A high-temperature-resistant DNA polymerase with high fidelity and high amplification efficiency, a preparation method thereof, a PCR amplification method and application thereof

Abstract

The application belongs to the technical field of molecular biology and protein engineering, and discloses a DNA polymerase with high fidelity and high amplification efficiency, a preparation method of the DNA polymerase, a PCR amplification method and application of the DNA polymerase. The amino acid sequence of the high-temperature-resistant DNA polymerase provided by the application is shown as SEQ ID NO:1. The high-temperature-resistant DNA polymerase has excellent mismatch correction capability and amplification performance, and can be well applied to in-vitro amplification of DNA, so that high-fidelity and high-efficiency amplification of DNA can be realized, and the application prospect is good.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology and protein engineering, and in particular relates to a DNA polymerase with high fidelity and high amplification efficiency, a preparation method thereof, a PCR amplification method and an application thereof. Background Art

[0002] Polymerase Chain Reaction (PCR) is a molecular biology technique that has rapidly become popular and widely used in recent decades. It simulates the DNA replication process in vivo in an in vitro environment. Under the action of DNA polymerase, by controlling the periodic change of the reaction temperature, it can achieve exponential growth of the target sequence in a short period of time and efficiently amplify the expected DNA fragment. PCR amplification has a wide range of applications, including but not limited to molecular cloning, genome sequencing, forensic identification, and diagnosis of genetic diseases and infectious diseases. It has not only promoted the development of precision medicine, but also accelerated the progress of research on the correlation between genes and genetic diseases. However, PCR amplification also faces many challenges and limitations. Factors such as DNA template quality, reaction system and reaction conditions will have a significant impact on the amplification process.

[0003] Among them, the performance of DNA polymerase is one of the important factors affecting the amplification rate and effect of PCR. The first high-temperature resistant DNA polymerase discovered was Taq DNA polymerase isolated by Rand K. Saiki from Thermus aquaticus YT-1. The discovery of this enzyme has significantly simplified the operational process of PCR amplification. However, existing DNA polymerases cannot effectively correct mismatched single nucleotides during DNA synthesis, have low fidelity, and mainly play the role of DNA damage repair and gene recombination in cells. They have poor extension ability and extension rate, and have great limitations. Summary of the Invention

[0004] The first object of the present invention is to provide a thermostable DNA polymerase. This thermostable DNA polymerase has excellent mismatch correction and amplification performance, can be well applied to in vitro DNA amplification to achieve high-fidelity and high-efficiency DNA amplification, and has good application prospects.

[0005] The second object of the present invention is to provide a method for preparing the above-mentioned high-temperature resistant DNA polymerase.

[0006] The third object of the present invention is to provide a PCR amplification method.

[0007] The fourth object of the present invention is to provide the use of the above-mentioned high-temperature resistant DNA polymerase in the field of DNA in vitro amplification.

[0008] Specifically, the amino acid sequence of the thermostable DNA polymerase provided by the present invention is shown in SEQ ID NO: 1.

[0009] The preparation method of the above-mentioned high-temperature resistant DNA polymerase provided by the present invention specifically comprises: S1, taking the coding gene of the high-temperature resistant DNA polymerase and introducing it into the host cell to obtain an engineered bacterium; S2, taking the engineered bacterium and fermenting it to obtain a fermentation culture liquid; S3, taking the fermentation culture liquid and purifying it to obtain the high-temperature resistant DNA polymerase.

[0010] Furthermore, in step S1, the gene encoding the thermostable DNA polymerase includes the nucleotide fragment shown in SEQ ID NO: 2.

[0011] Furthermore, in step S1, the host cell is selected from one or more of Escherichia coli BL21, Escherichia coli BL21 (DE3) and Escherichia coli BL21 Star (DE3).

[0012] Furthermore, in step S2, the fermentation treatment includes: taking the engineered bacteria and inoculating them into a fermentation medium for fermentation culture to obtain a seed liquid; taking an inducing expression agent and adding it to the seed liquid to induce expression to obtain a fermentation culture liquid.

