WO2025129707A1 - Dna聚合酶突变体及其应用 - Google Patents

Dna聚合酶突变体及其应用 Download PDF

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WO2025129707A1
WO2025129707A1 PCT/CN2023/141294 CN2023141294W WO2025129707A1 WO 2025129707 A1 WO2025129707 A1 WO 2025129707A1 CN 2023141294 W CN2023141294 W CN 2023141294W WO 2025129707 A1 WO2025129707 A1 WO 2025129707A1
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amino acid
dna polymerase
pcr
polymerase mutant
dna
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French (fr)
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丁孙家
张晓红
谢庆庆
郑越
董宇亮
章文蔚
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BGI Shenzhen Co Ltd
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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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]

Definitions

  • the present invention relates to the field of DNA polymerase, in particular to a DNA polymerase mutant and application thereof.
  • PCR technology is an important cornerstone of modern molecular biology, and Taq DNA polymerase is the classic main force of PCR technology.
  • Taq DNA polymerase belongs to the A family DNA polymerase, has good thermal stability, and is widely used in fields related to PCR technology.
  • Taq polymerase lacks 3'-5' proofreading activity, but has the characteristics of 5'-3' exonuclease activity and terminal addition of A. It is widely used in the field of molecular biology, such as direct PCR, allele detection, Sanger sequencing, fluorescent quantitative PCR, TA cloning, etc.
  • PCR is one of the fastest growing technologies in the molecular biology application market. New applications of PCR and new technologies of PCR are being developed and used in research and diagnosis.
  • Taq DNA polymerase With the widespread application of PCR technology, the study of the properties of Taq DNA polymerase is becoming increasingly important. However, Taq DNA polymerase also has some shortcomings. The amplification activity and inhibition resistance still need to be further improved. There is still a need for further improved DNA polymerase variants with various performances to meet the needs of DNA amplification, synthesis, detection, sequencing and other important technologies.
  • the main purpose of the present invention is to provide a DNA polymerase mutant and its application to solve the problem of low polymerization activity of wild-type DNA polymerase in the prior art.
  • a DNA polymerase mutant which has DNA polymerase activity and includes an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% homology with the amino acid sequence shown in SEQ ID NO: 1.
  • the DNA polymerase mutant has an amino acid substitution at at least one of the following sites relative to the amino acid sequence shown in SEQ ID NO: 1: D488, S577, S543, E388, E400, E397, T544, L549, V586, L670, L678, T664 and G59.
  • the amino acid substitution at the D488 site includes D488H, D488R or D488K; the amino acid substitution at the S543 site includes S543T; the amino acid substitution at the S577 site includes S577T; the amino acid substitution at the E388 site includes E388K; the amino acid substitution at the E400 site includes E400R; the amino acid substitution at the E397 site includes E397Q; the amino acid substitution at the T544 site includes T544Y, T544D, T544K, T544S ...
  • amino acid substitution at L549 site includes L549F, L549I, L549D or L549K; amino acid substitution at V586 site includes V586F; amino acid substitution at L670 site includes L670F; amino acid substitution at L678 site includes L678Y, L678W, L678I, L678F, L678K or L678D; amino acid substitution at T664 site includes T664F; amino acid substitution at G59 site includes G59W.
  • the mutation of the DNA polymerase mutant includes any one of the following amino acid mutation combinations: D488H, D488R, D488K, S577T, G59W, E388K, E397Q, E400R, S543T, T544Y, T544D, T544K, T544S, T544W, T544F, L549F, L549I, L549D, L549K, V586F, T664F, L670F, L678Y, L678K, L678D, L678I, L678F, G59W+E388K, G59W+E400R, G59W+D488K, G59W+S543T, G59W+S577T, E388K+E397Q, E388K+E400R, E388K+D488K, E388K+S543T, E38 8K+T544Y, E388K+L5
  • the mutation of the DNA polymerase mutant includes any one of the following amino acid mutation combinations: G59W, G59W+D488K, S543T, S577T, E400R, L549F, T664F, G59W+D488K+S543T, G59W+D488K+L549F, G59W+S577T, G59W+E388K, S543T+T664F, L678I, E388K+L549F, G59W+D488K, G59W+D488K+L549F, G59W+S577T or G59W+E388K, the PCR inhibitor resistance of the DNA polymerase mutant is better than the PCR inhibitor resistance of the amino acid sequence shown in SEQ ID NO:1.
  • a DNA molecule is provided, wherein the DNA molecule comprises a polynucleotide encoding the above DNA polymerase mutant.
  • a recombinant vector which comprises the above DNA molecule.
  • a host cell wherein the host cell contains the above DNA molecule or the above recombinant vector.
  • the host cell includes a prokaryotic cell or a eukaryotic cell; preferably, the prokaryotic cell includes Escherichia coli.
  • a PCR kit comprising the above DNA polymerase mutant.
  • the PCR kit also includes any one or more of the following components: 1) a buffer for providing a PCR amplification environment; 2) PCR primers; 3) a reagent for extracting target DNA.
  • a PCR method which comprises: using the above DNA polymerase mutant or the above PCR kit to perform PCR amplification on the target DNA.
  • PCR includes multiple PCR amplification; preferably, the PCR system contains PCR inhibitors; more preferably, the PCR inhibitors include one or more of heme, humic acid, tannic acid, EDTA, heparin, phenol, sodium dodecyl sulfate, hemin, urea, plant polysaccharides, bile salts, polystyrene, polypropylene, protease, bilirubin, bromophenol blue, calcium ions and iron ions.
  • the PCR inhibitors include one or more of heme, humic acid, tannic acid, EDTA, heparin, phenol, sodium dodecyl sulfate, hemin, urea, plant polysaccharides, bile salts, polystyrene, polypropylene, protease, bilirubin, bromophenol blue, calcium ions and iron ions.
  • the activity of DNA polymerase is one of the key factors to improve the efficiency of PCR amplification.
  • the above-mentioned DNA polymerase mutant has higher activity than the wild-type Taq DNA polymerase.
  • FIG. 1 shows a graph showing the agarose gel electrophoresis results according to Example 4 of the present invention.
  • FIG. 2 shows the agarose gel electrophoresis result according to Example 4 of the present invention.
  • FIG. 3 shows a graph showing the agarose gel electrophoresis results according to Example 4 of the present invention.
  • FIG. 4 shows a graph showing the agarose gel electrophoresis results according to Example 5 of the present invention.
  • DNA polymerase As mentioned in the background technology, with the widespread application of PCR technology, the study of the properties of DNA polymerase is becoming increasingly important. However, DNA polymerase also has some shortcomings, and the polymerization activity is difficult to meet the needs of practical applications, and it is urgent to improve DNA polymerase, so as to increase the DNA polymerization rate.
  • the polymerization rate, or the number of nucleotides incorporated per unit time is affected by many parameters, including the binding affinity of the substrate (dNTP, primer) and the catalytic efficiency (nucleotide transfer rate, pyrophosphate release and translocation step).
  • the inventor attempts to develop a novel DNA polymerase mutant with enhanced polymerization activity, and thus proposes a series of protection schemes of the present application.
  • a DNA polymerase mutant which has DNA polymerase activity and includes an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% homology with the amino acid sequence shown in SEQ ID NO: 1.
  • the above-mentioned DNA polymerase mutant has an amino acid replacement at at least one of the following sites relative to the amino acid sequence shown in SEQ ID NO: 1: D488, S577, S543, E388, E400, E397, T544, L549, V586, L670, L678, T664 and G59.
  • the amino acid substitution at the D488 site includes D488H, D488R or D488K; the amino acid substitution at the S543 site includes S543T; the amino acid substitution at the S577 site includes S577T; the amino acid substitution at the E388 site includes E388K; the amino acid substitution at the E400 site includes E400R; the amino acid substitution at the E397 site includes E397Q; the amino acid substitution at the T544 site includes T544Y, T544D, T544K, T544S, T544W or T544F;
  • the amino acid substitution at site 549 includes L549F, L549I, L549D or L549K; the amino acid substitution at site V586 includes V586F; the amino acid substitution at site L670 includes L670F; the amino acid substitution at site L678 includes L678Y, L678W, L678I, L678F, L678K or L678D; the amino acid substitution at
  • the mutation (replacement) of the DNA polymerase mutant includes any one of the following amino acid mutation combinations:
  • the mutation of the DNA polymerase mutant comprises any one of the following amino acid mutation combinations: G59W, D488K, G59W+D488K, S543T, E388K, E400R, E397Q, L549F, T664F, G59W+D488K+L549F, G59W+S577T, G59W+E388K, G59W+E400R, E400R+D488K, D488K+L549F, E388K+E397Q, E388K+T664F, S543T+T664F, E400R+T664F, L678I or E388K+L549F.
  • the improvement in the polymerase activity of the above-mentioned DNA polymerase mutant can produce more double-stranded DNA products per unit time compared to the wild-type DNA polymerase (SEQ ID NO: 1).
  • the mutation of the DNA polymerase mutant includes any one of the following amino acid mutation combinations: G59W+D488K, S543T, E400R, T544Y, L549F, G59W+D488K+L549F, G59W+S543T, G59W+S577T or G59W+E400R.
