WO2021127848A1 - 嵌合dna聚合酶及其应用 - Google Patents

嵌合dna聚合酶及其应用 Download PDF

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WO2021127848A1
WO2021127848A1 PCT/CN2019/127462 CN2019127462W WO2021127848A1 WO 2021127848 A1 WO2021127848 A1 WO 2021127848A1 CN 2019127462 W CN2019127462 W CN 2019127462W WO 2021127848 A1 WO2021127848 A1 WO 2021127848A1
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dna polymerase
peptide segment
homology
chimeric
terminus
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PCT/CN2019/127462
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English (en)
French (fr)
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安群
郭苗苗
席凤
郭斐
郑越
董宇亮
章文蔚
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深圳华大生命科学研究院
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Priority to PCT/CN2019/127462 priority Critical patent/WO2021127848A1/zh
Priority to CN201980102953.XA priority patent/CN114829593B/zh
Publication of WO2021127848A1 publication Critical patent/WO2021127848A1/zh

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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides

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  • the present invention relates to the field of biology. Specifically, the present invention relates to chimeric DNA polymerases and their applications.
  • DNA polymerase is an enzyme that uses a single strand of DNA as a template and four kinds of deoxynucleotides as a substrate to replicate and synthesize a new strand of DNA that is complementary to the template strand sequence from the 5'end.
  • DNA polymerase can add free nucleotides to the 3'end of the newly formed strand, resulting in the extension of the new strand in the 5' ⁇ 3' direction.
  • Some enzymes have 3' ⁇ 5' exonuclease activity, which can correct errors in newly synthesized DNA. If a mismatched base is generated during PCR amplification, it can cut it off.
  • Enzymes with correction functions have a lower error rate than ordinary DNA polymerases (such as Taq DNA polymerase), and are suitable for experiments that require high PCR fidelity, such as gene screening, sequencing, mutation detection, etc.
  • DNA polymerases are mainly divided into six families: A, B, C, D, X, and Y. At present, the heat-resistant DNA polymerases found all belong to the A family or the B family.
  • DNA polymerases belonging to the A family are derived from eubacteria, such as Taq (Thermus aquaticus), Tth (Thermus thermophilus), Tca (Thermus caldophilus), Tfl (Thermus flavus), Tfi (Thermus filiformis) and sources Bst (Bacillus stearothemophilis) of the genus Bacillus.
  • eubacteria such as Taq (Thermus aquaticus), Tth (Thermus thermophilus), Tca (Thermus caldophilus), Tfl (Thermus flavus), Tfi (Thermus filiformis) and sources Bst (Bacillus stearothemophilis) of the genus Bacillus.
  • the heat-resistant DNA polymerases belonging to the B family are all derived from archaea, such as Tli (Thermococcus litoralis) from Thermococcus, Pfu (Pyrococcus furiosus), KOD1 (Thermococcus kodacaraensis), Pwo (Pyrococcus woesei) , Tgo (Thermococcus gorgonarius) and Pab (Pyrococcus abyssi), etc.
  • archaea such as Tli (Thermococcus litoralis) from Thermococcus, Pfu (Pyrococcus furiosus), KOD1 (Thermococcus kodacaraensis), Pwo (Pyrococcus woesei) , Tgo (Thermococcus gorgonarius) and Pab (Pyrococcus abyssi), etc
  • the amino acid sequence of the polymerase is the basis of the functional structure of the polymerase. Based on the structure and function analysis of DNA polymerase, its various functions, such as catalytic activity, proofreading, nucleotide transfer, substrate binding, etc., have been assigned various domains. Taking archaeal DNA polymerase as an example, archaeal DNA polymerase is usually divided into five domains, namely N-terminal domain, exonucleic domain, palm domain, finger domain, and thumb domain. It is generally believed that the activity of DNA polymerase is related to the palm domain, finger domain and thumb domain.
  • the palm domain is the catalytic site of the polymerase; the thumb domain interacts with newly synthesized dsDNA and the introduced nucleotides; the finger domain is in the template Play a role in fixation and nucleotide specificity.
  • the exonucleodomain is related to 5' ⁇ 3' exonucleic activity, 3' ⁇ 5' exonucleic activity, or both, and is used to remove erroneously inserted bases.
  • the domains of DNA polymerase work closely with each other to complete the whole process of DNA amplification.
  • the present invention aims to solve at least one of the technical problems existing in the prior art at least to a certain extent.
  • the present invention proposes a chimeric DNA polymerase and a method for obtaining the same, isolated nucleic acids, constructs, recombinant cells or recombinant microorganisms, kits and applications thereof, the chimeric DNA polymerase has both high sustained synthesis ability , High elongation, thermal stability, strong resistance to salt and high fidelity performance, can meet the needs of DNA amplification, synthesis, detection, sequencing, etc., and has a wide range of application prospects.
  • DNA polymerase with correction function are offset by its relatively low continuous synthesis ability, and the yield of DNA amplification products is therefore reduced.
  • the Taq DNA polymerase in Family A DNA polymerase is represented by its high amplification efficiency but lacks fidelity.
  • DNA polymerases such as KOD/Pfu in Family B DNA polymerases are represented by DNA polymerases, which have disadvantages in both high sustained synthesis ability and high fidelity performance.
  • the inventors merged heterologous domains from different DNA polymerases to form a chimeric DNA polymerase.
  • the unique interactions within and between domains can easily form specific spatial structures and present corresponding functions. characteristic.
  • °N-7 (9°N) 7 DNA polymerases and 5 domains of each DNA polymerase were selected as the research objects, and they were screened with high sustained synthesis ability, high elongation, thermal stability, and salt resistance.
  • the chimeric DNA polymerase with high performance and high fidelity can meet the needs of DNA amplification, synthesis, detection, and sequencing, and has a wide range of application prospects.
  • the present invention proposes a chimeric DNA polymerase.
  • the chimeric DNA polymerase includes: a first peptide segment that has at least 80% homology with at least part of the amino acid sequence in the N-terminal domain of the KOD DNA polymerase
  • the second peptide segment, the N-terminus of the second peptide segment is connected to the C-terminus of the first peptide segment, and the second peptide segment has at least part of the amino acid sequence in the exonucleic acid domain of Pab DNA polymerase
  • the third peptide segment, the N-terminus of the third peptide segment is connected to the C-terminus of the second peptide segment, and the third peptide segment is in the N-terminal domain of KOD DNA polymerase
  • At least part of the amino acids of the fourth peptide fragment has at least 80% homology
  • the chimeric DNA polymerase may also have the following additional technical features:
  • the first peptide segment has at least 80% homology with the amino acid sequence at positions 1 to 130 of KOD DNA polymerase.
  • the second peptide segment has at least 80% homology with the 131-337 amino acid sequence of Pab DNA polymerase.
  • the third peptide segment has at least 80% homology with the amino acid sequence at positions 338 to 373 of KOD DNA polymerase.
  • the fourth peptide segment has at least 80% homology with the amino acid sequence at positions 374 to 448 of Pfu DNA polymerase.
  • the fifth peptide segment has at least 80% homology with the 449-500th amino acid sequence of Pab DNA polymerase.
  • the sixth peptide segment has at least 80% homology with the 501st to 591th amino acid sequence of Pfu DNA polymerase.
  • the seventh peptide has at least 80% homology with the amino acid sequence 591-774 of KOD DNA polymerase.
  • the chimeric DNA polymerase has an amino acid sequence shown in SEQ ID NO:1.
  • the invention provides an isolated nucleic acid.
  • the isolated nucleic acid encodes the aforementioned chimeric DNA polymerase.
  • the isolated nucleic acid according to the embodiments of the present invention can be used to encode a chimeric DNA polymerase with high sustained synthesis ability, high elongation, thermal stability, strong resistance to salt, and high fidelity. It meets the needs of DNA amplification, synthesis, detection, sequencing, etc., and has broad application prospects.
  • the isolated nucleic acid has a nucleotide sequence shown in SEQ ID NO: 2.
  • the present invention proposes a construct.
  • the construct contains the aforementioned isolated nucleic acid. Therefore, the constructs according to the embodiments of the present invention can express chimeric DNA polymerases that have high sustained synthesis ability, high elongation, thermal stability, strong salt resistance, high fidelity and other properties, and can satisfy DNA Amplification, synthesis, detection, sequencing and other needs, have a wide range of application prospects.
  • the present invention provides a recombinant cell or recombinant microorganism.
  • the recombinant cell or recombinant microorganism contains the aforementioned isolated nucleic acid.
