Taq enzyme mutants, methods of making and using same

Abstract

The application discloses a Taq enzyme mutant, a preparation method and application thereof. In the application, the Taq enzyme mutant comprises a mutant of an amino acid sequence shown in SEQ ID NO: 2, wherein the amino acid sequence is mutated at the following group of sites: P40, L125, G200, A335, G499, E634 and F769. The Taq enzyme mutant provided by the application has high amplification activity, more amplification products can be obtained compared with wild-type Taq enzyme under the same number of PCR cycles, and is resistant to whole blood and high salt and has low 5'-3' exonuclease activity.

Description

Technical Field

[0001] The embodiments of this invention relate to the field of PCR detection, and particularly to Taq enzyme mutants, their preparation methods, and applications. Background Technology

[0002] Taq polymerase is a thermostable DNA polymerase derived from the thermostable bacterium *Thermus aquaticus*, with a molecular weight of 94 kDa. Its optimal reaction temperature is 75-80°C in the presence of magnesium ions, and its active half-life at 95°C is 40 minutes. It possesses 5'-3' exonuclease activity. Due to its thermostable properties, it is widely used in polymerase chain reaction (PCR) and is the enzyme of choice for nucleic acid amplification and detection. Modern molecular biology detection technologies place increasingly higher demands on the sensitivity, accuracy, and robustness of PCR reactions, and wild-type Taq polymerase cannot fully meet the needs of practical applications.

[0003] However, wild-type Taq enzymes have poor tolerance to extreme conditions (such as high salt or whole blood), which limits their applicability and reduces their accuracy and sensitivity, failing to fully meet the requirements of modern molecular biology detection techniques for PCR reactions. Furthermore, when using dye-based PCR amplification, the wild-type Taq enzyme in the system exhibits 5'–3' exonuclease activity, leading to a decrease in amplification efficiency during dye-based PCR amplification.

[0004] Therefore, it is particularly important to develop a Taq enzyme mutant with low 5'–3' exonuclease activity and tolerance to whole blood and high salt. Summary of the Invention

[0005] The purpose of this invention is to provide a Taq enzyme mutant.

[0006] Another object of the present invention is to provide a nucleotide molecule encoding the above-mentioned Taq enzyme mutant.

[0007] Another object of the present invention is to provide a carrier.

[0008] Another object of the present invention is to provide a host cell.

[0009] Another object of the present invention is to provide a method for preparing a Taq enzyme mutant.

[0010] Another object of the present invention is to provide a kit containing a Taq enzyme mutant.

[0011] To address the aforementioned technical problems, the first aspect of this invention provides a Taq enzyme mutant, the Taq enzyme mutant comprising:

[0012] The amino acid sequence shown in SEQ ID NO:2 has mutations at the following sites: P40, L125, G200, A335, G499, E634, and F769.

[0013] In some preferred embodiments, the amino acid sequence is mutated at seven sites in the following group: P40W, L125I, G200M, A335V, G499K, E634G, and F769I.

[0014] In some preferred embodiments, the Taq enzyme mutant exhibits a resistance to sodium chloride of not less than 70 mM, more preferably not less than 80 mM, more preferably not less than 90 mM, more preferably not less than 100 mM, more preferably not less than 130 mM, more preferably not less than 150 mM, and most preferably not less than 180 mM.

[0015] In some preferred embodiments, the Taq enzyme mutant exhibits resistance to potassium chloride of not less than 100 mM, more preferably not less than 110 mM, more preferably not less than 120 mM, more preferably not less than 130 mM, more preferably not less than 140 mM, more preferably not less than 150 mM, more preferably not less than 180 mM, more preferably not less than 190 mM, and most preferably not less than 200 mM.

[0016] In some preferred embodiments, the Taq enzyme mutant exhibits resistance to EDTA whole blood of 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%, and most preferably not less than 50%.

[0017] In some preferred embodiments, the Taq enzyme mutant exhibits resistance to heparinized whole blood of not less than 1%, 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%, and most preferably not less than 35%. SEQ ID NO:1 (wild-type Taq enzyme DNA sequence is as follows):

[0018]

[0019] SEQ ID NO:2 (Wild-type Taq enzyme amino acid sequence is as follows):

[0020]

[0021] A second aspect of the invention also provides a nucleotide molecule that encodes a Taq enzyme mutant of the mutation described in the first aspect of the invention.