  • Multiplex PCR refers to a technique that simultaneously amplifies multiple targets through a single PCR reaction, and detects the amplified products in combination with certain detection methods to achieve diagnosis of multiple targets.
  • the advantage of multiplex PCR technology lies in its high efficiency. It can detect and identify multiple pathogens through a single amplification reaction. Compared with multiple single-plex detection, the cost is significantly reduced. It is widely used in scientific research and disease diagnosis. It is the most widely used multiple nucleic acid detection technology in clinical practice. It is currently mainly used for the detection and quantification of bacteria and viruses, single nucleotide polymorphism (SNP) typing, disease mutation genes, and promoter methylation detection. Since Chamberlain first proposed this concept in 1988, multiplex PCR technology has developed rapidly.
  • SNP single nucleotide polymorphism
  • ThermoFisher's multiplex PCR kit can achieve 20-plex amplification; PrimerPlex, iCubate2.0 and other software can realize the design and optimization of multiplex PCR primers and probes, thereby reducing the possibility of non-specific amplification; Liao et al. used fluorescent probe melting curve technology to successfully achieve the typing of 15 high-risk HPV types and the typing and analysis of 48 human single nucleotide polymorphisms.
  • multiplex PCR The basic principle of multiplex PCR is the same as that of conventional PCR. The difference is that two or more pairs of primers are added to the multiplex PCR reaction system. Each pair of primers binds to the corresponding part of the template and amplifies the PCR reaction of multiple nucleic acid fragments at the same time.
  • the multiplex PCR experiment is not simply to mix multiple pairs of specific primers into a reaction system.
  • the difficulty lies in the balanced amplification of each target in the system.
  • the amplification conditions may be incompatible between multiple targets, and non-specific amplification may exist under multiple primers. In order to achieve balanced amplification of each target, primers, reaction conditions, reaction systems, etc. are generally optimized.
  • DNA polymerase in addition to conventional PCR performance, also needs to have amplification uniformity and have equal preference for various primers. Otherwise, some products will be very large and can be easily detected, while some products will be completely reduced to the background.
  • the above-mentioned DNA polymerase mutant Compared with the wild-type DNA polymerase (SEQ ID NO: 1), the above-mentioned DNA polymerase mutant has better multiple amplification ability, that is, it meets the requirements for amplification rate in multiple PCR, and has amplification uniformity. It can perform the same PCR amplification with the same preference for different primers and target fragments, thereby achieving the effect of simultaneously amplifying multiple targets in one PCR reaction.
  • the mutation comprises any one of the following amino acid mutation combinations: G59W, G59W+D488K, S543T, S577T, E400R, L549F, T664F, G59W+D488K+S543T, G59W+D488K+L549F, G59W+S577T, G59W+E388K, S543T+T664F, L678I, E388K+L549F, G59W+D488K, G59W+D488K+L549F, G59W+S577T or G59W+E388K.
  • PCR is one of the most important tools in the field of molecular biology research. In addition to amplifying purified DNA templates, it also has a variety of complex templates. Most wild-type polymerases are restricted by PCR inhibitors in the amplification template, and their amplification ability is affected, such as humic acid in soil, various components in serum, and animal and plant tissues. This limitation This has hindered the more efficient and widespread application of PCR in various fields.
  • PCR-based methods and techniques such as clinical diagnosis, environmental testing, forensic identification, etc.
  • use various types of complex samples commonly used are blood, blood cards, oral mucosal cells (oral swabs), soil, plant tissues, etc.
  • inhibitory substances in these samples can interfere with PCR, causing false negative reactions or reduced sensitivity.
  • PCR analysis of blood samples is commonly used in clinical practice, including human health-related tests for diagnosing genetic diseases, viral and microbial infections, blood typing, and safe blood banks.
  • researchers In order to reduce the impact of inhibitors in blood on PCR detection, researchers have developed various laboratory procedures to pretreat samples.
  • DNA purification methods suitable for PCR include dialysis, Chelex 100 resin, dilution with cell lysis buffer, centrifugal recovery, washing with NaOH and adding bovine serum albumin.
  • these pretreatment steps are very time-consuming and cumbersome, and different methods need to be used for different samples. In the pretreatment process, some precious nucleic acid samples will be lost and inhibitors cannot be completely removed.
  • Adding additives to the PCR system is one of the strategies to improve the PCR results of complex samples, which can reduce the impact of PCR inhibitors in the sample. For example, the addition of bovine serum albumin or single-stranded DNA binding proteins can enhance the amplification ability of certain polymerases.
  • Modifying the DNA polymerase itself is also a feasible strategy for improving the ability of DNA polymerase to perform PCR in complex samples.
  • the use of a polymerase with good inhibition resistance can avoid the sample pretreatment step, save labor, avoid cross contamination, and achieve direct amplification of complex samples.
  • the above-mentioned DNA polymerase mutants are tolerant to common inhibitory substances such as heme, humic acid or tannic acid.
  • the use of the above-mentioned DNA polymerase can achieve direct PCR of DNA in the above-mentioned samples containing inhibitory substances, reducing the time and cost required for pretreatment and the loss and damage to DNA.
  • DNA polymerase mutants including but not limited to promoters for regulating transcription and translation levels, molecular tags for purifying proteins, signal peptides for localizing proteins, and other known protein sequences in the prior art.
  • the above-mentioned elements do not affect the activity of the DNA polymerase mutants, including but not limited to one or more of polymerization ability, inhibition resistance, or multiple PCR ability.
  • the homology in this specification refers to the "identity" between amino acid sequences, that is, the total ratio of the same type of amino acid residues in the amino acid sequence.
  • the homology of amino acid sequences can be determined using alignment programs such as BLAST (Basic Local Alignment Search Tool) and FASTA.
  • amino acid residues are listed as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine (Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr;
  • conservative amino acid substitutions include but are not limited to:
  • Hydrophobic amino acids (Ala, Cys, Gly, Pro, Met, Val, Ile, Leu) are replaced by other hydrophobic amino acids;
  • hydrophobic amino acids with bulky side chains (Phe, Tyr, Trp) are replaced by other hydrophobic amino acids with bulky side chains;
  • Amino acids with positively charged side chains are replaced by other amino acids with positively charged side chains;
  • Amino acids with polar, uncharged side chains (Ser, Thr, Asn, Gln) are replaced by other amino acids with polar, uncharged side chains.
  • a person skilled in the art may also perform conservative substitutions on amino acids according to amino acid substitution rules well known to those skilled in the art, such as the "blosum62 scoring matrix" in the prior art.
  • a DNA molecule is provided, wherein the DNA molecule comprises a polynucleotide encoding the above-mentioned DNA polymerase mutant.
  • the DNA molecule includes the nucleotide sequence shown in SEQ ID NO: 2.
  • nucleic acid sequences for expressing commonly used elements in the prior art near the above-mentioned DNA molecules include but are not limited to promoters for regulating transcription and translation levels, molecular tags for purifying proteins, signal peptides for localizing proteins, and other known sequences in the prior art.
  • a recombinant vector comprising the above-mentioned DNA molecule.
  • the DNA can encode the DNA polymerase mutant and can be connected to a recombinant vector to form a circular DNA. Both the DNA and the recombinant vector can be transcribed and translated under the action of RNA polymerase, ribosome, tRNA, etc. to obtain the DNA polymerase mutant.
  • the nucleotide sequence can be flexibly codon-optimized using existing technology to obtain a nucleotide sequence with higher transcription and translation efficiency.
  • a host cell wherein the host cell contains the above-mentioned DNA molecule or recombinant vector.
  • the host cell includes a prokaryotic cell or a eukaryotic cell; preferably, the prokaryotic cell includes Escherichia coli.
  • the recombinant vector can be replicated in the host cells, and the DNA molecules carried on the recombinant vector can also be transcribed and translated to obtain a large number of DNA polymerase mutants.
  • the host cells can be crushed, protein purified after crushing or other methods to obtain DNA polymerase mutants.
  • the host cell is a host cell of non-plant or non-animal origin, and the host cell does not have developmental omnipotence.
  • a PCR kit which includes the above-mentioned DNA polymerase mutant.
