WO2023098035A1 - Taq酶突变体及其制备方法和用途 - Google Patents

Taq酶突变体及其制备方法和用途 Download PDF

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WO2023098035A1
WO2023098035A1 PCT/CN2022/100275 CN2022100275W WO2023098035A1 WO 2023098035 A1 WO2023098035 A1 WO 2023098035A1 CN 2022100275 W CN2022100275 W CN 2022100275W WO 2023098035 A1 WO2023098035 A1 WO 2023098035A1
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taq enzyme
amino acid
enzyme mutant
taq
acid sequence
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French (fr)
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蒋析文
刘霭珊
张伟
谢晓成
陆雪兰
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广州达安基因股份有限公司
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    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1252—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
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Definitions

  • the embodiments of the present invention relate to the field of PCR detection, in particular to Taq enzyme mutants and their preparation methods and uses.
  • 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 optimum reaction temperature is 75-80°C, and its active half-life at 95°C For 40 minutes, with 5'-3' exonuclease activity. Because of its high temperature resistance, it is widely used in polymerase chain reaction (PCR), and is the preferred enzyme for nucleic acid amplification, detection and other reactions. Modern molecular biological detection technology has higher and higher requirements for the sensitivity, accuracy and durability of PCR reaction, and the wild-type Taq enzyme cannot fully meet the needs of practical applications.
  • the wild-type Taq enzyme has poor tolerance to extreme conditions (such as high salt or whole blood), which makes its scope of application smaller, accuracy and sensitivity lower, and fully meets the requirements of modern molecular biological detection technology for PCR reactions.
  • the wild-type Taq enzyme in the system has 5' ⁇ 3' exonuclease activity, which makes the amplification efficiency decrease when the dye method is used for PCR amplification.
  • the purpose of the present invention is to provide a Taq enzyme mutant.
  • Another object of the present invention is to provide a nucleotide molecule encoding the above Taq enzyme mutant.
  • Another object of the present invention is to provide a carrier.
  • Another object of the present invention is to provide a host cell.
  • Another object of the present invention is to provide a method for preparing a Taq enzyme mutant.
  • Another object of the present invention is to provide a kit containing Taq enzyme mutants.
  • the first aspect of the present invention provides a Taq enzyme mutant, and the Taq enzyme mutant comprises:
  • the amino acid sequence is mutated at any four or more positions selected from the group consisting of P40W, L125I, G200M, D335V, G499K, E634G and F769I.
  • the amino acid sequence is mutated at the following four sites: D335V, G499K, E634G and F769I; and the amino acid sequence is further mutated at any one or several sites selected from the group: P40W , L125I and G200M.
  • amino acid sequence is mutated at the following seven positions: P40W, L125I, G200M, D335V, G499K, E634G and F769I.
  • the resistance of the Taq enzyme mutant to sodium chloride is not lower than 70mM, more preferably not lower than 80mM, more preferably not lower than 90mM, more preferably not lower than 100mM, more preferably not lower than Below 130mM, more preferably not below 150mM, most preferably not below 180mM.
  • the resistance of the Taq enzyme mutant to potassium chloride is not lower than 100mM, more preferably not lower than 110mM, more preferably not lower than 120mM, more preferably not lower than 130mM, more preferably not lower than It is lower than 140 mM, more preferably not lower than 150 mM, more preferably not lower than 180 mM, more preferably not lower than 190 mM, most preferably not lower than 200 mM.
  • the resistance of the Taq enzyme mutant to EDTA whole blood is not less than 5%, more preferably not less than 7%, more preferably not less than 10%, more preferably not less than 20%, More preferably not less than 30%, more preferably not less than 40%, most preferably not less than 50%.
  • the resistance of the Taq enzyme mutant to heparin whole blood is not lower than 1%, more preferably not lower than 3%, more preferably not lower than 5%, more preferably not lower than 10%, More preferably not less than 20%, more preferably not less than 30%, most preferably not less than 35%.
  • SEQ ID NO:1 the wild-type Taq enzyme DNA sequence is as follows:
  • SEQ ID NO:2 (the wild-type Taq enzyme amino acid sequence is as follows):
  • any one or several mutations selected from the following group occur in the amino acid sequence:
  • the Taq enzyme mutant comprises an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:1; more preferably, the Taq enzyme mutant comprises an amino acid sequence identical to the amino acid sequence shown in SEQ ID NO:1
  • the amino acid sequence shown in: 1 has an amino acid sequence with at least 90% identity; more preferably, the Taq enzyme mutant includes an amino acid sequence with at least 95% identity with the amino acid sequence shown in SEQ ID NO: 1.
  • the second aspect of the present invention also provides a nucleotide molecule encoding the mutant Taq enzyme described in the first aspect of the present invention.
  • the third aspect of the present invention also provides a vector containing the nucleotide molecule described in the second aspect of the present invention.
  • the fourth aspect of the present invention also provides a host cell, which contains the nucleotide molecule of the second aspect of the present invention or has the nucleotide molecule of the second aspect of the present invention integrated into its chromosome.
  • the host cells are prokaryotic cells or eukaryotic cells.
  • the prokaryotic cell is Escherichia coli.
  • the eukaryotic cells are yeast cells.
  • the fifth aspect of the present invention also provides a kit containing the Taq enzyme mutant described in the first aspect of the present invention.
  • the sixth aspect of the present invention also provides a method for preparing the Taq enzyme mutant described in the first aspect of the present invention, the method comprising the steps of:
  • the seventh aspect of the present invention also provides the use of the kit described in the fifth aspect of the present invention, which is applied to DNA sequencing, DNA labeling, primer extension, amplification and the like.
  • the present invention has at least the following advantages:
  • the Taq enzyme mutant provided by the present invention has high amplification activity, and can obtain more amplification products than the wild-type Taq enzyme under the same number of PCR cycles;
  • the Taq enzyme mutant provided by the invention is resistant to whole blood and high salt at the same time;
  • the 5'-3' exonuclease activity of the Taq enzyme mutant provided by the present invention is low, and is suitable for the dye-based PCR system.
  • Fig. 1 is according to the target protein SDS-PAGE identification result figure in the embodiment of the present invention.
  • Fig. 2 is the electrophoresis diagram of Taq-aCb protein N1 column purification according to the embodiment of the present invention
  • Fig. 3 is according to the electrophoresis diagram of Taq-aCb protein Q column purification in the embodiment of the present invention
  • Fig. 4 is according to wild-type Taq enzyme and Taq-aCb anti-NaCl experimental result figure in the embodiment of the present invention
  • Fig. 5 is according to the result figure of Taq-aCb anti-NaCl experiment in the embodiment of the present invention.
  • Figure 6 is a graph showing the experimental results of wild-type Taq enzyme and Taq-aCb anti-KCl according to the embodiment of the present invention.
  • Fig. 7 is a graph showing the results of wild-type Taq enzyme and Taq-aCb anti-EDTA blood experiment according to the embodiment of the present invention.
  • Figure 8 is a diagram showing the results of wild-type Taq enzyme and Taq-aCb anti-heparin blood experiments according to the embodiment of the present invention.