[0022] A third aspect of the present invention also provides a carrier containing the nucleotide molecules described in the second aspect of the present invention.

[0023] A fourth aspect of the invention also provides a host cell containing the nucleotide molecules described in the second aspect of the invention or chromosomes integrated with the nucleotide molecules described in the second aspect of the invention.

[0024] In some preferred embodiments, the host cell is a prokaryotic cell or a eukaryotic cell.

[0025] In some preferred embodiments, the prokaryotic cell is Escherichia coli.

[0026] In some preferred embodiments, the eukaryotic cells are yeast cells.

[0027] A fifth aspect of the present invention also provides a kit containing the Taq enzyme mutant described in the first aspect of the present invention.

[0028] 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:

[0029] (i) Under suitable conditions, the host cells described in the fourth aspect of the invention are cultured to express the Taq enzyme mutant; and

[0030] (ii) Isolate the Taq enzyme mutant.

[0031] The seventh aspect of the present invention also provides the use of the kit described in the fifth aspect of the present invention for DNA sequencing, DNA labeling, primer extension, amplification, etc.

[0032] Compared with the prior art, the present invention has at least the following advantages:

[0033] (1) The Taq enzyme mutant provided by the present invention has high amplification activity and can obtain more amplification products compared with wild-type Taq enzyme under the same number of PCR cycles.

[0034] (2) The Taq enzyme mutant provided by this invention is resistant to both whole blood and high salt;

[0035] (3) The Taq enzyme mutants provided by this invention have low 5' to 3' exonuclease activity and are suitable for dye-based PCR systems.

[0036] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0037] One or more embodiments are illustrated by way of example with reference to the accompanying drawings, and these illustrative descriptions do not constitute a limitation on the embodiments.

[0038] Figure 1 This is a diagram showing the SDS-PAGE identification results of the target protein according to an embodiment of the present invention;

[0039] Figure 2 This is an electrophoresis image of Taq-aCb protein purified by N1 column according to an embodiment of the present invention;

[0040] Figure 3 This is an electrophoresis image of Taq-aCb protein purified by Q column according to an embodiment of the present invention;

[0041] Figure 4 This is a diagram showing the experimental results of wild-type Taq enzyme and Taq-aCb resistance to NaCl according to embodiments of the present invention;

[0042] Figure 5 This is a diagram showing the experimental results of Taq-aCb resistance to NaCl according to an embodiment of the present invention;

[0043] Figure 6 This is a diagram showing the experimental results of wild-type Taq enzyme and Taq-aCb resistance to KCl according to embodiments of the present invention;

[0044] Figure 7 This is a diagram showing the results of blood experiments on wild-type Taq enzyme and Taq-aCb anti-EDTA according to embodiments of the present invention;

[0045] Figure 8 This is a graph showing the results of blood experiments on wild-type Taq enzyme and Taq-aCb antiheparin according to embodiments of the present invention;

[0046] Figure 9 This is the RFU-cycle curve diagram according to Embodiment 8 of the present invention. Detailed Implementation

[0047] Wild-type Taq enzymes are difficult to amplify under extreme conditions, exhibiting very low polymerization activity under conditions such as high salinity and whole blood. While existing techniques have attempted to mutate wild-type Taq enzymes to enhance their tolerance to extreme conditions, the results have been minimal. Through extensive and in-depth research, the inventors of this invention have used site-directed mutagenesis to screen for high-performance Taq enzyme mutants with good polymerization activity and excellent tolerance to high salinity and whole blood, making them suitable for clinical use. Furthermore, the Taq enzyme mutants screened in this invention exhibit low 5'–3' exonuclease activity, making them highly suitable for dye-based PCR systems.

[0048] Some preferred embodiments of the present invention provide a Taq enzyme mutant, the Taq enzyme mutant comprising:

[0049] An amino acid sequence having at least 70% identity with the amino acid sequence shown in SEQ ID NO:2, wherein the amino acid sequence is mutated at the following sites: P40, L125, G200, A335, G499, E634 and F769.

[0050] In some preferred embodiments, the Taq enzyme mutant exhibits a resistance to sodium chloride of not less than 70 mM, more preferably not less than 80 mM, more preferably not less than 90 mM, more preferably not less than 100 mM, more preferably not less than 130 mM, more preferably not less than 150 mM, and most preferably not less than 180 mM.

