A thermostable DNA polymerase mutant with high amplification activity

Abstract

A thermostable DNA polymerase mutant with high amplification activity. Using protein directed evolution technology, a random mutation library was constructed for the polymerase active domain targeting Taq enzyme. By gradually adding screening pressure, unsuitable mutations were naturally eliminated, and mutations with advantageous traits gradually accumulated. Finally, a series of amino acid sites and their mutations that play a key role in the amplification performance and polymerization performance of Taq enzyme were screened out, and a Taq enzyme mutant with high amplification performance was obtained.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology. Specifically, the present invention relates to a thermostable DNA polymerase mutant with high amplification activity. Background Art

[0002] Taq enzyme is a heat-resistant DNA polymerase derived from the heat-resistant bacterium Thermus aquaticus, with a molecular weight of 94KDa. In the presence of magnesium ions, its optimal reaction temperature is 75-80°C, and its half-life of activity at 95°C is 40 minutes. It has 5'-3' exonuclease activity. Due to its heat-resistant characteristics, it is widely used in polymerase chain reaction (PCR) and is the first choice enzyme for nucleic acid amplification, detection and other reactions. Commercial Taq enzyme is cloned and expressed using the Escherichia coli prokaryotic expression system. Modern molecular biological detection techniques have increasingly high requirements for the sensitivity, accuracy, and durability of PCR reactions, and wild-type Taq enzyme cannot fully meet the actual application needs.To make it more suitable for use in specific technologies, many attempts have been made to mutate and modify the Taq enzyme sequence. For example, adding a DNA-binding domain to make it have stronger elongation activity (Wang Y (2004). A novel strategy to engineer DNA polymerases for enhanced processivity and improved performance in vitro. Nucleic Acids Res 32, 1197–1207)); making it have higher fidelity by site-directed mutagenesis and deleting domains (Suzuki M, Yoshida S, Adman ET, Blank A, Loeb LA (2000) Thermus Aquaticus DNA polymerase I mutants with altered fidelity. Interacting mutations in the O-Helix. J Biol Chem 275:32728–32735), higher DNA polymerase activity (Mutant Taq DNA polymerases with improved elongation ability as a useful reagent for genetic engineering. Front Microbiol 5:461. doi:10.3389 / fmicb.2014.00461), tolerance to high concentrations of inhibitors (Zhang Z, Kermekchiev MB, Barnes WM (2010) Direct DNA amplification from crude clinical samples using a PCR enhancer cocktail and novel mutants of Taq. J Mol Diagn 12:152–161), and reduced 5'-3' exonuclease activity (Vainshtein I, Atrazhev A, Eom SH, Elliott JF, Wishart DS, Malcolm BA (1996) Peptide rescue of an N-Terminal truncation of the Stoffel fragment of Taq DNA polymerase. Protein Sci 5:51785–51792).

[0003] The modification of Taq enzyme mainly has the following several ways. 1: Adding domains to endow it with new properties. For example, adding a single-stranded DNA-binding domain (SSB) or the DNA-binding protein Sso7 can enhance the binding ability of Taq enzyme to primers and template DNA, endowing it with stronger extension ability and continuous synthesis ability, which is suitable for the amplification reaction of long-fragment DNA. However, adding domains will directly increase the molecular weight of Taq enzyme, which may reduce the solubility and stability of Taq enzyme, and decrease the yield of prokaryotic expression production. 2: Removing unnecessary domains from Taq enzyme. For example, deleting the 5'-3' exonuclease domain (the first 280 amino acids at the N-terminus of Taq enzyme) can make Taq enzyme retain only the nucleic acid polymerase active region, reducing the possibility of primer and template DNA degradation caused by high-concentration Taq enzyme, so as to achieve the purpose of improving the polymerization activity of Taq enzyme. However, the Taq enzyme mutant obtained by this method lacks 5'-3' exonuclease activity and is not suitable for quantitative PCR reactions based on TaqMan probe method, and its applicable range is limited. 3: Site-directed mutagenesis method. Site-directed mutagenesis is carried out on the amino acids at the active site, magnesium ion binding site, and DNA binding site to improve the affinity of each site for substrates, templates, and primers, thereby improving the tolerance to various inhibitors. Due to the complexity of protein structure, some amino acids far from the active site may also affect the overall structure of the enzyme. Therefore, only mutating the amino acids at specific active center sites is difficult to modify the enzyme as a whole. Moreover, existing computer simulation technologies are difficult to predict the impact of mutations at each site on the overall structure. The workload of preparing mutants and screening mutants by site-directed mutagenesis is very large and the efficiency is low, and some sites that may have a significant impact on activity cannot be identified. Summary of the Invention

[0004] An object of the present invention is to provide a thermostable DNA polymerase mutant with high amplification activity.

[0005] In a first aspect of the present invention, a mutant DNA polymerase is provided, wherein the mutant DNA polymerase is mutated at one or more sites selected from the group consisting of: V453, F495, E507, K508, T509, A518, S624, Y672, E734, R737, F749, T757, L764, H785, and the amino acid residue numbering is based on the numbering shown in SEQ ID NO.2.

[0006] In another preferred embodiment, the activity of the mutant DNA polymerase is at least 1.5 times that of the wild-type DNA polymerase (SEQ ID NO.: 2); preferably at least 2 times; more preferably at least 3 times.

[0007] In another preferred example, the amino acid sequence of the wild-type DNA polymerase is as shown in SEQ ID NO.:2.

[0008] In another preferred example, the amino acid sequence of the mutant DNA polymerase has at least 80% homology compared to SEQ ID NO.2; more preferably, at least 90% homology; most preferably, at least 95% homology; such as at least 96%, 97%, 98%, 99% homology.

[0009] In another preferred example, the mutant DNA polymerase is selected from the following group of mutants 1-20:

[0010]

[0011]

[0012] In another preferred example, the number of mutation sites in the mutant DNA polymerase is 1-4, preferably 2 or 3.

[0013] In another preferred example, the mutant DNA polymerase is selected from each specific mutant enzyme in Table 2.

[0014] In another preferred example, the mutant DNA polymerase includes the mutation sites of each specific mutant enzyme in Table 2.

[0015] In another preferred example, the mutant DNA polymerase is mutated on the basis of the wild-type DNA polymerase shown in SEQ ID NO.:2, and the mutant DNA polymerase includes mutation sites selected from the following group:

[0016] (1) E507A, K508L, E734E, F749K;

[0017] (2) K508L, V453A, R737K

[0018] (3) E734G

[0019] (4) F749G, K508L, L764K

[0020] (5) E507Q, T757S

[0021] (6) H785G

[0022] (7) S624T, F749V

[0023] (8) E734F, F749V

[0024] (9) K508L, R737W, Y672R

[0025] (10)E507H, H785L

[0026] (11)A518Q, E734M

[0027] (12)F495R, F749T

[0028] (13)K508L, F749T, E734F

[0029] (14)R737P, S624K

[0030] (15)T757W, V453G, E507M

[0031] (16)F749E, H785G, F495G

[0032] (17)E734F, Y672P

[0033] (18)T509L, H785K

[0034] (19)E734G, T757S, L764Q; and

[0035] (20)K508L, V453A, A518Q.

[0036] In a second aspect of the present invention, there is provided a polynucleotide molecule encoding the mutant DNA polymerase as described in the first aspect of the present invention.

[0037] In a third aspect of the present invention, there is provided a vector containing the nucleic acid molecule as described in the second aspect of the present invention.

[0038] In a fourth aspect of the present invention, there is provided a host cell containing the vector as described in the first aspect of the present invention or having the nucleic acid molecule as described in the second aspect of the present invention integrated into its chromosome.

[0039] In another preferred embodiment, the host cell is a prokaryotic cell or a eukaryotic cell.

[0040] In another preferred embodiment, the prokaryotic cell is Escherichia coli.

[0041] In another preferred embodiment, the eukaryotic cell is a yeast cell.

[0042] In a fifth aspect of the present invention, there is provided a method for preparing the mutant DNA polymerase as described in the first aspect of the present invention, comprising the steps of:

[0043] (i) culturing the host cell as described in the fourth aspect of the present invention under suitable conditions to express the mutant DNA polymerase; and

[0044] (ii) Isolate the mutated DNA polymerase described above.

[0045] In another preferred embodiment, the temperature for culturing the host cell in step (i) is 20°C - 40°C; preferably 25°C - 37°C, such as 35°C.

[0046] In the sixth aspect of the present invention, a kit is provided, which contains the mutated DNA polymerase described in the first aspect of the present invention.