  • Fig. 9 is a graph of RFU-cycle in Example 8 according to the present invention.
  • the wild-type Taq enzyme is difficult to amplify under some extreme conditions, that is, under some extreme conditions, its polymerization activity is very low, such as high-salt, whole blood environment.
  • the prior art attempts to mutate the wild-type Taq enzyme to enhance its tolerance to extreme conditions, little success has been achieved.
  • the present inventors have screened out Taq enzyme mutants with good performance by using enzyme site-directed mutagenesis technology, which have good polymerization activity, good tolerance to high salt and whole blood, and are suitable for clinical use.
  • the Taq enzyme mutant screened by the present invention has low 5'-3' exonuclease activity and is very suitable for the dye method PCR system.
  • Some preferred embodiments of the present invention provide a kind of Taq enzyme mutant, described Taq enzyme mutant comprises:
  • any one or several mutations selected from the following group occur in the amino acid sequence:
  • the amino acid sequence is mutated at any four or more positions selected from the group consisting of P40W, L125I, G200M, D335V, G499K, E634G and F769I.
  • the amino acid sequence is mutated at the following four sites: D335V, G499K, E634G and F769I; and the amino acid sequence is further mutated at any one or several sites selected from the group: P40W , L125I and G200M.
  • amino acid sequence is mutated at the following seven positions: P40W, L125I, G200M, D335V, G499K, E634G and F769I.
  • the resistance of the Taq enzyme mutant to sodium chloride is not lower than 70mM, more preferably not lower than 80mM, more preferably not lower than 90mM, more preferably not lower than 100mM, more preferably not lower than Below 130mM, more preferably not below 150mM, most preferably not below 180mM.
  • the resistance of the Taq enzyme mutant to potassium chloride is not lower than 100mM, more preferably not lower than 110mM, more preferably not lower than 120mM, more preferably not lower than 130mM, more preferably not lower than It is lower than 140 mM, more preferably not lower than 150 mM, more preferably not lower than 180 mM, more preferably not lower than 190 mM, most preferably not lower than 200 mM.
  • the resistance of the Taq enzyme mutant to EDTA whole blood is not less than 5% (the percentage here refers to the percentage of the plasma volume accounting for the total volume of the PCR solution system), more preferably not less than 7% %, more preferably not less than 10%, more preferably not less than 20%, more preferably not less than 30%, more preferably not less than 40%, most preferably not less than 50%.
  • the resistance of the Taq enzyme mutant to heparin whole blood is not less than 1% (the percentage here refers to the percentage of the plasma volume accounting for the total volume of the PCR solution system), more preferably not less than 3% %, more preferably not less than 5%, more preferably not less than 10%, more preferably not less than 20%, more preferably not less than 30%, most preferably not less than 35%.
  • SEQ ID NO:1 (the wild-type Taq enzyme DNA sequence is as follows):
  • SEQ ID NO:2 (the wild-type Taq enzyme amino acid sequence is as follows):
  • the Taq enzyme mutant comprises an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:1; more preferably, the Taq enzyme mutant comprises an amino acid sequence identical to the amino acid sequence shown in SEQ ID NO:1
  • the amino acid sequence shown in: 1 has an amino acid sequence with at least 90% identity; more preferably, the Taq enzyme mutant includes an amino acid sequence with at least 95% identity with the amino acid sequence shown in SEQ ID NO: 1.
  • Some preferred embodiments of the present invention provide a nucleotide molecule encoding the mutant Taq enzyme mutant described in the first aspect of the present invention.
  • Some preferred embodiments of the present invention provide a vector containing the nucleotide molecule described in the second aspect of the present invention.
  • Some preferred embodiments of the present invention provide a host cell, which contains the nucleotide molecule of the second aspect of the present invention or has the nucleotide molecule of the second aspect of the present invention integrated into its chromosome.
  • the host cells are prokaryotic cells or eukaryotic cells.
  • the prokaryotic cell is Escherichia coli.
  • the eukaryotic cells are yeast cells.
  • kits containing the Taq enzyme mutant described in the first aspect of the present invention provide a kit containing the Taq enzyme mutant described in the first aspect of the present invention.
  • Some preferred embodiments of the present invention provide a method for preparing the Taq enzyme mutant described in the first aspect of the present invention, the method comprising steps:
  • kits described in the fifth aspect of the present invention which is applied to DNA sequencing, DNA labeling, primer extension, amplification and the like.
  • amino acid in its broadest sense refers to any compound and/or substance that can be incorporated into a polypeptide chain.
  • the amino acid has the general structure H2N-C(H)(R)-COOH.
  • the amino acid is a naturally occurring amino acid.
  • the amino acid is a synthetic amino acid; in some embodiments, the amino acid is a D-amino acid; in some embodiments, the amino acid is an L-amino acid.
  • Standard amino acid refers to any of the twenty standard L-amino acids commonly found in naturally occurring peptides.
  • Non-standard amino acid refers to any amino acid other than a standard amino acid, whether prepared synthetically or obtained from a natural source.
  • synthetic amino acid includes chemically modified amino acids, including but not limited to salts, amino acid derivatives (such as amides), and/or substitutions.
  • Amino acids including carboxyl and/or amino terminal amino acids in peptides, can be modified by methylation, amidation, acetylation and/or substitution with other chemicals without adversely affecting their activity. Amino acids can have disulfide bonds.
  • amino acid is used interchangeably with “amino acid residue” and may refer to free amino acids and/or amino acid residues of peptides.
  • mutation refers to changes introduced into a parental sequence, including but not limited to substitutions, insertions, deletions (including truncations). Consequences of mutations include, but are not limited to, the development of new properties, properties, functions, phenotypes or traits not found in the protein encoded by the parental sequence. , the term “mutant” refers to a modified protein that exhibits altered characteristics when compared to the parent protein.
  • % homology is used interchangeably herein with the term “% identity” and refers to the nucleic acid encoding any one of the polypeptides of the invention when aligned using a sequence alignment program The level of nucleic acid or amino acid sequence identity between sequences or amino acid sequences of polypeptides of the invention.
  • nucleotide refers to a monomeric unit of DNA or RNA constructed from sugar moieties (pentose sugars), phosphate esters, and nitrogen-containing heterocyclic bases.
  • the base is attached to the sugar moiety via the glycosidic carbon (1' carbon of the pentose sugar), and the combination of base and sugar is a nucleoside.
  • a nucleoside contains a phosphate group bonded to the 3'- or 5'-position of a pentose sugar, it is called a nucleotide.
  • a sequence of operably linked nucleotides is generally referred to herein as a "base sequence” or a “nucleotide sequence juxtaposition”, and is expressed herein in such a form that its left-to-right direction is from the 5'-end to the Regular orientation of the 3'-end.
  • vector refers to a nucleic acid construct designed for transfer between different host cells.
  • Expression vector refers to a vector capable of incorporating and expressing a heterologous DNA fragment in a foreign cell.
  • Many prokaryotic and eukaryotic expression vectors are commercially available. The selection of suitable expression vectors is within the knowledge of those skilled in the art.