[0051] In some preferred embodiments, the Taq enzyme mutant exhibits resistance to potassium chloride of not less than 100 mM, more preferably not less than 110 mM, more preferably not less than 120 mM, more preferably not less than 130 mM, more preferably not less than 140 mM, more preferably not less than 150 mM, more preferably not less than 180 mM, more preferably not less than 190 mM, and most preferably not less than 200 mM.

[0052] In some preferred embodiments, the Taq enzyme mutant exhibits resistance to EDTA whole blood of not less than 5% (here, the percentage refers to the percentage of plasma volume to 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%, and most preferably not less than 50%.

[0053] In some preferred embodiments, the Taq enzyme mutant exhibits resistance to heparinized whole blood of not less than 1% (here, the percentage refers to the percentage of plasma volume to 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%, and most preferably not less than 35%.

[0054] SEQ ID NO:1 (Wild-type Taq enzyme DNA sequence is as follows):

[0055]

[0056]

[0057] SEQ ID NO:2 (Wild-type Taq enzyme amino acid sequence is as follows):

[0058]

[0059] Some preferred embodiments of the present invention provide a nucleotide molecule that encodes a Taq enzyme mutant of the mutation described in the first aspect of the present invention.

[0060] Some preferred embodiments of the present invention provide a carrier containing the nucleotide molecules described in the second aspect of the present invention.

[0061] Some preferred embodiments of the present invention provide a host cell containing the nucleotide molecules described in the second aspect of the present invention or chromosomes integrated with the nucleotide molecules described in the second aspect of the present invention.

[0062] In some preferred embodiments, the host cell is a prokaryotic cell or a eukaryotic cell.

[0063] In some preferred embodiments, the prokaryotic cell is Escherichia coli.

[0064] In some preferred embodiments, the eukaryotic cells are yeast cells.

[0065] Some preferred embodiments of the present invention provide a kit containing the Taq enzyme mutant described in the first aspect of the present invention.

[0066] 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 the steps of:

[0067] (i) Under suitable conditions, the host cells described in the fourth aspect of the invention are cultured to express the Taq enzyme mutant; and

[0068] (ii) Isolate the Taq enzyme mutant.

[0069] Some preferred embodiments of the present invention provide the use of the kit described in the fifth aspect of the present invention for DNA sequencing, DNA labeling, primer extension, amplification, etc.

[0070] the term

[0071] As used herein, the term "amino acid" in its broadest sense refers to any compound and / or substance that can be incorporated into a polypeptide chain. In some embodiments, an amino acid has the universal structure H₂N-C(H)(R)-COOH. In some embodiments, an amino acid is a naturally occurring amino acid. In some embodiments, an amino acid is a synthetic amino acid; in some embodiments, an amino acid is a D-amino acid; in some embodiments, an 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 synthetically prepared or obtained from a natural source. As used herein, "synthetic amino acid" includes chemically modified amino acids, including but not limited to salts, amino acid derivatives (such as amides), and / or substitutes. Amino acids, including carboxyl and / or amino-terminal amino acids in peptides, can be modified by methylation, amidation, acetylation, and / or substitution with other chemical substances without adversely affecting their activity. Amino acids may have disulfide bonds. The term "amino acid" is used interchangeably with "amino acid residue" and can refer to free amino acids and / or amino acid residues of peptides. Whether the term refers to free amino acids or peptide residues will be self-evident from the context in which it is used. It should be noted that all amino acid residue sequences herein are represented by formulas with a conventional orientation from the amino terminus to the carboxyl terminus.

[0072] As used herein, the term "mutation" refers to an alteration introduced into a parental sequence, including but not limited to substitution, insertion, and deletion (including truncation). The consequences of a mutation include, but are not limited to, the emergence of novel characteristics, properties, functions, phenotypes, or traits not found in proteins encoded by the parental sequence. The term "mutant" refers to a modified protein that exhibits altered characteristics compared to a parental protein.

[0073] As used herein, the term “% homology” is used interchangeably with the term “% identity” and refers to the level of nucleic acid or amino acid sequence identity between nucleic acid sequences encoding any of the polypeptides of the present invention or amino acid sequences of the polypeptides of the present invention when aligned using a sequence alignment procedure.