  • the PCR kit further includes any one or more of the following components: 1) a buffer for providing a PCR amplification environment; 2) PCR primers; 3) a reagent for extracting target DNA.
  • PCR primers include but are not limited to universal primers for amplifying specific target DNA, including but not limited to 16S universal amplification primers for prokaryotic bacteria, 18S universal amplification primers for eukaryotic bacteria, ITS universal amplification primers for fungi, or other PCR primers designed for specific organisms or specific target fragments.
  • the above buffer is the common PCR buffer in the prior art, which is used to provide reaction conditions suitable for the above DNA polymerase mutant.
  • the DNA polymerase mutant of the present application is similar to the wild-type Taq DNA polymerase and can perform PCR reaction in the PCR buffer of the prior art. Those skilled in the art can also flexibly optimize the components in the above PCR buffer to obtain a buffer that is more suitable for this DNA polymerase mutant.
  • the above reagent for extracting target DNA can extract DNA from the target sample and provide an amplification template for the subsequent PCR reaction.
  • a PCR method comprises: using the above-mentioned DNA polymerase mutant or the above-mentioned kit to perform PCR amplification on the target DNA.
  • PCR amplification includes multiplex PCR amplification; preferably, the PCR system contains a PCR inhibitor; more preferably, the PCR inhibitor includes but is not limited to one or more of heme, humic acid, tannic acid, EDTA, heparin, phenol, sodium dodecyl sulfate, hemin, urea, plant polysaccharides, bile salts, polystyrene, polypropylene, protease, bilirubin, bromophenol blue, calcium ions and iron ions.
  • the PCR inhibitor includes but is not limited to one or more of heme, humic acid, tannic acid, EDTA, heparin, phenol, sodium dodecyl sulfate, hemin, urea, plant polysaccharides, bile salts, polystyrene, polypropylene, protease, bilirubin, bromophenol blue, calcium ions and iron
  • a PCR reaction can be performed on a reaction system containing multiple inhibitors.
  • Plasmids containing sequences encoding different DNA polymerase mutants were constructed by means of site-directed mutagenesis PCR, as shown in Example 1 for details.
  • the constructed plasmids containing sequences encoding different DNA polymerase mutants were then transferred into E. coli for culture and induced expression, and finally the DNA polymerase was purified by heat treatment and affinity chromatography to obtain Taq DNA polymerases containing different mutations, as shown in Example 2 for details.
  • the obtained mutants were tested for polymerase activity, inhibition resistance and multiple PCR, and superior mutants with improved performance were obtained. At the same time, the superior mutation sites were combined and superimposed to obtain combined mutants with improved performance.
  • the wild-type Taq DNA polymerase was modified to obtain an advantageous mutant, whose enzyme activity/inhibition resistance/multiple PCR ability was significantly improved compared with the wild type.
  • the improvement of polymerase activity can increase the polymerization rate and quickly obtain a large number of products; the improvement of inhibition resistance allows it to directly amplify complex templates without pretreatment of the template, saving steps and simple operation; the improvement of multiple amplification ability allows a single round of PCR to amplify multiple targets at the same time, improving efficiency.
  • the gene sequence of the wild-type Taq DNA polymerase is shown in SEQ ID NO:2, and the amino acid sequence encoded by it is shown in SEQ ID NO:1.
  • the expression plasmid pET-28a(+)-Taq containing the wild-type Taq DNA polymerase coding sequence was commissioned to Beijing Liuhe BGI Gene Technology Co., Ltd. for gene synthesis and vector construction. Among them, six histidine residues (6 ⁇ His) were fused to the N-terminus of the amino acid sequence to facilitate protein purification.
  • the single-site mutant was introduced into the site-directed mutation by PCR amplification using Q5 High-Fidelity DNA Polymerase (purchased from New England Biolabs, catalog number M0491V) on the basis of the wild-type Taq DNA polymerase expression plasmid using the above-mentioned primer pairs.
  • Q5 High-Fidelity DNA Polymerase purchased from New England Biolabs, catalog number M0491V
  • the specific reaction system is shown in Table 3 below.
  • Combination mutation refers to a mutant with two or more mutations compared to the wild-type Taq DNA polymerase.
  • Combination mutation is constructed by using the corresponding primers to first perform site-directed mutation at one mutation site, obtain the mutant product, and then perform a second site-directed mutation on the mutant product, and so on until a multi-site mutant compared to the wild-type Taq DNA polymerase is obtained.
  • the PCR reaction system preparation and PCR reaction conditions are as above.
  • the annealing temperature can be adjusted according to the Tm value of the primer.
  • the wild-type and mutant plasmids were transformed into BL21 (DE3) competent cells (purchased from Tiangen Biotechnology Co., Ltd., catalog number CB105), and then a single colony was picked and placed in 2 mL of LB medium containing kanamycin resistance (50 ⁇ g/mL), and cultured overnight at 37°C at 200 rpm/min.
  • the collected bacteria were resuspended in a cell resuspension solution (50 mM Tris-HCl, 500 mM NaCl, 5% Glycerol, pH 7.8). 100 ⁇ L 10 mg/mL lysozyme was added to the resuspended bacteria and incubated at 37 °C for 30 min, then heat treated at 75 °C for 40 min. Centrifuged at 14000 rpm for 10 min, and the supernatant was collected.
  • a cell resuspension solution 50 mM Tris-HCl, 500 mM NaCl, 5% Glycerol, pH 7.8
  • 100 ⁇ L 10 mg/mL lysozyme was added to the resuspended bacteria and incubated at 37 °C for 30 min, then heat treated at 75 °C for 40 min. Centrifuged at 14000 rpm for 10 min, and the supernatant was collected.
  • Wash buffer 2 (50 mM Tris-HCl, 500 mM NaCl, 5% Glycerol, 50 mM Imidazole, pH 7.8) was added in an amount equivalent to the volume of the supernatant, centrifuged at 100 g for 30 s. The filtrate was discarded. Add Elution buffer (50mM Tris-HCl, 500mM NaCl, 5% Glycerol, 500mM Imidazole, pH7.8) for elution, 200 ⁇ L each time, elute twice, and collect the eluate. After the collected eluates are combined, they are concentrated and replaced with a 0.5mL 50KDa ultrafiltration tube (purchased from Millipore, catalog number UFC505096).
  • the purified wild-type Taq DNA polymerase and its mutants were tested for polymerase activity.
  • the activity was tested using M13 ssDNA (NEB, catalog number N4040S) bound to a primer as a template-primer complex.
  • N4040S M13 ssDNA
  • the DNA chain was extended to obtain an extended double-stranded DNA product.
  • the activity of the DNA polymerase was calculated by detecting the amount of double-stranded DNA generated.
  • the sequence of the bound primer was: 5'-AGCGAACCTCCCGACTTGCGGGAGG-3' (SEQ ID NO: 65).
  • the formula of the 10 ⁇ PCR buffer used in the present invention was: 100mM Tris-HCl, 500mM KCl, 15mM MgCl 2 , 25% Glycerol, 0.5mg/mL BSA (bovine serum albumin).
  • the reaction system for Taq DNA polymerase activity detection is shown in Table 5.
  • the prepared reaction system was placed in a PCR instrument. After reacting at 72°C for 5 minutes, 0.5 ⁇ L of 0.5 M EDTA was added to terminate the reaction. The amount of double-stranded DNA was detected using Qubit dsDNA HS Assay Kit (purchased from Thermo Fisher Scientific, catalog number Q32854). The polymerization activity of the mutant relative to the wild type was calculated (the polymerization activity of the wild type was taken as 1), as shown in Table 6.
  • the purified wild-type Taq DNA polymerase and its mutants were subjected to 8-fold human housekeeping gene amplification tests.
  • the template for 8-plex amplification is human genomic DNA (human gDNA), which was extracted from Expi293F TM cells (purchased from Thermo Fisher Scientific, Catalog No. A14527) using a DNA extraction kit (purchased from Tiangen Biochemical Technology Co., Ltd., Catalog No. DP304-02).
  • the amplification primers and target fragment sizes are shown in Table 7 below.
  • the PCR reaction system for 8-plex amplification of human housekeeping genes is shown in Table 8 below.
  • the purified wild-type Taq DNA polymerase and its mutants were tested by PCR in an inhibitor-containing system.
  • the PCR system contained inhibitors (12 ⁇ M heme or 1.6 ⁇ g/mL humic acid or 1.2 ⁇ g/mL tannic acid).
  • the specific components of the PCR system are described in Table 10 below.
  • Mut5 (G59W+D488K), Mut19 (G59W+D488K+L549F), Mut21 (G59W+S577T), and Mut22 (G59W+E388K) all showed improved tolerance to the three inhibitors.
  • the Taq DNA polymerase mutant disclosed in the present application has enhanced polymerization activity; it has enhanced inhibition resistance and can perform PCR reactions with templates mixed with inhibitory substances, avoiding the need for template extraction, purification and other treatments before the PCR reaction; the above-mentioned DNA polymerase mutant can also perform multiplex PCR. Using the above-mentioned DNA polymerase mutant, more products can be obtained in the same time, or PCR reactions with templates mixed with inhibitory substances can be performed, avoiding the need for template extraction, purification and other treatments before the PCR reaction, or performing multiplex PCR. This mutant has significant improvements in DNA polymerization activity, inhibition resistance and multiplex amplification compared to the wild type.