  • the recombinant cell or recombinant microorganism according to the embodiment of the present invention can express a chimeric DNA polymerase that has high sustained synthesis ability, high elongation, thermal stability, strong salt resistance, high fidelity and other properties, and can It meets the needs of DNA amplification, synthesis, detection, sequencing, etc., and has broad application prospects.
  • the present invention provides a method for obtaining the aforementioned chimeric DNA polymerase.
  • the method includes: culturing the aforementioned recombinant cell or recombinant microorganism under conditions suitable for the expression of the chimeric DNA polymerase, so as to obtain the chimeric DNA polymerase.
  • the method according to the embodiments of the present invention can obtain a chimeric DNA polymerase with high sustained synthesis ability, high elongation, thermal stability, strong resistance to salt, and high fidelity, which can satisfy DNA amplification , Synthesis, detection, sequencing and other needs, has a wide range of application prospects.
  • the present invention provides a kit.
  • the kit contains the aforementioned chimeric DNA polymerase, the isolated nucleic acid, the construct, the recombinant cell or the recombinant microorganism. Therefore, the use of the kit according to the embodiment of the present invention to amplify DNA has the advantages of high amplification product yield and high amplification accuracy, and is suitable for wide production and application.
  • the present invention proposes the use of the aforementioned chimeric DNA polymerase, isolated nucleic acid, construct, recombinant cell or recombinant microorganism or kit in DNA amplification. Therefore, the amplified DNA has the advantages of high yield of amplified products and strong amplification accuracy, and is suitable for a wide range of production and applications.
  • the chimeric DNA polymerase, isolated nucleic acid, construct, recombinant cell or recombinant microorganism or kit is used for gene screening, sequencing or mutation detection.
  • Figure 1 shows a schematic diagram of the structure of a chimeric DNA polymerase according to an embodiment of the present invention
  • Figure 2 shows an enzyme expression and purification electrophoresis diagram according to an embodiment of the present invention
  • Figure 3 shows a comparison electrophoresis diagram of the amplification performance of fragments with different amplified lengths according to an embodiment of the invention
  • Figure 4 shows an electrophoresis diagram of thermal stability comparison according to an embodiment of the present invention
  • Fig. 5 shows a comparison electrophoresis diagram of amplification of different templates according to an embodiment of the present invention.
  • first and second are only used for descriptive purposes, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of indicated technical features. Thus, the features defined with “first” and “second” may explicitly or implicitly include one or more of these features. Further, in the description of the present invention, unless otherwise specified, “plurality” means two or more.
  • the present invention provides a chimeric DNA polymerase and a method for obtaining the same, isolated nucleic acids, constructs, recombinant cells or recombinant microorganisms, kits and applications thereof, which will be described in detail below.
  • the present invention proposes a chimeric DNA polymerase.
  • the chimeric DNA polymerase includes: a first peptide segment that has at least 80% homology with at least part of the amino acid sequence in the N-terminal domain of the KOD DNA polymerase; A dipeptide segment, the N-terminus of the second peptide segment is connected to the C-terminus of the first peptide segment, and the second peptide segment has at least 80% homology with at least part of the amino acid sequence in the exonucleic acid domain of Pab DNA polymerase; The third peptide segment, the N-terminus of the third peptide segment is connected to the C-terminus of the second peptide segment, and the third peptide segment has at least 80% homology with at least part of the amino acids in the N-terminal domain of KOD DNA polymerase; Four peptides, the N-terminal of the fourth peptide is connected to the C-terminal of the
  • the fifth peptide fragment has at least 80% homology with at least part of the amino acids in the finger domain of Pab DNA polymerase; the sixth peptide fragment, the sixth The N-terminus of the peptide is connected to the C-terminus of the fifth peptide.
  • the sixth peptide has at least 80% homology with at least part of the amino acids in the palm domain of Pfu DNA polymerase; the seventh peptide is the seventh peptide.
  • the N-terminus is connected to the C-terminus of the sixth peptide segment, and the seventh peptide segment has at least 80% homology with at least part of the amino acids in the thumb domain of KOD DNA polymerase.
  • thermostable family A and family B DNA polymerases among the six major types of DNA polymerases, and analyzed the amplification of each enzyme. Enhance performance characteristics and select chimeric preparations. After polymerase structure analysis, sequence analysis and consideration of amplification fidelity and other requirements, the amino acid sequences of the corresponding domains of the following 7 DNA polymerases are determined, and the selection is made through different chimerization of 2, 3, 4 or 5 enzymes. Potential new chimeric DNA polymerase.
  • the seven DNA polymerases are all archaea B family DNA polymerases, which are derived from Pyrococcus furiosus (Pfu), Thermococcus kodacaraensis (KOD), Pyrococcus woesei (Pwo), Thermococcus gorgonarius (Tgo), Pyrococcus abyssi (Pab), Pyrococcus species GB-D (Deep vent) and Thermococcus sp. 9°N-7(9°N).
  • Different combinations of 5 domains of the above 7 DNA polymerases are chimerized. That is, as a chimeric polymerase, there are 7 candidates for each domain. Therefore, there are (5 7 -7) chimeric modes in total for the chimeric enzymes.
  • the following optimization analysis was carried out in terms of performance: First, the performance characteristics of the above seven DNA polymerases were compared and analyzed. For example, Pfu and Pab DNA polymerases have better fidelity performance than other enzymes. Therefore, first consider that the exonucleic acid domain in the chimeric combination is the chimeric combination of the corresponding nucleoside sequences of the two DNA polymerases; similarly, according to the research and analysis of the amplification performance and thermal stability of each enzyme, the selection is suitable as a palm The candidate sequences of finger domain, thumb domain and N-terminal domain further narrow the chimeric combination.
  • the corresponding nucleotide sequence is obtained based on the amino acid sequence of the candidate chimeric polymerase, and constructed in an expression vector, the candidate chimeric polymerase is induced to express and purified in the expression cell, and the candidate chimeric polymerase is compared by comparing each chimeric polymerase.
  • the expression performance, fidelity, thermal stability, and synthesis ability of the enzyme are finally screened to obtain the optimal chimeric DNA polymerase of the present invention.
  • the structure of the chimeric DNA polymerase is shown in Figure 1. It combines high sustained synthesis ability, high elongation, thermal stability, strong salt resistance and high fidelity performance, which can meet DNA amplification, synthesis, detection, and sequencing. And other needs, has a wide range of application prospects.
  • Pab DNA polymerase amino acid sequence (SEQ ID NO: 3):
  • the first peptide segment has at least 80% homology with the amino acid sequence of positions 1 to 130 of KOD DNA polymerase.
  • the amino acid sequence of positions 1 to 130 of KOD DNA polymerase is the first gray marker sequence in the sequence shown in SEQ ID NO: 5, which is a partial sequence in the N-terminal domain of KOD DNA polymerase.
  • the second peptide segment has at least 80% homology with the 131-337th amino acid sequence of Pab DNA polymerase.
  • the amino acid sequence at positions 131 to 337 of Pab DNA polymerase is the first gray marker sequence in the sequence shown in SEQ ID NO: 3, which is a partial sequence in the exonucleic domain of Pab DNA polymerase.
  • the third peptide segment has at least 80% homology with the 338-373 amino acid sequence of KOD DNA polymerase.
  • the amino acid sequence at positions 338 to 373 of KOD DNA polymerase is the second gray marker sequence in the sequence shown in SEQ ID NO: 5, which is a partial sequence in the N-terminal domain of KOD DNA polymerase.
  • the fourth peptide segment has at least 80% homology with the amino acid sequence at positions 374 to 448 of Pfu DNA polymerase.
  • the amino acid sequence at positions 374 to 448 of Pfu DNA polymerase is the first gray marker sequence in the sequence shown in SEQ ID NO: 4, which is a partial sequence in the palm domain of Pfu DNA polymerase.
  • the fifth peptide segment has at least 80% homology with the 449-500th amino acid sequence of Pab DNA polymerase.
  • the amino acid sequence at positions 449 to 500 of the Pab DNA polymerase is the second gray marker sequence in the sequence shown in SEQ ID NO: 3, which is a partial sequence of the Pab DNA polymerase finger domain.
  • the sixth peptide segment has at least 80% homology with the amino acid sequence at positions 501 to 591 of Pfu DNA polymerase.
  • the amino acid sequence at positions 501 to 591 of Pfu DNA polymerase is the second gray marker sequence in the sequence shown in SEQ ID NO: 4, which is a partial sequence in the palm domain of Pfu DNA polymerase.
  • the seventh peptide has at least 80% homology with the amino acid sequence 591-774 of KOD DNA polymerase.
