity DNA polymerase with high fidelity and high robustness, especially in the splicing and assembly of long DNA fragments, exhibiting strong stability and low error rate, and possessing high practical value. Summary of the Invention

[0006] The purpose of this invention is to provide a high-fidelity DNA polymerase, its preparation method, and its application.

[0007] To achieve the above-mentioned objectives, the technical solution of the present invention is as follows:

[0008] On one hand, the present invention provides a high-fidelity DNA polymerase, wherein the high-fidelity DNA polymerase is formed by fusing a DNA-binding protein with a Pfu DNA polymerase, wherein 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.

[0009] In another aspect, the present invention provides a DNA molecule encoding the aforementioned high-fidelity DNA polymerase.

[0010] Specifically, the nucleotide sequence of the DNA molecule is shown in SEQ ID NO:8.

[0011] In another aspect, the present invention provides a recombinant expression vector, which is obtained by cloning the above-mentioned DNA molecule into an expression vector.

[0012] Specifically, the expression vector is a prokaryotic expression vector;

[0013] Furthermore, the prokaryotic expression vector is a pET series vector.

[0014] Furthermore, the expression vector is pET-28a.

[0015] In another aspect, the present invention provides a recombinant engineered cell line, which is obtained by converting the above-mentioned recombinant expression vector into engineered cells.

[0016] Specifically, the engineered cells are Escherichia coli cells.

[0017] Furthermore, the engineered cells are Escherichia coli DE3 cells.

[0018] In another aspect, the present invention provides the application of the above-mentioned high-fidelity DNA polymerase, the above-mentioned DNA molecule, the above-mentioned recombinant expression vector, or the above-mentioned recombinant engineered cell line in DNA amplification, DNA splicing and assembly, gene cloning, and NGS sequencing library preparation.

[0019] In another aspect, the present invention provides a method for preparing the above-mentioned high-fidelity DNA polymerase, comprising the following steps:

[0020] S1. Construct a recombinant expression plasmid containing the nucleotide sequence shown in SEQ ID NO:8;

[0021] S2. Transform the protein expression plasmid into the host cell, induce expression, and obtain bacterial cells;

[0022] S3. Destroy the bacterial cells, centrifuge and collect the supernatant, purify and dialyze to obtain high-fidelity DNA polymerase.

[0023] Specifically, the expression plasmid in step S1 is pET-28a.

[0024] Specifically, the host cell mentioned in step S2 is a BL21(DE3) competent cell.

[0025] According to some embodiments of the present invention, the specific steps for inducing expression are as follows: the transformed bacterial culture is spread on a solid LB culture plate containing kanamycin and cultured overnight at 37°C. Single colonies are picked and inoculated into LB medium containing kanamycin and cultured with shaking at 37°C and 220 rpm until the OD600 value reaches 0.8-1.0. IPTG solution is added to the culture medium to a final concentration of 1 mM. The bacterial culture is then further induced at 37°C for 2 hours.

[0026] Specifically, in step S3, the cell destruction involves adding lysis buffer to the cells, resuspending them, and then adding lysozyme solution.

[0027] According to some embodiments of the present invention, the lysis buffer comprises 50 mM Tris HCl (pH 8.0), 500 mM NaCl, 0.1% NP-40, and 0.1% Triton X-100.

[0028] Furthermore, the concentration of the lysozyme solution is 100 mg / ml.

[0029] Specifically, step S3 includes placing the enzymatically digested bacterial suspension in a 75°C water bath for 1 hour before centrifugation.

[0030] Specifically, the centrifugation conditions in step S3 are 4°C, 16904g, and centrifugation for 10 minutes.

[0031] Specifically, in step S3, after centrifugation, the supernatant is collected, filtered, and glycerol and benzalkonium chloride are added to the filtrate.

[0032] Specifically, nickel column purification is used in step S3.

[0033] In another aspect, the present invention provides a genome-packaged product containing the aforementioned high-fidelity DNA polymerase.

[0034] The beneficial effects of this invention are as follows:

[0035] This invention provides a high-fidelity DNA polymerase with high enzyme activity (84.10±2.76) and the ability to assemble long DNA fragments. It can reduce the number of mismatched bases and achieve an assembly error rate as low as 0.29%. This invention, based on the engineering modification of DNA polymerase to improve its performance, has important research value for genome assembly technology. Attached Figure Description

[0036] Figure 1 This is a schematic diagram of the structural domains of four candidate fusion proteins.