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Abstract

本发明提供了一种DNA聚合酶突变体及其应用。其中，上述DNA聚合酶突变体具有DNA聚合酶活性，包括与SEQ ID NO：1所示的氨基酸序列具有至少70％、至少75％、至少80％、至少85％、至少90％、至少95％或至少99％同源性的氨基酸序列。能够解决现有技术中野生型DNA聚合酶聚合活性低的问题，适用于DNA聚合酶领域。

Description

DNA聚合酶突变体及其应用 技术领域
本发明涉及DNA聚合酶领域，具体而言，涉及一种DNA聚合酶突变体及其应用。
背景技术
PCR技术是现代分子生物学的重要基石，而Taq DNA聚合酶是PCR技术的经典主力军。Taq DNA聚合酶属于A家族DNA聚合酶，具有良好的热稳定性，被广泛用于PCR技术相关的领域。Taq聚合酶缺乏3’-5’校正活性，但具有5’-3’外切活性和末端加A的特点，广泛应用于分子生物学领域，如直接PCR、等位基因检测、Sanger测序、荧光定量PCR、TA克隆等。目前，PCR是分子生物学应用市场增长最快的技术之一。PCR的新应用和PCR的新技术正在开发并用于研究和诊断领域，随着PCR技术的普遍应用，对Taq DNA聚合酶性质的研究日渐重要。但Taq DNA聚合酶也存在一些不足之处，扩增活性、耐抑制能力等仍然有待进一步提高，仍然需要进一步改进的DNA聚合酶变体，兼具各方面性能以满足DNA扩增、合成、检测、测序以及其他重要技术的需要。
发明内容
本发明的主要目的在于提供一种DNA聚合酶突变体及其应用，以解决现有技术中野生型DNA聚合酶聚合活性低的问题。
为了实现上述目的，根据本发明的第一个方面，提供了一种DNA聚合酶突变体，该DNA聚合酶突变体具有DNA聚合酶活性，包括与SEQ ID NO：1所示的氨基酸序列具有至少70％、至少75％、至少80％、至少85％、至少90％、至少95％或至少99％同源性的氨基酸序列。
进一步地，DNA聚合酶突变体相对于SEQ ID NO：1所示的氨基酸序列具有以下至少一个位点的氨基酸替换：D488、S577、S543、E388、E400、E397、T544、L549、V586、L670、L678、T664和G59。
进一步地，D488位点的氨基酸替换包括D488H、D488R或D488K；S543位点的氨基酸替换包括S543T；S577位点的氨基酸替换包括S577T；E388位点的氨基酸替换包括E388K；E400位点的氨基酸替换包括E400R；E397位点的氨基酸替换包括E397Q；T544位点的氨基酸替换包括T544Y、T544D、T544K、T544S、T544W或T544F；L549位点的氨基酸替换包括L549F、L549I、L549D或L549K；V586位点的氨基酸替换包括V586F；L670位点的氨基酸替换包括L670F；L678位点的氨基酸替换包括L678Y、L678W、L678I、L678F、L678K或L678D；T664位点的氨基酸替换包括T664F；G59位点的氨基酸替换包括G59W。
进一步地，DNA聚合酶突变体的突变包括如下任意一种氨基酸突变组合：D488H、D488R、D488K、S577T、G59W、E388K、E397Q、E400R、S543T、T544Y、T544D、T544K、T544S、 T544W、T544F、L549F、L549I、L549D、L549K、V586F、T664F、L670F、L678Y、L678K、L678D、L678I、L678F、G59W+E388K、G59W+E400R、G59W+D488K、G59W+S543T、G59W+S577T、E388K+E397Q、E388K+E400R、E388K+D488K、E388K+S543T、E388K+T544Y、E388K+L549F、E388K+S577T、E388K+L670F、E388K+T664F、E388K+L678Y、E397Q+D488K、E397Q+T664F、E400R+D488K、E400R+T664F、D488K+S543T、D488K+T544Y、D488K+L549F、D488K+S577T、D488K+V586F、D488K+T664F、D488K+L670F、D488K+L678Y、S543T+T664F、S577T+T664F、V586F+T664F、T664F+L670F、T664F+L678W、T664F+L678Y、G59W+D488K+S543T或G59W+D488K+L549F；优选地，DNA聚合酶突变体的突变包括如下任意一种氨基酸突变组合：G59W、D488K、G59W+D488K、S543T、E388K、E400R、E397Q、L549F、T664F、G59W+D488K+L549F、G59W+S577T、G59W+E388K、G59W+E400R、E400R+D488K、D488K+L549F、E388K+E397Q、E388K+T664F、S543T+T664F、E400R+T664F、L678I或E388K+L549F，DNA聚合酶突变体的DNA聚合活性优于SEQ ID NO：1所示的氨基酸序列的DNA聚合活性；优选地，DNA聚合酶突变体的突变包括如下任意一种氨基酸突变组合：G59W+D488K、S543T、E400R、T544Y、L549F、G59W+D488K+L549F、G59W+S543T、G59W+S577T或G59W+E400R，DNA聚合酶突变体的多重扩增能力优于SEQ ID NO：1所示的氨基酸序列的多重扩增能力；优选地，DNA聚合酶突变体的突变包括如下任意一种氨基酸突变组合：G59W、G59W+D488K、S543T、S577T、E400R、L549F、T664F、G59W+D488K+S543T、G59W+D488K+L549F、G59W+S577T、G59W+E388K、S543T+T664F、L678I、E388K+L549F、G59W+D488K、G59W+D488K+L549F、G59W+S577T或G59W+E388K，DNA聚合酶突变体的耐PCR抑制剂能力优于SEQ ID NO：1所示的氨基酸序列的耐PCR抑制剂能力。
为了实现上述目的，根据本发明的第二个方面，提供了一种DNA分子，该DNA分子包含编码上述DNA聚合酶突变体的多核苷酸。
为了实现上述目的，根据本发明的第三个方面，提供了一种重组载体，该重组载体包含有上述DNA分子。
为了实现上述目的，根据本发明的第四个方面，提供了一种宿主细胞，该宿主细胞内含有上述DNA分子或上述重组载体。
进一步地，宿主细胞包括原核细胞或真核细胞；优选地，原核细胞包括大肠杆菌。
为了实现上述目的，根据本发明的第五个方面，提供了一种PCR试剂盒，该PCR试剂盒包括上述DNA聚合酶突变体。
进一步地，PCR试剂盒中还包括如下任意一种或多种成分：1)用于提供PCR扩增环境的缓冲液；2)PCR引物；3)用于提取目标DNA的试剂。
为了实现上述目的，根据本发明的第六个方面，提供了一种PCR方法，该PCR方法包括：利用上述DNA聚合酶突变体或上述PCR试剂盒，对目标DNA进行PCR扩增。
进一步地，PCR包括多重PCR扩增；优选地，PCR的体系中含有PCR抑制剂；更优选地，PCR抑制剂包括血红素、腐殖酸、单宁酸、EDTA、肝素、苯酚、十二烷基磺酸钠、氯高铁血红素、尿素、植物多糖、胆酸盐、聚苯乙烯、聚丙烯、蛋白酶、胆红素、溴酚蓝、钙离子和铁离子中的一种或多种。
DNA聚合酶的活性是提高PCR扩增效率的关键因素之一，应用本发明的技术方案，上述DNA聚合酶突变体相较于野生型Taq DNA聚合酶，具有更高的活性。
附图说明
构成本申请的一部分的说明书附图用来提供对本发明的进一步理解，本发明的示意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定。在附图中：
图1示出了根据本发明实施例4的琼脂糖凝胶电泳结果图。
图2示出了根据本发明实施例4的琼脂糖凝胶电泳结果图。
图3示出了根据本发明实施例4的琼脂糖凝胶电泳结果图。
图4示出了根据本发明实施例5的琼脂糖凝胶电泳结果图。
具体实施方式
需要说明的是，在不冲突的情况下，本申请中的实施例及实施例中的特征可以相互组合。下面将结合实施例来详细说明本发明。
如背景技术所提到的，随着PCR技术的普遍应用，对DNA聚合酶性质的研究日渐重要。但DNA聚合酶也存在一些不足之处，聚合活性难以满足实际应用的需求，亟待对DNA聚合酶进行改进，从而提高DNA聚合速率。聚合速率，或单位时间内掺入的核苷酸数量(在指定的反应条件下，包括温度、pH、离子强度等)，受到许多参数的影响，包括底物的结合亲和力(dNTP、引物)和催化效率(核苷酸转移速率、焦磷酸盐释放和易位步骤)。在本申请中发明人尝试开发一种新型的DNA聚合酶突变体，具有增强的聚合活性，因而提出了本申请的一系列保护方案。
在本申请第一种典型的实施方式中，提供了一种DNA聚合酶突变体，该DNA聚合酶突变体具有DNA聚合酶活性，包括与SEQ ID NO：1所示的氨基酸序列具有至少70％、至少75％、至少80％、至少85％、至少90％、至少95％或至少99％同源性的氨基酸序列。
SEQ ID NO：1：