  • the amino acid sequence at positions 591 to 774 of KOD DNA polymerase is the third gray marker sequence in the sequence shown in SEQ ID NO: 5, which is a partial sequence in the thumb domain of KOD DNA polymerase.
  • the chimeric DNA polymerase obtained has high continuous synthesis ability, high elongation, thermal stability, strong resistance to salt, and high fidelity, and can meet the needs of DNA amplification, synthesis, detection, and sequencing. , Has a wide range of application prospects.
  • the chimeric DNA polymerase has an amino acid sequence shown in SEQ ID NO:1. Therefore, the chimeric DNA polymerase according to the embodiment of the present invention has the performances of high continuous synthesis ability, high elongation, thermal stability, strong salt resistance and high fidelity, and can satisfy DNA amplification, synthesis, and detection. , Sequencing and other needs, has a wide range of application prospects.
  • the invention provides an isolated nucleic acid.
  • the isolated nucleic acid encodes the aforementioned chimeric DNA polymerase.
  • the isolated nucleic acid according to the embodiments of the present invention can be used to encode a chimeric DNA polymerase with high sustained synthesis ability, high elongation, thermal stability, strong resistance to salt, and high fidelity. It meets the needs of DNA amplification, synthesis, detection, sequencing, etc., and has broad application prospects.
  • the nucleic acid encoding the first peptide segment has at least 80% homology with the nucleotide sequence 1 to 390 of KOD DNA polymerase.
  • the first to 390th position of the nucleotide sequence encoding KOD DNA polymerase is the first gray marker sequence in the sequence shown in SEQ ID NO: 8, which is part of the sequence encoding the N-terminal domain of KOD DNA polymerase .
  • the nucleic acid encoding the second peptide segment has at least 80% homology with the 391st-1011th nucleotide sequence of Pab DNA polymerase.
  • the 391st-1011th sequence of the nucleotide sequence encoding Pab DNA polymerase is the first gray marker sequence in the sequence shown in SEQ ID NO: 6, which is the partial sequence encoding the Pab DNA polymerase exonucleic acid domain .
  • the nucleic acid encoding the third peptide segment has at least 80% homology with the 1012 to 1119 nucleotide sequence of KOD DNA polymerase.
  • the 1012 to 1119th sequence of the nucleotide sequence encoding KOD DNA polymerase is the second gray marker sequence in the sequence shown in SEQ ID NO: 8, which is part of the sequence encoding the N-terminal domain of KOD DNA polymerase .
  • the nucleic acid encoding the fourth peptide segment has at least 80% homology with the 1120 to 1344 nucleotide sequence of Pfu DNA polymerase.
  • the sequence at positions 1120 to 1344 of the nucleotide sequence encoding Pfu DNA polymerase is the first gray marker sequence in the sequence shown in SEQ ID NO: 7, which is a partial sequence encoding the palm domain of Pfu DNA polymerase.
  • the nucleic acid encoding the fifth peptide segment has at least 80% homology with the nucleotide sequence 1345 to 1500 of Pab DNA polymerase.
  • the sequence at positions 1345 to 1500 of the nucleotide sequence encoding Pab DNA polymerase is the second gray marker sequence in the sequence shown in SEQ ID NO: 6, which is a partial sequence in the finger domain encoding Pab DNA polymerase.
  • the nucleic acid encoding the sixth peptide segment has at least 80% homology with the 1501-1773 nucleotide sequence of Pfu DNA polymerase.
  • the 1501 to 1773 sequence of the nucleotide sequence encoding Pfu DNA polymerase is the second gray marker sequence in the sequence shown in SEQ ID NO: 7, which is a partial sequence encoding the Pfu DNA polymerase palm domain.
  • the nucleic acid encoding the seventh peptide segment has at least 80% homology with the 1771st to 2325th nucleotide sequence of KOD DNA polymerase.
  • the 1771st to 2325th sequence of the nucleotide sequence encoding KOD DNA polymerase is the third gray marker sequence in the sequence shown in SEQ ID NO: 8, which is a partial sequence of the thumb domain encoding KOD DNA polymerase.
  • the chimeric DNA polymerase obtained has high continuous synthesis ability, high elongation, thermal stability, strong resistance to salt, and high fidelity, and can meet the needs of DNA amplification, synthesis, detection, and sequencing. , Has a wide range of application prospects.
  • the isolated nucleic acid has a nucleotide sequence shown in SEQ ID NO: 2.
  • Pab DNA polymerase nucleotide sequence (SEQ ID NO: 6):
  • Pfu DNA polymerase nucleotide sequence (SEQ ID NO: 7):
  • the present invention proposes a construct.
  • the construct contains the aforementioned isolated nucleic acid. Therefore, the constructs according to the embodiments of the present invention can express chimeric DNA polymerases that have high sustained synthesis ability, high elongation, thermal stability, strong salt resistance, high fidelity and other properties, and can satisfy DNA Amplification, synthesis, detection, sequencing and other needs, have a wide range of application prospects.
  • the present invention provides a recombinant cell or recombinant microorganism.
  • the recombinant cell or recombinant microorganism contains the aforementioned isolated nucleic acid.
  • the recombinant cell or recombinant microorganism according to the embodiment of the present invention can express a chimeric DNA polymerase that has high sustained synthesis ability, high elongation, thermal stability, strong salt resistance, high fidelity and other properties, and can It meets the needs of DNA amplification, synthesis, detection, sequencing, etc., and has a wide range of application prospects.
  • the present invention provides a method for obtaining the aforementioned chimeric DNA polymerase.
  • the method includes: culturing the aforementioned recombinant cell or recombinant microorganism under conditions suitable for the expression of the chimeric DNA polymerase, so as to obtain the chimeric DNA polymerase.
  • the method according to the embodiments of the present invention can obtain a chimeric DNA polymerase with high sustained synthesis ability, high elongation, thermal stability, strong resistance to salt, and high fidelity, which can satisfy DNA amplification , Synthesis, detection, sequencing and other needs, has a wide range of application prospects.
  • the present invention provides a kit.
  • the kit contains the aforementioned chimeric DNA polymerase, isolated nucleic acid, construct, recombinant cell or recombinant microorganism. Therefore, the use of the kit according to the embodiment of the present invention to amplify DNA has the advantages of high amplification product yield and strong amplification accuracy, and is suitable for a wide range of production and applications.
  • kit of the present invention may also contain other reagents commonly used in the art, such as buffers, primers, nucleoside triphosphates, etc., which can be flexibly selected according to actual conditions, and the present invention is not strictly limited.
  • the present invention proposes the use of the aforementioned chimeric DNA polymerase, isolated nucleic acid, construct, recombinant cell or recombinant microorganism or kit in DNA amplification. Therefore, the amplified DNA has the advantages of high yield of amplified products and strong amplification accuracy, and is suitable for a wide range of production and applications.
  • the chimeric DNA polymerase, isolated nucleic acid, construct, recombinant cell or recombinant microorganism or kit is used for gene screening, sequencing or mutation detection. Because the above-mentioned DNA polymerase has the properties of high continuous synthesis ability, high elongation, thermal stability, strong resistance to salt, and high fidelity, it can be effectively applied to genes that require high amplification yield and fidelity. Screening, sequencing or mutation detection.
  • Example 1 Design and construction of chimeric DNA polymerase
  • Pfu, Pab and KOD DNA Polymerase with different phenotypic characteristics, in: thermal stability, and having in all fidelity DNA polymerase, the lowest probability of error, Pfu DNA Polymerase error rate of about 2.0 ⁇ 10 - 6 .
  • Pab DNA polymerase also exhibits a low probability of amplification errors and has higher thermal stability.
  • KOD is a DNA polymerase with high amplification ability. The amplification speed is 2 times that of Taq DNA polymerase and 6 times that of Pfu DNA polymerase, and the amplification yield is higher ( ⁇ 300nts).
  • the chimeric DNA polymerase used in this experiment is a chimeric combination of Pfu, Pab and KOD DNA polymerase (shown in Figure 1). Specifically, the nucleotide sequence of the N-terminal domain of the KOD enzyme (1 ⁇ 390 and 1012 ⁇ 1119) ) And thumb domain nucleotide sequence (1771 ⁇ 2325), Pab exonucleic acid domain nucleotide sequence (391 ⁇ 1011) and finger domain sequence (1345 ⁇ 1500), Pfu palm domain nucleotide sequence (1120 ⁇ 1344 and 1501 ⁇ 1773) were constructed between the XhoI/BamHI restriction sites of the prokaryotic expression vector pET28a, and the vector carrying the above nucleotide sequence was transformed into E. coli BL21 (DE3), and the expression was obtained after culture Strains.