[0037] Figure 2 The results show the expression, purification, and identification of four candidate fusion proteins. The positive control was bovine serum albumin (BSA), with a molecular weight of 66.5 kDa. The theoretical molecular weights of the four proteins (excluding the His tag) calculated using Snapgene software are: hfDNAP-1 97,805.86 Da; hfDNAP-2 97,622.91 Da; hfDNAP-3 97,674.66 Da; hfDNAP-4 97,598.75 Da.

[0038] Figure 3 DNA polymerase activity of four candidate fusion proteins.

[0039] Figure 4 Agarose gel electrophoresis image of 4.9k kDa DNA products assembled from four candidate fusion proteins.

[0040] Figure 5 This study compares the hfDNAP-3 enzyme obtained through screening with wild-type and commercially available high-fidelity DNA polymerases in assembling other long DNA fragments. 1: Long DNA fragment one, 3804 bp in length; 2: Long DNA fragment two, 3877 bp in length; 3: Long DNA fragment three, 3836 bp in length. Detailed Implementation

[0041] To make the technical means, creative features, and achieved objectives and effects of this invention easier to understand, the invention is further illustrated below with specific embodiments. However, the following embodiments are merely preferred embodiments of this invention and not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments described herein without creative effort are all within the protection scope of this invention. Unless otherwise specified, the operating methods and equipment used in the following embodiments are conventional operating methods, and the materials and equipment used in each embodiment are the same.

[0042] Example 1: Design and preparation of high-fidelity DNA polymerase

[0043] 1. Design

[0044] Using the structure of the Sso7d protein (PDB ID: 1BNZ) as the query structure, the Foldseek protein structure search tool was used to perform structural homology searches in multiple protein structure databases (including AF50, AFDB-proteome, AFDB-swissport, Cath50, GMGCL, MGnify_ESM30, and PDB100). No more than 1000 matching records were retained from each database, resulting in a total of 3748 candidate protein structures. From the Foldseek alignment results, based on the starting position of the structural homology region, no more than 20 amino acid residues were extended from both ends to extract the corresponding protein amino acid sequence fragments.

[0045] The obtained candidate proteins were filtered as follows: records with a bitscore less than 50 in the Foldseek alignment results were removed; records with a probability value (prob) less than 0.5 were removed; and records with a query coverage (qcov) of less than 50% were removed. The preliminarily screened homologous protein sequences were then aligned with the Sso7d prototype protein sequence, and records with sequence similarity exceeding 75% were removed, retaining homologous proteins with sequence novelty.

[0046] Based on the key residue features of DNA recognition and binding, the homologous proteins obtained from the initial screening were further evaluated and screened, ultimately identifying four candidate DNA-binding proteins with the following sequences:

[0047] The amino acid sequence of DNA-binding protein 1 is shown in SEQ ID NO:1:

[0048] MAEFIVVHENGEERIVNLAWVEEIRPDDGRAVFYYAFQGAGYMEQDSIKTDEPYNAVKRMIWR.

[0049] The amino acid sequence of DNA-binding protein 2 is shown in SEQ ID NO:2:

[0050] MKYLTLKYTDEKYKINLDKITMVQIREGYICITFDAHNISEIYENECSNFFEIKKILENL.

[0051] The amino acid sequence of DNA-binding protein 3 is shown in SEQ ID NO:3:

[0052] MRLYRFTNEHGQDAALNIDKIGDMHQDRKDVHVRWGGAYQETTRIPNTTLEELINNLKLLGES.

[0053] The amino acid sequence of DNA-binding protein 4 is shown in SEQ ID NO:4:

[0054] MAFIKVKDKKTKEDTIINTNMICRISRNKNGYTVFFSSGNVGAAYYEYDEDNAKKIFDAIGVSL.

[0055] Nucleotide sequence design and synthesis:

[0056] Sequence design: The amino acid sequence of wild-type Pfu DNA polymerase is shown in SEQ ID NO:5. Four candidate DNA-binding proteins are fused to the C-terminus of Pfu DNA polymerase via a linker sequence. Figure 1 The four fusion proteins are candidate high-fidelity DNA polymerases (denoted as hfDNAP-1, hfDNAP-2, hfDNAP-3, and hfDNAP-4), and their nucleotide sequences, generated and optimized based on their amino acid sequences, are shown in SEQ ID NO:6-9.