[0013] Furthermore, in step S2, the cell inoculation amount of the fermentation culture is 10 4 ~10 6 / mL, the temperature is 35-40℃, and the incubation time is 12-48h.

[0014] Furthermore, in step S2, the expression inducing agent is selected from one or more of isopropylthiogalactoside, lactose and arabinose.

[0015] Furthermore, in step S2, the amount of the inducing agent added for inducing expression is 0.05 to 0.2 mmol / L, the temperature is 25 to 30° C., and the time is 16 to 18 hours.

[0016] Furthermore, in step S3, the purification treatment includes: taking the lysate and adding it to the fermentation culture medium for ultrasonic lysis treatment to obtain a lysis product; taking the lysis product and performing centrifugation treatment, Ni-NTA column chromatography treatment and dialysis treatment to obtain the high-temperature resistant DNA polymerase.

[0017] Furthermore, in step S3, the lysis solution includes 2-10 mg / mL lysozyme and 0.01-0.1 mol / L PBS buffer, and the pH of the PBS buffer is 7-8.

[0018] Furthermore, in step S3, the temperature of the ultrasonic lysis treatment is 0-5°C, and the ultrasonic power is 50-200W.

[0019] In the PCR amplification method provided by the present invention, the above-mentioned high-temperature resistant DNA polymerase is used to perform PCR amplification on a DNA template.

[0020] The present invention provides application of the high-temperature resistant DNA polymerase in the field of DNA in vitro amplification.

[0021] Beneficial effects:

[0022] The specific amino acid composition of the high-temperature resistant DNA polymerase provided by the present invention enables it to have excellent binding force for DNA, can well bind to the DNA template, and can quickly undergo conformational changes, thereby well catalyzing the formation of peptide bonds and achieving movement, thereby endowing the high-temperature resistant DNA polymerase with excellent amplification ability, and can achieve high-efficiency and long-fragment amplification of DNA; at the same time, the high-temperature resistant DNA polymerase also has good 3'-5' nuclease activity, can effectively correct mismatched single nucleotides during the DNA synthesis process, has excellent fidelity performance, can be well applied to in vitro DNA amplification, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a gel electrophoresis diagram of the high-temperature resistant DNA polymerase provided in Example 1 of the present invention. DETAILED DESCRIPTION

[0024] To obtain a thermostable DNA polymerase with excellent mismatch correction and amplification performance, the inventors of the present invention, through extensive and in-depth research, creatively obtained a thermostable DNA polymerase. The amino acid sequence of the thermostable DNA polymerase is shown in SEQ ID NO: 1.

[0025] In the present invention, the high-temperature resistant DNA polymerase belongs to a type of KOD DNA polymerase, which has excellent thermal stability, amplification performance and mismatch correction ability, and can be well applied to PCR amplification to achieve high-efficiency and accurate in vitro amplification of DNA.

[0026] To obtain the aforementioned thermostable DNA polymerase, the present invention also provides a method for preparing the thermostable DNA polymerase. The method specifically comprises: S1, introducing the gene encoding the thermostable DNA polymerase into a host cell to obtain an engineered bacterium; S2, fermenting the engineered bacterium to obtain a fermentation broth; and S3, purifying the fermentation broth to obtain the thermostable DNA polymerase.

[0027] In the present invention, in step S1, the gene encoding the thermostable DNA polymerase preferably includes SEQ ID NO: 2. At this time, the gene encoding the thermostable DNA polymerase is codon-optimized and can be efficiently expressed in the host cell to achieve high-efficiency production of the thermostable DNA polymerase.

[0028] In the present invention, in step S1, the host cell is a type of cell commonly used in the field of genetic engineering, which has a protein expression system for synthesizing the high-temperature resistant DNA polymerase. Those skilled in the art can make adaptive choices according to actual needs, and the present invention does not impose any special limitations on it.

[0029] In some specific embodiments, specific examples of the host cell include but are not limited to: the host cell is selected from one or more of Escherichia coli BL21, Escherichia coli BL21 (DE3) and Escherichia coli BL21Star (DE3).