  • the chimeric DNA polymerase exhibits a high amplification ability similar to that of KOD, and has a lower probability of amplification mismatch than KOD.
  • Fermentation cell processing the mass volume ratio of cell weight (g) to cell suspension A (ml) (20mM Tris, 300mM NaCl, 20mM Imidazole, 5% Glycerol, pH8.0) is 1:20 weight Suspend the bacteria, and ultrasonically disrupt them. The crushed supernatant was collected by centrifugation at 12000 rpm for 20 minutes, and after denaturation in a water bath at 75°C for 30 minutes, the supernatant was recovered by centrifugation at 12000 rpm for 20 minutes.
  • Ni column purification filter the recovered supernatant through a 0.22 ⁇ m filter device. After washing the balanced Ni column with bacterial resuspension solution A, inject the above 0.22 ⁇ m filter solution, adjust the concentration of imidazole in the eluent (20mM Tris, 300mM NaCl, 5% Glycerol, 500mM Imidazole, pH 8.0) for gradient washing After removal, the fraction from the column was collected, and the active fraction was analyzed by SDS-PAGE. The fractions where the pure target protein was observed on the Coomassie-stained SDS-PAGE gel were combined.
  • Anion column purification combine the above fractions and pass the sample through the ion column to control the endonuclease residue and nucleic acid residue in the sample.
  • the combined samples were dialyzed into Buffer C (20mM Tris, 100mM NaCl, 5% Glycerol, pH 8.0), and the concentration of salt ions in Buffer D (20mM Tris, 500 mM NaCl, 5% Glycerol, pH 8.0) was adjusted for gradient Elution, collecting the elution column fraction is the new chimeric DNA polymerase.
  • the obtained sample was dialyzed to a preservation system (20mM Tris, 200mM KCl, 50% Glycerol, 0.2mM EDTA, 2mM DTT, 0.001% Tween20, 0.001% NP40, pH 8.0).
  • the purified enzymes are shown in Figure 2, and all target proteins with higher purity were obtained.
  • Example 3 Amplification performance of chimeric DNA polymerase
  • the chimeric DNA polymerase obtained in Example 2 of the present invention is amplified using lambda DNA as a template, and the amplified fragment is 2Kb-8Kb.
  • the extension time of Pfu DN polymerase is 60s/kb
  • the extension time of Pab, KOD and chimeric enzymes is 30s/kb.
  • the primer sequences used are as follows:
  • lam2K-R CGTCTGTTCATCGTCGTGGCGGCCCATAATAATCT (SEQ ID NO: 10)
  • lam8K-R CGGGAATACGACGGTTACCCACCACAAGCACG (SEQ ID NO: 12)
  • the LacIQZ a gene was amplified with Pab DNA polymerase, Pfu DNA polymerase, KOD DNA polymerase, and chimeric enzymes, and the amplified fragments and vectors were digested with XbaI/NcoI.
  • the vector fragments were enzymatically connected, the ligation mixture was transformed into E. coli DH5a and the cells were inoculated on LB-Amp-X-gal plates for culture. Count the number of blue colonies, white colonies, and the total number of colonies, and calculate the fidelity.
  • the sequence of LacIQZ a gene amplification primer is as follows:
  • Lac-F GTTTTCCCAGTCACGAC (SEQ ID NO: 13)
  • Lac-R GGTATCTTTATAGTCCTGTCG (SEQ ID NO: 14)
  • the fidelity calculation formula is:
  • the amplification primer sequence is as follows:
  • Ecoli-F AGAGTTTGATCMTGGCTCAG (SEQ ID NO: 15)
  • Ecoli-R CGGTTACCTTGTTACGACTT (SEQ ID NO: 16)
  • Example 6 Amplification of chimeric DNA polymerase under low template conditions
  • E. coli gDNA 10ng, 1ng, and 100pg of E. coli gDNA were added to the amplification system respectively, and amplified with Pfu, Pab, KOD and chimeric enzymes, and the PCR products were analyzed by agarose gel.
  • Pfu, Pab, KOD and chimeric enzymes 10ng, 1ng, and 100pg of E. coli gDNA were added to the amplification system respectively, and amplified with Pfu, Pab, KOD and chimeric enzymes, and the PCR products were analyzed by agarose gel.