  • the term "host cell” refers to a cell invaded by a gene of interest, wherein the gene of interest can invade the cell in a form bound to a carrier (such as a virus, chromosome or plasmid) to achieve replication.
  • a carrier such as a virus, chromosome or plasmid
  • chromosomal integration refers to the integration by means of homologous recombination of multiple copies of a gene of interest at well-defined sites.
  • a preferred method of synthesis is the asymmetric PCR method.
  • the asymmetric PCR method uses a pair of primers of unequal amount to produce a large amount of single-stranded DNA (ssDNA) after PCR amplification.
  • the pair of primers are respectively called non-restricted primers and restricted primers, and their ratio is generally 50-100:1.
  • the amplified product is mainly double-stranded DNA, but when the restrictive primer (low concentration primer) is consumed, the PCR guided by the non-restrictive primer (high concentration primer) will Produces large amounts of single-stranded DNA.
  • Primers for PCR can be appropriately selected based on the sequence information of the present invention disclosed herein, and can be synthesized by conventional methods. Amplified DNA/RNA fragments can be separated and purified by conventional methods such as by gel electrophoresis.
  • Taq enzyme mutant of the present invention can be expressed or produced by conventional recombinant DNA technology, comprising steps:
  • an expression vector containing the coding DNA sequence of the Taq enzyme mutant of the present invention and appropriate transcription/translation control signals preferably a commercially available vector: pET28. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology and the like. Said DNA sequence can be operably linked 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.
  • the expression vector preferably contains one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells.
  • the recombinant vector includes: a promoter, a target gene and a terminator in the direction from 5' to 3'. If necessary, the recombinant vector can also include the following elements: protein purification tag; 3' polynucleotide signal; non-translated nucleic acid sequence; transport and targeting nucleic acid sequence; selection marker (antibiotic resistance gene, fluorescent protein, etc.) ; an enhancer; or an operator.
  • the expression vector can be bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus or other vectors. In general, any plasmid and vector can be used as long as it can replicate and be stable in the host.
  • Those skilled in the art can use well-known methods to construct a vector 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 recombination technology and the like.
  • the expression vector of the present invention can be used to transform appropriate host cells, so that the host can transcribe the target RNA or express the target protein.
  • Host cells can be prokaryotic cells, such as Escherichia coli, Corynebacterium glutamicum, Brevibacterium flavum, Streptomyces, Agrobacterium; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as plant cells .
  • prokaryotic cells such as Escherichia coli, Corynebacterium glutamicum, Brevibacterium flavum, Streptomyces, Agrobacterium
  • lower eukaryotic cells such as yeast cells
  • higher eukaryotic cells such as plant cells .
  • the host When the host is a prokaryotic organism (such as Escherichia coli), it can be treated with CaCl2 or electroporation.
  • the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods (such as microinjection, electroporation, liposome packaging, etc.). Transformation of plants can also use methods such as Agrobacterium transformation or biolistic transformation, such as leaf disk method, immature embryo transformation method, flower bud soaking method and the like.
  • Transformed plant cells, tissues or organs can be regenerated into plants by conventional methods, so as to obtain transgenic plants.
  • Step 1 Using the wild-type Taq enzyme expression vector as a template, use the QuikChange Lightning multi-site mutation kit to prepare the Taq-aCb mutant plasmid.
  • the wild-type expression vector is preserved by Guangzhou Daan Gene Co., Ltd., and the vector is pET28a, N-terminal Add His6 tag. All mutant plasmids were sent for detection and sequencing, and the sequencing results showed that the mutant vector was constructed successfully.
  • Step 2 Transform Escherichia coli BL21(DE3) with the recombinant plasmid
  • step 2 inoculate it in TB medium containing 100 ⁇ g/mL kana-resistance aseptically, culture it with shaking at 220 rpm at 37°C until the OD600 is between 0.6 and 0.8, and induce it with IPTG (final concentration is 0.1 mM), placed at 37°C and 18°C for shaking culture overnight, and the group without IPTG was used as a control, cultured at 37°C for 3 hours, and each group of experiments was repeated once.
  • the samples were sonicated for identification by SDS-PAGE, and the results are shown in Figure 1.
  • 1.5 L of bacterial liquid was cultured in shake flasks of TB medium, and the expression conditions were consistent with 3) expression of the target protein.
  • the bacteria were collected by centrifugation, and the wet weight of the two kinds of protein bacteria was about 30g. Weigh about 4g of bacteria, add 35ml Lysis Buffer and resuspend on ice. After sonication, centrifuge at 20,000 rpm at 4° C. for 30 minutes, take the supernatant, and filter it with a needle filter of 0.22 ⁇ m to obtain the supernatant.
  • the supernatant was subjected to Ni-column affinity chromatography, and 0-60% Buffer B was used for linear elution, and the eluate from the main peak was taken for ion exchange elution.
  • the purification column used was HisTrapTM Q-HP, and 0-60% Buffer C for linear elution.
  • Electropherograms are shown in Figure 2 and Figure 3. Calculate the expression content of the target protein, Taq-aCb is 1.46mg/mL, and the concentration of the solution used is as follows:
  • Buffer B 50mM Tris, 50mM NaCl, 500mM Imidazole, 5% Glycerol, pH8.5;
  • Lysis Buffer 50mM Tris, 300mM NaCl, 5% Glycerol, pH8.5;
  • Buffer C 100mM Tris, 1M NaCl, 10% Glycerol, pH8.5.
  • Embodiment 2 Taq-aCb anti-NaCl performance test
  • the resistance of Taq-aCb to NaCl was determined using wild-type Taq enzyme as a positive control.
  • the PCR reaction system was prepared according to the recipe in Table 2, in which RV and M4 primers were purchased from TaKaRa, 5X Fast Taq Buffer, and pUC19 plasmid were preserved by Guangzhou Daan Gene Co., Ltd.
  • the amount of enzyme added per 10 ⁇ L of PCR reaction system was 5 U.
  • the amplification experiment was carried out with the pUC19 plasmid as a template, and the PCR product was subjected to 2% agarose gel electrophoresis.
  • Reagent BufferA BufferB RV (10pmol) 1.2 ⁇ L 1.2 ⁇ L M4 (10pmol) 1.2 ⁇ L 1.2 ⁇ L pUC19plasmid 6 ⁇ L 6 ⁇ L 5X Fast Taq Buffer 12 ⁇ L 12 ⁇ L Wild-type Taq/Taq 2C2/Taq 2C2 Mut 30U 30U 1M NaCl 6 ⁇ L 0 ⁇ L ddH 2 O 31.6 ⁇ L 37.6 ⁇ L
  • 1-11 are respectively 0, 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100mM NaCl concentrations of wild-type Taq enzyme PCR product lanes; 12-22 are respectively 0, 10, The PCR product lanes of Taq-aCb at 20, 30, 40, 50, 60, 70, 80, 90, and 100 mM NaCl concentrations show that Taq-aCb has better NaCl resistance than wild type.