在一种优选的实施例中，上述DNA聚合酶突变体且相对于SEQ ID NO：1所示的氨基酸序列具有以下至少一个位点的氨基酸替换：D488、S577、S543、E388、E400、E397、T544、L549、V586、L670、L678、T664和G59。
在一种优选的实施例中，D488位点的氨基酸替换包括D488H、D488R或D488K；S543位点的氨基酸替换包括S543T；S577位点的氨基酸替换包括S577T；E388位点的氨基酸替换包括E388K；E400位点的氨基酸替换包括E400R；E397位点的氨基酸替换包括E397Q；T544位点的氨基酸替换包括T544Y、T544D、T544K、T544S、T544W或T544F；L549位点的氨基酸替换包括L549F、L549I、L549D或L549K；V586位点的氨基酸替换包括V586F；L670位点的氨基酸替换包括L670F；L678位点的氨基酸替换包括L678Y，L678W，L678I，L678F，L678K或L678D；T664位点的氨基酸替换包括T664F；G59位点的氨基酸替换包括G59W；其中，数字前字母代表原始氨基酸，数字后字母代表突变氨基酸。
在一种优选的实施例中，DNA聚合酶突变体的突变(替换)包括如下任意一种氨基酸突变组合：
G59W、E388K、E397Q、E400R、D488H、D488R、D488K、S543T、T544Y、T544D、T544K、T544S、T544W、T544F、L549F、L549I、L549D、L549K、S577T、V586F、T664F、L670F、L678Y、L678K、L678D、L678I、L678F、G59W+E388K、G59W+E400R、G59W+D488K、G59W+S543T、G59W+S577T、E388K+E397Q、E388K+E400R、E388K+D488K、E388K+S543T、E388K+T544Y、E388K+L549F、E388K+S577T、E388K+L670F、E388K+T664F、E388K+L678Y、E397Q+D488K、E397Q+T664F、E400R+D488K、E400R+T664F、D488K+S543T、D488K+T544Y、D488K+L549F、D488K+S577T、D488K+V586F、D488K+T664F、D488K+L670F、D488K+L678Y、S543T+T664F、S577T+T664F、V586F+T664F、T664F+L670F、T664F+L678W、T664F+L678Y、G59W+D488K+S543T或G59W+D488K+L549F；
优选地，DNA聚合酶突变体的突变包括如下任意一种氨基酸突变组合：G59W、D488K、G59W+D488K、S543T、E388K、E400R、E397Q、L549F、T664F、G59W+D488K+L549F、G59W+S577T、G59W+E388K、G59W+E400R、E400R+D488K、D488K+L549F、E388K+E397Q、E388K+T664F、S543T+T664F、E400R+T664F、L678I或 E388K+L549F。
上述DNA聚合酶突变体的聚合酶活性的提高，能够在单位时间内获得相较于野生型DNA聚合酶(SEQ ID NO：1)更多的双链DNA产物。
优选地，DNA聚合酶突变体的突变包括如下任意一种氨基酸突变组合：G59W+D488K、S543T、E400R、T544Y、L549F、G59W+D488K+L549F、G59W+S543T、G59W+S577T或G59W+E400R。
多重PCR是指通过一次PCR反应同时对多个靶标进行扩增，结合一定的检测手段对扩增产物进行检测从而实现对多个靶标进行诊断的技术。多重PCR技术的优势在于它的高效性，通过一次扩增反应即可实现对多种病原体进行检测和鉴别，相较多个单重检测而言成本显著降低，广泛应用于科学研究和疾病诊断等领域，是临床应用最广泛的多重核酸检测技术，目前主要用于细菌、病毒的检测与定量、单核苷酸多态性(single nucleotide polymorphism，SNP)分型、疾病突变基因、启动子甲基化的检测。自Chamberlain于1988年首次提出这个概念以来，多重PCR技术迅速发展，ThermoFisher公司的多重PCR试剂盒能实现20重扩增；PrimerPlex、iCubate2.0等软件可以实现多重PCR引物与探针的设计与优化，从而降低非特异性扩增的可能；Liao等利用荧光探针熔解曲线技术成功地实现了对15种高危型HPV的分型以及实现了对48种人类单核苷酸多态性的分型和分析。
多重PCR基本原理与常规PCR相同，区别在于多重PCR反应体系加入两对及以上的引物，各对引物分别结合在模板相对应部位，同时扩增出多个核酸片段的PCR反应。但多重PCR实验并不是简单的将多对特异性引物混合成一个反应体系，其难点在于体系中各靶标的均衡扩增。多重PCR中可能多个靶点之间扩增条件不兼容，及多引物下可能存在的非特异扩增。为使每个靶标能均衡扩增，一般会对引物、反应条件、反应体系等进行优化。DNA聚合酶作为多重PCR应用中的核心原料，除了具有常规的PCR性能外，还需要具有扩增均一性，对各种引物具有相等的偏好性，否则就会出现部分产物量非常多能够被很容易的检测到，而有部分产物彻底沦为背景的结果。
上述DNA聚合酶突变体相较于野生型DNA聚合酶(SEQ ID NO：1)，具有较好的多重扩增能力，即满足了多重PCR中对于扩增速率的要求，又具有扩增均一性，能够对不同的引物和目标片段进行偏好相同的PCR扩增，从而实现在一次PCR反应中实现同时对多个靶标进行扩增的效果。
优选地，突变包括如下任意一种氨基酸突变组合：G59W、G59W+D488K、S543T、S577T、E400R、L549F、T664F、G59W+D488K+S543T、G59W+D488K+L549F、G59W+S577T、G59W+E388K、S543T+T664F、L678I、E388K+L549F、G59W+D488K、G59W+D488K+L549F、G59W+S577T或G59W+E388K。
PCR作为目前分子生物学研究领域最重要的工具之一，除了对提纯的DNA模板进行扩增外，还会有各式各样的复杂模板。大部分野生型聚合酶受到扩增模板中PCR抑制剂的限制，扩增能力受到影响，例如土壤中的腐殖酸、血清中的各种成分、动植物组织等。这一局限性 阻碍了PCR更高效、更广泛地应用于多种领域。
一些基于PCR的方法技术，如：临床诊断、环境检测、法医鉴定等，会用到各种类型的复杂样本，常用的有血液、血卡、口腔黏膜细胞(口腔拭子)、土壤、植物组织等。这些样本中都存在着可以干扰PCR的抑制物质，从而引起假阴性反应或灵敏度的降低。血液样本的PCR分析在临床上较为常用，包括用于诊断遗传疾病、病毒和微生物感染、血型和安全血库的人类健康相关测试。为了减少血液中抑制剂对PCR检测的影响，研究人员已经开发了各种实验室程序对样本进行预处理。适用于PCR的DNA纯化方法包括透析、Chelex 100树脂、用细胞裂解液稀释、离心回收、用NaOH洗涤并添加牛血清蛋白等。而这些预处理步骤十分耗时且繁琐，对不同的样本还需使用不同的方法，而预处理过程中还会损失部分宝贵的核酸样品且无法完全去除抑制剂。在PCR体系中添加添加剂是改善复杂样品PCR结果的策略之一，其可以减小样品中PCR抑制剂的影响。例如，添加牛血清蛋白或单链DNA结合蛋白可以增强某些聚合酶的扩增能力。
而对DNA聚合酶本身进行改造也是一种可行的用于提高DNA聚合酶在复杂样品中进行PCR能力的策略。对于一些复杂样本，使用耐抑制能力好的聚合酶可以免于样本预处理步骤，节省劳动力，避免交叉污染，并实现对复杂样本的直接扩增。上述DNA聚合酶突变体对血红素、腐殖酸或单宁酸等常见的抑制物质具有耐受能力，利用上述DNA聚合酶能够实现对于上述含有抑制物质的样本中的DNA进行直接PCR，减少前处理所需的时间、成本和对于DNA的损失和破坏。
本领域技术人员能够灵活在上述DNA聚合酶突变体上增加现有技术中常用元件，上述元件包括但不限于调控转录翻译水平的启动子，用于纯化蛋白的分子标签，用于定位蛋白的信号肽等现有技术中的已知的蛋白序列。上述元件不影响DNA聚合酶突变体的活性，包括但不限于聚合能力、耐抑制能力或多重PCR能力中的一种或多种。
上述氨基酸突变均在本申请实施例中进行试验探究，相较于具有SEQ ID NO：1所示的氨基酸序列的母本，均具有DNA聚合酶活性。与上述氨基酸序列具有70％、75％、80％、85％、90％、95％、96％、97％、98％、99％、99.5％或99.9％或以上同源性、且具有相同提高酶活性作用能力的蛋白质。
本说明书中的同源性(Identity)是指氨基酸序列之间的“同一性”，即氨基酸序列中的种类相同的氨基酸残基的比率的总计。氨基酸序列的同源性可以利用BLAST(Basic Local Alignment Search Tool)、FASTA等比对程序来确定。
与上述提供的蛋白具有70％、75％、80％、85％、90％、95％、99％以上(比如85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、98.5％、99％、99.5％、99.6％、99.7％、99.8％以上，甚至99.9％以上)同源性且具有相同功能的蛋白质，其活性位点、活性口袋、活性机制、蛋白结构等均和上述蛋白质大概率相同，为通过氨基酸突变获得的同源蛋白。
具有上述同源性序列的获得，可以通过氨基酸的取代、替换等来实现。取代、替换等规则，一般情况下，性质类似的氨基酸之间相互替换后的效果也类似。为方便描述，此处先将氨基酸残基缩写罗列如下：丙氨酸(Ala；A)、天冬酰胺(Asn；N)、天冬氨酸(Asp；D)、精氨酸(Arg；R)、半胱氨酸(Cys；C)、谷氨酸(Glu；E)、谷氨酰胺(Gln；Q)、甘氨酸(Gly；G)、组氨酸(His；H)、异亮氨酸(Ile；I)、亮氨酸(Leu；L)、赖氨酸(Lys；K)、蛋氨酸(Met；M)、苯丙氨酸(Phe；F)、脯氨酸(Pro；P)，丝氨酸(Ser；S)、苏氨酸(Thr；T)、色氨酸(Trp；W)、酪氨酸(Tyr；Y)和缬氨酸(Val；V)。
氨基酸的取代或替换，例如，在上述同源蛋白中，可发生保守的氨基酸替换。“保守的氨基酸替换”包括但不限于：
疏水性氨基酸(Ala、Cys、Gly、Pro、Met、Val、Ile、Leu)被其他疏水性氨基酸取代；
侧链粗大的疏水性氨基酸(Phe、Tyr、Trp)被其他侧链粗大的疏水性氨基酸取代；
侧链带正电的氨基酸(Arg、His、Lys)被其他侧链带正电的氨基酸取代；
侧链有极性不带电的氨基酸(Ser、Thr、Asn、Gln)被其他侧链有极性不带电的氨基酸取代。
本领域技术人员也可以根据现有技术中的“blosum62评分矩阵”等本领域技术人员熟知的氨基酸替换规则对氨基酸进行保守替换。
在本申请第二种典型的实施方式中，提供了一种DNA分子，该DNA分子包含编码上述DNA聚合酶突变体的多核苷酸。
优选地，DNA分子包括SEQ ID NO：2所示的核苷酸序列。
SEQ ID NO：2：