[0030] In the present invention, in step S1, the method for introducing the gene encoding the high-temperature resistant DNA polymerase into the host cell is the exogenous gene introduction technology conventionally used in the field of genetic engineering, which is limited to the ability to enable the gene encoding the high-temperature resistant DNA polymerase to enter the host cell without causing too much adverse effect on the host cell. Those skilled in the art can make adaptive choices according to actual needs, and the present invention does not impose any special limitation on it.

[0031] In the present invention, in step S2, the fermentation treatment is to expand the culture of the host cells and induce the host cells to express the high-temperature resistant DNA polymerase, specifically comprising: taking the engineered bacteria and inoculating them into a fermentation medium for fermentation culture to obtain a seed liquid; taking an inducing expression agent and adding it to the seed liquid to induce expression to obtain a fermentation culture liquid.

[0032] In the present invention, the fermentation medium is a type of bacterial culture medium commonly used in the biological field, and is limited to the host cells being able to carry out normal life activities such as growth and reproduction. Those skilled in the art can make adaptive choices according to actual needs. The present invention does not impose any particular limitation on it. Specific examples include but are not limited to: one or more of LB medium, MS medium and EMB medium.

[0033] In some specific embodiments, the fermentation culture conditions include: the cell inoculation amount is preferably 10 4 ~10 6 Particles / mL, such as 1×10 4 / mL, 1.5×10 4 / mL, 1.8×10 4 / mL, 1×10 5 / mL, 1.5×105 / mL, 1×10 6 cells / mL or any value therebetween; the temperature is preferably 35-40°C, such as 35°C, 36°C, 38.5°C, 39°C, 40°C or any value therebetween; the culture time is preferably 12-48h, such as 12h, 18h, 24h, 30h, 36h, 40h, 48h or any value therebetween.

[0034] In the present invention, the inducible expression agent is a type of substance that can induce the expression of exogenous genes in host cells with a prokaryotic protein expression system such as Escherichia coli. Those skilled in the art can make adaptive choices based on actual needs. The present invention does not impose any particular limitation on it. Specific examples include but are not limited to: one or more of isopropylthiogalactoside, lactose and arabinose.

[0035] In some specific embodiments, the conditions for inducing expression include: the addition amount of the inducing expression agent is preferably 0.05-0.2 mmol / L, such as 0.05 mmol / L, 0.08 mmol / L, 0.1 mmol / L, 0.13 mmol / L, 0.15 mmol / L, 0.18 mmol / L, 0.2 mmol / L or any value therebetween; the temperature is preferably 25-30°C, such as 25°C, 28°C, 29°C, 30°C or any value therebetween; the time is preferably 16-18 h, such as 16 h, 16.5 h, 17 h, 18 h or any value therebetween.

[0036] In the present invention, in step S3, the purification treatment is to separate the high-temperature resistant DNA polymerase from the fermentation culture medium and remove impurities therein, thereby obtaining a high-purity high-temperature resistant polymerase, specifically comprising: taking a lysate and adding it to the fermentation culture medium for ultrasonic lysis treatment to obtain a lysate; taking the lysate and performing centrifugation, Ni-NTA column chromatography and dialysis treatment to obtain the high-temperature resistant DNA polymerase.

[0037] In the present invention, the lysis solution is a type of substance commonly used in the biological field to destroy the cell membrane structure of the host cell to release intracellular substances. Those skilled in the art can make adaptive choices according to the needs of the example, and the present invention does not impose any special limitation on it.

[0038] In some specific embodiments, the lysis solution specifically comprises lysozyme and a PBS buffer having a pH of 7 to 8. The concentration of the lysozyme is preferably 2 to 10 mg / mL, such as 2 mg / mL, 2.3 mg / mL, 2.8 mg / mL, 3 mg / mL, 4 mg / mL, 6 mg / mL, 8 mg / mL, 10 mg / mL, or any value therebetween; and the concentration of the PBS buffer is preferably 0.01 to 0.1 mol / L, such as 0.01 mol / L, 0.03 mol / L, 0.05 mol / L, 0.08 mol / L, 0.1 mol / L, or any value therebetween.