  • 1 to 9 are the PCR product lanes of Taq-aCb at concentrations of 110, 120, 130, 140, 150, 160, 170, 180, and 190 Mm NaCl respectively, and 10 is the positive control.
  • the resistance of wild-type Taq enzyme to NaCl is 70mM, and the resistance of Taq-aCb to NaCl has reached 180mM, so the anti-NaCl performance of Taq enzyme mutant is significantly improved compared with wild-type Taq enzyme.
  • Embodiment 3 Taq-aCb anti-KCl performance test
  • the resistance of Taq-aCb to KCl was tested by the same method as in Example 2, and the electrophoresis result of the amplified product is shown in FIG. 6 .
  • 1-11 are respectively 50, 80, 100, 150, 160, 170, 180, 190, 200, 250, 300mM KCl concentration of wild-type Taq enzyme PCR product swimming lanes
  • 13-23 are respectively 50, 80, PCR product swimming lanes of Taq-aCb under 100, 150, 160, 170, 180, 190, 200, 250, 300mM KCl concentrations; 12, 24 are positive controls.
  • the resistance of the wild-type Taq enzyme to KCl is 100mM, and the resistance of the Taq mutant enzyme Taq-aCb to NaCl has reached 200mM, so the anti-KCl performance of the Taq enzyme mutant is significantly improved compared with the wild-type Taq enzyme .
  • Embodiment 4 Taq-aCb anti-EDTA whole blood performance test
  • 1 ⁇ 10 are respectively 5, 10, 15, 20, 25, 30, 35, 40, 45, 50% (V/V) EDTA blood concentration lower wild-type Taq enzyme PCR product swimming lane; 11 ⁇ 20 respectively These are PCR product lanes of Taq-aCb at 5, 10, 15, 20, 25, 30, 35, 40, 45, 50% (V/V) EDTA blood concentrations.
  • the resistance of wild-type Taq enzyme to EDTA whole blood is 5% (volume fraction is calculated according to the percentage of the blood volume added to PCR accounting for the total volume of PCR system), and Taq mutant enzyme Taq-aCb reaches EDTA whole blood resistance Therefore, the anti-EDTA whole blood performance of the Taq enzyme mutant was significantly improved compared with the wild-type Taq enzyme.
  • Embodiment 5 Taq-aCb anti-heparin whole blood performance test
  • 1 to 10 are respectively 5, 10, 15, 20, 25, 30, 35, 40, 45, 50% (V/V) heparin blood concentrations of wild-type Taq enzyme PCR product swimming lanes; 11 to 20 respectively The lanes are PCR product lanes of Taq-aCb at 5, 10, 15, 20, 25, 30, 35, 40, 45, 50% (V/V) heparin blood concentrations.
  • the resistance of the wild-type Taq enzyme to heparin whole blood is 0% (the volume fraction is calculated according to the percentage of the blood volume added to PCR in the total volume of the PCRT system), and the resistance of Taq mutant enzyme Taq-aCb to heparin whole blood reaches Compared with the wild-type Taq enzyme, the anti-heparin whole blood performance of the Taq enzyme mutant was significantly improved.
  • the 5'-3' exonuclease activity was detected by fluorescent probe PCR method, and the wild-type Taq enzyme (ThermoFisher Scientific) was used as a positive control, diluted to 1U, 2U, 3U, 4U, 5U, and Taq-aCb was also diluted to the same active concentration.
  • Table 5 where 10X Taq Buffer was purchased from TaRaKa:
  • PCR reaction conditions 95°C for 10 minutes, (95°C for 10 seconds, 55°C for 30 seconds to read fluorescence) ⁇ 40 cycles, with the reaction cycle as the X-axis, and the fluorescence value (RFU) corresponding to each cycle as the Y-axis, as RFU-cycle graph.