[0047] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. Detailed Embodiments

[0048] Through extensive and in-depth research, the inventors of the present invention applied protein directed evolution technology to construct a random mutation library for the polymerase active domain targeting Taq enzyme. By gradually adding screening pressure, unsuitable mutations were naturally eliminated, and mutations with advantageous traits gradually accumulated. Finally, a series of amino acid sites and their mutations that play a key role in the amplification performance and polymerization performance of Taq enzyme were screened out, and Taq enzyme mutants with high amplification performance were obtained. On this basis, the present invention was completed.

[0049] Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, as such methods and conditions can vary. It should also be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to be restrictive, and the scope of the present invention will be limited only by the appended claims.

[0050] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs. As used herein, when referring to a specifically recited numerical value, the term "about" means that the value can vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0051] Although any methods and materials similar or equivalent to those described in the present invention can be used in the implementation or testing of the present invention, preferred methods and materials are exemplified herein.

[0052] Taq enzyme

[0053] Taq enzyme is widely used in polymerase chain reaction (PCR) and is the enzyme of choice for reactions such as nucleic acid amplification and detection. Commercial Taq enzyme is cloned and expressed using the Escherichia coli prokaryotic expression system.

[0054] The DNA sequence of wild-type Taq enzyme is as follows:

[0055] ATGCGTGGCATGCTGCCGCTTTTCGAGCCTAAGGGACGCGTTCTTCTTGTGGATGGACATCAT

[0056] CTGGCGTACCGTACCTTTCATGCCCTGAAGGGCCTGACCACTTCGCGTGGGGAACCCGTGCAAGCAG

[0057] TTTATGGATTCGCCAAATCGTTACTTAAGGCTCTGAAGGAGGATGGTGATGCGGTCATTGTTGTGTT

[0058] CGACGCAAAAGCTCCCTCGTTCCGTCACGAGGCCTACGGCGGCTATAAAGCTGGGCGTGCACCCACA

[0059] CCTGAGGATTTTCCCCGGCAACTTGCTTTGATAAAGGAATTAGTAGACCTGTTAGGCCTGGCGCGGT

[0060] TAGAAGTGCCGGGTTACGAAGCAGATGACGTCTTGGCTAGTTTAGCGAAAAAGGCTGAAAAAGAGGG

[0061] ATATGAAGTGCGGATCCTGACCGCGGATAAAGATCTGTATCAACTGTTGTCCGACCGTATTCACGTG

[0062] CTTCATCCGGAGGGCTACTTGATAACCCCGGCTTGGCTGTGGGAGAAATATGGGCTGCGTCCAGATC

[0063] AGTGGGCTGATTATCGTGCACTTACAGGCGATGAATCTGATAATCTTCCCGGCGTCAAGGGGATTGG

[0064] TGAGAAAACCGCCCGTAAACTTTTGGAGGAGTGGGGCAGCTTGGAGGCGCTGTTGAAGAATCTGGAT

[0065] CGTTTGAAACCCGCTATACGGGAAAAAATCTTGGCGCACATGGACGACTTAAAACTGTCTTGGGACC

[0066] TGGCGAAAGTTCGTACTGATTTGCCGCTGGAGGTCGACTTTGCGAAGCGTCGCGAGCCCGATCGTGA

[0067] ACGTCTTCGCGCATTTCTGGAGCGTTTAGAATTTGGCTCCCTGTTGCATGAGTTTGGTTTGCTTGAA

[0068] AGCCCGAAGGCACTTGAGGAAGCTCCTTGGCCTCCGCCTGAGGGCGCTTTTGTCGGATTTGTCTTGA

[0069] GCCGTAAAGAACCGATGTGGGCGGACTTACTGGCCCTTGCTGCTGCTCGTGGGGGTCGCGTGCATCG

[0070] CGCACCGGAGCCATACAAAGCACTTCGTGACCTTAAAGAAGCCCGTGGCTTGTTGGCAAAAGATTTA

[0071] AGTGTCCTGGCTTTACGCGAGGGCTTGGGCTTACCACCGGGAGATGATCCGATGCTTTTGGCCTATC

[0072] TGCTGGACCCGAGCAACACGACTCCAGAGGGCGTTGCCCGTCGTTATGGCGGAGAATGGACGGAGGA

[0073] GGCGGGAGAGCGCGCAGCGTTAAGCGAGCGTCTGTTTGCTAATCTGTGGGGACGCTTAGAGGGAGAG

[0074] GAGCGCCTGTTGTGGTTGTACCGTGAAGTGGAACGGCCGCTGAGTGCAGTGTTAGCTCACATGGAAG

[0075] CAACCGGGGTGCGGCTGGACGTTGCGTATTTGCGTGCGCTGTCGTTAGAGGTCGCGGAGGAAATAGC

[0076] CCGTCTGGAGGCCGAAGTATTCCGTTTGGCTGGCCATCCTTTCAACCTGAACAGTCGGGATCAGCTG

[0077] GAACGTGTACTTTTTGATGAACTGGGGCTGCCCGCCATCGGCAAAACCGAAAAAACCGGCAAACGTA

[0078] GCACCTCTGCGGCAGTGCTGGAAGCGTTACGTGAAGCTCATCCGATTGTGGAGAAAATTCTGCAATA

[0079] TCGCGAATTGACGAAACTGAAGAGCACCTATATTGATCCGCTGCCAGACTTAATTCACCCCCGTACC

[0080] GGACGGTTGCATACCCGCTTCAACCAGACCGCGACGGCGACAGGGCGGCTGAGTAGCAGCGATCCGA

[0081] ACCTGCAAAACATTCCCGTGCGTACCCCGCTGGGTCAGCGTATTCGCCGTGCTTTCATTGCCGAGGA

[0082] AGGCTGGCTGCTGGTCGCGCTGGACTACTCGCAAATCGAATTGCGTGTGTTGGCCCACCTGTCGGGC

[0083] GACGAAAACTTAATACGCGTGTTTCAAGAAGGTCGTGACATACATACTGAAACCGCGTCCTGGATGT

[0084] TTGGAGTCCCACGGGAGGCTGTCGATCCTCTTATGCGTCGTGCCGCCAAAACAATTAACTTCGGAGT

[0085] TCTGTACGGCATGTCGGCACATCGTTTATCACAGGAACTGGCGATTCCGTATGAAGAAGCGCAGGCC

[0086] TTCATAGAACGTTATTTCCAATCATTCCCCAAGGTGCGGGCCTGGATTGAGAAGACCCTGGAAGAGG

[0087] GCCGTCGTCGTGGCTATGTAGAGACTCTGTTCGGACGTCGGCGGTATGTACCCGATCTTGAGGCCCG

[0088] TGTGAAGTCCGTTCGTGAGGCAGCAGAACGTATGGCGTTTAACATGCCAGTCCAGGGCACAGCGGCG

[0089] GACCTGATGAAATTAGCTATGGTTAAGCTGTTTCCGCGTTTGGAAGAAATGGGCGCTCGTATGCTGT

[0090] TACAGGTTCATGACGAGTTAGTATTAGAAGCACCGAAGGAGCGTGCCGAAGCCGTGGCCCGGTTAGC

[0091] CAAAGAGGTAATGGAAGGCGTCTACCCCCTTGCAGTCCCGCTTGAAGTCGAAGTTGGCATAGGGGAA

[0092] GACTGGTTATCTGCGAAGGAA(SEQ ID NO.:1)

[0093] The amino acid sequence of wild-type Taq enzyme is as follows:

[0094] MRGMLPLFEPKGRVLLVDGHHLAYRTFHALKGLTTSRGEPVQAVYGFAKSLLKALKEDGDAVIVVFDAKAPSFRHEAYGGYKAGRAPTPEDFPRQLALIKELVDLLGLARLEVPGYEADDVLASLAKKAEKEGYEVRILTADKDLYQLLSDRIHVLHPEGYLITPAWLWEKYGLRPDQWADYRALTGDESDNLPGVKGIGEKTARKLLEEWGSLEALLKNLDRLKPAIREKILAHMDDLKLSWDLAKVRTDLPLEVDFAKRREPDRERLRAFLERLEFGSLLHEFGLLESPKALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDLKEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFANLWGRLEGEERLLWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEEIARLEAEVFRLAGHPFNLNSRDQLERVLFDELGLPAIGKTEKTGKRSTSAAVLEALREAHPIVEKILQYRELTKLKSTYIDPLPDLIHPRTGRLHTRFNQTATATGRLSSSDPNLQNIPVRTPLGQRIRRAFIAEEGWLLVALDYSQIELRVLAHLSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPLMRRAAKTINFGVLYGMSAHRLSQELAIPYEEAQAFIERYFQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTAADLMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDWLSAKE(SEQ ID NO.:2)

[0095] Through directed evolution, the present invention screens out amino acid sites and their mutation modes that are highly related to the amplification activity of Taq enzyme. The related mutant amino acid sites include: V453, F495, E507, K508, T509, A518, S624, Y672, E734, R737, F749, T757, L764, and H785. The amino acid residue numbers are based on SEQ ID NO.:2. Mutating the above amino acid sites into any other amino acids can obtain Taq enzyme mutants with higher activity. Preferred mutation forms include: E507A / Q / H / M, K508L, E734G / F / M, F749K / G / V / T / E, L764K / Q, V453A / G, R737K / W / P, T757S / W, H785G / L / K, S624T / K, Y672R / P, A518Q, F495G / R, T509L.