[0074] As used herein, the term "nucleotide" refers to a monomeric unit of DNA or RNA consisting of a sugar moiety (pentose), a phosphate ester, and a nitrogenous heterocyclic base. The base is linked to the sugar moiety via a glycosidic carbon (the 1' carbon of the pentose), and the combination of base and sugar is a nucleoside. When a nucleoside contains a phosphate ester group bonded to the 3' or 5' position of the pentose, it is called a nucleotide. The sequence of operable linked nucleotides is generally referred to herein as a "base sequence" or "nucleotide sequence in tandem," and in this form, the left-to-right direction is the conventional 5'-to-3'-end orientation.

[0075] As used herein, the term "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 fragment of heterologous DNA in foreign cells. Many prokaryotic and eukaryotic expression vectors are commercially available. The selection of a suitable expression vector is within the knowledge of those skilled in the art.

[0076] As used in this article, the term "host cell" refers to a cell invaded by a target gene, which can invade the cell in the form of a vector (such as a virus, chromosome, or plasmid) to replicate.

[0077] As used in this article, the term "chromosomal integration" refers to the integration of multiple copies of a target gene into a sufficiently defined site through homologous recombination.

[0078] Preparation of Taq enzyme mutants

[0079] The Taq enzyme gene sequence of the present invention can be obtained by conventional methods used by those skilled in the art, such as fully artificial synthesis or PCR synthesis. A preferred synthesis method is asymmetric PCR. Asymmetric PCR uses unequal amounts of a pair of primers, resulting in a large amount of single-stranded DNA (ssDNA) after PCR amplification. This pair of primers is referred to as the non-restriction primer and the restriction 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 after the restriction primer (low concentration primer) is consumed, the PCR guided by the non-restriction 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 using conventional methods. The amplified DNA / RNA fragments can be separated and purified using conventional methods such as gel electrophoresis.

[0080] The Taq enzyme mutant of the present invention can be expressed or produced using conventional recombinant DNA technology, including the following steps:

[0081] (1) Transform or transduce suitable host cells using a polynucleotide encoding the protein of the present invention, or using a recombinant expression vector containing the polynucleotide;

[0082] (2) Culture the host cells in a suitable culture medium;

[0083] (3) Isolate and purify the target protein from the culture medium or cells to obtain the Taq enzyme mutant.

[0084] Methods well known to those skilled in the art can be used to construct expression vectors containing the coding DNA sequence of the Taq enzyme mutant of the present invention and suitable transcription / translation control signals, preferably commercially available vectors such as pET28. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. The DNA sequence can be efficiently ligated to an appropriate promoter in the expression vector to guide mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. Furthermore, the expression vector preferably contains one or more selective marker genes to provide phenotypic traits for selecting transformed host cells.

[0085] The recombinant vector includes, in the 5' to 3' direction: a promoter, a target gene, and a terminator. If desired, the recombinant vector may also include the following elements: a protein purification tag; a 3' polynucleotide signal; a non-translated nucleic acid sequence; a transport and targeting nucleic acid sequence; a selection marker (antibiotic resistance gene, fluorescent protein, etc.); an enhancer; or an operator.

[0086] The methods used to prepare recombinant vectors are well known to those skilled in the art. Expression vectors can be bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses, or other vectors. In short, any plasmid and vector can be used as long as it can replicate and remain stable within the host.

[0087] Those skilled in the art can construct vectors containing the promoter and / or target gene sequence of this invention using well-known methods. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc.

[0088] The expression vector of this invention can be used to transform suitable host cells to enable the host to transcribe target RNA or express 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. Those skilled in the art will understand how to select appropriate vectors and host cells. Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote (such as *E. coli*), it can be treated with CaCl2 or electroporation. When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate co-precipitation, conventional mechanical methods (such as microinjection, electroporation, liposome packaging, etc.). Transformation of plants can also be performed using methods such as *Agrobacterium* transformation or gene gun transformation, for example, leaf disc transformation, embryo transformation, flower bud soaking, etc. Transformed plant cells, tissues, or organs can be regenerated into plants using conventional methods to obtain transgenic plants.

[0089] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments without specific conditions are generally performed according to conventional conditions as described in *Molecular Cloning Laboratory Guide* by Sambrook J. et al. (translated by Huang Peitang et al., Beijing: Science Press, 2002), or according to the manufacturer's recommendations. Unless otherwise stated, percentages and parts are weight percentages and parts by weight. Unless otherwise specified, the experimental materials and reagents used in the following embodiments are commercially available.