  • RFU-cycle graph Take the known enzyme concentration as the X-axis, and the initial slope corresponding to the RFU-cycle graph as the Y-axis to fit the standard curve, and the slope of the obtained linear regression equation can represent the activity of the 5'-3' exonuclease. The result is shown in Figure 9.
  • the 5' ⁇ 3' exonuclease activity of Taq-aCb is 66% of the wild type Taq enzyme, and after mutation, the 5' ⁇ 3' exonuclease activity of Taq-aCb is decreased compared with the wild type . It is basically the same as the wild-type Taq enzyme.

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Abstract

提供了一种Taq酶突变体及其制备方法和用途。所述Taq酶突变体包括：与SEQ ID NO:2所示的氨基酸序列具有至少70％同一性的氨基酸序列；所述氨基酸序列在选自下组的一个或多个位点发生突变：P40、L125、G200、D335、G499、E634、F769或其组合。所提供的Taq酶突变体具有高扩增活性，在同等PCR循环次数下，相比野生型Taq酶可以得到更多的扩增产物、同时耐全血和高盐且5'～3'外切酶活性低。

Description

Taq酶突变体及其制备方法和用途
相关申请交叉引用
本专利申请要求于2021年11月30日提交的、申请号为202111442628.3、发明名称为“Taq酶突变体及其制备方法和用途”的中国专利申请的优先权，上述申请的全文以引用的方式并入本文中。
技术领域
本发明实施例涉及PCR检测领域，特别涉及Taq酶突变体及其制备方法和用途。
背景技术
Taq酶是一种来源于耐热性细菌Thermus aquaticus的耐热性DNA聚合酶，分子量94KDa，在镁离子存在的条件下，其最适反应温度为75-80℃，在95℃下的活性半衰期为40分钟，具有5’-3’核酸外切酶活性。由于其具有耐高温的特性，因此广泛用于聚合酶链式反应(PCR)，是核酸扩增、检测等反应的首选用酶。现代分子生物检测技术对PCR反应的灵敏度、精确度、耐用性要求越来越高，野生型Taq酶无法完全满足实际应用的需求。
然而，野生型Taq酶对于极端条件(例如高盐或全血)耐受性差，使得其适用范围变小，准确度和灵敏度下降，完全满足现代分子生物检测技术对PCR反应的要求。此外，使用染料法进行PCR扩增时，系统中野生型Taq酶具有5’～3’外切酶活性，使得染料法进行PCR扩增时扩增效率下降。
因此，开发一种5’～3’外切酶活性低的，且耐全血和高盐的Taq酶突变体尤为重要。
发明内容
本发明的目的在于提供一种Taq酶突变体。
本发明的另一目的在于提供一种编码上述Taq酶突变体的核苷酸分子。
本发明的另一目的在于提供一种载体。
本发明的另一目的在于提供一种宿主细胞。
本发明的另一目的在于提供一种Taq酶突变体的制备方法。
本发明的另一目的在于提供一种含有Taq酶突变体的试剂盒。
为解决上述技术问题，本发明第一方面提供了一种Taq酶突变体，所述Taq酶突变体包括：
与SEQ ID NO:2所示的氨基酸序列具有至少70％同一性的氨基酸序列，所述氨基酸序列在选自下组的一个或多个位点发生突变：P40、L125、G200、D335、G499、E634、F769或其组合。
在一些优选的方案中，所述氨基酸序列在选自下组的任四个或四个以上位点发生突变：P40W、L125I、G200M、D335V、G499K、E634G和F769I。
在一些优选的方案中，所述氨基酸序列在下组四个位点发生突变D335V,G499K,E634G和F769I；并且所述氨基酸序列还在选自下组的任一个或几个位点发生突变：P40W、L125I和G200M。
在一些优选的方案中，所述氨基酸序列在下组七个位点发生突变P40W、L125I、G200M、D335V、G499K、E634G和F769I。
在一些优选的方案中，所述Taq酶突变体对氯化钠的抗性不低于70mM,，更优选不低于80mM，更优选不低于90mM，更优选不低于100mM，更优选不低于130mM，更优选不低于150mM，最优选不低于180mM。
在一些优选的方案中，所述Taq酶突变体对氯化钾的抗性不低于100mM,，更优选不低于110mM，更优选不低于120mM，更优选不低于130mM，更优选不低于140mM，更优选不低于150mM，更优选不低于180mM，更优选不低于190mM，最优选不低200mM。
在一些优选的方案中，所述Taq酶突变体对EDTA全血的抗性不低于5％，更优选不低于7％，更优选不低于10％，更优选不低于20％，更优选不低于30％，更优选不低于40％，最优选不低于50％。
在一些优选的方案中，所述Taq酶突变体对肝素全血的抗性不低于1％，更优选不低于3％，更优选不低于5％，更优选不低于10％，更优选不低于20％，更优选不低于30％，最优选不低于35％。SEQ ID NO:1(野生型Taq酶DNA序列如下)：
Figure PCTCN2022100275-appb-000001
Figure PCTCN2022100275-appb-000002
Figure PCTCN2022100275-appb-000003
SEQ ID NO:2(野生型Taq酶氨基酸序列如下)：
Figure PCTCN2022100275-appb-000004
在一些优选的方案中，所述氨基酸序列发生选自下组的任一种或几种突变：
P40W、L125I、G200M、D335V、G499K、E634G和F769I。
在一些优选的方案中，所述Taq酶突变体包括与SEQ ID NO:1所示的氨基酸序列具有至少80％同一性的氨基酸序列；更优选地，所述Taq酶突变体包括与SEQ ID NO:1所示的氨基酸序列具有至少90％同一性的氨基酸序列；更优选地，所述Taq酶突变体包括与SEQ ID NO:1所示的氨基酸序列具有至少95％同一性的氨基酸序列。
本发明的第二方面还提供了一种核苷酸分子，所述核苷酸分子编码本发明第一方面所述的的突的Taq酶突变体。
本发明的第三方面还提供了一种含有本发明第二方面所述的核苷酸分子的载体。
本发明的第四方面还提供了一种宿主细胞，所述宿主细胞含有本发明第二方面所述的核苷酸分子或染色体整合有本发明第二方面所述的核苷酸分 子。
在一些优选的方案中，所述宿主细胞为原核细胞、或真核细胞。
在一些优选的方案中，所述原核细胞为大肠杆菌。
在一些优选的方案中，所述真核细胞为酵母细胞。
本发明的第五方面还提供了一种含有本发明第一方面所述的Taq酶突变体的试剂盒。
本发明第六方面还提供了一种制备本发明第一方面所述的Taq酶突变体的方法，所述方法包括步骤：
(i)在适合的条件下，培养本发明第四方面所述的宿主细胞，从而表达出所述的Taq酶突变体；和
(ii)分离所述的Taq酶突变体。
本发明的第七方面还提供了本发明的第五方面所述的试剂盒的用途，应用于DNA测序，DNA标记，引物延伸，扩增等。
本发明相对于现有技术而言，至少具有下述优点：
(1)本发明提供的Taq酶突变体具有高扩增活性，在同等PCR循环次数下，相比野生型Taq酶可以得到更多的扩增产物；
(2)本发明提供的Taq酶突变体同时耐全血和高盐；