[0096] Through directed evolution technology, the present invention screens out amino acid sites and their mutation modes that are highly related to the activity of Taq enzyme from a random mutation library. The number of mutants is 10 times that of site-directed mutagenesis 5 times, which is more conducive to screening out mutant sites with synergistic effects, and these sites cannot be predicted by existing computer simulation technologies. Moreover, based on the principle of directed evolution, the accumulated advantageous traits are the most adaptable to the added screening conditions, so the obtained mutants must also be the optimal individuals among all mutants.

[0097] In a preferred embodiment of the present invention, the activity of the mutant DNA polymerase provided by the present invention is at least 1.2 times that of the wild-type DNA polymerase (SEQ ID NO.:2); preferably at least 1.3 times; more preferably at least 1.5 times, such as more than 2 times.

[0098] In a preferred embodiment of the present invention, the activity test methods of the mutant DNA polymerase and the wild-type DNA polymerase (SEQ ID NO.:2) are as follows:

[0099] PCR reaction solution: 100 pg of pET28a vector

[0100] 10 ng of Taq enzyme mutant or wild-type Taq enzyme

[0101] 2 μl of 10X Taq enzyme reaction solution (100 mM Tris, 500 mM KCl, 100 mM (NH4)2SO4, pH 8.0)

[0102] 4 pmol of pET28_F primer

[0103] 4 pmol of pET28_R primer

[0104] 2 μL of dNTPs (2.5 mM)

[0105] Make up to 20 μL with ddH₂O

[0106] PCR program: 95°C for 5 minutes, 30 cycles (95°C for 15 seconds, 55°C for 15 seconds, 72°C for 10 seconds), 4°C∞

[0107] Purify the PCR product by ethanol precipitation, measure the absorbance of the product at 260 nm, and calculate the total amount (ng) of the PCR product corresponding to each cycle number.

[0108] Among them, the following primer pair is used in the PCR reaction:

[0109] pET28_F primer: TACGGTTAACCCTTTGAATCA (SEQ ID NO.: 9)

[0110] pET28_R primer: GTTACCTGGTTAAACTGTACT (SEQ ID NO.: 10).

[0111] Divide the total amount of the PCR product obtained using the Taq enzyme mutant by the total amount of the PCR product obtained using the wild-type Taq enzyme, and the resulting value is the activity multiple of the Taq enzyme mutant compared to the wild-type Taq enzyme.

[0112] Those of ordinary skill in the art can obtain the Taq enzyme gene sequence of the present invention by conventional methods, such as total artificial synthesis or PCR synthesis. A preferred synthesis method is asymmetric PCR. Asymmetric PCR uses unequal amounts of a pair of primers, and after PCR amplification, a large amount of single-stranded DNA (ssDNA) is produced. This pair of primers are respectively called the non-limiting primer and the limiting primer, and their ratio is generally 50 - 100∶1. In the first 10 - 15 cycles of the PCR reaction, the amplification product is mainly double-stranded DNA, but when the limiting primer (low-concentration primer) is consumed, the PCR guided by the non-limiting primer (high-concentration primer) will produce a large amount of single-stranded DNA. The primers used for PCR can be appropriately selected according to the sequence information of the present invention disclosed herein and can be synthesized by conventional methods. The amplified DNA / RNA fragments can be separated and purified by conventional methods such as gel electrophoresis.

[0113] The Taq enzyme of the present invention can be expressed or produced by conventional recombinant DNA techniques, including the steps of:

[0114] (1) Transform or transduce a suitable host cell with the polynucleotide encoding the protein of the present invention, or with a recombinant expression vector containing the polynucleotide;

[0115] (2) Culture the host cell in a suitable medium;

[0116] (3) Isolate and purify the target protein from the culture medium or cells to obtain Taq enzyme.

[0117] Methods well-known to those skilled in the art can be used to construct an expression vector containing the coding DNA sequence of the Taq enzyme of the present invention and appropriate transcriptional / translational control signals, preferably a commercially available vector: pET28. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombinant technology, etc. The DNA sequence can be effectively ligated to an appropriate promoter in the expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. In addition, the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selecting transformed host cells.

[0118] The recombinant vector includes, in the 5' to 3' direction: a promoter, a target gene, and a terminator. If necessary, the recombinant vector may further include the following elements: a protein purification tag; a 3' polyadenylation signal; an untranslated nucleic acid sequence; a transport and targeting nucleic acid sequence; a selectable marker (antibiotic resistance gene, fluorescent protein, etc.); an enhancer; or an operator.

[0119] Methods for preparing recombinant vectors are well-known to those of ordinary skill in the art. The expression vector can be a bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus, or other vector. In short, any plasmid and vector can be adopted as long as it can replicate and be stable in the host.

[0120] Those of ordinary skill in the art can use well-known methods to construct vectors containing the promoter and / or target gene sequence of the present invention. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombinant technology, etc.

[0121] The expression vector of the present invention can be used to transform a suitable host cell so that the host transcribes the target RNA or expresses the target protein. The host cell can be a prokaryotic cell, such as Escherichia coli, Corynebacterium glutamicum, Brevibacterium flavum, Streptomyces, Agrobacterium; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a plant cell. Those of ordinary skill in the art are well aware of how to select an appropriate vector and host cell. Transforming a host cell with recombinant DNA can be carried out by conventional techniques well-known to those skilled in the art. When the host is a prokaryote (such as Escherichia coli), it can be treated with the CaCl2 method or by electroporation. When the host is a eukaryote, the following DNA transfection methods can be selected: calcium phosphate co-precipitation method, conventional mechanical methods (such as microinjection, electroporation, liposome packaging, etc.). For plants, methods such as Agrobacterium transformation or gene gun transformation can also be used, such as the leaf disc method, immature embryo transformation method, flower bud soaking method, etc. The transformed plant cells, tissues or organs can be regenerated into plants by conventional methods to obtain transgenic plants.

[0122] The term "operably linked" refers to linking a target gene to be transcribed and expressed to its control sequence in a conventional manner in the art for expression.

[0123] Cultivation of engineered bacteria and fermentation production of target protein

[0124] After obtaining the engineered cells, the engineered cells can be cultured under suitable conditions to express the protein encoded by the gene sequence of the present invention. Depending on the host cell, the culture medium used in the culture can be selected from various conventional media and cultured under conditions suitable for the growth of the host cell. When the host cell grows to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature shift or chemical induction), and the cells are cultured for a further period of time.

[0125] In the present invention, conventional fermentation conditions can be employed. Representative conditions include (but are not limited to):

[0126] (a) In terms of temperature, the fermentation and induction temperature of Taq enzyme is maintained at 25-37 °C;

[0127] (b) In terms of the pH value during the induction period, the pH during the induction period is controlled at 3-9;

[0128] (c) In terms of dissolved oxygen (DO), the DO is controlled at 10-90%, and the maintenance of dissolved oxygen can be solved by introducing a mixed gas of oxygen / air;

[0129] (d) In terms of feeding, the types of feed should include carbon sources such as glycerol, methanol, glucose, etc., and can be fed individually or in combination;

[0130] (e) Regarding the IPTG concentration during the induction period, conventional induction concentrations can be used in the present invention. Generally, the IPTG concentration is controlled at 0.1 - 1.5 mM;

[0131] (f) Regarding the induction time, there is no particular limitation. Generally, it is 2 - 20 hours, preferably 5 - 15 hours.

[0132] The target protein Taq enzyme of the present invention exists inside Escherichia coli cells. The host cells are collected by a centrifuge, and then the host cells are disrupted by high pressure, mechanical force, enzymatic digestion of the cell wall, or other cell disruption methods to release the recombinant protein. The preferred method is high pressure. The host cell lysate can be preliminarily purified by methods such as flocculation, salting out, and ultrafiltration, and then subjected to chromatography, ultrafiltration, etc. for purification, or directly subjected to chromatography purification.