[0090] Unless otherwise specified, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. It should be noted that the terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the exemplary embodiments of this application.

[0091] Example 1: Preparation of Taq enzyme mutants Taq-aCb and Taq-aCb Mut

[0092] Steps for preparing Taq enzyme mutants

[0093] Step 1: Using the wild-type Taq enzyme expression vector as a template, the Taq-aCb mutant plasmid was prepared using the QuikChange Lightning multisite mutation kit. The wild-type expression vector was preserved by Guangzhou DaAn Gene Co., Ltd., and the vector was pET28a with a His6 tag added to the N-terminus. All mutant plasmids were sequenced, and the sequencing results showed that the mutant vector was successfully constructed.

[0094] Step 2: Transform recombinant plasmid into E. coli BL21(DE3)

[0095] Take 1 μL of plasmid and add it to 30 μL of competent E. coli BL21(DE3) cells under ice bath conditions. Incubate on ice for 20 minutes, then heat shock at 42°C for 45 seconds, immediately place on ice for 2 minutes, add 400 μL of antibiotic-free SOC medium, and incubate at 37°C with shaking at 220 rpm for 50 minutes. Spread 100 μL of the bacterial culture evenly onto LB agar plates containing 100 μg / mL kanamycin and incubate overnight at 37°C.

[0096] Step 3: Expression of the target protein

[0097] Single clones from step 2 were aseptically inoculated into TB medium containing 100 μg / mL kanamycin resistance and cultured at 37°C with shaking at 220 rpm until the OD600 was between 0.6 and 0.8. Induction with IPTG (final concentration 0.1 mM) was then performed, followed by overnight incubation at 37°C and 18°C ​​with shaking. A control group without IPTG was incubated at 37°C for 3 hours. Each experiment was repeated once. Samples were ultrasonically disrupted and analyzed by SDS-PAGE. Results are shown below. Figure 1 .

[0098] according to Figure 1 At 18℃, it can be expressed in large quantities in the supernatant of TB medium, with the amount of soluble protein accounting for more than 90% of the total amount of the target protein and the molecular weight of the protein being approximately 100 kDa.

[0099] Step 4: Purification of Taq-aCb

[0100] 1.5 L of bacterial culture was cultured in shake flasks in TB medium, with expression conditions consistent with those for the target protein in step 3). The bacterial cells were collected by centrifugation; the wet weight of the two protein cells was approximately 30 g. Approximately 4 g of the bacterial cells were weighed and resuspended in 35 ml of Lysis Buffer on ice. After sonication and centrifugation at 20,000 rpm, 4°C for 30 minutes, the supernatant was collected and filtered through a 0.22 μm syringe filter. The supernatant was subjected to Ni-column affinity chromatography, with linear elution using 0–60% Buffer B. The eluent containing the main elution peak was then subjected to ion exchange chromatography using a HisTrap™ Q-HP column, with linear elution using 0–60% Buffer C.

[0101] Electrophoresis image as shown Figure 2 and Figure 3 As shown. The expression level of the target protein, Taq-aCb, was calculated to be 1.46 mg / mL. The concentrations of the solutions used are shown below:

[0102] Buffer B: 50mM Tris, 50mM NaCl, 500mM Imidazole, 5% Glycerol, pH8.5;

[0103] Lysis Buffer: 50mM Tris, 300mM NaCl, 5% Glycerol, pH8.5;

[0104] Buffer C: 100mM Tris, 1M NaCl, 10% Glycerol, pH8.5.

[0105] Example 2: Taq-aCb NaCl resistance test

[0106] Prepare different NaCl concentration gradient solutions (the NaCl concentration gradient configurations are shown in Table 1 below), and prepare Buffer A and Buffer B respectively (the formulations of Buffer A and Buffer B are shown in Table 1 below). Mix Buffer A and Buffer B in different proportions to form NaCl concentration gradient solutions.

[0107] Table 1

[0108]

[0109] The resistance of Taq-aCb to NaCl was determined using wild-type Taq enzyme as a positive control.

[0110] The PCR reaction system was prepared according to the formula in Table 2. RV and M4 primers were purchased from TaKaRa, and 5X Fast Taq Buffer and pUC19 plasmid were stored by Guangzhou DaAn Gene Co., Ltd. 5 U of enzyme was added to every 10 μL of the PCR reaction system. Amplification experiments were performed using the pUC19 plasmid as a template, and the PCR products were subjected to 2% agarose gel electrophoresis.