  • Example 3 for the amplification system and procedures, and refer to Example 5 for the sequence of the amplification primers. The results are shown in Figure 5.
  • the chimeric enzyme can amplify the target product, and the yield of the target product is better.

Abstract

提供嵌合DNA聚合酶，其包括：第一肽段，所述第一肽段与KOD DNA聚合酶的N-端域中的至少部分氨基酸序列具有至少80％同源性；第二肽段，所述第二肽段与Pab DNA聚合酶的核酸外切域中的至少部分氨基酸序列具有至少80％同源性；第三肽段，所述第三肽段与KOD DNA聚合酶的N-端域中的至少部分氨基酸具有至少80％同源性；第四肽段，所述第四肽段与Pfu DNA聚合酶的掌域中的至少部分氨基酸具有至少80％同源性；第五肽段，所述第五肽段与Pab DNA聚合酶的指域中的至少部分氨基酸具有至少80％同源性；第六肽段，所述第六肽段与Pfu DNA聚合酶的掌域中的至少部分氨基酸具有至少80％同源性；第七肽段，所述第七肽段与KOD DNA聚合酶的拇指域中的至少部分氨基酸具有至少80％同源性。

Description

嵌合DNA聚合酶及其应用 技术领域
本发明涉及生物领域。具体地，本发明涉及嵌合DNA聚合酶及其应用。
背景技术
DNA聚合酶是以DNA单链为模板，以4种脱氧核苷酸为底物，由5’端开始复制合成一条与模板链序列互补的DNA新链的酶。DNA聚合酶可以将游离核苷酸添加到新形成链的3’端，从而导致新链在5’→3’方向的延伸。某些酶具有3’→5’核酸外切酶的活性，可以校正新合成DNA中的错误。PCR扩增过程中如果产生了错配碱基，它可以将其切掉，在错误碱基切除以后，聚合酶可以重新插入正确的碱基并继续复制，从而保证了扩增的准确性。具有校正功能的酶比普通DNA聚合酶(如Taq DNA聚合酶)具有更低的错误率，它适合对PCR保真性要求较高的实验，如基因筛选、测序、突变检测等。
DNA聚合酶主要分为六个家族：A、B、C、D、X以及Y。目前，发现的耐热DNA聚合酶均属于A家族或B家族。属于A家族DNA聚合酶均来源于真细菌，如来源于栖热属的Taq(Thermus aquaticus)、Tth(Thermus thermophilus)、Tca(Thermus caldophilus)、Tfl(Thermus flavus)、Tfi(Thermus filiformis)以及来源于芽孢菌属的Bst(Bacillus stearothemophilis)。属于B家族的耐热DNA聚合酶均来源于古细菌，如来源于高温球菌属的Tli(Thermococcus litoralis)、焦热球菌属的Pfu(Pyrococcus furiosus)、KOD1(Thermococcus kodacaraensis)、Pwo(Pyrococcus woesei)、Tgo(Thermococcus gorgonarius)以及Pab(Pyrococcus abyssi)等。
聚合酶的氨基酸序列是聚合酶功能结构的基础。基于DNA聚合酶的结构功能分析，其各种功能，如催化活性、校对、核苷酸转移、底物结合等，已分配有各种结构域。以古菌DNA聚合酶为例，古菌DNA聚合酶通常分为5个结构域，分别为N-端域、核酸外切域、掌域、指域及拇指域。一般认为，DNA聚合酶活性与掌域、指域及拇指域有关，掌域是聚合酶的催化部位；拇指域与新合成的dsDNA相互作用并与引入的核苷酸相互作用；指域在模板固定和核苷酸特异性中发挥作用。核酸外切域与5’→3’核酸外切活性、3’→5’核酸外切活性或两者有关，用来除去错误插入的碱基。DNA聚合酶各域彼此紧密配合以完成DNA扩增全过程。
然而，目前的DNA聚合酶仍有待研究。
发明内容
本发明旨在至少在一定程度上解决现有技术中存在的技术问题至少之一。为此，本发明提出了一种嵌合DNA聚合酶及其获得方法、分离的核酸、构建体、重组细胞或者重组微生物、试剂盒及其应用，该嵌合DNA聚合酶兼具高持续合成能力、高延伸率、热稳定性、对盐的抗性强以及高保真性等性能，可以满足DNA扩增、合成、检测、测序等需要，具有广泛的应用前景。
需要说明的是，本发明是基于发明人的下列发现而完成：
目前，具有校正功能的DNA聚合酶的优点被它的相对低的持续合成能力所抵消，DNA扩增产物的产率因而降低。以家族A DNA聚合酶中的Taq DNA聚合酶为代表，其扩增效率较高，但缺乏保真性。以家族B DNA聚合酶中的KOD/Pfu等DNA聚合酶为代表，其在同时兼具高持续合成能力及高保真性能中具有劣势。
有鉴于此，发明人合并来自不同DNA聚合酶的异源域以形成嵌合DNA聚合酶，利用不同异源域融合时各域内及域间独特的相互作用易形成特异空间结构并呈现相应的功能特性。具体地，发明人以Pyrococcus furiosus(Pfu)、Thermococcus kodacaraensis(KOD)，Pyrococcus woesei(Pwo)、Thermococcus gorgonarius(Tgo)、Pyrococcus abyssi(Pab)、Pyrococcus species GB-D(Deep vent)及Thermococcus sp.9°N-7(9°N)7种DNA聚合酶及每种DNA聚合酶5种结构域作为研究对象，筛选出了兼具高持续合成能力、高延伸率、热稳定性、对盐的抗性强以及高保真性等性能的嵌合DNA聚合酶，可以满足DNA扩增、合成、检测、测序等需要，具有广泛的应用前景。
为此，在本发明的一个方面，本发明提出了一种嵌合DNA聚合酶。根据本发明的实施例，所述嵌合DNA聚合酶包括：第一肽段，所述第一肽段与KOD DNA聚合酶的N-端域中的至少部分氨基酸序列具有至少80％同源性；第二肽段，所述第二肽段的N端与所述第一肽段的C端相连，所述第二肽段与Pab DNA聚合酶的核酸外切域中的至少部分氨基酸序列具有至少80％同源性；第三肽段，所述第三肽段的N端与所述第二肽段的C端相连，所述第三肽段与KOD DNA聚合酶的N-端域中的至少部分氨基酸具有至少80％同源性；第四肽段，所述第四肽段的N端与所述第三肽段的C端相连，所述第四肽段与Pfu DNA聚合酶的掌域中的至少部分氨基酸具有至少80％同源性；第五肽段，所述第五肽段的N端与所述第四肽段的C端相连，所述第五肽段与Pab DNA聚合酶的指域中的至少部分氨基酸具有至少80％同源性；第六肽段，所述第六肽段的N端与所述第五肽段的C端相连，所述第六肽段与Pfu DNA聚合酶的掌域中的至少部分氨基酸具有至少80％同源性；第七肽段，所述第七肽段的N端与所述第六肽段的C端相连，所述第七肽段与KOD DNA聚合酶的拇指域中 的至少部分氨基酸具有至少80％同源性。
发明人以Pyrococcus furiosus(Pfu)、Thermococcus kodacaraensis(KOD)，Pyrococcus woesei(Pwo)、Thermococcus gorgonarius(Tgo)、Pyrococcus abyssi(Pab)、Pyrococcus species GB-D(Deep vent)及Thermococcus sp.9°N-7(9°N)7种DNA聚合酶及每种DNA聚合酶5种结构域作为研究对象，筛选出了兼具高持续合成能力、高延伸率、热稳定性、对盐的抗性强以及高保真性等性能的嵌合DNA聚合酶，可以满足DNA扩增、合成、检测、测序等需要，具有广泛的应用前景。
根据本发明的实施例，嵌合DNA聚合酶还可以具有下列附加技术特征：
根据本发明的实施例，所述第一肽段与KOD DNA聚合酶的第1～130位氨基酸序列具有至少80％同源性。
根据本发明的实施例，所述第二肽段与Pab DNA聚合酶的第131～337位氨基酸序列具有至少80％同源性。
根据本发明的实施例，所述第三肽段与KOD DNA聚合酶的第338～373位氨基酸序列具有至少80％同源性。
根据本发明的实施例，所述第四肽段与Pfu DNA聚合酶的第374～448位氨基酸序列具有至少80％同源性。
根据本发明的实施例，所述第五肽段与Pab DNA聚合酶的第449～500位氨基酸序列具有至少80％同源性。
根据本发明的实施例，所述第六肽段与Pfu DNA聚合酶的第501～591位氨基酸序列具有至少80％同源性。
根据本发明的实施例，所述第七肽段与KOD DNA聚合酶的第591～774位氨基酸序列具有至少80％同源性。
根据本发明的实施例，所述嵌合DNA聚合酶具有SEQ ID NO：1所示的氨基酸序列。
在本发明的另一方面，本发明提出了一种分离的核酸。根据本发明的实施例，所述分离的核酸编码前面所述嵌合DNA聚合酶。由此，利用根据本发明实施例的分离的核酸可以编码得到兼具高持续合成能力、高延伸率、热稳定性、对盐的抗性强以及高保真性等性能的嵌合DNA聚合酶，能够满足DNA扩增、合成、检测、测序等需要，具有广泛的应用前景。
根据本发明的实施例，所述分离的核酸具有SEQ ID NO：2所示的核苷酸序列。