[0133] Chromatography techniques include cation exchange chromatography, anion exchange chromatography, gel filtration chromatography, hydrophobic chromatography, affinity chromatography, etc. Common chromatography methods include:

[0134] 1. Anion exchange chromatography:

[0135] Anion exchange chromatography media include (but are not limited to): Q-Sepharose, DEAE-Sepharose. If the salt concentration of the fermentation sample is high and affects the binding to the ion exchange medium, the salt concentration needs to be reduced before ion exchange chromatography. The sample can be balanced and the buffer solution replaced by means such as dilution, ultrafiltration, dialysis, and gel filtration chromatography until it is similar to the corresponding ion exchange column equilibrium solution system, and then loaded for gradient elution of salt concentration or pH.

[0136] 2. Hydrophobic chromatography:

[0137] Hydrophobic chromatography media include (but are not limited to): Phenyl-Sepharose, Butyl-Sepharose, Octyle-Sepharose. The salt concentration of the sample is increased by adding NaCl, (NH4)2SO4, etc., and then loaded, and eluted by reducing the salt concentration. Hydrophobic chromatography is used to remove miscellaneous proteins with significantly different hydrophobicities.

[0138] 3. Gel filtration chromatography

[0139] Hydrophobic chromatography media include (but are not limited to): Sephacryl, Superdex, Sephadex types. The buffer system is replaced or further purified by gel filtration chromatography.

[0140] 4. Affinity chromatography

[0141] Affinity chromatography media include (but are not limited to): HiTrap TMHeparin HP Columns。

[0142] 5. Membrane filtration

[0143] The ultrafiltration medium includes: organic membranes such as polysulfone membranes, inorganic membranes such as ceramic membranes, and metal membranes. Purification and concentration can be achieved through membrane filtration.

[0144] The main advantages of the present invention are as follows:

[0145] (1) The amount of product amplified by the thermostable DNA polymerase mutant with high amplification activity of the present invention is significantly higher than that of the wild-type Taq enzyme under the same number of PCR cycles.

[0146] (2) The time required for the thermostable DNA polymerase mutant with high amplification activity of the present invention to amplify the same amount of product under the same conditions is significantly shorter than that of the wild-type Taq enzyme. Therefore, the detection efficiency can be significantly improved.

[0147] The present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specified detailed conditions in the following embodiments are usually carried out under conventional conditions such as those described in "Molecular Cloning: A Laboratory Manual" by Sambrook. J et al. (translated by Huang Peitang et al., Beijing: Science Press, 2002), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight. The experimental materials and reagents used in the following embodiments can be obtained from commercial sources without special instructions.

[0148] Example 1: Construction of Taq enzyme random mutation plasmid

[0149] The DNA sequence of the polymerase active domain of Taq enzyme (amino acid coding sequence at positions 423 - 831) was amplified by low-fidelity PCR (Error-PCR) with a mutation incidence of 0.3%, and then ligated to the remaining coding sequence of Taq enzyme (amino acid sequence at positions 1 - 423) and cloned into the pET28a prokaryotic expression vector to obtain the Taq enzyme random mutation plasmid. The specific steps are as follows:

[0150] 1) Using the Taq-pET28a plasmid as a template, primer T(1 - 423) was designed to amplify the Taq(1 - 423) fragment.

[0151] Taq(1-423)DNA Seq

[0152] ATGCGTGGCATGCTGCCGCTTTTCGAGCCTAAGGGACGCGTTCTTCTTGTGGATGGACATCATCTGGCGTACCGTACCTTTCATGCCCTGAAGGGCCTGACCACTTCGCGTGGGGAACCCGTGCAAGCAGTTTATGGATTCGCCAAATCGTTACTTAAGGCTCTGAAGGAGGATGGTGATGCGGTCATTGTTGTGTTCGACGCAAAAGCTCCCTCGTTCCGTCACGAGGCCTACGGCGGCTATAAAGCTGGGCGTGCACCCACACCTGAGGATTTTCCCCGGCAACTTGCTTTGATAAAGGAATTAGTAGACCTGTTAGGCCTGGCGCGGTTAGAAGTGCCGGGTTACGAAGCAGATGACGTCTTGGCTAGTTTAGCGAAAAAGGCTGAAAAAGAGGGATATGAAGTGCGGATCCTGACCGCGGATAAAGATCTGTATCAACTGTTGTCCGACCGTATTCACGTGCTTCATCCGGAGGGCTACTTGATAACCCCGGCTTGGCTGTGGGAGAAATATGGGCTGCGTCCAGATCAGTGGGCTGATTATCGTGCACTTACAGGCGATGAATCTGATAATCTTCCCGGCGTCAAGGGGATTGGTGAGAAAACCGCCCGTAAACTTTTGGAGGAGTGGGGCAGCTTGGAGGCGCTGTTGAAGAATCTGGATCGTTTGAAACCCGCTATACGGGAAAAAATCTTGGCGCACATGGACGACTTAAAACTGTCTTGGGACCTGGCGAAAGTTCGTACTGATTTGCCGCTGGAGGTCGACTTTGCGAAGCGTCGCGAGCCCGATCGTGAACGTCTTCGCGCATTTCTGGAGCGTTTAGAATTTGGCTCCCTGTTGCATGAGTTTGGTTTGCTTGAAAGCCCGAAGGCACTTGAGGAAGCTCCTTGGCCTCCGCCTGAGGGCGCTTTTGTCGGATTTGTCTTGAGCCGTAAAGAACCGATGTGGGCGGACTTACTGGCCCTTGCTGCTGCTCGTGGGGGTCGCGTGCATCGCGCACCGGAGCCATACAAAGCACTTCGTGACCTTAAAGAAGCCCGTGGCTTGTTGGCAAAAGATTTAAGTGTCCTGGCTTTACGCGAGGGCTTGGGCTTACCACCGGGAGATGATCCGATGCTTTTGGCCTATCTGCTGGACCCGAGCAACACGACTCCAGAGGGCGTTGCCCGTCGTTATGGCGGAGAATGGACGGAGGAGGCGGGAGAGCGCGCAGCGTTAAGCGAGCGTCTGTTTGCTAATCTGTGGGGACGCTTAGAGGGAGAG(SEQ ID NO.:3)

[0153] T1-423_PF: 5'ATATCATATGCGTGGCATGCTGCCGCTTTT 3'(SEQ ID NO.:4)

[0154] T1-423_PR: 5'GCATGAATTCCGTCTCCTCTCCCTCTAAGC 3'(SEQ ID NO.:5)

[0155] PCR reaction system and procedure:

[0156] Taq-pET28a plasmid 10 ng

[0157] T1-423_PF primer 4 pmol

[0158] T1-423_PR primer 4 pmol

[0159] 2.5 mM dNTP 2 ul

[0160] 10X reaction buffer 2 ul

[0161] KAPA HiFi DNA Polymerase 5 U

[0162] ddH2O to make up the total volume to 20 ul

[0163] PCR procedure: 3 minutes at 95°C, (30 seconds at 95°C, 30 seconds at 60°C, 1 minute at 72°C) × 25 cycles, 3 minutes at 72°C, store at 4°C

[0164] The PCR product was purified and recovered using a DNA gel extraction kit, digested with NdeI and XhoI, ligated into the pET28a vector, and the sequence was confirmed by sequencing. The resulting plasmid was named Taq(1-423)-pET28

[0165] 2) Using the Taq-pET28a plasmid as a template, Clontech PCR RandomMutagenesis Kit (TaKaRa PT3393-2, Dalian) was used to design primers (TMu_F / R) to amplify the Taq(423-822) fragment

[0166] Taq(423-832)DNA Seq

[0167] GGAGAGGAGCGCCTGTTGTGGTTGTACCGTGAAGTGGAACGGCCGCTGAGTGCAGTGTTAGCTCACATGGAAGCAACCGGGGTGCGGCTGGACGTTGCGTATTTGCGTGCGCTGTCGTTAGAGGTCGCGGAGGAAATAGCCCGTCTGGAGGCCGAAGTATTCCGTTTGGCTGGCCATCCTTTCAACCTGAACAGTCGGGATCAGCTGGAACGTGTACTTTTTGATGAACTGGGGCTGCCCGCCATCGGCAAAACCGAAAAAACCGGCAAACGTAGCACCTCTGCGGCAGTGCTGGAAGCGTTACGTGAAGCTCATCCGATTGTGGAGAAAATTCTGCAATATCGCGAATTGACGAAACTGAAGAGCACCTATATTGATCCGCTGCCAGACTTAATTCACCCCCGTACCGGACGGTTGCATACCCGCTTCAACCAGACCGCGACGGCGACAGGGCGGCTGAGTAGCAGCGATCCGAACCTGCAAAACATTCCCGTGCGTACCCCGCTGGGTCAGCGTATTCGCCGTGCTTTCATTGCCGAGGAAGGCTGGCTGCTGGTCGCGCTGGACTACTCGCAAATCGAATTGCGTGTGTTGGCCCACCTGTCGGGCGACGAAAACTTAATACGCGTGTTTCAAGAAGGTCGTGACATACATACTGAAACCGCGTCCTGGATGTTTGGAGTCCCACGGGAGGCTGTCGATCCTCTTATGCGTCGTGCCGCCAAAACAATTAACTTCGGAGTTCTGTACGGCATGTCGGCACATCGTTTATCACAGGAACTGGCGATTCCGTATGAAGAAGCGCAGGCCTTCATAGAACGTTATTTCCAATCATTCCCCAAGGTGCGGGCCTGGATTGAGAAGACCCTGGAAGAGGGCCGTCGTCGTGGCTATGTAGAGACTCTGTTCGGACGTCGGCGGTATGTACCCGATCTTGAGGCCCGTGTGAAGTCCGTTCGTGAGGCAGCAGAACGTATGGCGTTTAACATGCCAGTCCAGGGCACAGCGGCGGACCTGATGAAATTAGCTATGGTTAAGCTGTTTCCGCGTTTGGAAGAAATGGGCGCTCGTATGCTGTTACAGGTTCATGACGAGTTAGTATTAGAAGCACCGAAGGAGCGTGCCGAAGCCGTGGCCCGGTTAGCCAAAGAGGTAATGGAAGGCGTCTACCCCCTTGCAGTCCCGCTTGAAGTCGAAGTTGGCATAGGGGAAGACTGGTTATCTGCGAAGGAATAA(SEQ ID NO.:6)