[0057] SEQ ID NO:5:

[0058] 。

[0059] SEQ ID NO:6:

[0060]

[0061] SEQ ID NO:7:

[0062]

[0063] SEQ ID NO:8:

[0064]

[0065] SEQ ID NO:9:

[0066]

[0067] 2. Plasmid vector construction:

[0068] Four genes were synthesized and assembled according to SEQ ID NO:6-9. They were constructed in the open reading frame region of the pET-28a expression vector (P0023, Miaoling Biotechnology). The four plasmid DNAs were transformed into BL21(DE3) competent cells. The transformed bacterial culture was plated on solid LB agar plates containing kanamycin and cultured overnight at 37°C. Single colonies were picked and verified using Sanger sequencing. Single colonies with completely correct sequences were selected, and the corresponding plasmids and bacterial cultures were preserved.

[0069] 3. Prepare the solution:

[0070] 1) Isopropyl-β-D-thiogalactoside (IPTG) solution (1M): Weigh 2.38g IPTG and add it to 10mL of enzyme-free water. After dissolving, filter it through a 0.22µm filter membrane and store it at -20℃ for later use.

[0071] 2) Lysis buffer: 50mM Tris HCl (pH 8.0), 500mM NaCl, 0.1% NP-40 and 0.1% Triton X-100.

[0072] 3) Imidazole solution stock solution (2M): Weigh 1.36g of imidazole and add it to 10mL of enzyme-free water. After dissolving, adjust the pH to 8 with hydrochloric acid and store at 4℃ for later use.

[0073] 4) Gradient elution buffer:

[0074] 10mM elution buffer: 50mM Tris HCl (pH 8.0), 500mM NaCl / 100mM NaCl, 0.05% NP-40, 5% glycerol, 5mM β-mercaptoethanol, 1mM benzalkonium chloride and 10mM imidazole.

[0075] 20mM elution buffer: 50mM Tris HCl (pH 8.0), 100mM NaCl, 0.05% NP-40, 5% glycerol, 5mM β-mercaptoethanol, 1mM benzalkonium chloride and 20mM imidazole.

[0076] 50mM elution buffer: 50mM Tris HCl (pH 8.0), 100mM NaCl, 0.05% NP-40, 5% glycerol, 5mM β-mercaptoethanol, 1mM benzalkonium chloride and 50mM imidazole.

[0077] 300mM elution buffer: 50mM Tris HCl (pH 8.0), 100mM NaCl, 0.05% NP-40, 5% glycerol, 5mM β-mercaptoethanol, 1mM benzalkonium chloride and 300mM imidazole.

[0078] 5) Storage solution: 20mM Tris HCl (pH 8.0), 500mM NaCl.

[0079] 4. Protein-induced expression:

[0080] The four monoclonal bacterial cultures that were correctly sequenced were revived and inoculated into 200 mL of LB medium containing 50 μg / mL kanamycin. The cultures were then incubated with shaking at 37°C and 220 rpm until OD (outlet count) was reached. 600 When the pH reaches 0.8-1.0, add IPTG solution to the culture medium to a final concentration of 1 mM. Continue to induce culture at 37°C for 2 hours, then collect the bacterial cells by centrifugation.

[0081] 5. Protein purification:

[0082] The bacterial cells were washed twice with PBS, centrifuged, and the supernatant was discarded. 4 mL of lysis buffer was added to the bacterial pellet, and the cells were resuspended thoroughly. 20 µL of lysozyme solution (100 mg / mL) was added, and the mixture was thoroughly mixed. The mixture was placed in a four-dimensional rotary mixer and rotated for 15 min at room temperature. The enzymatically digested bacterial suspension was heated in a 75°C water bath for 1 h, centrifuged at 16000 × g for 10 min at 4°C, and the supernatant was collected. The mixture was filtered through a 0.22 µm filter membrane. 400 µL of glycerol and 1.4 µL of 100 mM benzidine were added to the filtrate, and the mixture was thoroughly mixed before use. Purification was performed using a nickel column (Ni-NTA). The nickel column was equilibrated with lysis buffer. 20 µL of imidazole stock solution (2 M) was added to the cell lysis buffer, mixed, and then bound to the nickel column. The target protein was eluted sequentially with a gradient of elution buffers from low to high concentration.