进一步地，本领域技术人员能够灵活在上述DNA分子附近增加用于表达现有技术中常用元件的核酸序列，上述元件包括但不限于调控转录翻译水平的启动子，用于纯化蛋白的分子标签，用于定位蛋白的信号肽等现有技术中的已知的序列。
在本申请第三种典型的实施方式中，提供了一种重组载体，该重组载体包含有上述DNA分子。
上述DNA能够编码上述DNA聚合酶突变体，并能够连接在重组载体上形成环状DNA。上述DNA和重组载体均能在RNA聚合酶、核糖体、tRNA等的作用下，进行转录、翻译，获得上述DNA聚合酶突变体。针对DNA分子或重组载体的不同的宿主种类，可以利用现有技术灵活对核苷酸序列进行密码子优化，从而获得转录、翻译效率更高的核苷酸序列。
在本申请第四种典型的实施方式中，提供了一种宿主细胞，该宿主细胞内含有上述DNA分子或重组载体。
在一种优选的实施例中，宿主细胞包括原核细胞或真核细胞；优选地，原核细胞包括大肠杆菌。
利用上述宿主细胞，能够在宿主细胞中进行重组载体的复制，也能够将重组载体上携带的DNA分子进行转录、翻译，获得大量DNA聚合酶突变体。利用现有技术，对宿主细胞进行破碎、破碎后蛋白纯化或其他方式，能够获得DNA聚合酶突变体。该宿主细胞为非植物来源或非动物来源的宿主细胞，该宿主细胞不具有发育全能性。
在本申请第五种典型的实施方式中，提供了一种PCR试剂盒，该PCR试剂盒包括上述DNA聚合酶突变体。
在一种优选的实施例中，PCR试剂盒中还包括如下任意一种或多种成分：1)用于提供PCR扩增环境的缓冲液；2)PCR引物；3)用于提取目标DNA的试剂。
上述PCR引物，包括但不限于用于扩增特定目标DNA的通用引物，包括但不限于针对原核细菌的16S通用扩增引物、针对真核细菌的18S通用扩增引物、针对真菌的ITS通用扩增引物或其他对于特定生物或特定目标片段设计的PCR引物。
上述缓冲液即现有技术中常见的PCR buffer，用于提供适合上述DNA聚合酶突变体的反应条件。本申请的DNA聚合酶突变体与野生型的Taq DNA聚合酶类似，能够在现有技术的PCR buffer中进行PCR反应，本领域技术人员也可以对上述PCR buffer中的成分进行灵活优化，从而获得更适宜此种DNA聚合酶突变体的缓冲液。
上述用于提取目标DNA的试剂，能够从目标样本中提取获得DNA，为后续的PCR反应提供扩增模板。
在本申请第六种典型的实施方式中，提供了一种PCR方法，该PCR方法包括：利用上述DNA聚合酶突变体或上述试剂盒，对目标DNA进行PCR扩增。
在一种优选的实施例中，PCR扩增包括多重PCR扩增；优选地，PCR体系中含有PCR抑制剂；更优选地，PCR抑制剂包括但不限于血红素、腐殖酸、单宁酸、EDTA、肝素、苯酚、十二烷基磺酸钠、氯高铁血红素、尿素、植物多糖、胆酸盐、聚苯乙烯、聚丙烯、蛋白酶、胆红素、溴酚蓝、钙离子和铁离子中的一种或多种。
利用上述DNA聚合酶突变体、上述试剂盒和上述PCR方法中的任一种，均能对含有多种抑制剂的反应体系进行PCR反应。
下面将结合具体的实施例来进一步详细解释本申请的有益效果。
通过定点突变PCR的手段构建了含有编码不同DNA聚合酶突变体序列的质粒，详情见实施例1。然后将构建好的含有编码不同DNA聚合酶突变体序列的质粒转入E.Coli中进行培养并诱导表达，最后通过热处理、亲和层析的方法对DNA聚合酶进行纯化，从而得到含有不同突变的Taq DNA聚合酶，具体见实施例2。对所得突变体进行聚合酶活性、耐抑制和多重PCR测试，获得了性能提升的优势突变体。同时，对优势突变位点进行组合叠加并获得性能提升的组合突变体。
对野生型Taq DNA聚合酶改造，获得优势突变体，其酶活/耐抑制能力/多重PCR能力较野生型显著提升。聚合酶活性的提高能够提高聚合速率，快速获得大量产物；耐抑制能力的提升允许其对复杂模板进行直扩，不需对模板进行预处理，节省工序，操作简单；多重扩增能力的提升允许单轮PCR同时对多个靶标进行扩增，提高效率。
实施例1 Taq DNA聚合酶及其突变体表达质粒的构建
具体实施步骤如下：
(1)野生型Taq DNA聚合酶表达质粒的构建
野生型的Taq DNA聚合酶的基因序列如SEQ ID NO：2所示，其编码的氨基酸序列如SEQ ID NO：1所示。
SEQ ID NO：2：
SEQ ID NO：1：