[0168] TMu_F: 5'GGAGAGGAGCGCCTGTTGTGGTTGT 3'(SEQ ID NO.:7)

[0169] TMu_R: 5'TTATTCCTTCGCAGATAACCAGTCT 3'(SEQ ID NO.:8)

[0170] PCR reaction system and procedure:

[0171]

[0172] 95°C for 3 minutes, (95°C for 30 seconds, 60°C for 30 seconds, 68°C for 2 minutes) X 25 cycles, 68°C for 5 minutes, store at 4°C

[0173] The PCR product was digested with BsmBI and XhoI, and then ligated to the Taq(1 - 423)-pET28 plasmid digested with BsmBI and XhoI. The ligation product was transformed into the BL21(DE3) expression host strain, and the number of transformants was counted.

[0174] Example 2: Expression and directed evolution screening of Taq enzyme mutants

[0175] The Taq enzyme mutant plasmid was transformed into the BL21(DE3) expression strain to induce the expression of the Taq enzyme mutant library. The BL21(DE3) induced expression bacteria containing the Taq enzyme mutant library were dispersed and encapsulated with the emulsion PCR system for PCR reaction to amplify the DNA containing the Taq enzyme mutant fragment. Then the DNA fragment amplified by emulsion PCR was subjected to high-fidelity PCR secondary amplification with Taq enzyme specific primers, and the amplified DNA product was recloned into the pET28a expression vector to complete one screening process. After that, the screening process of emulsion PCR - secondary high-fidelity PCR - cloning into the pET28a expression vector was repeated, and at the same time, the extension time of emulsion PCR in each screening was gradually shortened to accumulate the mutant population with high extension activity and high amplification activity. The specific steps are as follows:

[0176] 1) Take the transformants obtained in Example 1, inoculate them into LB medium, shake culture at 37°C for 6 hours, add isopropyl β-D-thiogalactopyranoside (IPTG) with a final concentration of 0.1 mM, and induce culture at 37°C for 3 hours. Centrifuge to collect the bacterial cells, wash the bacterial cells twice with ddH2O, and finally resuspend the bacterial cells with ddH2O. Measure the optical absorbance value (OD600 value) of the bacterial cell solution at 600 nm, and dilute it to OD600 = 1.0 with ddH2O

[0177] 2) Prepare the oil phase solution

[0178] Tween-80 200 ul

[0179] Triton X-100 25 ul

[0180] Mineral oil 10 ml

[0181] Combine the above 3 reagents and mix well

[0182] 3) Preparation of the aqueous phase reaction solution

[0183] Dilute the bacterial cell resuspension with OD600 = 1.0 prepared in step 1) 100 times with ddH2O to prepare the following reaction solution

[0184] 10X Taq enzyme reaction solution (100 mM Tris, 500 mM KCl, 100 mM (NH4)2SO4, pH 8.0) 13 ul

[0185]

[0186] pET28_F primer: TACGGTTAACCCTTTGAATCA (SEQ ID NO.: 9)

[0187] pET28_R primer: GTTACCTGGTTAAACTGTACT (SEQ ID NO.: 10)

[0188] 4) Preparation of emulsion system

[0189] Take 200 ul of aqueous phase + 400 ul of oil phase and mix them in a 2 ml tube. Vortex at high speed for 10 minutes. Take 5 PCR tubes and dispense 100 ul of the mixed solution into each tube. PCR program: 95°C for 5 minutes, (95°C for 30 seconds, 55°C for 30 seconds, 72°C for 2 minutes) X 25 cycles, 72°C for 5 minutes, 4°C ∞

[0190] 5) Transfer the emulsion PCR product to a 1.5 ml tube, add 166 ul of water-saturated ether, vortex for 30 seconds, centrifuge at 12000 rpm for 10 minutes, transfer away the lower liquid phase, let it stand at room temperature for 10 minutes for the ether to evaporate, extract and purify the liquid phase product using the phenol-chloroform method, and then precipitate the product with ethanol overnight for recovery.

[0191] 6) High-fidelity PCR secondary amplification product

[0192] Using the product from step 4) as a template, perform PCR secondary amplification

[0193]

[0194] The PCR program is as follows: 95°C for 5 minutes, 20 cycles X (95°C for 30 seconds, 62°C for 30 seconds, 72°C for 2 minutes), 72°C for 5 minutes, 4°C ∞

[0195] Taq_F primer: ATGCGTGGCATGCTGCCGCTTTTCGAGCCTAAGGGACG (SEQ ID NO.: 11)

[0196] Taq_R primer: TTCCTTCGCAGATAACCAGTCTTCCCCTATGCCAACTTCGAC (SEQ ID NO.: 12)

[0197] 7) The PCR products were purified using a DNA product purification and recovery kit and then religated to the pET28a expression vector. Thus, one round of screening was completed.

[0198] 8) The transformants with the religated pET28a vector were subjected to steps (1)-(6) repeatedly, and the conditions of emulsion PCR were changed according to the program in the following table to gradually apply selection pressure to the mutant library.

[0199] Second round of screening: 95°C for 5 minutes, (95°C for 30 seconds, 55°C for 30 seconds, 72°C for 1.5 minutes) × 25 cycles, 72°C for 5 minutes, 4°C ∞

[0200] Third round of screening: 95°C for 5 minutes, (95°C for 30 seconds, 55°C for 30 seconds, 72°C for 1 minute) × 20 cycles, 72°C for 5 minutes, 4°C ∞

[0201] Fourth round of screening: 95°C for 5 minutes, (95°C for 30 seconds, 55°C for 30 seconds, 72°C for 30 seconds) × 15 cycles, 72°C for 5 minutes, 4°C ∞

[0202] After 4 rounds of screening, the obtained Taq enzyme mutant transformants entered Example 3 of high-throughput screening. Example 3: High-throughput screening of Taq enzyme mutants

[0203] Randomly pick 384 monoclonal colonies from the mutant library obtained in Example 2. After culturing and inducing expression, test their amplification activities using high-throughput PCR reactions, and select 20 mutants with high amplification activities. The specific steps are as follows:

[0204] 1) Pick 384 monoclonal colonies and inoculate them into LB medium. Culture at 37°C for 6 hours, add isopropyl β-D-thiogalactoside (IPTG) with a final concentration of 0.1 mM, and induce culture at 37°C for 3 hours.

[0205] 2) Centrifuge to collect the bacteria after induced culture, add a lysis solution containing 0.1 mg / ml lysozyme (50 mM Tris, 50 mM NaCl, 5% glycerol, pH 8.5), resuspend the bacteria, incubate at 37°C for 10 minutes, and heat at 75°C for 30 minutes. Then centrifuge at 12000 rpm for 10 minutes and take the supernatant.

[0206] 3) Take a 96-well PCR plate and add the following reaction components to each well.

[0207] 10X Taq enzyme reaction solution (100 mM Tris, 500 mM KCl, 100 mM (NH4)2SO4, pH 8.0) 2 μl

[0208] pET28_F primer 4 pmol

[0209] pET28_R primer 4 pmol

[0210] dNTPs (2.5 mM) 2 uL

[0211] 1 uL of the treated supernatant

[0212] Make up to 20 uL with ddH2O

[0213] PCR program: 95°C for 5 minutes, 20 cycles of X (95°C for 30 seconds, 62°C for 30 seconds, 72°C for 60 seconds), 4°C ∞

[0214] Take 5 uL of the PCR product for agarose gel electrophoresis, compare the yields of the PCR products of the supernatants prepared from each monoclonal, and select the 20 monoclonal with the highest yields. The amplification yields of the mutants are 1.2 to 2 times that of the wild-type amplification yield.