在本发明的又一方面，本发明提出了一种构建体。根据本发明的实施例，所述构建体含有前面所述分离的核酸。由此，利用根据本发明实施例的构建体可以表达兼具高持续合 成能力、高延伸率、热稳定性、对盐的抗性强以及高保真性等性能的嵌合DNA聚合酶，能够满足DNA扩增、合成、检测、测序等需要，具有广泛的应用前景。
在本发明的又一方面，本发明提出了一种重组细胞或者重组微生物。根据本发明的实施例，所述重组细胞或者重组微生物含有前面所述分离的核酸。由此，根据本发明实施例的重组细胞或者重组微生物可以表达兼具高持续合成能力、高延伸率、热稳定性、对盐的抗性强以及高保真性等性能的嵌合DNA聚合酶，能够满足DNA扩增、合成、检测、测序等需要，具有广泛的应用前景。
在本发明的又一方面，本发明提出了一种获得前面所述嵌合DNA聚合酶的方法。根据本发明的实施例，所述方法包括：在适于所述嵌合DNA聚合酶表达的条件下，培养前面所述的重组细胞或者重组微生物，以便获得所述嵌合DNA聚合酶。由此，根据本发明实施例的方法可以获得兼具高持续合成能力、高延伸率、热稳定性、对盐的抗性强以及高保真性等性能的嵌合DNA聚合酶，能够满足DNA扩增、合成、检测、测序等需要，具有广泛的应用前景。
在本发明的又一方面，本发明提出了一种试剂盒。根据本发明的实施例，所述试剂盒含有前面所述嵌合DNA聚合酶、所述分离的核酸、所述构建体、所述重组细胞或者重组微生物。由此，利用根据本发明实施例的试剂盒扩增DNA具有扩增产物产率高和扩增准确性强等优点，适于广泛生产和应用。
在本发明的又一方面，本发明提出了前面所述嵌合DNA聚合酶、分离的核酸、构建体、重组细胞或者重组微生物或试剂盒在DNA扩增中的用途。由此，扩增DNA具有扩增产物产率高和扩增准确性强等优点，适于广泛生产和应用。
根据本发明的实施例，所述嵌合DNA聚合酶、分离的核酸、构建体、重组细胞或者重组微生物或者试剂盒用于基因筛选、测序或突变检测。
本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本发明的实践了解到。
附图说明
本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中：
图1显示了根据本发明一个实施例的嵌合DNA聚合酶的结构示意图；
图2显示了根据本发明一个实施例的酶表达纯化电泳图；
图3显示了根据本发明一个实施例的不同扩增长度片段扩增性能比较电泳图；
图4显示了根据本发明一个实施例的热稳定性比较电泳图；
图5显示了根据本发明一个实施例的不同模板扩增比较电泳图。
具体实施方式
下面详细描述本发明的实施例。下面描述的实施例是示例性的，仅用于解释本发明，而不能理解为对本发明的限制。
需要说明的是，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括一个或者更多个该特征。进一步地，在本发明的描述中，除非另有说明，“多个”的含义是两个或两个以上。
本发明提出了嵌合DNA聚合酶及其获得方法、分离的核酸、构建体、重组细胞或者重组微生物、试剂盒及其应用，下面将分别对其进行详细描述。
嵌合DNA聚合酶
在本发明的一个方面，本发明提出了嵌合DNA聚合酶。根据本发明的实施例，该嵌合DNA聚合酶包括：第一肽段，该第一肽段与KOD DNA聚合酶的N-端域中的至少部分氨基酸序列具有至少80％同源性；第二肽段，该第二肽段的N端与第一肽段的C端相连，第二肽段与Pab DNA聚合酶的核酸外切域中的至少部分氨基酸序列具有至少80％同源性；第三肽段，第三肽段的N端与第二肽段的C端相连，第三肽段与KOD DNA聚合酶的N-端域中的至少部分氨基酸具有至少80％同源性；第四肽段，第四肽段的N端与第三肽段的C端相连，第四肽段与Pfu DNA聚合酶的掌域中的至少部分氨基酸具有至少80％同源性；第五肽段，第五肽段的N端与第四肽段的C端相连，第五肽段与Pab DNA聚合酶的指域中的至少部分氨基酸具有至少80％同源性；第六肽段，第六肽段的N端与第五肽段的C端相连，第六肽段与Pfu DNA聚合酶的掌域中的至少部分氨基酸具有至少80％同源性；第七肽段，第七肽段的N端与第六肽段的C端相连，第七肽段与KOD DNA聚合酶的拇指域中的至少部分氨基酸具有至少80％同源性。
发明人为了研究出具有高持续合成能力及改善保真性能的DAN聚合酶，首先，在6大类DNA聚合酶中，聚焦具有热稳定性的家族A及家族B DNA聚合酶，分析各酶扩增性能特点并挑选嵌合备体。经聚合酶结构分析、序列分析并考虑扩增保真性等需求，确定由以下7种DNA聚合酶的相应各域氨基酸序列，通过2种、3种、4种或5种酶不同嵌合以筛选具有潜能的新型嵌合DNA聚合酶。7种DNA聚合酶均是古细菌B家族DNA聚合酶，分别来源于Pyrococcus furiosus(Pfu)、Thermococcus kodacaraensis(KOD)，Pyrococcus woesei (Pwo)、Thermococcus gorgonarius(Tgo)、Pyrococcus abyssi(Pab)、Pyrococcus species GB-D(Deep vent)及Thermococcus sp.9°N-7(9°N)。上述7种DNA聚合酶5种结构域的不同组合嵌合。即作为嵌合聚合酶的每个结构域均有7种候选，因此，构成的嵌合酶总共有(5 7-7)种嵌合方式。
为了进一步缩小上述嵌合库，从性能方面进行了如下优化分析：首先，比较分析上述7种DNA聚合酶的性能特点，如Pfu、Pab两种DNA聚合酶在保真性能方面较其他酶更具优势，因此首先考虑嵌合组合中核酸外切域为该两种DNA聚合酶相应核苷序列的嵌合组合；同样地，依据各酶扩增性能、热稳定性的研究分析，筛选适宜作为掌指域、拇指域及N-端域的候选序列，进一步缩小嵌合组合。通过上述方式的筛选，并基于候选嵌合聚合酶氨基酸序列获得相应的核苷酸序列，并构建于表达载体中，于表达菌体中诱导表达并纯化候选嵌合聚合酶，通过比较各嵌合酶的表达性能、保真性、热稳定性、合成能力，最终筛选获得本发明最优的嵌合DNA聚合酶。该嵌合DNA聚合酶结构参见图1，其兼具高持续合成能力、高延伸率、热稳定性、对盐的抗性强以及高保真性等性能，可以满足DNA扩增、合成、检测、测序等需要，具有广泛的应用前景。
Pab DNA聚合酶氨基酸序列(SEQ ID NO：3)：
Figure PCTCN2019127462-appb-000001
Pfu DNA聚合酶氨基酸序列(SEQ ID NO：4)：
Figure PCTCN2019127462-appb-000002
Figure PCTCN2019127462-appb-000003
KOD DNA聚合酶氨基酸序列(SEQ ID NO：5)：
Figure PCTCN2019127462-appb-000004
根据本发明的实施例，第一肽段与KOD DNA聚合酶的第1～130位氨基酸序列具有至少80％同源性。KOD DNA聚合酶的第1～130位氨基酸序列为SEQ ID NO：5所示序列中的第一段灰色标记序列，即为KOD DNA聚合酶N-端域中的部分序列。
根据本发明的实施例，第二肽段与Pab DNA聚合酶的第131～337位氨基酸序列具有至少80％同源性。Pab DNA聚合酶的第131～337位氨基酸序列为SEQ ID NO：3所示序列中的第一段灰色标记序列，即为Pab DNA聚合酶核酸外切域中的部分序列。
根据本发明的实施例，第三肽段与KOD DNA聚合酶的第338～373位氨基酸序列具有至少80％同源性。KOD DNA聚合酶的第338～373位氨基酸序列为SEQ ID NO：5所示序列中的第二段灰色标记序列，即为KOD DNA聚合酶N-端域中的部分序列。
根据本发明的实施例，第四肽段与Pfu DNA聚合酶的第374～448位氨基酸序列具有至少80％同源性。Pfu DNA聚合酶的第374～448位氨基酸序列为SEQ ID NO：4所示序列中的第一段灰色标记序列，即为Pfu DNA聚合酶的掌域中的部分序列。
根据本发明的实施例，第五肽段与Pab DNA聚合酶的第449～500位氨基酸序列具有至少80％同源性。Pab DNA聚合酶的第449～500位氨基酸序列为SEQ ID NO：3所示序列中的第二段灰色标记序列，即为Pab DNA聚合酶指域中的部分序列。
根据本发明的实施例，第六肽段与Pfu DNA聚合酶的第501～591位氨基酸序列具有至少80％同源性。Pfu DNA聚合酶的第501～591位氨基酸序列为SEQ ID NO：4所示序列中的第二段灰色标记序列，即为Pfu DNA聚合酶的掌域中的部分序列。
根据本发明的实施例，第七肽段与KOD DNA聚合酶的第591～774位氨基酸序列具有至少80％同源性。KOD DNA聚合酶的第591～774位氨基酸序列为SEQ ID NO：5所示序列中的第三段灰色标记序列，即为KOD DNA聚合酶的拇指域中的部分序列。
由此，获得的嵌合DNA聚合酶兼具高持续合成能力、高延伸率、热稳定性、对盐的抗性强以及高保真性等性能，可以满足DNA扩增、合成、检测、测序等需要，具有广泛的应用前景。