(3)本发明提供的Taq酶突变体5’～3’外切酶活性低，适用于染料法PCR系统。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
一个或多个实施例通过与之对应的附图中的图片进行示例性说明，这些示例性说明并不构成对实施例的限定。
图1是根据本发明实施例中目的蛋白SDS-PAGE鉴定结果图；
图2是根据本发明实施例中Taq-aCb蛋白N1柱纯化电泳图；
图3是根据本发明实施例中Taq-aCb蛋白Q柱纯化电泳图；
图4是根据本发明实施例中野生型Taq酶和Taq-aCb抗NaCl实验结果 图；
图5是根据本发明实施例中Taq-aCb抗NaCl实验结果图；
图6是根据本发明实施例中野生型Taq酶和Taq-aCb抗KCl实验结果图；
图7是根据本发明实施例中野生型Taq酶和Taq-aCb抗EDTA血液实验结果图；
图8是根据本发明实施例中野生型Taq酶和Taq-aCb抗肝素血液实验结果图；
图9是根据本发明实施例8中RFU-循环曲线图。
具体实施方式
野生型Taq酶难以在一些极端条件下进行扩增，即在一些极端条件下，其聚合活性很低，例如高盐、全血的环境。虽然现有技术中尝试对野生型Taq酶进行突变，以增强其对极端条件的耐受性，但收效甚微。本发明人通过广泛而深入的研究，应用酶定点突变技术，筛选出性能良好的Taq酶突变体，其聚合活性好，对高盐和全血有很好的耐受性，适宜临床使用。此外，本发明筛选出的Taq酶突变体的5’～3’外切酶活性低，十分适用于染料法PCR系统。
本发明的一些优选的实施方式提供了一种Taq酶突变体，所述Taq酶突变体包括：
与SEQ ID NO:2所示的氨基酸序列具有至少70％同一性的氨基酸序列，所述氨基酸序列在选自下组的一个或多个位点发生突变：P40、L125、G200、D335、G499、E634、F769或其组合。
在一些优选的方案中，所述氨基酸序列发生选自下组的任一种或几种突变：
P40W、L125I、G200M、D335V、G499K、E634G和F769I。
在一些优选的方案中，所述氨基酸序列在选自下组的任四个或四个以上位点发生突变：P40W、L125I、G200M、D335V、G499K、E634G和F769I。
在一些优选的方案中，所述氨基酸序列在下组四个位点发生突变D335V,G499K,E634G和F769I；并且所述氨基酸序列还在选自下组的任一个或几个位点发生突变：P40W、L125I和G200M。
在一些优选的方案中，所述氨基酸序列在下组七个位点发生突变P40W、L125I、G200M、D335V、G499K、E634G和F769I。
在一些优选的方案中，所述Taq酶突变体对氯化钠的抗性不低于70mM,，更优选不低于80mM，更优选不低于90mM，更优选不低于100mM，更优选不低于130mM，更优选不低于150mM，最优选不低于180mM。
在一些优选的方案中，所述Taq酶突变体对氯化钾的抗性不低于100mM,，更优选不低于110mM，更优选不低于120mM，更优选不低于130mM，更优选不低于140mM，更优选不低于150mM，更优选不低于180mM，更优选不低于190mM，最优选不低200mM。
在一些优选的方案中，所述Taq酶突变体对EDTA全血的抗性不低于5％(这里的百分比指的是血浆体积占PCR溶液体系总体积的百分比)，更优选不低于7％，更优选不低于10％，更优选不低于20％，更优选不低于30％，更优选不低于40％，最优选不低于50％。
在一些优选的方案中，所述Taq酶突变体对肝素全血的抗性不低于1％(这里的百分比指的是血浆体积占PCR溶液体系总体积的百分比)，更优选不低于3％，更优选不低于5％，更优选不低于10％，更优选不低于20％，更优选不低于30％，最优选不低于35％。
SEQ ID NO:1(野生型Taq酶DNA序列如下)：
Figure PCTCN2022100275-appb-000005
Figure PCTCN2022100275-appb-000006
SEQ ID NO:2(野生型Taq酶氨基酸序列如下)：
Figure PCTCN2022100275-appb-000007
在一些优选的方案中，所述Taq酶突变体包括与SEQ ID NO:1所示的氨基酸序列具有至少80％同一性的氨基酸序列；更优选地，所述Taq酶突变体包括与SEQ ID NO:1所示的氨基酸序列具有至少90％同一性的氨基酸序列；更优选地，所述Taq酶突变体包括与SEQ ID NO:1所示的氨基酸序列具有至少95％同一性的氨基酸序列。
本发明的一些优选的实施方式提供了一种核苷酸分子，所述核苷酸分子编码本发明第一方面所述的的突的Taq酶突变体。
本发明的一些优选的实施方式提供了一种含有本发明第二方面所述的核苷酸分子的载体。
本发明的一些优选的实施方式提供了一种宿主细胞，所述宿主细胞含有本发明第二方面所述的核苷酸分子或染色体整合有本发明第二方面所述的核苷酸分子。
在一些优选的方案中，所述宿主细胞为原核细胞、或真核细胞。
在一些优选的方案中，所述原核细胞为大肠杆菌。
在一些优选的方案中，所述真核细胞为酵母细胞。
本发明的一些优选的实施方式提供了一种含有本发明第一方面所述的Taq酶突变体的试剂盒。
本发明的一些优选的实施方式提供了一种制备本发明第一方面所述的Taq酶突变体的方法，所述方法包括步骤：
(i)在适合的条件下，培养本发明第四方面所述的宿主细胞，从而表达出所述的Taq酶突变体；和
(ii)分离所述的Taq酶突变体。
本发明的一些优选的实施方式提供了本发明的第五方面所述的试剂盒的用途，应用于DNA测序，DNA标记，引物延伸，扩增等。
术语
如本文所用，术语“氨基酸”在其最广泛的含义中是指可掺入多肽链中的任何化合物和/或物质。在一些实施方案中，氨基酸具有通用结构H2N-C(H)(R)-COOH。在一些实施方案中，氨基酸是天然存在的氨基酸。在一些实施方案中，氨基酸是合成氨基酸；在一些实施方案中，氨基酸是D-氨基酸；在一些实施方案中，氨基酸是L-氨基酸。“标准氨基酸”是指通常在天然存在的肽中发现的二十种标准L-氨基酸中的任一种。“非标准氨基酸”是指除了标准氨基酸以外的任何氨基酸，无论是合成制备的还是从天然来源获得的。如本文所用，“合成氨基酸”包括经化学修饰的氨基酸，包括但不限于盐、氨基酸衍生物(诸如酰胺)和/或取代物。氨基酸，包括肽中的羧基和/或氨基末端氨基酸，可以通过甲基化、酰胺化、乙酰化和/或用其它化学物质取代进行修饰，而不对其活性产生不利影响。氨基酸可以具有二硫键。术语“氨基酸”可与“氨基酸残基”可互换使用，并且可以指游离氨基酸和/或肽的氨基酸残基。无论该术语是指游离氨基酸还是肽的残基，从使用该术语的上下文中都将是显而易见的。应当注意，所有氨基酸残基序列在本文中都由左右取向为氨基末端到羧基末端的常规方向的式表示。
如本文所用，术语“突变”是指引入亲本序列的改变，包括但不眼于取代、插入、缺失(包括截短)。突变的后果包括但不限于在由亲本序列编码的蛋白中未发现的新特性、性质、功能、表型或性状的产生。，术语"突变体"是指经修饰的蛋白，当与亲本蛋白相比时，所述经修饰的蛋白呈现改变的特 征。
如本文所用，术语“％同源性”在本文中与术语“％同一性”可互换地使用，并且是指当使用序列比对程序进行比对时，编码本发明多肽的任何一个的核酸序列或本发明多肽的氨基酸序列之间的核酸或氨基酸序列同一性的水平。
如本文所用，术语“核苷酸”是指，自糖部分(戊糖)、磷酸酯以及含氮杂环碱基构成的DNA或RNA的单体单元。碱基经由糖苷碳(戊糖的1’碳)而连接至糖部分，并且碱基和糖的组合是核苷。当核苷包含键合至戊糖的3'-或5'-位置的磷酸酯基团时，它被称作核苷酸。可操作连接的核苷酸的序列在本文中通常称作"碱基序列"或"核苷酸序列并列，并且在本文中这样的形式表示，其左方向到右方向是在5'-末端至3'-末端的常规方向。