[0215] Example 4: Confirmation of the mutation sites of the dominant Taq enzyme mutants

[0216] Perform DNA sequence sequencing on the Taq enzyme mutants selected in Example 3 to determine the mutation situation of their amino acid sequences, and count the high-frequency mutation sites and their mutation forms.

[0217] Table 1

[0218]

[0219]

[0220] Sequence the 20 mutants with better amplification activity, and count their amino acid mutation situations as shown in the above table. It can be seen that: V453, F495, E507, K508, T509, A518, S624, Y672, E734, R737, F749, T757, L764, H785 appear frequently in the 20 mutants, proving that their mutations have a significant impact on the amplification activity of Taq enzyme.

[0221] Example 5 Comparison between the mutant Taq enzyme after mutation and the wild-type Taq enzyme

[0222] Take the Taq enzyme mutants, after expression and purification, perform the following amplification ability test with the wild-type Taq enzyme:

[0223] 100 pg of pET28a vector

[0224] 10 ng of Taq enzyme mutant / wild-type Taq enzyme

[0225] 2 uL of 10X Taq enzyme reaction solution (100 mM Tris, 500 mM KCl, 100 mM (NH4)2SO4, pH 8.0)

[0226] 4 pmol of pET28_F primer

[0227] 4 pmol of pET28_R primer

[0228] 2 μL of dNTPs (2.5 mM)

[0229] Make up to 20 μL with ddH₂O

[0230] PCR program: 95°C for 5 minutes, n cycles of X (95°C for 15 seconds, 55°C for 15 seconds, 72°C for 10 seconds), 4°C ∞

[0231] Prepare the above reaction solution, perform PCR amplification for 15, 20, 25, and 30 cycles, purify the PCR product by ethanol precipitation, measure the absorbance of the product at 260 nm, and calculate the total amount (ng) of the PCR product corresponding to each cycle number. The results are as follows:

[0232] Table 2

[0233]

[0234]

[0235] As can be seen from the above results, the amount of products amplified by Taq enzyme mutants 1 to 20 is significantly higher than that of wild-type Taq enzyme under the same number of PCR cycles. Among them, the amount of products obtained by mutant 1 after 20 cycles of amplification is equivalent to that of wild-type Taq enzyme after 30 cycles of amplification; under the condition of 30 amplification cycles, the amount of products obtained by mutant 1 reaches more than 2.5 times that of wild-type Taq enzyme.

[0236] Example 6 Application of the mutated Taq enzyme in the fluorescence quantitative PCR detection kit for novel coronavirus SARS-CoV-2

[0237] Select Taq enzyme mutants 1#, 6#, and 17#, and prepare the reaction system according to the following table

[0238] 17 μL of NC (ORF1ab / N) PCR reaction solution A

[0239] 10 ng of Taq enzyme mutant / wild-type Taq enzyme

[0240] 200 U of MMLV reverse transcriptase

[0241] 20 U of RNase Inhibitor

[0242] 5 μL of nucleic acid extract of NC (ORF1ab / N) positive control product

[0243] Among them, the NC(ORF1ab / N) PCR reaction solution A and the nucleic acid extract of the NC(ORF1ab / N) positive control product are both provided by the 2019 novel coronavirus (2019-nCoV) ORF1ab N nucleic acid detection kit (PCR-fluorescent probe method) (Daan Gene Co., Ltd., Sun Yat-sen University). The MMLV reverse transcriptase and RNase Inhibitor are both prepared by Daan Gene Co., Ltd., Sun Yat-sen University. The PCR program is set as follows:

[0244]

[0245] The ct values of each Taq enzyme mutant and wild-type Taq enzyme for amplifying NC(ORF1ab / N) positive control products with different concentration gradients are as follows:

[0246]

[0247] It can be seen from the above results that the performance of the Taq enzyme mutant in the SARS-CoV-2 fluorescence quantitative PCR detection kit is significantly improved compared with that of the wild-type Taq enzyme.