含有野生型Taq DNA聚合酶编码序列的表达质粒pET-28a(+)-Taq委托北京六合华大基因科技有限公司进行基因合成和载体构建。其中，氨基酸序列的N端融合了6个组氨酸残基(6×His)以利于蛋白的纯化。
(2)突变体Taq DNA聚合酶表达质粒的构建
根据理性设计筛选的突变位点设计正反向突变引物对，引物序列如下表1所示。
表1

构建的单点或组合突变体如下表2所示。
表2

单位点突变体使用Q5 High-Fidelity DNA Polymerase(购自New England Biolabs公司，货号M0491V)在野生型Taq DNA聚合酶表达质粒的基础上以上述引物对通过PCR扩增引入定点突变，具体的反应体系如下表3所示。
表3
PCR反应条件如下表4所示。
表4
反应结束后，加入1μL DpnI(NEB，货号R0176V)于37℃消化2h，然后取5μL消化后的产物转化到E.Coli感受态细胞DH5α(购自天根生物科技有限公司，货号CB101)。然后从平板上挑取单克隆进行培养后提取质粒，质粒寄送到北京六合华大基因科技有限公司进行Sanger测序，再通过测序比对分析得到的突变体是否正确。
组合突变指相对于野生型的Taq DNA聚合酶有两或多个位点发生突变的突变体。组合突变的构建是利用相应引物先进行一个突变位点的定点突变，获得突变产物后再对该突变产物进行第二个定点突变，以此类推直至得到相较于野生型Taq DNA聚合酶的多位点突变体。PCR反应体系配制和PCR反应条件如上，对于部分引物可根据引物的Tm值调整退火温度。
实施例2 Taq DNA聚合酶及其突变体的诱导表达和纯化
野生型及突变体质粒转化到BL21(DE3)感受态细胞(购自天根生物科技有限公司，货号CB105)，然后挑取单菌落于2mL含卡那霉素抗性(50μg/mL)LB培养基中，于37℃在200rpm/min的转速下过夜培养。次日取200μL菌液转接于10mL含卡那霉素抗性(50μg/mL)的LB培养基中，于37℃在200rpm/min的转速下培养至OD600在0.6～0.8，按照终浓度为0.5mM的量加入诱导剂IPTG，并继续在37℃ 200rpm/min转速下培养4h进行诱导表达。培养液最后使用8000rpm/min的条件离心5min收集诱导后的菌体。
收集的菌体使用细胞重悬液(50mM Tris-HCl，500mM NaCl，5％Glycerol(甘油)，pH7.8)进行重悬。在重悬菌体中加入100μL 10mg/mL溶菌酶于37℃孵育30min，再转至75℃热处理40min。14000rpm离心10min，收集上清液。
使用His SpinTrap柱(购自Cytiva，货号28401353)进行纯化。将上清液每次500μL加入至His SpinTrap柱中，100g离心30s，弃过滤液。加入等同于上清液体积的Wash buffer 1(50mM Tris-HCl，500mM NaCl，5％Glycerol，30mM Imidazole，pH7.8)，100g离心30s，弃过滤液。加入等同于上清液体积的Wash buffer 2(50mM Tris-HCl，500mM NaCl，5％Glycerol，50mM Imidazole(咪唑)，pH7.8)，100g离心30s。弃过滤液。加入Elution buffer(50mM Tris-HCl，500mM NaCl，5％Glycerol，500mM Imidazole，pH7.8)进行洗脱，每次200μL，洗脱2次，收集洗脱液。收集的洗脱液合并后用0.5mL 50KDa超滤管(购自Millipore，货号UFC505096)进行浓缩换液。7000rpm离心5min，弃滤液。加入500μL Dialysis Buffer(40mM Tris-HCl，200mM KCl，2mM DTT，0.2mM EDTA-Na2，5％Glycerol，pH7.8)，7000rpm离心3～5min，弃滤液，重复5-6次，收集内管酶液。蛋白定量后进行酶储(含50％ Glycerol)，酶储浓度0.1mg/mL。之后制备成浓度为0.05mg/mL的热启动形式，用于后续的测定和分析。热启动抗体及使用方法参考专利申请CN114685671A。
实施例3 Taq DNA聚合酶及其突变体的聚合活性测定及分析
对纯化得到的野生型Taq DNA聚合酶及其突变体进行聚合酶活性的测定。活性测定使用结合有引物的M13 ssDNA(NEB，货号N4040S)为模板-引物复合体。在DNA聚合酶的作用下发生DNA链的延伸，得到延长的双链DNA产物。通过检测生成的双链DNA量来计算DNA聚合酶的活性。其中结合的引物序列为：5’-AGCGAACCTCCCGACTTGCGGGAGG-3’(SEQ ID NO：65)。本发明中所用的10×PCR缓冲液的配方均为：100mM Tris-HCl，500mM KCl，15mM MgCl2，25％Glycerol，0.5mg/mL BSA(牛血清白蛋白)。
Taq DNA聚合酶活性检测的反应体系如下表5所示。
表5
将上述配好的反应体系置于PCR仪中，72℃反应5min后加入0.5μL的0.5M EDTA终止反应，然后用Qubit dsDNA HS Assay Kit(购自赛默飞公司，货号Q32854))检测双链DNA的量，通过计算得到突变体相对于野生型的聚合活性(以野生型的聚合活性为1)如表6所示。
表6

活性测试结果显示，相对酶活数值大于1的表明相较于野生型的Taq DNA聚合酶，突变型Taq DNA聚合酶的聚合活性提高。突变体Mut1(G59W)、Mut4(D488K)、Mut5(G59W+D488K)、Mut6(S543T)、Mut8(E388K)、Mut9(E400R)、Mut10(E397Q)、Mut12(L549F)、Mut17(T664F)、Mut19(G59W+D488K+L549F)、Mut21(G59W+S577T)、Mut22(G59W+E388K)、Mut23(G59W+E400R)、Mut27(E400R+D488K)、Mut30(D488K+L549F)、Mut38(E388K+E397Q)、Mut42(E388K+T664F)、Mut43(S543T+T664F)、Mut45(E400R+T664F)、Mut58(L678I)、Mut63(E388K+L549F)聚合活性较野生型具有不同程度的提高。
实施例4 Taq DNA聚合酶及其突变体的多重PCR测试及分析
为了获得能够应用于多重PCR扩增的DNA聚合酶突变体，分别对纯化得到的野生型Taq DNA聚合酶及其突变体进行8重的人源持家基因扩增测试。
8重扩增的模板为人的基因组DNA(人gDNA)，使用DNA提取试剂盒(购自天根生化科技有限公司，货号DP304-02)从Expi293FTM细胞(购自赛默飞世尔科技公司，货号A14527)中提取获得，扩增引物及目的片段大小如下表7所示。
表7