[0111] Table 2

[0112] reagents BufferA BufferB RV (10 pmol) 1.2μL 1.2μL M4 (10 pmol) 1.2μL 1.2μL pUC19plasmid 6μL 6μL 5X Fast Taq Buffer 12μL 12μL Wild type Taq / Taq 2C2 / Taq 2C2 Mut 30U 30U 1M NaCl 6μL 0μL <![CDATA[ddH2O]]> 31.6μL 37.6μL

[0113] Reaction conditions: 95℃ for 2 minutes, (95℃ for 15 seconds, 44℃ for 15 seconds, 72℃ for 1 minute) × 30 cycles, 72℃ for 1 minute. Electrophoresis results of the amplified products are as follows. Figure 4 .

[0114] like Figure 4Lanes 1-11 represent the PCR product lanes of wild-type Taq enzyme at NaCl concentrations of 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 mM; lanes 12-22 represent the PCR product lanes of Taq-aCb at NaCl concentrations of 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 mM. It can be seen that Taq-aCb has good NaCl resistance compared to the wild type.

[0115] Continue increasing the NaCl concentration in the PCR reaction system to 110mM-190mM. The electrophoresis results of the amplified products are as follows: Figure 5 As shown.

[0116] like Figure 5 Lanes 1-9 represent the PCR products of Taq-aCb at NaCl concentrations of 110, 120, 130, 140, 150, 160, 170, 180, and 190 Mm, respectively, while lane 10 is the positive control.

[0117] according to Figure 5 The wild-type Taq enzyme showed a resistance of 70 mM to NaCl, while the Taq-aCb enzyme showed a resistance of 180 mM to NaCl. Therefore, the Taq enzyme mutant exhibited significantly improved resistance to NaCl compared to the wild-type Taq enzyme.

[0118] Example 3: Taq-aCb KCl resistance test

[0119] Different KCl concentration gradient solutions were prepared as described in Example 2 (Table 3), with NaCl in Buffer A replaced by KCl.

[0120] Table 3

[0121] KCl concentration / mM 0 10 20 30 40 50 60 70 80 90 100 Buffer A / μL 0 1 2 3 4 5 6 7 8 9 10 Buffer B / μL 10 9 8 7 6 5 4 3 2 1 0

[0122] The resistance of Taq-aCb to KCl was tested using the same method as in Example 2. The electrophoresis results of the amplified products are as follows: Figure 6 .

[0123] like Figure 6 Lanes 1-11 represent the PCR product lanes of wild-type Taq enzyme at concentrations of 50, 80, 100, 150, 160, 170, 180, 190, 200, 250, and 300 mM KCl, respectively; lanes 13-23 represent the PCR product lanes of Taq-aCb at concentrations of 50, 80, 100, 150, 160, 170, 180, 190, 200, 250, and 300 mM KCl, respectively; lanes 12 and 24 are positive controls.

[0124] according to Figure 6The wild-type Taq enzyme showed resistance to KCl of 100 mM, while the Taq mutant Taq-aCb showed resistance to NaCl of 200 mM. Therefore, the Taq enzyme mutant showed significantly improved resistance to KCl compared to the wild-type Taq enzyme.

[0125] Example 4: Taq-aCb anti-EDTA whole blood performance test

[0126] Different whole blood concentration gradient solutions were prepared as described in Example 2 (Table 4). NaCl in Buffer A was replaced with EDTA whole blood, and the volume fraction was calculated based on the volume of blood added to the PCR reaction system.

[0127] Table 4

[0128]

[0129]

[0130] The resistance of Taq-aCb to EDTA whole blood was tested using the same method as in Example 2. The electrophoresis results of the amplified products are as follows: Figure 7 .

[0131] like Figure 7 Lanes 1-10 represent the PCR product lanes of wild-type Taq enzyme at blood concentrations of 5, 10, 15, 20, 25, 30, 35, 40, 45, and 50% (V / V) EDTA, respectively; lanes 11-20 represent the PCR product lanes of Taq-aCb at blood concentrations of 5, 10, 15, 20, 25, 30, 35, 40, 45, and 50% (V / V) EDTA, respectively.