[0248] All the documents mentioned in the present invention are cited in this application as references, just as if each document is cited separately as a reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application. Sequence Listing <110> Daan Gene Co., Ltd., Guangzhou <120> A Thermostable DNA Polymerase Mutant with High Amplification Activity <130> P220174 <160> 12 <170> PatentIn version 3.5 <210> 1 <211> 2496 <212> DNA <213> Artificial sequence <400> 1 atgcgtggca tgctgccgct tttcgagcct aagggacgcg ttcttcttgt ggatggacat 60 catctggcgt accgtacctt tcatgccctg aagggcctga ccacttcgcg tggggaaccc 120 gtgcaagcag tttatggatt cgccaaatcg ttacttaagg ctctgaagga ggatggtgat 180 gcggtcattg ttgtgttcga cgcaaaagct ccctcgttcc gtcacgaggc ctacggcggc 240 tataaagctg ggcgtgcacc cacacctgag gattttcccc ggcaacttgc tttgataaag 300 gaattagtag acctgttagg cctggcgcgg ttagaagtgc cgggttacga agcagatgac 360 gtcttggcta gtttagcgaa aaaggctgaa aaagagggat atgaagtgcg gatcctgacc 420 gcggataaag atctgtatca actgttgtcc gaccgtattc acgtgcttca tccggagggc 480 tacttgataa ccccggcttg gctgtgggag aaatatgggc tgcgtccaga tcagtgggct 540 gattatcgtg cacttacagg cgatgaatct gataatcttc ccggcgtcaa ggggattggt 600 gagaaaaccg cccgtaaact tttggaggag tggggcagct tggaggcgct gttgaagaat 660 ctggatcgtt tgaaacccgc tatacgggaa aaaatcttgg cgcacatgga cgacttaaaa 720 ctgtcttggg acctggcgaa agttcgtact gatttgccgc tggaggtcga ctttgcgaag 780 cgtcgcgagc ccgatcgtga acgtcttcgc gcatttctgg agcgtttaga atttggctcc 840 ctgttgcatg agtttggttt gcttgaaagc ccgaaggcac ttgaggaagc tccttggcct 900 ccgcctgagg gcgcttttgt cggatttgtc ttgagccgta aagaaccgat gtgggcggac 960 ttactggccc ttgctgctgc tcgtgggggt cgcgtgcatc gcgcaccgga gccatacaaa 1020 gcacttcgtg accttaaaga agcccgtggc ttgttggcaa aagatttaag tgtcctggct 1080 ttacgcgagg gcttgggctt accaccggga gatgatccga tgcttttggc ctatctgctg 1140 gacccgagca acacgactcc agagggcgtt gcccgtcgtt atggcggaga atggacggag 1200 gaggcgggag agcgcgcagc gttaagcgag cgtctgtttg ctaatctgtg gggacgctta 1260 gagggagagg agcgcctgtt gtggttgtac cgtgaagtgg aacggccgct gagtgcagtg 1320 ttagctcaca tggaagcaac cggggtgcgg ctggacgttg cgtatttgcg tgcgctgtcg 1380 ttagaggtcg cggaggaaat agcccgtctg gaggccgaag tattccgttt ggctggccat 1440 cctttcaacc tgaacagtcg ggatcagctg gaacgtgtac tttttgatga actggggctg 1500 cccgccatcg gcaaaaccga aaaaaccggc aaacgtagca cctctgcggc agtgctggaa 1560 gcgttacgtg aagctcatcc gattgtggag aaaattctgc aatatcgcga attgacgaaa 1620 ctgaagagca cctatattga tccgctgcca gacttaattc acccccgtac cggacggttg 1680 catacccgct tcaaccagac cgcgacggcg acagggcggc tgagtagcag cgatccgaac 1740 ctgcaaaaca ttcccgtgcg taccccgctg ggtcagcgta ttcgccgtgc tttcattgcc 1800 gaggaaggct ggctgctggt cgcgctggac tactcgcaaa tcgaattgcg tgtgttggcc 1860 cacctgtcgg gcgacgaaaa cttaatacgc gtgtttcaag aaggtcgtga catacatact 1920 gaaaccgcgt cctggatgtt tggagtccca cgggaggctg tcgatcctct tatgcgtcgt 1980 gccgccaaaa caattaactt cggagttctg tacggcatgt cggcacatcg tttatcacag 2040 gaactggcga ttccgtatga agaagcgcag gccttcatag aacgttattt ccaatcattc 2100 cccaaggtgc gggcctggat tgagaagacc ctggaagagg gccgtcgtcg tggctatgta 2160 gagactctgt tcggacgtcg gcggtatgta cccgatcttg aggcccgtgt gaagtccgtt 2220 cgtgaggcag cagaacgtat ggcgtttaac atgccagtcc agggcacagc ggcggacctg 2280 atgaaattag ctatggttaa gctgtttccg cgtttggaag aaatgggcgc tcgtatgctg 2340 ttacaggttc atgacgagtt agtattagaa gcaccgaagg agcgtgccga agccgtggcc 2400 cggttagcca aagaggtaat ggaaggcgtc tacccccttg cagtcccgct tgaagtcgaa 2460 gttggcatag gggaagactg gttatctgcg aaggaa 2496 <210> 2 <211> 832 <212> PRT <213> Artificial sequence <400> 2 Met Arg Gly Met Leu Pro Leu Phe Glu Pro Lys Gly Arg Val Leu Leu 1 5 10 15 Val Asp Gly His His Leu Ala Tyr Arg Thr Phe His Ala Leu Lys Gly 20 25 30 Leu Thr Thr Ser Arg Gly Glu Pro Val Gln Ala Val Tyr Gly Phe Ala 35 40 45 Lys Ser Leu Leu Lys Ala Leu Lys Glu Asp Gly Asp Ala Val Ile Val 50 55 60 Val Phe Asp Ala Lys Ala Pro Ser Phe Arg His Glu Ala Tyr Gly Gly 65 70 75 80 Tyr Lys Ala Gly Arg Ala Pro Thr Pro Glu Asp Phe Pro Arg Gln Leu 85 90 95 Ala Leu Ile Lys Glu Leu Val Asp Leu Leu Gly Leu Ala Arg Leu Glu 100 105 110 Val Pro Gly Tyr Glu Ala Asp Asp Val Leu Ala Ser Leu Ala Lys Lys 115 120 125 Ala Glu Lys Glu Gly Tyr Glu Val Arg Ile Leu Thr Ala Asp Lys Asp 130 135 140 Leu Tyr Gln Leu Leu Ser Asp Arg Ile His Val Leu His Pro Glu Gly 145 150 155 160 Tyr Leu Ile Thr Pro Ala Trp Leu Trp Glu Lys Tyr Gly Leu Arg Pro 165 170 175 Asp Gln Trp Ala Asp Tyr Arg Ala Leu Thr Gly Asp Glu Ser Asp Asn 180 185 190 Leu Pro Gly Val Lys Gly Ile Gly Glu Lys Thr Ala Arg Lys Leu Leu 195 200 205 Glu Glu Trp Gly Ser Leu Glu Ala Leu Leu Lys Asn Leu Asp Arg Leu 210 215 220 Lys Pro Ala Ile Arg Glu Lys Ile Leu Ala His Met Asp Asp Leu Lys 225 230 235 240 Leu Ser Trp Asp Leu Ala Lys Val Arg Thr Asp Leu Pro Leu Glu Val 245 250 255 Asp Phe Ala Lys Arg Arg Glu Pro Asp Arg Glu Arg Leu Arg Ala Phe 260 265 270 Leu Glu Arg Leu Glu Phe Gly Ser Leu Leu His Glu Phe Gly Leu Leu 275 280 285 Glu Ser Pro Lys Ala Leu Glu Glu Ala Pro Trp Pro Pro Pro Glu Gly 290 295 300 Ala Phe Val Gly Phe Val Leu Ser Arg Lys Glu Pro Met Trp Ala Asp 305 310 315 320 Leu Leu Ala Leu Ala Ala Ala Arg Gly Gly Arg Val His Arg Ala Pro 325 330 335 Glu Pro Tyr Lys Ala Leu Arg Asp Leu Lys Glu Ala Arg Gly Leu Leu 340 345 350 Ala Lys Asp Leu Ser Val Leu Ala Leu Arg Glu Gly Leu Gly Leu Pro 355 360 365 Pro Gly Asp Asp Pro Met Leu Leu Ala Tyr Leu Leu Asp Pro Ser Asn 370 375 380 Thr Thr Pro Glu Gly Val Ala Arg Arg Tyr Gly Gly Glu Trp Thr Glu 385 390 395 400 Glu Ala Gly Glu Arg Ala Ala Leu Ser Glu Arg Leu Phe Ala Asn Leu 405 410 415 Trp Gly Arg Leu Glu Gly Glu Glu Arg Leu Leu Trp Leu Tyr Arg Glu 420 425 430 Val Glu Arg Pro Leu Ser Ala Val Leu Ala His Met Glu Ala Thr Gly 435 440 445 Val Arg Leu Asp Val Ala Tyr Leu Arg Ala Leu Ser Leu Glu Val Ala 450 455 460 Glu Glu Ile Ala Arg Leu Glu Ala Glu Val Phe Arg Leu Ala Gly His 465 470 475 480 Pro Phe Asn Leu Asn Ser Arg Asp Gln Leu Glu Arg Val Leu Phe Asp 485 490 495 Glu Leu Gly Leu Pro Ala Ile Gly Lys Thr Glu Lys Thr Gly Lys Arg 500 505 510 Ser Thr Ser Ala Ala Val Leu Glu Ala Leu Arg Glu Ala His Pro Ile 515 520 525 Val Glu Lys Ile Leu Gln Tyr Arg Glu Leu Thr Lys Leu Lys Ser Thr 530 535 540 Tyr Ile Asp Pro Leu Pro Asp Leu Ile His Pro Arg Thr Gly Arg Leu 545 550 555 560 His Thr Arg Phe Asn Gln Thr Ala Thr Ala Thr Gly Arg Leu Ser Ser 565 570 575 Ser Asp Pro Asn Leu Gln Asn Ile Pro Val Arg Thr Pro Leu Gly Gln 580 585 590 Arg Ile Arg Arg Ala Phe Ile Ala Glu Glu Gly Trp Leu Leu Val Ala 595 600 605 Leu Asp Tyr Ser Gln Ile Glu Leu Arg Val Leu Ala His Leu Ser Gly 610 615 620 Asp Glu Asn Leu Ile Arg Val Phe Gln Glu Gly Arg Asp Ile His Thr 625 630 635 640 Glu Thr Ala Ser Trp Met Phe Gly Val Pro Arg Glu Ala Val Asp Pro 645 650 655 Leu Met Arg Arg Ala Ala Lys Thr Ile Asn Phe Gly Val Leu Tyr Gly 660 665 670 Met Ser Ala His Arg Leu Ser Gln Glu Leu Ala Ile Pro Tyr Glu Glu 675 680 685 Ala Gln Ala Phe Ile Glu Arg Tyr Phe Gln Ser Phe Pro Lys Val Arg 690 695 700 Ala Trp Ile Glu Lys Thr Leu Glu Glu Gly Arg Arg Arg Gly Tyr Val 705 710 715 720 Glu Thr Leu Phe Gly Arg Arg Arg Tyr Val Pro Asp Leu Glu Ala Arg 725 730 735 Val Lys Ser Val Arg Glu Ala Ala Glu Arg Met Ala Phe