[0039] In the present invention, the ultrasonic lysis treatment is intended to destroy the cell structure of the host cells and release the high-temperature resistant DNA polymerase. It is a technique commonly used in the field of genetic engineering, and is limited to achieving protein release without destroying protein activity. Those skilled in the art can make adaptive choices based on actual needs, and the present invention does not impose any special limitations on it.

[0040] In some specific embodiments, the conditions for ultrasonic lysis include: the temperature is preferably 0-5°C, such as 0°C, 0.5°C, 1°C, 2°C, 4°C, 5°C or any value therebetween; the ultrasonic power is preferably 50-200W, such as 50W, 80W, 100W, 150W, 200W or any value therebetween.

[0041] In the present invention, the centrifugation treatment is a technique commonly used in the biological field, which is limited to achieving the separation between the high-temperature resistant DNA polymerase and other impurities with large molecular weight differences. Those skilled in the art can make adaptive choices according to actual needs, and the present invention does not impose any special limitations on it.

[0042] In the present invention, the Ni-NTA column chromatography treatment is an affinity chromatography technology that utilizes nickel ions to bind to a histidine tag (His-tag). It is a technology commonly used in the biological field, and is limited to separating target proteins with His tags from complex biological samples. Those skilled in the art can make adaptive choices based on actual needs, and the present invention does not impose any particular limitation on it.

[0043] In the present invention, the dialysis treatment is a technology that utilizes the pore size of a semipermeable membrane to achieve separation of small molecules and proteins. It is a technology commonly used in the biological field. Those skilled in the art can make adaptive choices based on actual needs, and the present invention does not impose any special limitations on it.

[0044] The present invention also provides a PCR amplification method. The PCR amplification method uses the above-mentioned high-temperature resistant DNA polymerase to catalyze in vitro DNA amplification under periodic temperature changes, and specifically comprises: using the high-temperature resistant DNA polymerase to perform PCR amplification on a DNA template.

[0045] In the present invention, the PCR amplification is a technique commonly used in the biological field, which is limited to achieving rapid and large-scale amplification of DNA templates. Those skilled in the art can make adaptive choices according to actual needs, and the present invention does not impose any special limitations on it.

[0046] The present invention also provides the use of the thermostable DNA polymerase in in vitro DNA amplification. More specifically, specific examples of the use of the thermostable DNA polymerase in in vitro DNA amplification include, but are not limited to, one or more of: nucleic acid product production, molecular cloning, genome sequencing, and nucleic acid detection.

[0047] The embodiments of the present invention are described in detail below. The examples of the embodiments are intended to explain the present invention and are not to be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in this area or according to the product specifications. Reagents or instruments used that do not specify the manufacturer are all conventional products that can be obtained commercially.

[0048] The reagents and sources used in the following examples include:

[0049] The lysis solution included 4 mg / mL lysozyme and 0.01 mol / L PBS, pH = 7.4.

[0050] The treatment buffer included 0.1 mmol / L EDTA, 50 mmol / L KCl, 1 mmol / L DTT, 50% (v / v) glycerol and 50 mmol / L Tris-HCl, pH=8.0.

[0051] KOD DNA polymerase was purchased from Beijing Biolabs, product number BTN101002.

[0052] Bacterial Transformation Kit (Thermo Fisher, catalog number K2711).

[0053] PCR buffer (Shanghai Chuangsai Technology, catalog number PM11657).

[0054] DNA Ladder (Thermo Fisher Scientific, catalog number SM0331).

[0055] Example 1

[0056] This example is used to illustrate a thermostable DNA polymerase and a preparation method thereof. The amino acid sequence of the thermostable DNA polymerase is shown in SEQ ID NO: 1. The preparation method specifically includes:

[0057] 1. Construction of engineered bacteria

[0058] (1) The codons of the Escherichia coli expression system were optimized according to the amino acid sequence of the high-temperature resistant DNA polymerase to obtain a gene encoding the high-temperature resistant DNA polymerase, the specific nucleotide sequence of which is shown in SEQ ID NO: 2, and BamHⅠ and HindIIIⅠ restriction sites were added to both ends of the encoding gene, which was then synthesized by a chemical synthesis company; finally, the obtained gene encoding the high-temperature resistant DNA polymerase was recombined into the pET16a vector using BamHⅠ and HindIIIⅠ enzymes to obtain a recombinant plasmid.