[0083] 6. Protein analysis, quantification, and storage:

[0084] The eluted fractions were subjected to SDS-PAGE electrophoresis and Coomassie Brilliant Blue staining. Based on the staining results, fractions with high target protein purity were collected for overnight dialysis. After dialysis, the fractions were concentrated using a 50 kDa ultrafiltration tube. The concentrate was collected for BCA quantification, and the purity was determined by SDS-PAGE electrophoresis and Coomassie Brilliant Blue staining. Figure 2 After being packaged, the product should be stored at -80°C to avoid repeated freeze-thaw cycles.

[0085] Example 2 Polymerase Activity Assay

[0086] The purified protein was uniformly diluted to 0.01 μg / μL, and the activity of the purified mutant enzyme was detected using a fluorescent polymerase activity assay kit (Biotium, USA, catalog number: #29051). The assay procedure was performed according to the kit instructions. Phusion® Ultra Fidelity DNA Polymerase (NEB, USA, catalog number: M0530S) was used to construct a standard curve. The results are as follows: Figure 3 As shown, 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. Among them, hfDNAP-3 had the highest polymerase activity, and its amino acid sequence is shown in SEQ ID NO:10.

[0087] SEQ ID NO:10

[0088] MILDVDYITEEGKPVIRLFKKENGKFKIEHDRTFRPYIYALLRDDSKIEEVKKITGERHGKIVRIVDVEKVEKKFLGKPITVWKLYLEHPQDVPTIREKVREHPAVVDIFEYDIPFAKRYLIDKGLIPMEGEEELKILAFDIETLYHEGEEFGKGPIIMISYADENEAKVITWKNIDLPYVEVVSSEREMIKRFLRIIREKDPDIIVTYNGDSFDFPYLAKRAEKLGIKLTIGRDGSEPKMQRIGDMTAVEVKGRIHFDLYHVITRTINLPTYTLEAVYEAIFGKPKEKVYADEIAKAWESGENLERVAKYSMEDAKATYELGKEFLPMEIQLSRLVGQPLWDVSRSSTGNLVEWFLLRKAYERNEVAPNKPSEEEYQRRLRESYTGGFVKEPEKGLWENIVYLDFRALYPSIIITHNVSPDTLNLEGCKNYDIAPQVGHKFCKDIPGFIPSLLGHLLEERQKIKTKMKETQDPIEKILLDYRQKAIKLLANSFYGYYGYAKARWYCKECAESVTAWGRKYIELVWKELEEKFGFKVLYIDTDGLYATIPGGESEEIKKKALEFVKYINSKLPGLLELEYEGFYKRGFFVTKKRYAVIDEEGKVITRGLEIVRRDWSEIAKETQARVLETILKHGDVEEAVRIVKEVIQKLANYEIPPEKLAIYEQITRPLHEYKAIGPHVAVAKKLAAKGVKIKPGMVIGYIVLRGDGPISNRAILAEEYDPKKHKYDAEYYIENQVLPAVLRILEGFGYRKEDLRYQKTRQVGLTSWLNIKKSGTGGGGRLYRFTNEHGQDAALNIDKIGDMHQDRKDVHVRWGGAYQETTRIPNTTLEELINNLKLLGES。

[0089] Example 34. Detection of the error rate of 9k DNA fragment assembly

[0090] A DNA sequence of 4914 bp was selected, as shown in SEQ ID NO:11. Oligonucleotide sequences (SEQ ID NO:12-SEQ ID NO:137) for assembling this DNA fragment were designed using DNAWorks software. Each sequence was synthesized and mixed to prepare an Oligo Mix solution (10 µM). Six groups were set up for the experiment: the Phu-WT group used laboratory-expressed and purified Phusion high-fidelity DNA polymerase; the hfDNAP group used four candidate high-fidelity DNA polymerases; and the Positive Control group used imported commercial reagent Phusion® ultra-fidelity DNA polymerase (NEB Corporation, catalog number: M0530S).