如本文所用，术语“载体”是指设计用于在不同宿主细胞之间转移的核酸构建体。“表达载体”是指能够在外来细胞中掺入并表达异源DNA片段的载体。许多原核和真核表达载体是可商购的。合适的表达载体的选择在本领域技术人员的知识范围内。
如本文所用，术语“宿主细胞”是指被目的基因侵入的细胞，其中目的基因可以以结合到载体(如病毒、染色体或质粒)上的形式侵入细胞，实现复制。
如本文所用，术语“染色体整合”是指通过同源重组将目的基因的多拷贝整合到充分限定的位点的整合方式。
Taq酶突变体的制备
本领域的普通技术人员可以使用的常规方法获得本发明的Taq酶基因序列，例如全人工合成或PCR法合成。一种优选的合成法为不对称PCR法。不对称PCR法是用不等量的一对引物，PCR扩增后产生大量的单链DNA(ssDNA)。这对引物分别称为非限制引物与限制性引物，其比例一般为50-100∶1。在PCR反应的最初10-15个循环中，其扩增产物主要是双链DNA，但当限制性引物(低浓度引物)消耗完后，非限制性引物(高浓度引物)引导的PCR就会产生大量的单链DNA。用于PCR的引物可根据本文所公开的本发明的序列信息适当地选择，并可用常规方法合成。可用常规方法如通过凝胶 电泳分离和纯化扩增的DNA/RNA片段。
本发明的Taq酶突变体可以通过常规的重组DNA技术进行表达或生产，包括步骤：
(1)用编码本发明蛋白的多核苷酸，或用含有该多核苷酸的重组表达载体转化或转导合适的宿主细胞；
(2)在合适的培养基中培养宿主细胞；
(3)从培养基或细胞中分离、纯化目的蛋白质，从而获得Taq酶突变体。
本领域的技术人员熟知的方法能用于构建含本发明Taq酶突变体的编码DNA序列和合适的转录/翻译控制信号的表达载体，优选市售的载体：pET28。这些方法包括体外重组DNA技术、DNA合成技术、体内重组技术等。所述的DNA序列可有效连接到表达载体中的适当启动子上，以指导mRNA合成。表达载体还包括翻译起始用的核糖体结合位点和转录终止子。此外，表达载体优选包含一个或多个选择性标记基因，以提供用于选择转化的宿主细胞的表型性状。
所述重组载体在5'到3'方向上包括：启动子，目的基因和终止子。如果需要，所述重组载体还可以包括以下元件：蛋白纯化标签；3'多聚核苷酸化信号；非翻译核酸序列；转运和靶向核酸序列；选择标记(抗生素抗性基因、荧光蛋白等)；增强子；或操作子。
用于制备重组载体的方法是本领域普通技术人员所熟知的。表达载体可以是细菌质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒或其他载体。总之，只要其能够在宿主体内复制和稳定，任何质粒和载体都可以被采用。
本领域普通技术人员可以采用熟知的方法构建含有本发明启动子和/或目的基因序列的载体。这些方法包括体外重组DNA技术、DNA合成技术、体内重组技术等。
本发明的表达载体，可以用于转化适当的宿主细胞，以使宿主转录目的RNA或表达目的蛋白质。宿主细胞可以是原核细胞，如大肠杆菌、谷氨酸棒杆菌、黄色短杆菌、链霉菌属、农杆菌：或是低等真核细胞，如酵母细胞；或是高等真核细胞，如植物细胞。本领域一般技术人员都清楚如何选择适当 的载体和宿主细胞。用重组DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。当宿主为原核生物(如大肠杆菌)时，可以用CaCl2法处理，也可用电穿孔法进行。当宿主是真核生物，可选用如下的DNA转染方法：磷酸钙共沉淀法，常规机械方法(如显微注射、电穿孔、脂质体包装等)。转化植物也可使用农杆菌转化或基因枪转化等方法，例如叶盘法、幼胚转化法、花芽浸泡法等。对于转化的植物细胞、组织或器官可以用常规方法再生成植株，从而获得转基因的植物。
为使本发明实施例的目的、技术方案和优点更加清楚，下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，下列实施例中未注明详细条件的实验方法，通常按照常规条件如美国Sambrook.J等著《分子克隆实验室指南》(黄培堂等译，北京：科学出版社，2002年)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。以下实施例中所用的实验材料和试剂如无特别说明均可从市售渠道获得。
除非另有指明，本文所用的技术和科学术语具有与本申请所属技术领域的普通技术人员通常理解的相同含义，需要注意的是，本文所用的术语仅为了描述具体实施方式，而非意图限制本申请的示例性实施方式。
实施例1、Taq酶突变体Taq-aCb和Taq-aCb Mut的制备
Taq酶突变体制备的步骤，
步骤1：以野生型Taq酶表达载体为模板，利用QuikChange Lightning多位点突变试剂盒制备Taq-aCb突变质粒，其中野生型表达载体由广州达安基因股份有限公司保存，载体为pET28a，N端添加His6标签。突变质粒均送检测序，测序结果表明突变载体构建成功。
步骤2：重组质粒转化大肠杆菌BL21(DE3)
取1μL质粒，在冰浴条件下，加入到30μL大肠杆菌感受态BL21(DE3)中，冰浴放置20分钟，42℃水浴热激45秒，立刻冰上放置2分钟，加入400μL不含抗生素的SOC培养基，37℃、220rpm振荡培养50分钟。取100μL菌液均匀涂布到含100μg/mL卡那抗性的LB平板上，37℃培养箱培养过夜。
步骤3：目的蛋白表达
挑取步骤2中的单克隆，无菌操作接种于含100μg/mL卡那抗性的TB培养基中，37℃220rpm振荡培养至OD600在0.6至0.8之间，IPTG进行诱导(终浓度为0.1mM)，分别放置于37℃和18℃振荡培养过夜，不加IPTG组作为对照，37℃培养3小时，每组实验重复一次。取样超声破碎进行SDS-PAGE鉴定，结果见图1。
根据图1，18℃条件下，在TB培养基中即可在上清液中大量可溶性表达，可溶性蛋白量占目的蛋白总量的90％以上，蛋白分子量约为100Kda。
步骤4：Taq-aCb的纯化
TB培养基摇瓶培养1.5L菌液，表达条件与3)目的蛋白表达一致。离心收集菌体，两种蛋白菌体湿重约为30g。取称取约4g菌体，加入35ml Lysis Buffer在冰上重悬。超声破碎后离心，20000rpm，4℃离心30分钟，取上清，0.22μm针式过滤器过滤得到上清液。上清液过Ni-柱亲和层析，0～60％Buffer B进行线性洗脱，取洗脱主峰洗脱液进离子交换洗脱，所用纯化柱为HisTrapTM Q-HP，用0-60％Buffer C进行线性洗脱。
电泳图如图2和图3所示。计算目的蛋白表达含量，Taq-aCb为1.46mg/mL，其中，所用溶液浓度如下所示：
Buffer B:50mM Tris,50mM NaCl,500mM Imidazole,5％Glycerol,pH8.5；
Lysis Buffer:50mM Tris,300mM NaCl,5％Glycerol,pH8.5；
Buffer C:100mM Tris,1M NaCl,10％Glycerol,pH8.5。
实施例2、Taq-aCb抗NaCl性能测试
配制不同的NaCl浓度梯度溶液(NaCl浓度梯度配置如下表1所示)，分别配制Buffer A和Buffer B(Buffer A和Buffer B的配方如下表1所示)，通过将Buffer A和Buffer B以不同比例混合形成NaCl浓度梯度溶液。
表1
Figure PCTCN2022100275-appb-000008
Figure PCTCN2022100275-appb-000009
以野生型Taq酶为阳性对照测定Taq-aCb对NaCl的抗性。
按照表2的配方配制PCR反应体系，其中RV和M4引物购自TaKaRa，5X Fast Taq Buffer、pUC19质粒由广州达安基因股份有限公司保存。每10μL的PCR反应体系加入的酶量为5U。以pUC19质粒为模板进行扩增实验，PCR产物进行2％琼脂糖凝胶电泳。
表2
试剂 BufferA BufferB