[0059] (2) The recombinant plasmid was transformed into Escherichia coli BL21 (DE3) competent cells using a bacterial transformation kit according to the instructions, and spread on a solid LB plate containing 100 mg / mL ampicillin. The cells were cultured at 37°C overnight to obtain single colonies that could grow. Some cells from the single colonies were taken for identification, and the engineered bacteria that had successfully introduced the recombinant plasmid were screened.

[0060] 2. Fermentation treatment of engineered bacteria

[0061] (1) According to 10 5 The engineered bacteria were selected at an inoculum size of 100 mg / mL and inoculated into LB liquid culture medium containing 100 mg / mL ampicillin, and cultured at 37° C. for 12 h to obtain seed solution.

[0062] (2) IPTG was added to the seed solution at a dosage of 0.1 mmol / L and cultured at 28°C for 18 h to obtain a fermentation culture solution.

[0063] 3. Purification of thermostable DNA polymerase

[0064] (1) Take 50 mL of fermentation culture medium and centrifuge it at 4°C and 6000 rpm for 15 min. Remove the supernatant. Take 10 mL of lysis solution pre-cooled to 4°C and add it to the precipitate for resuspending. Then, ultrasonically lyse the precipitate in an ice bath using an ultrasonic disruptor (ultrasonication for 27 seconds, interval for 3 seconds, ultrasonic power of 100 W) to obtain a clear and transparent lysate.

[0065] (2) Take the lysate and centrifuge it at 4°C and 10,000 r / min for 30 min, collect the supernatant to obtain a crude enzyme solution; take an equal volume of 1× PBS and mix it evenly with the crude enzyme solution to obtain a mixed solution; take 600 μL of the mixed solution each time and add it to the Ni-NTA column, centrifuge it at 750 r / min for 30 s, and remove the supernatant; after the mixed solution is added, use 1× PBS to wash the Ni-NTA column 5 times; take the treatment buffer and add it to the Ni-NTA column and let it stand for 30 s, centrifuge it at 4°C and 750 r / min for 30 s, collect the eluate to obtain a pre-purified enzyme solution; take the pre-purified enzyme solution and add it to the dialysis membrane bag, and place the dialysis membrane bag in a beaker filled with treatment buffer, dialyze it at 4°C for 12 h, and change the treatment buffer every 4 h; transfer the solution in the dialysis membrane to a centrifuge tube, add 5 μL of MgCl2 (1 mol / L) and 10 μL of DNase I (10U / μL), incubate at 37℃ for 20min, heat at 70℃ for 10min, then centrifuge at 14000r / min for 5min, collect the supernatant to obtain pure enzyme solution. Take 1μL of pure enzyme solution for gel electrophoresis, the results are as follows Figure 1 shown.

[0066] Depend on Figure 1 The test results show that the molecular weight of the thermostable DNA polymerase is 86.22 kD and has a high purity.

[0067] Example 2

[0068] This example is used to illustrate the catalytic activity and thermal stability of the thermostable DNA polymerase provided in Example 1. Commercially available KOD DNA polymerase is used as a control. The thermostable DNA polymerase and commercially available KOD DNA polymerase are mixed with nuclease-free water to prepare a test enzyme solution with a concentration of 0.05 μg / μL. The test enzyme solution is incubated at 90° C. for 0 h, 2 h, 4 h, 6 h, 8 h, and 10 h, respectively, and the following tests are performed:

[0069] (1) Take 1 μL of the enzyme solution to be tested, use salmon sperm DNA as template / primer, and perform the enzyme-catalyzed reaction at 74°C for 30 min. Immediately place on ice to terminate the reaction.

[0070] The reaction system of the enzyme-catalyzed reaction includes 1 ng of salmon sperm DNA, 200 μmol / L of dNTPs, 1 μCi [α-32P] of dCTP, 20 mmol / L of Tris-HCl, 2 mmol / L of MgCl2, 5 mmol / L of (NH4)2SO4, 10 mmol / L of KCl, 0.1 g / L of BSA and 0.05% of Triton X-100, pH 8.0.