根据本发明的实施例，嵌合DNA聚合酶具有SEQ ID NO：1所示的氨基酸序列。由此，根据本发明实施例的嵌合DNA聚合酶兼具高持续合成能力、高延伸率、热稳定性、对盐的抗性强以及高保真性等性能，可以满足DNA扩增、合成、检测、测序等需要，具有广泛的应用前景。
Figure PCTCN2019127462-appb-000005
分离的核酸
在本发明的另一方面，本发明提出了一种分离的核酸。根据本发明的实施例，该分离的核酸编码前面所述嵌合DNA聚合酶。由此，利用根据本发明实施例的分离的核酸可以编码得到兼具高持续合成能力、高延伸率、热稳定性、对盐的抗性强以及高保真性等性能的嵌合DNA聚合酶，能够满足DNA扩增、合成、检测、测序等需要，具有广泛的应用前景。
根据本发明的实施例，编码第一肽段的核酸与KOD DNA聚合酶的第1～390位核苷酸序列具有至少80％同源性。编码KOD DNA聚合酶的核苷酸序列的第1～390位序列为SEQ  ID NO：8所示序列中的第一段灰色标记序列，即为编码KOD DNA聚合酶N-端域中的部分序列。
根据本发明的实施例，编码第二肽段的核酸与Pab DNA聚合酶的第391～1011位核苷酸序列具有至少80％同源性。编码Pab DNA聚合酶的核苷酸序列的第391～1011位序列为SEQ ID NO：6所示序列中的第一段灰色标记序列，即为编码Pab DNA聚合酶核酸外切域中的部分序列。
根据本发明的实施例，编码第三肽段的核酸与KOD DNA聚合酶的第1012～1119位核苷酸序列具有至少80％同源性。编码KOD DNA聚合酶的核苷酸序列的第1012～1119位序列为SEQ ID NO：8所示序列中的第二段灰色标记序列，即为编码KOD DNA聚合酶N-端域中的部分序列。
根据本发明的实施例，编码第四肽段的核酸与Pfu DNA聚合酶的第1120～1344位核苷酸序列具有至少80％同源性。编码Pfu DNA聚合酶的核苷酸序列的第1120～1344位序列为SEQ ID NO：7所示序列中的第一段灰色标记序列，即为编码Pfu DNA聚合酶掌域中的部分序列。
根据本发明的实施例，编码第五肽段的核酸与Pab DNA聚合酶的第1345～1500位核苷酸序列具有至少80％同源性。编码Pab DNA聚合酶的核苷酸序列的第1345～1500位序列为SEQ ID NO：6所示序列中的第二段灰色标记序列，即为编码Pab DNA聚合酶指域中的部分序列。
根据本发明的实施例，编码第六肽段的核酸与Pfu DNA聚合酶的第1501～1773位核苷酸序列具有至少80％同源性。编码Pfu DNA聚合酶的核苷酸序列的第1501～1773位序列为SEQ ID NO：7所示序列中的第二段灰色标记序列，即为编码Pfu DNA聚合酶掌域中的部分序列。
根据本发明的实施例，编码第七肽段的核酸与KOD DNA聚合酶的第1771～2325位核苷酸序列具有至少80％同源性。编码KOD DNA聚合酶的核苷酸序列的第1771～2325位序列为SEQ ID NO：8所示序列中的第三段灰色标记序列，即为编码KOD DNA聚合酶拇指域中的部分序列。
由此，获得的嵌合DNA聚合酶兼具高持续合成能力、高延伸率、热稳定性、对盐的抗性强以及高保真性等性能，可以满足DNA扩增、合成、检测、测序等需要，具有广泛的应用前景。
根据本发明的实施例，所述分离的核酸具有SEQ ID NO：2所示的核苷酸序列。
Figure PCTCN2019127462-appb-000006
Figure PCTCN2019127462-appb-000007
Pab DNA聚合酶核苷酸序列(SEQ ID NO：6)：
Figure PCTCN2019127462-appb-000008
Pfu DNA聚合酶核苷酸序列(SEQ ID NO：7)：
Figure PCTCN2019127462-appb-000009
KOD DNA聚合酶核苷酸序列(SEQ ID NO：8)：
Figure PCTCN2019127462-appb-000010
本领域技术人员能够理解的是，前面针对嵌合DNA聚合酶所描述的特征和优点，同样 适用于该分离的核酸，在此不再赘述。
构建体
在本发明的又一方面，本发明提出了一种构建体。根据本发明的实施例，该构建体含有前面所述分离的核酸。由此，利用根据本发明实施例的构建体可以表达兼具高持续合成能力、高延伸率、热稳定性、对盐的抗性强以及高保真性等性能的嵌合DNA聚合酶，能够满足DNA扩增、合成、检测、测序等需要，具有广泛的应用前景。
本领域技术人员能够理解的是，前面针对分离的核酸所描述的特征和优点，同样适用于该构建体，在此不再赘述。
重组细胞或者重组微生物
在本发明的又一方面，本发明提出了一种重组细胞或者重组微生物。根据本发明的实施例，该重组细胞或者重组微生物含有前面所述分离的核酸。由此，根据本发明实施例的重组细胞或者重组微生物可以表达兼具高持续合成能力、高延伸率、热稳定性、对盐的抗性强以及高保真性等性能的嵌合DNA聚合酶，能够满足DNA扩增、合成、检测、测序等需要，具有广泛的应用前景。
本领域技术人员能够理解的是，前面针对分离的核酸所描述的特征和优点，同样适用于该重组细胞或者重组微生物，在此不再赘述。
获得嵌合DNA聚合酶的方法
在本发明的又一方面，本发明提出了一种获得前面所述嵌合DNA聚合酶的方法。根据本发明的实施例，该方法包括：在适于嵌合DNA聚合酶表达的条件下，培养前面所述的重组细胞或者重组微生物，以便获得嵌合DNA聚合酶。由此，根据本发明实施例的方法可以获得兼具高持续合成能力、高延伸率、热稳定性、对盐的抗性强以及高保真性等性能的嵌合DNA聚合酶，能够满足DNA扩增、合成、检测、测序等需要，具有广泛的应用前景。
本领域技术人员能够理解的是，前面针对嵌合DNA聚合酶所描述的特征和优点，同样适用于该获得嵌合DNA聚合酶的方法，在此不再赘述。
试剂盒
在本发明的又一方面，本发明提出了一种试剂盒。根据本发明的实施例，该试剂盒含有前面所述嵌合DNA聚合酶、分离的核酸、构建体、重组细胞或者重组微生物。由此，利 用根据本发明实施例的试剂盒扩增DNA具有扩增产物产率高和扩增准确性强等优点，适于广泛生产和应用。
需要说明的是，本发明的试剂盒中还可以含有其他本领域常用的试剂，例如缓冲液、引物、核苷三磷酸等，具体可以根据实际情况灵活选择，本发明不作严格限定。
本领域技术人员能够理解的是，前面针对嵌合DNA聚合酶、分离的核酸、构建体、重组细胞或者重组微生物所描述的特征和优点，同样适用于该试剂盒，在此不再赘述。
用途
在本发明的又一方面，本发明提出了前面所述嵌合DNA聚合酶、分离的核酸、构建体、重组细胞或者重组微生物或试剂盒在DNA扩增中的用途。由此，扩增DNA具有扩增产物产率高和扩增准确性强等优点，适于广泛生产和应用。
根据本发明的实施例，嵌合DNA聚合酶、分离的核酸、构建体、重组细胞或者重组微生物或者试剂盒用于基因筛选、测序或突变检测。由于上述DNA聚合酶兼具高持续合成能力、高延伸率、热稳定性、对盐的抗性强以及高保真性等性能，可以有效地应用于对扩增产率和保真要求较高的基因筛选、测序或突变检测。
本领域技术人员能够理解的是，前面针对嵌合DNA聚合酶、分离的核酸、构建体、重组细胞或者重组微生物或试剂盒所描述的特征和优点，同样适用于该用途，在此不再赘述。
下面将结合实施例对本发明的方案进行解释。本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1：嵌合DNA聚合酶的设计与构建
Pfu、Pab和KOD DNA聚合酶具有不同的表型特征，表现在：在所有热稳定性且具有保真性的DNA聚合酶中，Pfu DNA聚合酶的错误几率最低，错误率约为2.0×10 -6。Pab DNA聚合酶同样表现低的扩增错误几率，且具有更高的热稳定性能。KOD是具有高扩增能力的DNA聚合酶，扩增速度是Taq DNA聚合酶的2倍、Pfu DNA聚合酶的6倍，扩增产量较高(～300nts)。
本试验的嵌合DNA聚合酶是Pfu、Pab和KOD DNA聚合酶的嵌合组合(图1所示)，具体地，KOD酶的N-端域核苷酸序列(1～390和1012～1119)及拇指域核苷酸序列(1771～2325)、Pab的核酸外切域核苷酸序列(391～1011)及指域序列(1345～1500)、Pfu的掌域核苷酸序列(1120～1344和1501～1773)构建到原核表达载体pET28a的XhoI/BamHI限制性酶切位点间， 将携带有上述核苷酸序列的载体转入大肠杆菌BL21(DE3)中，经培养后，获得表达菌株。该嵌合DNA聚合酶呈现出与KOD相近的高扩增能力，并具有比KOD更低的扩增错配几率。
实施例2：嵌合DNA聚合酶的发酵表达与纯化
1、发酵表达：将实施例1获得的表达菌株以1:100比例放大接种至含有卡那霉素的液体LB培养基中，37℃，220rpm震荡培养到OD600＝0.6时，加入0.5mM IPTG，16℃，220rpm低温诱导表达过夜(16h)。6000rpm离心8min，收集菌体沉淀。