Asn Met Pro 740 745 750 Val Gln Gly Thr Ala Ala Asp Leu Met Lys Leu Ala Met Val Lys Leu 755 760 765 Phe Pro Arg Leu Glu Glu Met Gly Ala Arg Met Leu Leu Gln Val His 770 775 780 Asp Glu Leu Val Leu Glu Ala Pro Lys Glu Arg Ala Glu Ala Val Ala 785 790 795 800 Arg Leu Ala Lys Glu Val Met Glu Gly Val Tyr Pro Leu Ala Val Pro 805 810 815 Leu Glu Val Glu Val Gly Ile Gly Glu Asp Trp Leu Ser Ala Lys Glu 820 825 830 <210> 3 <211> 1269 <212> DNA <213> Artificial sequence <400> 3 atgcgtggca tgctgccgct tttcgagcct aagggacgcg ttcttcttgt ggatggacat 60 atgcgtggca tgctgccgct tttcgagcct aagggacgcg ttcttcttgt ggatggacat 60 catctggcgt accgtacctt tcatgccctg aagggcctga ccacttcgcg tggggaaccc 120 catctggcgt accgtacctt tcatgccctg aagggcctga ccacttcgcg tggggaaccc 120 gtgcaagcag tttatggatt cgccaaatcg ttacttaagg ctctgaagga ggatggtgat 180 gtgcaagcag tttatggatt cgccaaatcg ttacttaagg ctctgaagga ggatggtgat 180 gcggtcattg ttgtgttcga cgcaaaagct ccctcgttcc gtcacgaggc ctacggcggc 240 gcggtcattg ttgtgttcga cgcaaaagct ccctcgttcc gtcacgaggc ctacggcggc 240 tataaagctg ggcgtgcacc cacacctgag gattttcccc ggcaacttgc tttgataaag 300 tataaagctg ggcgtgcacc cacacctgag gattttcccc ggcaacttgc tttgataaag 300 gaattagtag acctgttagg cctggcgcgg ttagaagtgc cgggttacga agcagatgac 360 gaattagtag acctgttagg cctggcgcgg ttagaagtgc cgggttacga agcagatgac 360 gtcttggcta gtttagcgaa aaaggctgaa aaagagggat atgaagtgcg gatcctgacc 420 gtcttggcta gtttagcgaa aaaggctgaa aaagagggat atgaagtgcg gatcctgacc 420 gcggataaag atctgtatca actgttgtcc gaccgtattc acgtgcttca tccggagggc 480 gcggataaag atctgtatca actgttgtcc gaccgtattc acgtgcttca tccggagggc 480 tacttgataa ccccggcttg gctgtgggag aaatatgggc tgcgtccaga tcagtgggct 540 tacttgataa ccccggcttg gctgtgggag aaatatgggc tgcgtccaga tcagtgggct 540 gattatcgtg cacttacagg cgatgaatct gataatcttc ccggcgtcaa ggggattggt 600 gattatcgtg cacttacagg cgatgaatct gataatcttc ccggcgtcaa ggggattggt 600 gagaaaaccg cccgtaaact tttggaggag tggggcagct tggaggcgct gttgaagaat 660 gagaaaaccg cccgtaaact tttggaggag tggggcagct tggaggcgct gttgaagaat 660 ctggatcgtt tgaaacccgc tatacgggaa aaaatcttgg cgcacatgga cgacttaaaa 720 ctggatcgtt tgaaacccgc tatacgggaa aaaatcttgg cgcacatgga cgacttaaaa 720 ctgtcttggg acctggcgaa agttcgtact gatttgccgc tggaggtcga ctttgcgaag 780 cgtcgcgagc ccgatcgtga acgtcttcgc gcatttctgg agcgtttaga atttggctcc 840 ctgttgcatg agtttggttt gcttgaaagc ccgaaggcac ttgaggaagc tccttggcct 900 ccgcctgagg gcgcttttgt cggatttgtc ttgagccgta aagaaccgat gtgggcggac 960 ttactggccc ttgctgctgc tcgtgggggt cgcgtgcatc gcgcaccgga gccatacaaa 1020 gcacttcgtg accttaaaga agcccgtggc ttgttggcaa aagatttaag tgtcctggct 1080 ttacgcgagg gcttgggctt accaccggga gatgatccga tgcttttggc ctatctgctg 1140 gacccgagca acacgactcc agagggcgtt gcccgtcgtt atggcggaga atggacggag 1200 gaggcgggag agcgcgcagc gttaagcgag cgtctgtttg ctaatctgtg gggacgctta 1260 gagggagag 1269 <210> 4 <211> 30 <212> DNA <213> Artificial sequence <400> 4 atatcatatg cgtggcatgc tgccgctttt 30 <210> 5 <211> 30 <212> DNA <213> Artificial sequence <400> 5 gcatgaattc cgtctcctct ccctctaagc 30 <210> 6 <211> 1236 <212> DNA <213> Artificial sequence <400> 6 ggagaggagc gcctgttgtg gttgtaccgt gaagtggaac ggccgctgag tgcagtgtta 60 gctcacatgg aagcaaccgg ggtgcggctg gacgttgcgt atttgcgtgc gctgtcgtta 120 gaggtcgcgg aggaaatagc ccgtctggag gccgaagtat tccgtttggc tggccatcct 180 ttcaacctga acagtcggga tcagctggaa cgtgtacttt ttgatgaact ggggctgccc 240 gccatcggca aaaccgaaaa aaccggcaaa cgtagcacct ctgcggcagt gctggaagcg 300 ttacgtgaag ctcatccgat tgtggagaaa attctgcaat atcgcgaatt gacgaaactg 360 aagagcacct atattgatcc gctgccagac ttaattcacc cccgtaccgg acggttgcat 420 acccgcttca accagaccgc gacggcgaca gggcggctga gtagcagcga tccgaacctg 480 caaaacattc ccgtgcgtac cccgctgggt cagcgtattc gccgtgcttt cattgccgag 540 gaaggctggc tgctggtcgc gctggactac tcgcaaatcg aattgcgtgt gttggcccac 600 gaaggctggc tgctggtcgc gctggactac tcgcaaatcg aattgcgtgt gttggcccac 600 ctgtcgggcg acgaaaactt aatacgcgtg tttcaagaag gtcgtgacat acatactgaa 660 ctgtcgggcg acgaaaactt aatacgcgtg tttcaagaag gtcgtgacat acatactgaa 660 accgcgtcct ggatgtttgg agtcccacgg gaggctgtcg atcctcttat gcgtcgtgcc 720 accgcgtcct ggatgtttgg agtcccacgg gaggctgtcg atcctcttat gcgtcgtgcc 720 gccaaaacaa ttaacttcgg agttctgtac ggcatgtcgg cacatcgttt atcacaggaa 780 gccaaaacaa ttaacttcgg agttctgtac ggcatgtcgg cacatcgttt atcacaggaa 780 ctggcgattc cgtatgaaga agcgcaggcc ttcatagaac gttatttcca atcattcccc 840 ctggcgattc cgtatgaaga agcgcaggcc ttcatagaac gttatttcca atcattcccc 840 aaggtgcggg cctggattga gaagaccctg gaagagggcc gtcgtcgtgg ctatgtagag 900 aaggtgcggg cctggattga gaagaccctg gaagagggcc gtcgtcgtgg ctatgtagag 900 actctgttcg gacgtcggcg gtatgtaccc gatcttgagg cccgtgtgaa gtccgttcgt 960 actctgttcg gacgtcggcg gtatgtaccc gatcttgagg cccgtgtgaa gtccgttcgt 960 gaggcagcag aacgtatggc gtttaacatg ccagtccagg gcacagcggc ggacctgatg 1020 gaggcagcag aacgtatggc gtttaacatg ccagtccagg gcacagcggc ggacctgatg 1020 aaattagcta tggttaagct gtttccgcgt ttggaagaaa tgggcgctcg tatgctgtta 1080 aaattagcta tggttaagct gtttccgcgt ttggaagaaa tgggcgctcg tatgctgtta 1080 caggttcatg acgagttagt attagaagca ccgaaggagc gtgccgaagc cgtggcccgg 1140 caggttcatg acgagttagt attagaagca ccgaaggagc gtgccgaagc cgtggcccgg 1140 ttagccaaag aggtaatgga aggcgtctac ccccttgcag tcccgcttga agtcgaagtt 1200 ttagccaaag aggtaatgga aggcgtctac ccccttgcag tcccgcttga agtcgaagtt 1200 ggcatagggg aagactggtt atctgcgaag gaataa 1236 ggcatagggg aagactggtt atctgcgaag gaataa 1236 <210> 7 <210> 7 <211> 25 <211> 25 <212> DNA <213> Artificial sequence <400> 7 ggagaggagc gcctgttgtg gttgt 25 <210> 8 <211> 25 <212> DNA <213> Artificial sequence <400> 8 ttattccttc gcagataacc agtct 25 <210> 9 <211> 21 <212> DNA <213> Artificial sequence <400> 9 tacggttaac cctttgaatc a 21 <210> 10 <211> 21 <212> DNA <213> Artificial sequence <400> 10 gttacctggt taaactgtac t 21 <210> 11 <211> 38 <212> DNA <213> Artificial sequence <400> 11 atgcgtggca tgctgccgct tttcgagcct aagggacg 38 <210> 12 <211> 42 <212> DNA <213> Artificial sequence <400> 12 ttccttcgca gataaccagt cttcccctat gccaacttcg ac 42

Claims

1. A kit, characterized in that, The kit contains a mutant DNA polymerase, and the kit is a fluorescence quantitative PCR detection kit for SARS-CoV-2, the novel coronavirus. The mutant DNA polymerase is mutated on the basis of the wild-type DNA polymerase shown in SEQ ID NO.2, and the mutation sites of the mutant DNA polymerase are: E507A, K508L, and F749K.

2. Use of a mutant DNA polymerase, characterized in that, For the preparation of a fluorescence quantitative PCR detection kit for SARS-CoV-2, the novel coronavirus, the mutant DNA polymerase is mutated on the basis of the wild-type DNA polymerase shown in SEQ ID NO.2, and the mutation sites of the mutant DNA polymerase are: E507A, K508L, and F749K.

3. A non-diagnostic method for detecting the novel coronavirus SARS-CoV-2, characterized in that, The mutant DNA polymerase is used in the method. The mutant DNA polymerase is mutated on the basis of the wild-type DNA polymerase shown in SEQ ID NO.2, and the mutation sites of the mutant DNA polymerase are: E507A, K508L, and F749K.

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