RV(10pmol) 1.2μL 1.2μL
M4(10pmol) 1.2μL 1.2μL
pUC19 plasmid 6μL 6μL
5X Fast Taq Buffer 12μL 12μL
野生型Taq/Taq 2C2/Taq 2C2 Mut 30U 30U
1M NaCl 6μL 0μL
ddH 2O 31.6μL 37.6μL
反应条件：95℃2分钟，(95℃15秒，44℃15秒，72℃1分钟)×30个循环，72℃1分钟，扩增产物电泳结果如图4。
如图4，1～11分别为0、10、20、30、40、50、60、70、80、90、100mM NaCl浓度下野生型Taq酶PCR产物泳道；12～22分别为0、10、20、30、40、50、60、70、80、90、100mM NaCl浓度下Taq-aCb的PCR产物泳道，可见Taq-aCb相比野生型具有很好的NaCl抗性。
继续提高PCR反应体系中的NaCl浓度至110mM-190mM，扩增产物电泳结果如图5所示。
如图5，1～9分别为110、120、130、140、150、160、170、180、190Mm NaCl浓度下Taq-aCb的PCR产物泳道，10为正对照。
根据图5，野生型Taq酶对NaCl的抗性为70mM,Taq-aCb对NaCl的抗 性达到了180mM，因此Taq酶突变体与野生型Taq酶相比其抗NaCl性能显著提高。
实施例3、Taq-aCb抗KCl性能测试
参考实施例2中配制不同的KCl浓度梯度溶液(表3)，将BufferA中NaCl替换成KCl。
表3
KCl浓度/mM 0 10 20 30 40 50 60 70 80 90 100
BufferA/μL 0 1 2 3 4 5 6 7 8 9 10
BufferB/μL 10 9 8 7 6 5 4 3 2 1 0
按照与实施例2相同的方法测试Taq-aCb对KCl的抗性，扩增产物电泳结果如图6。
如图6，1～11分别为50、80、100、150、160、170、180、190、200、250、300mM KCl浓度下野生型Taq酶PCR产物泳道，13～23分别为50、80、100、150、160、170、180、190、200、250、300mM KCl浓度下Taq-aCb的PCR产物泳道；12、24为正对照。
根据图6，野生型Taq酶对KCl的抗性为100mM，Taq突变酶Taq-aCb对NaCl的抗性均达到了200mM，因此Taq酶突变体与野生型Taq酶相比其抗KCl性能显著提高。
实施例4、Taq-aCb抗EDTA全血性能测试
参考实施例2中配制不同的全血浓度梯度溶液(表4)，将将BufferA中NaCl替换成EDTA全血，按加入PCR反应体系的血液体积计算体积分数。
表4
Figure PCTCN2022100275-appb-000010
按照与实施例2相同的方法测试Taq-aCb对EDTA全血的抗性，扩增产物电泳结果如图7。
如图7，1～10分别为5、10、15、20、25、30、35、40、45、50％(V/V)EDTA血液浓度下野生型Taq酶PCR产物泳道；11～20分别为5、10、15、20、25、30、35、40、45、50％(V/V)EDTA血液浓度下Taq-aCb的PCR产物泳道。
根据图7，野生型Taq酶对EDTA全血的抗性为5％(按加入PCR的血液体积占PCR体系总体积的百分比计算体积分数)，Taq突变酶Taq-aCb对EDTA全血抗性达到了50％，因此Taq酶突变体与野生型Taq酶相比其抗EDTA全血性能显著提高。
实施例5、Taq-aCb抗肝素全血性能测试
参考实施例2中配制不同的全血浓度梯度溶液(表5)，将BufferA中NaCl替换成肝素全血，按加入PCR反应体系的血液体积计算体积分数。
表5
Figure PCTCN2022100275-appb-000011
按照与实施例2相同的方法测试Taq-aCb对肝素全血的抗性，扩增产物电泳结果如图8。
如图8，1～10分别为5、10、15、20、25、30、35、40、45、50％(V/V)肝素血液浓度下野生型Taq酶PCR产物泳道；11～20分别为5、10、15、20、25、30、35、40、45、50％(V/V)肝素血液浓度下Taq-aCb的PCR产物泳 道。
根据图8，野生型Taq酶对肝素全血的抗性为0％(按加入PCR的血液体积占PCRT体系总体积的百分比计算体积分数)，Taq突变酶Taq-aCb对肝素全血抗性达到了35％，因此Taq酶突变体与野生型Taq酶相比其抗肝素全血性能显著提高。
实施例6、Taq-aCb 5’～3’外切酶活性测试
5’～3’外切酶活性采用荧光探针PCR法检测，以野生型Taq酶(ThermoFisher Scientific)为阳性对照，稀释至1U、2U、3U、4U、5U，Taq-aCb同样稀释至相同的活性浓度。配制反应体系如下表5(其中10X Taq Buffer购自TaRaKa)：
表5
Figure PCTCN2022100275-appb-000012
PCR反应条件：95℃10分钟，(95℃10秒，55℃30秒读取荧光)×40个循环，以反应循环为X轴，每个循环对应的荧光值(RFU)为Y轴，作RFU-循环曲线图。以已知的酶浓度为X轴，RFU-循环曲线图对应的初始斜率为Y轴拟合标准曲线，得到的线性回归方程的斜率可表示5`～3`外切酶活性大小。结果如图9。
根据图9，Taq-aCb的5’～3’外切酶活性为野生型Taq酶的66％，经突变后，Taq-aCb的5’～3’外切酶活性相比野生型有所下降。与野生型Taq酶基本一致。
本领域的普通技术人员可以理解，上述各实施方式是实现本发明的具体实施例，而在实际应用中，可以在形式上和细节上对其作各种改变，而不偏离本发明的精神和范围。

Claims (10)

  1. 一种Taq酶突变体，其特征在于，所述Taq酶突变体包括：
    与SEQ ID NO:2所示的氨基酸序列具有至少70％同一性的氨基酸序列；
    所述氨基酸序列在选自下组的一个或多个位点发生突变：P40、L125、G200、D335、G499、E634、F769或其组合。
  2. 根据权利要求1所述的Taq酶突变体，其特征在于，所述氨基酸序列发生选自下组的任一种或几种突变：P40W、L125I、G200M、D335V、G499K、E634G和F769I。
  3. 根据权利要求2所述的Taq酶突变体，其特征在于，所述氨基酸序列在选自下组的任四个或四个以上位点发生突变：P40W、L125I、G200M、D335V、G499K、E634G和F769I。
  4. 根据权利要求1至3任一项所述的Taq酶突变体，其特征在于，所述Taq酶突变体对氯化钠的抗性不低于70mM；
    和/或，所述Taq酶突变体对氯化钾的抗性不低于100mM。
  5. 根据权利要求1至3任一项所述的Taq酶突变体，其特征在于，所述Taq酶突变体对EDTA全血的抗性不低于5％；
    和/或，所述Taq酶突变体对肝素全血的抗性不低于1％。
  6. 一种核苷酸分子，其特征在于，所述核苷酸分子编码如权利要求1至5任一项所述的Taq酶突变体。
  7. 一种载体，其特征在于，所述载体含有权利要求6所述的核苷酸分子。
  8. 一种宿主细胞，其特征在于，所述宿主细胞包括如权利要求7所述的载体或染色体整合有如权利要求5所述的核酸分子。
  9. 一种试剂盒，其特征在于，所述试剂盒包括如权利要求求1至5任一项所述的Taq酶突变体。
  10. 一种制备如权利要求1所述的Taq酶突变体的方法，其特征在于，所述方法包括步骤：
    (i)在适合的条件下，培养权利要求8所述的宿主细胞，从而表达出所述的Taq酶突变体；和
    (ii)分离所述的Taq酶突变体。
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