[0091] SEQ ID NO:11:

[0092]

[0093] Prepare 10×PCR Buffer: 100mM Tris-HCl (pH 8.9), 500mM KCl, 15mM MgCl2. The first-round reaction mixture consisted of: 2.0μL 10×PCR Buffer, 1.6μL dNTPs (2.5mM each), 2μL hfDNAP protein, 4.0μL Loligos Mix, and ddH2O to a final volume of 20μL. The reaction program was: 95℃ for 3 min; 98℃ for 10 s, 60℃ for 20 s, 72℃ for 2 min / cycle, for a total of 30 cycles; 72℃ for 5 min; 4℃ infinity. The second-round reaction system consisted of: 5.0 μL 10×PCR Buffer, 4.0 μL dNTP (2.5 mM each), 5 μL hfDNAP protein, 1 µL of the first-round PCA product, 1.5 µL Primer F (10 µM), 1.5 µL Primer R (10 µM), and enzyme-free ultrapure water to a final volume of 50 μL. The reaction procedure 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.

[0094] After the reaction was completed, the assembled products were subjected to agarose gel electrophoresis, and the results are as follows: Figure 4 As shown, only the Phu-WT, hf-DNAP3, and Positive Control groups showed target bands. The assembly products from these three groups were recovered and ligated with the vector pUC57 (Beijing Bomeide Gene Technology Co., Ltd., catalog number: CL118) to construct plasmids. The ligation system consisted of 5.0 μL 2×NEB Builder Buffer, 100 ng DNA, 100 ng plasmid DNA, and ddH2O to a final volume of 10.0 μL. The mixture was incubated at 50°C for 1 h in a metal bath. The ligation products were then transformed into NEB10β competent cells and cultured on solid LB plates containing ampicillin at 37°C for 16–18 h. Six single colonies were picked and Sanger sequenced. Error types and error rates were analyzed (Tables 1–3).

[0095] Table 1 Statistical analysis of hf-DNAP-1 assembly error types

[0096]

[0097] Table 2 Statistical analysis of hfDNAP-3 assembly error types

[0098]

[0099] Table 3. Statistical Analysis of NEB Phusion® Ultra-Fidelity DNA Polymerase Assembly Error Types

[0100]

[0101] Example 4: DNA Assembly Stability Test

[0102] To test the stability or general applicability of hfDNAP-3 in DNA assembly, different sequences were selected for assembly according to the method in Example 3, and the presence of the target length band was identified by agarose gel electrophoresis. The CDS sequence (NM_004006.3) of the human DMD gene (NCBI Gene ID: 1756) was selected and divided into three segments: DNA long fragment one, 3804 bp in length; DNA long fragment two, 3877 bp in length; and DNA long fragment three, 3836 bp in length. The experiment was conducted in four groups: the Phu-WT group, using laboratory-expressed and purified Phusion high-fidelity DNA polymerase; the hfDNAP-3 group, using the screened high-fidelity DNA polymerase hfDNAP-3; the NEB Phu group, using imported commercial reagent Phusion® ultra-fidelity DNA polymerase (NEB Corporation, USA, catalog number: M0530S); and the NEB Q5 group, using imported commercial reagent Q5® ultra-fidelity DNA polymerase (NEB Corporation, USA, catalog number: M0491S). Each group was assembled using the corresponding high-fidelity enzyme to assemble the three fragments described above. The assembly reaction and procedure were the same as in Example 3. The gel electrophoresis results of the assembly products are shown below. Figure 5 As shown, hfDNAP-3 can successfully assemble three target fragments with low levels of non-specific assembly products and superior assembly stability compared to existing high-fidelity DNA polymerases and commercially available ultra-fidelity DNA polymerases.

[0103] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

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

10.

2. A DNA molecule encoding the high-fidelity DNA polymerase of claim 1.

3. The DNA molecule of claim 2, wherein, The nucleotide sequence of the DNA molecule is shown as SEQ ID NO:

8.

4. A recombinant expression vector, characterized in that, The DNA molecule of any one of claims 2-3 is cloned into an expression vector.

5. A recombinant engineered cell line, characterized in that, The recombinant expression vector of claim 4 is transformed into an engineered cell.

6. The recombineering cell strain of claim 5, wherein, The engineered cell is an E. coli cell.

7. The high-fidelity DNA polymerase of claim 1, the DNA molecule of any one of claims 2-3, the recombinant expression vector of claim 4, or the recombinant engineered cell strain of any one of claims 5-6 is used in DNA amplification, DNA assembly, gene cloning, NGS sequencing library preparation.

8. The method of producing a high-fidelity DNA polymerase according to claim 1, wherein, 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.

9. A product of a gene assembly, characterized in that, The product comprises the high-fidelity DNA polymerase of claim 1.

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