[0071] (2) 5 μL of the reaction product was added to an ion exchange paper disc. After the spot dried, it was rinsed three times with 2×SSC buffer (containing 300 mmol / L NaCl and 30 mmol / L Na3C6H5O7, pH 7.0). Finally, it was washed once with anhydrous ethanol after an ice bath and dried. The incorporated radioactive material was measured using a liquid scintillation counter. The amount of enzyme that catalyzes the incorporation of 10 nmol of dNTPs into DNA was defined as 1 unit of enzyme activity. The results are shown in Table 1.

[0072] Table 1.

[0073]

[0074] As shown in the test results in Table 1, compared with commercially available KOD DNA polymerase, the thermostable DNA polymerase provided in Example 1 of the present invention has an initial enzyme activity of 19.32 U / μL before incubation at 90°C, and still has an enzyme activity of 8.99 U / μL after incubation at 90°C for 10 h, demonstrating both excellent catalytic activity and thermal stability.

[0075] Example 3

[0076] This example illustrates the amplification performance of the thermostable DNA polymerase provided in Example 1. Commercially available KOD DNA polymerase was used as a control. The thermostable DNA polymerase and commercially available KOD DNA polymerase were mixed with nuclease-free water to prepare a test enzyme solution with a concentration of 0.05 μg / μL. The following tests were performed:

[0077] (1) Human genomic DNA was used as a template and PCR amplification was performed using the primers shown in Table 2 to obtain PCR amplification products with lengths of 1 kb, 2 kb, 4 kb, 8 kb, and 16 kb.

[0078] Table 2.

[0079]

[0080]

[0081] The PCR reaction program included: 30 cycles of pre-denaturation at 95°C for 3 minutes, denaturation at 95°C for 15 seconds, annealing at 58°C for 15 seconds, extension at 72°C for 2 minutes, and extension at 72°C for 5 minutes. The specific PCR reaction system is shown in Table 3.

[0082] Table 3.

[0083] substance concentration Add Volume Human genomic DNA 4mg / L 2.5 μL dATP 10mmol / L 0.5μL dTTP 10mmol / L 0.5μL dCTP 10mmol / L 0.5μL dGTP 10mmol / L 0.5μL PCR amplification buffer - 2.5 μL BSA 1mg / L 2.5 μL Primer pUC19F 10nmol / L 0.5μL Primer pUC19R 10nmol / L 0.5μL Enzyme solution to be tested 0.05 μg / μL 1 μL <![CDATA[ddH2O]]> - Make up the total volume to 25 μL

[0084] (2) 0.5 μL of each amplified product and DNA Ladder were respectively taken for gel electrophoresis. The grayscale of the gel electrophoresis bands was analyzed using Image J and normalized with reference to the total intensity of the DNA Ladder bands to obtain the normalized intensity of each band. The results are shown in Table 4.

[0085] Table 4.

[0086]

[0087] The test results shown in Table 4 show that, compared with the commercially available KOD DNA polymerase, the thermostable DNA polymerase provided in Example 1 of the present invention has an excellent amplification rate and also has good amplification ability for long DNA fragments.

[0088] Example 4

[0089] This example is used to illustrate the fidelity performance of the thermostable DNA polymerase provided in Example 1. Using commercially available KOD DNA polymerase as a control, the thermostable DNA polymerase and commercially available KOD DNA polymerase were mixed with nuclease-free water to prepare a test enzyme solution with a concentration of 0.05 μg / μL. The test was performed using the blue-white screening assay, specifically including:

[0090] (1) Using plasmid pUC19 as a template, PCR amplification was performed using primers pUC19F (nucleotide sequence shown in SEQ ID NO: 9) and pUC19R (nucleotide sequence shown in SEQ ID NO: 10) to obtain a PCR amplification product. The PCR reaction procedure and PCR reaction system were the same as those in Example 3.