[0132] according to Figure 7 The wild-type Taq enzyme showed a 5% resistance to EDTA in whole blood (calculated as a percentage of the total volume of blood added to the PCR system), while the Taq mutant Taq-aCb showed a 50% resistance to EDTA in whole blood. Therefore, the Taq enzyme mutant showed a significantly improved resistance to EDTA in whole blood compared to the wild-type Taq enzyme.

[0133] Example 5: Taq-aCb antiheparin whole blood performance test

[0134] Different whole blood concentration gradient solutions were prepared as described in Example 2 (Table 5). NaCl in Buffer A was replaced with heparinized whole blood, and the volume fraction was calculated based on the volume of blood added to the PCR reaction system.

[0135] Table 5

[0136]

[0137] The resistance of Taq-aCb to heparin whole blood was tested using the same method as in Example 2. The electrophoresis results of the amplified products are as follows: Figure 8 .

[0138] like Figure 8 Lanes 1-10 represent the PCR product lanes of wild-type Taq enzyme at blood concentrations of 5, 10, 15, 20, 25, 30, 35, 40, 45, and 50% (V / V) heparin, respectively; lanes 11-20 represent the PCR product lanes of Taq-aCb at blood concentrations of 5, 10, 15, 20, 25, 30, 35, 40, 45, and 50% (V / V) heparin, respectively.

[0139] according to Figure 8 The wild-type Taq enzyme showed 0% resistance to heparin in whole blood (calculated as the volume fraction of blood added to the PCR system relative to the total volume of the PCR-T system), while the Taq mutant Taq-aCb showed 35% resistance to heparin in whole blood. Therefore, the Taq enzyme mutant showed significantly improved resistance to heparin in whole blood compared to the wild-type Taq enzyme.

[0140] Example 6: Taq-aCb 5'-3' exonuclease activity test

[0141] The activities of 5'–3' exonucleases were detected using fluorescent probe PCR, with wild-type Taq enzyme (Thermo Fisher Scientific) as a positive control, diluted to 1 U, 2 U, 3 U, 4 U, and 5 U. Taq-aCb was also diluted to the same activity concentration. The reaction system is shown in Table 5 below (10X Taq Buffer was purchased from TaRaKa):

[0142] Table 5

[0143] reagents Mixing amount 10X Taq Buffer 2.5ul 25mM MgCl2 4.5ul 2.5mM dNTPs 0.2ul Probe2 (10 pmol) 0.5ul DNA polymerase test 2 (10 pmol) 0.25μl ddH2O 16.05μL

[0144] PCR reaction conditions: 95℃ for 10 minutes (95℃ for 10 seconds, 55℃ for 30 seconds for fluorescence reading) × 40 cycles. An RFU-cycle curve was plotted with the reaction cycle as the X-axis and the corresponding fluorescence value (RFU) for each cycle as the Y-axis. A standard curve was fitted with the known enzyme concentration as the X-axis and the initial slope of the RFU-cycle curve as the Y-axis. The slope of the resulting linear regression equation represents the activity of the 5'–3' exonuclease. Results are as follows: Figure 9 .

[0145] according to Figure 9 The 5'-3' exonuclease activity of Taq-aCb is 66% of that of wild-type Taq enzyme. After mutation, the 5'-3' exonuclease activity of Taq-aCb is lower than that of wild-type. It is basically consistent with wild-type Taq enzyme.

[0146] Those skilled in the art will understand that the above embodiments are specific examples of implementing the present invention, and in practical applications, various changes in form and detail may be made without departing from the spirit and scope of the present invention.

Claims

1. A mutant of Taq enzyme, characterized in that, The Taq enzyme mutant is only mutated at P40W, L125I, G200M, A335V, G499K, E634G and F769I based on the amino acid sequence shown in SEQ ID NO:

2.

2. A nucleotide molecule, characterized in that, The nucleotide molecule encodes the Taq enzyme mutant as claimed in claim 1.

3. An expression vector, characterized by, The expression vector contains the nucleotide molecule as claimed in claim 2.

4. A host cell, characterized in that, The host cell contains the expression vector as claimed in claim 3 or the chromosome of the host cell is integrated with the nucleotide molecule as claimed in claim 2.

5. A kit characterized in that, The kit comprises the Taq enzyme mutant as claimed in claim 1.

6. A method of preparing the mutant Taq enzyme of claim 1, wherein, The method comprises the following steps: (i) culturing the host cell as claimed in claim 4 under suitable conditions so as to express the Taq enzyme mutant; and (ii) isolating the Taq enzyme mutant.

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