人源持家基因8重扩增的PCR反应体系如下表8所示。
表8
人源持家基因8重扩增的PCR条件如下表9所示。
表9
人源持家基因8重扩增完成后，加入5μL 6x DNA Loading Buffer(60mM Tris-HCl，60mM EDTA，60％Glycerol，0.15％(m/V)OrangeG)后充分混匀后用2％的琼脂糖胶进行电泳分析，挑选出能扩增出所有条带或条带较野生型多、或产量提高、或均一性提高的突变体。而图中有些条带出现了拖尾现象，是由于存在某些非特异性扩增的现象，和DNA聚合酶的多重扩增能力没有关系，可以不必考虑。具体结果见图1、图2和图3，图中WT表示野生型DNA聚合酶(SEQ ID NO：1)对应的条带，M为marker条带。
图1、图2和图3结果表明，琼脂糖凝胶电泳图的扩增条带较亮或者扩增条带增多，表明相较于野生型的DNA聚合酶，突变型DNA聚合酶的多重扩增能力增强。突变体Mut5(G59W+D488K)、Mut6(S543T)、Mut9(E400R)、Mut11(T544Y)、Mut12(L549F)、 Mut19(G59W+D488K+L549F)、Mut20(G59W+S543T)、Mut21(G59W+S577T)和Mut23(G59W+E400R)应用于多重PCR中，扩增效果明显改善。
实施例5 Taq DNA聚合酶及其突变体的耐抑制能力测试
为了获得耐抑制能力提高的DNA聚合酶突变体，对纯化得到的野生型Taq DNA聚合酶及其突变体在含抑制剂的体系中进行PCR测试。PCR体系含抑制剂(12μM血红素或1.6μg/mL腐殖酸或1.2μg/mL单宁酸)。PCR体系具体成分如下表10所述。
表10
PCR反应条件如下表11所示。
表11
扩增完成后，加入5μL 6x DNA Loading Buffer后充分混匀后用2％的琼脂糖胶进行电泳分析，挑选出能扩增出目标条带的突变体。具体结果见附图4。其中，WT进行两组实验，一组不含抑制剂，一组同其他突变体一样含抑制剂。图中有些条带出现了拖尾现象，是由于存在某些非特异性扩增的现象，和DNA聚合酶的耐抑制能力没有关系，可以不必考虑。
图4结果表明，琼脂糖凝胶电泳图中出现扩增条带的，表明相较于野生型的Taq DNA聚合酶，突变型Taq DNA聚合酶的耐抑制能力增强。突变体Mut1(G59W)、Mut5(G59W+D488K)、Mut6(S543T)、Mut7(S577T)、Mut9(E400R)、Mut12(L549F)、Mut17(T664F)、Mut18(G59W+D488K+S543T)、Mut19(G59W+D488K+L549F)、Mut21(G59W+S577T)、Mut22(G59W+E388K)、Mut43(S543T+T664F)、Mut58(L678I)、Mut63(E388K+L549F)对至少一种抑制剂的耐受性提高。其中，Mut5(G59W+D488K)、Mut19(G59W+D488K+L549F)、Mut21(G59W+S577T)、Mut22(G59W+E388K)对三种抑制剂的耐受性均提高。
从以上的描述中，可以看出，本发明上述的实施例实现了如下技术效果：
本申请中公开的Taq DNA聚合酶突变体，具有增强的聚合活性；具有增强的耐抑制性能可以进行混有抑制物质模板的PCR反应，免于PCR反应前的模板提取、纯化等处理；上述DNA聚合酶突变体还能够进行多重PCR。利用上述DNA聚合酶突变体，相同时间内可以获得更多的产物，或进行混有抑制物质模板的PCR反应，免于PCR反应前的模板提取、纯化等处理，或进行多重PCR。此种突变体相较于野生型在DNA聚合活性、耐抑制能力、多重扩增方面显著提高。
以上所述仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (12)

  1. 一种DNA聚合酶突变体，其特征在于，所述DNA聚合酶突变体具有DNA聚合酶活性，包括与SEQ ID NO：1所示的氨基酸序列具有至少70％、至少75％、至少80％、至少85％、至少90％、至少95％或至少99％同源性的氨基酸序列。
  2. 根据权利要求1所述的DNA聚合酶突变体，其特征在于，所述DNA聚合酶突变体相对于SEQ ID NO：1所示的氨基酸序列具有以下至少一个位点的氨基酸替换：
    D488、S577、S543、E388、E400、E397、T544、L549、V586、L670、L678、T664和G59。
  3. 根据权利要求2所述的DNA聚合酶突变体，其特征在于，
    D488位点的氨基酸替换包括D488H、D488R或D488K；
    S543位点的氨基酸替换包括S543T；
    S577位点的氨基酸替换包括S577T；
    E388位点的氨基酸替换包括E388K；
    E400位点的氨基酸替换包括E400R；
    E397位点的氨基酸替换包括E397Q；
    T544位点的氨基酸替换包括T544Y、T544D、T544K、T544S、T544W或T544F；
    L549位点的氨基酸替换包括L549F、L549I、L549D或L549K；
    V586位点的氨基酸替换包括V586F；
    L670位点的氨基酸替换包括L670F；
    L678位点的氨基酸替换包括L678Y、L678W、L678I、L678F、L678K或L678D；
    T664位点的氨基酸替换包括T664F；
    G59位点的氨基酸替换包括G59W。
  4. 根据权利要求3所述的DNA聚合酶突变体，其特征在于，所述DNA聚合酶突变体的突变包括如下任意一种氨基酸突变组合：
    D488H、D488R、D488K、S577T、G59W、E388K、E397Q、E400R、S543T、T544Y、T544D、T544K、T544S、T544W、T544F、L549F、L549I、L549D、L549K、V586F、T664F、L670F、L678Y、L678K、L678D、L678I、L678F、G59W+E388K、G59W+E400R、G59W+D488K、G59W+S543T、G59W+S577T、E388K+E397Q、E388K+E400R、E388K+D488K、E388K+S543T、E388K+T544Y、E388K+L549F、E388K+S577T、E388K+L670F、E388K+T664F、E388K+L678Y、E397Q+D488K、 E397Q+T664F、E400R+D488K、E400R+T664F、D488K+S543T、D488K+T544Y、D488K+L549F、D488K+S577T、D488K+V586F、D488K+T664F、D488K+L670F、D488K+L678Y、S543T+T664F、S577T+T664F、V586F+T664F、T664F+L670F、T664F+L678W、T664F+L678Y、G59W+D488K+S543T或G59W+D488K+L549F；
    优选地，所述DNA聚合酶突变体的突变包括如下任意一种氨基酸突变组合：G59W、D488K、G59W+D488K、S543T、E388K、E400R、E397Q、L549F、T664F、G59W+D488K+L549F、G59W+S577T、G59W+E388K、G59W+E400R、E400R+D488K、D488K+L549F、E388K+E397Q、E388K+T664F、S543T+T664F、E400R+T664F、L678I或E388K+L549F，所述DNA聚合酶突变体的DNA聚合活性优于所述SEQ ID NO：1所示的氨基酸序列的所述DNA聚合活性；
    优选地，所述DNA聚合酶突变体的突变包括如下任意一种氨基酸突变组合：G59W+D488K、S543T、E400R、T544Y、L549F、G59W+D488K+L549F、G59W+S543T、G59W+S577T或G59W+E400R，所述DNA聚合酶突变体的多重扩增能力优于所述SEQ ID NO：1所示的氨基酸序列的所述多重扩增能力；
    优选地，所述DNA聚合酶突变体的突变包括如下任意一种氨基酸突变组合：G59W、G59W+D488K、S543T、S577T、E400R、L549F、T664F、G59W+D488K+S543T、G59W+D488K+L549F、G59W+S577T、G59W+E388K、S543T+T664F、L678I、E388K+L549F、G59W+D488K、G59W+D488K+L549F、G59W+S577T或G59W+E388K，所述DNA聚合酶突变体的耐PCR抑制剂能力优于所述SEQ ID NO：1所示的氨基酸序列的所述耐PCR抑制剂能力。
  5. 一种DNA分子，其特征在于，所述DNA分子包含编码权利要求1至4中任一项所述的DNA聚合酶突变体的多核苷酸。
  6. 一种重组载体，其特征在于，所述重组载体包含有权利要求5所述的DNA分子。
  7. 一种宿主细胞，其特征在于，所述宿主细胞内含有权利要求5所述的DNA分子或权利要求6所述的重组载体。
  8. 根据权利要求7所述的宿主细胞，其特征在于，所述宿主细胞包括原核细胞或真核细胞；
    优选地，所述原核细胞包括大肠杆菌。
  9. 一种PCR试剂盒，其特征在于，所述PCR试剂盒包括权利要求1至4中任一项所述的DNA聚合酶突变体。
  10. 根据权利要求9所述的PCR试剂盒，其特征在于，所述PCR试剂盒中还包括如下任意一种或多种成分：
    1)用于提供PCR扩增环境的缓冲液；
    2)PCR引物；
    3)用于提取目标DNA的试剂。
  11. 一种PCR方法，其特征在于，所述PCR方法包括：利用权利要求1至4中任一项所述的DNA聚合酶突变体或权利要求9或10所述的PCR试剂盒，对目标DNA进行PCR扩增。
  12. 根据权利要求11所述的PCR方法，其特征在于，所述PCR包括多重PCR扩增；
    优选地，所述PCR的体系中含有PCR抑制剂；
    更优选地，所述PCR抑制剂包括血红素、腐殖酸、单宁酸、EDTA、肝素、苯酚、十二烷基磺酸钠、氯高铁血红素、尿素、植物多糖、胆酸盐、聚苯乙烯、聚丙烯、蛋白酶、胆红素、溴酚蓝、钙离子和铁离子中的一种或多种。
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