[0091] (2) The PCR amplified product was transformed into Escherichia coli DH5α competent cells and spread onto a solid LB plate containing 100 mg / mL ampicillin. The plate was cultured overnight at 37°C to obtain single colonies. The number of blue colonies and white colonies on the plate culture medium was recorded, where blue colonies were normal and white colonies were mutants. The mismatch rate was calculated and the results are shown in Table 5.

[0092] Table 5.

[0093] Group Thermostable DNA polymerase Commercially purchased KOD DNA polymerase <![CDATA[Mismatch rate (×10 -6 )]]> 3.8 12.5

[0094] The test results shown in Table 5 show that compared with the commercially available KOD DNA polymerase, the mismatch rate of the high-temperature resistant DNA polymerase provided in Example 1 of the present invention for PCR amplification of plasmid pUC19 is only 3.8×10 -6 , with excellent fidelity performance.

[0095] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention without departing from the principles and purpose of the present invention.

[0096] The amino acid and nucleotide sequences involved in the present invention are shown in Table 6.

[0097] Table 6.

[0098]

[0099]

[0100]

Claims

1. A thermostable DNA polymerase, characterized in that The amino acid sequence of the high-temperature resistant DNA polymerase is shown in SEQ ID NO:

1.

2. The method for preparing the high temperature resistant DNA polymerase according to claim 1, wherein The preparation method comprises: S1, introducing the coding gene of the high-temperature resistant DNA polymerase into a host cell to obtain an engineered bacterium; S2, fermenting the engineered bacterium to obtain a fermentation broth; S3, purifying the fermentation broth to obtain the high-temperature resistant DNA polymerase.

3. The method for preparing the high temperature resistant DNA polymerase according to claim 2, wherein In step S1, the gene encoding the thermostable DNA polymerase is a nucleotide fragment as shown in SEQ ID NO:

2.

4. The method for preparing the high temperature resistant DNA polymerase according to claim 2, wherein In step S1, the host cell is selected from one or more of Escherichia coli BL21, Escherichia coli BL21 (DE3) and Escherichia coli BL21 Star (DE3).

5. The method for preparing the high temperature resistant DNA polymerase according to claim 2, wherein In step S2, the fermentation treatment includes: taking the engineered bacteria and inoculating them into a fermentation medium for fermentation culture to obtain a seed solution; taking an inducing expression agent and adding it into the seed solution to induce expression to obtain a fermentation culture solution.

6. The method for preparing the high temperature resistant DNA polymerase according to claim 5, characterized in that: The cell inoculation amount of the fermentation culture is 10 4 ~10 6 / mL, the temperature is 35~40℃, and the incubation time is 12~48h.

7. The method for preparing a high-temperature resistant DNA polymerase according to claim 5, wherein: The expression inducing agent is selected from one or more of isopropylthiogalactoside, lactose and arabinose.

8. The method for preparing a high-temperature resistant DNA polymerase according to claim 5, wherein The induction agent for inducing expression is added in an amount of 0.05-0.2 mmol / L, the temperature is 25-30° C., and the time is 16-18 hours.

9. The method for preparing a high-temperature resistant DNA polymerase according to claim 2, wherein: In step S3, the purification process includes: taking the lysate and adding it to the fermentation culture medium for ultrasonic lysis to obtain a lysate; taking the lysate and performing centrifugation, Ni-NTA column chromatography and dialysis to obtain the high temperature resistant DNA polymerase.

10. The method for preparing a high-temperature resistant DNA polymerase according to claim 9, wherein: The lysis solution includes 2-10 mg / mL lysozyme and 0.01-0.1 mol / L PBS buffer, and the pH of the PBS buffer is 7-8.

11. The method for preparing a high-temperature resistant DNA polymerase according to claim 9, wherein: The temperature of the ultrasonic lysis treatment is 0-5°C, and the ultrasonic power is 50-200W.

12. A PCR amplification method for non-therapeutic or non-diagnostic purposes, characterized in that: The PCR amplification method comprises: performing PCR amplification on a DNA template using the high-temperature resistant DNA polymerase according to claim 1.

13. Use of the thermostable DNA polymerase according to claim 1 in in vitro DNA amplification for non-therapeutic or non-diagnostic purposes.

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