2、发酵菌体处理：以菌体重量(g)与菌体重悬A液(ml)(20mM Tris，300mM NaCl，20mM Imidazole，5％Glycerol,pH8.0)的质量体积比为1:20重悬菌体，并超声破碎处理。12000rpm离心20min收集破碎上清液，75℃水浴锅中变性30min后，12000rpm离心20min回收上清液。
3、Ni柱纯化：将回收上清液通过0.22μm过滤装置过滤。在用菌体重悬A液洗涤平衡Ni柱后，进样上述0.22μm过滤液，调节洗脱液(20mM Tris，300mM NaCl，5％Glycerol,500mM Imidazole，pH8.0)中咪唑的浓度进行梯度洗脱，收集来自柱的级分，并通过SDS-PAGE将活性级分进行分析。将在考马斯染色的SDS-PAGE凝胶上观察到纯净的目标蛋白的级分合并。
4、阴离子柱纯化：将上述级分合并样品过离子柱，控制样品中核酸内切酶残留及核酸残留。级分合并样品透析至Buffer C(20mM Tris，100mM NaCl，5％Glycerol，pH8.0)中，调节Buffer D(20mM Tris，500mM NaCl，5％Glycerol，pH8.0)中盐离子的浓度进行梯度洗脱，收集洗脱柱级分即为新型嵌合DNA聚合酶。将获得样品透析至保存体系(20mM Tris，200mM KCl，50％Glycerol，0.2mM EDTA，2mM DTT，0.001％Tween20，0.001％NP40，pH8.0)。各纯化获得酶见图2所示，均获得纯度较高的目标蛋白。
实施例3：嵌合DNA聚合酶的扩增性能
将本发明实施例2获得的嵌合DNA聚合酶以λDNA为模板进行扩增，扩增片段为2Kb-8Kb。其中，Pfu DN聚合酶延伸时间为60s/kb，Pab、KOD及嵌合酶的延伸时间为30s/kb。
所用引物序列如下：
lam-F：CCTCTGTCGTTTCCTTTCTCTGTTTTTGTCCGTGG(SEQ ID NO：9)
lam2K-R：CGTCTGTTCATCGTCGTGGCGGCCCATAATAATCT(SEQ ID NO：10)
lam4K-R：GCACTCTTTCTCGTAGGTACTCAGTCCGGCTTCT(SEQ ID NO：11)
lam8K-R：CGGGAATACGACGGTTACCCACCACAAGCACG(SEQ ID NO：12)
扩增反应程序及体系如表2。结果见图3所示。图3表明，与Pfu及Pab聚合酶比较，新型嵌合DNA聚合酶扩增的目标产物产量更高，具有更佳的扩增效果；与KOD聚合酶比较，嵌合酶扩增效果与其相当。
表2扩增反应条件
Figure PCTCN2019127462-appb-000011
实施例4：嵌合DNA聚合酶扩增保真性分析
分别用Pab DNA聚合酶、Pfu DNA聚合酶、KOD DNA聚合酶及嵌合酶扩增LacIQZ a基因，用XbaI/NcoI双酶切扩增片段及载体，分别胶回收酶切后的扩增片段及载体片段并酶连，连接混合液转化至大肠杆菌DH5a并将细胞接种到LB-Amp-X-gal平板上培养。统计蓝色菌落、白色菌落的数目以及菌落总数，计算保真度。
LacIQZ a基因扩增引物序列如下：
Lac-F：GTTTTCCCAGTCACGAC(SEQ ID NO：13)
Lac-R：GGTATCTTTATAGTCCTGTCG(SEQ ID NO：14)
保真度计算公式为：
Figure PCTCN2019127462-appb-000012
结果见下表3所示。上述结果表明，与KOD相比，嵌合酶具有提高的扩增保真性能。
表3菌落数及错配率
  白色菌落数 蓝色菌落数 总菌落数 错配率(×10 -6)
Pfu 183 9290 9473 1.41
Kod 472 7319 7791 4.51
Pab 157 8763 8920 1.28
嵌合酶 151 8105 8256 1.33
实施例5：嵌合DNA聚合酶热稳定性测定
在98℃下温浴Pab、Pfu、KOD及嵌合酶每种酶0、30、60、90、120或180分钟。温浴完成后，以上述各酶扩增E.coli gDNA，用琼脂糖凝胶分析PCR产物。
扩增引物序列如下：
Ecoli-F：AGAGTTTGATCMTGGCTCAG(SEQ ID NO：15)
Ecoli-R：CGGTTACCTTGTTACGACTT(SEQ ID NO：16)
扩增体系与扩增程序参照实施例3。结果见图4所示。
结果显示，在98℃下预温浴2h小时后，Pfu DNA聚合酶没有观测到扩增片段；在98℃下预温浴3h小时，Pab DNA聚合酶亦未观测到扩增片段；在测试的所有时间点，嵌合酶均能够扩增到PCR产物。
实施例6：嵌合DNA聚合酶低模板条件扩增
分别于扩增体系中添加10ng、1ng、100pg的E.coli gDNA，以Pfu、Pab、KOD及嵌合酶进行扩增，用琼脂糖凝胶分析PCR产物。扩增体系及扩增程序参照实施例3，扩增引物序列见实施例5。结果见图5所示。三种模板添加量情况下，嵌合酶均能扩增出目标产物，且目标产物产量较佳。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。

Claims (17)

  1. 一种嵌合DNA聚合酶，其特征在于，包括：
    第一肽段，所述第一肽段与KOD DNA聚合酶的N-端域中的至少部分氨基酸序列具有至少80％同源性；
    第二肽段，所述第二肽段的N端与所述第一肽段的C端相连，所述第二肽段与Pab DNA聚合酶的核酸外切域中的至少部分氨基酸序列具有至少80％同源性；
    第三肽段，所述第三肽段的N端与所述第二肽段的C端相连，所述第三肽段与KOD DNA聚合酶的N-端域中的至少部分氨基酸具有至少80％同源性；
    第四肽段，所述第四肽段的N端与所述第三肽段的C端相连，所述第四肽段与Pfu DNA聚合酶的掌域中的至少部分氨基酸具有至少80％同源性；
    第五肽段，所述第五肽段的N端与所述第四肽段的C端相连，所述第五肽段与Pab DNA聚合酶的指域中的至少部分氨基酸具有至少80％同源性；
    第六肽段，所述第六肽段的N端与所述第五肽段的C端相连，所述第六肽段与Pfu DNA聚合酶的掌域中的至少部分氨基酸具有至少80％同源性；
    第七肽段，所述第七肽段的N端与所述第六肽段的C端相连，所述第七肽段与KOD DNA聚合酶的拇指域中的至少部分氨基酸具有至少80％同源性。
  2. 根据权利要求1所述的嵌合DNA聚合酶，其特征在于，所述第一肽段与KOD DNA聚合酶的第1～130位氨基酸序列具有至少80％同源性。
  3. 根据权利要求1所述的嵌合DNA聚合酶，其特征在于，所述第二肽段与Pab DNA聚合酶的第131～337位氨基酸序列具有至少80％同源性。
  4. 根据权利要求1所述的嵌合DNA聚合酶，其特征在于，所述第三肽段与KOD DNA聚合酶的第338～373位氨基酸序列具有至少80％同源性。
  5. 根据权利要求1所述的嵌合DNA聚合酶，其特征在于，所述第四肽段与Pfu DNA聚合酶的第374～448位氨基酸序列具有至少80％同源性。
  6. 根据权利要求1所述的嵌合DNA聚合酶，其特征在于，所述第五肽段与Pab DNA聚合酶的第449～500位氨基酸序列具有至少80％同源性。
  7. 根据权利要求1所述的嵌合DNA聚合酶，其特征在于，所述第六肽段与Pfu DNA聚合酶的第501～591位氨基酸序列具有至少80％同源性。
  8. 根据权利要求1所述的嵌合DNA聚合酶，其特征在于，所述第七肽段与KOD DNA聚合酶的第591～774位氨基酸序列具有至少80％同源性。
  9. 根据权利要求1所述的嵌合DNA聚合酶，其特征在于，所述嵌合DNA聚合酶具有SEQ ID NO：1所示的氨基酸序列。
  10. 一种分离的核酸，其特征在于，所述分离的核酸编码权利要求1～9任一项所述嵌合DNA聚合酶。
  11. 根据权利要求10所述的分离的核酸，其特征在于，所述分离的核酸具有SEQ ID NO：2所示的核苷酸序列。
  12. 一种构建体，其特征在于，含有权利要求10或11所述分离的核酸。
  13. 一种重组细胞或者重组微生物，其特征在于，含有权利要求10或11所述分离的核酸。
  14. 一种获得权利要求1～9任一项所述嵌合DNA聚合酶的方法，其特征在于，包括：
    在适于所述嵌合DNA聚合酶表达的条件下，培养权利要求13所述的重组细胞或者重组微生物，以便获得所述嵌合DNA聚合酶。
  15. 一种试剂盒，其特征在于，含有权利要求1～9任一项所述嵌合DNA聚合酶、权利要求10或11所述分离的核酸、权利要求12所述构建体、权利要求13所述重组细胞或者重组微生物。
  16. 权利要求1～9任一项所述嵌合DNA聚合酶、权利要求10或11所述分离的核酸、权利要求12所述构建体、权利要求13所述重组细胞或者重组微生物或者权利要求15所述试剂盒在DNA扩增中的用途。
  17. 根据权利要求16所述的用途，其特征在于，所述嵌合DNA聚合酶、分离的核酸、构建体、重组细胞或者重组微生物或者试剂盒用于基因筛选、测序或突变检测。
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