Recombinant protein and application thereof

Abstract

Provided are a recombinant protein having improved amplification performance, a nucleic acid encoding the recombinant protein, a vector comprising the nucleic acid, a kit comprising the protein, the nucleic acid or the vector, a method for preparing the recombinant protein, and a method for amplifying a target DNA using the recombinant protein. As a chimeric A family DNA polymerase, the recombinant protein has improved continuous synthesis capability, the amplification performance of the recombinant protein is significantly superior to that of a wild A family DNA polymerase, and the thermal stability of the recombinant protein is higher than that of a B family DNA polymerase.

Description

Recombinant proteins and their applications Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a recombinant protein, a nucleic acid encoding the recombinant protein, a vector comprising the nucleic acid, a kit comprising the protein, nucleic acid or vector, a method for preparing the recombinant protein, and a method for amplifying a target DNA using the recombinant protein. Background Art

[0002] The DNB-SEQ sequencing method typically uses phi29 DNA polymerase for rolling circle amplification (RCA) and multiple displacement amplification (MDA) reactions. Although phi29 DNA polymerase is a B-family polymerase with extremely high strand displacement activity and sustainable synthesis capacity, it has poor thermal stability and is easily inactivated. Therefore, it must be stored at low temperatures under harsh conditions and has a short shelf life. Furthermore, phi29 DNA polymerase has strong nonspecific binding ability and is difficult to elute, resulting in a strong fluorescence background during sequencing.

[0003] Bst polymerase is an A-family polymerase with high temperature resistance, and its optimal reaction temperature is 60-65°C. Therefore, whether in its preparation or application process, A-family polymerases have better tolerance to temperature and also have higher chain displacement activity. In sequencing, A-family polymerases are easier to elute than phi29 polymerases and perform better in complex systems. However, compared with B-family DNA polymerases (such as phi29, Pfu, and Kod polymerases), the thumb domain of A-family polymerases binds to the DNA template in a smaller area, resulting in weaker sustainable amplification capabilities of A-family polymerases, including Bst polymerase.

[0004] Therefore, it is necessary to research and develop A family polymerases with high processivity.

[0005] Summary of the Invention

[0006] The present invention aims to solve one of the technical problems in the related art at least to a certain extent.

[0007] To this end, embodiments of the present invention provide a recombinant protein having an amino acid sequence that enhances the interaction between a DNA polymerase and a target DNA during an amplification reaction, a nucleic acid encoding the recombinant protein, a vector comprising the nucleic acid, a kit comprising the protein, nucleic acid, or vector, a method for preparing the recombinant protein, and a method for amplifying a target DNA using the recombinant protein. The recombinant protein provided in embodiments of the present invention is a chimeric A-family DNA polymerase with improved processivity, enhanced thermal stability, polymerization activity, and DNA double-strand affinity, and can be used to amplify a target DNA.

[0008] In a first aspect, an embodiment of the present invention provides a recombinant protein, the amino acid sequence of which is obtained by replacing the homologous sequence in the wild-type A family DNA polymerase with an amino acid sequence that can enhance the interaction between the DNA polymerase and the target DNA in an amplification reaction.

[0009] In some embodiments, the A-family DNA polymerase is selected from the group consisting of E. coli DNA polymerase I, T3 DNA polymerase, T5 DNA polymerase, T7 DNA polymerase, Taq DNA polymerase, Bsu DNA polymerase, and Bst DNA polymerase.

[0010] In some embodiments, the A family DNA polymerase is selected from at least one of Bst DNA polymerase having the amino acid sequence shown in SEQ ID NO: 1, Taq DNA polymerase having the amino acid sequence shown in SEQ ID NO: 9, Escherichia coli DNA polymerase having the amino acid sequence shown in SEQ ID NO: 10, and Bsu DNA polymerase having the amino acid sequence shown in SEQ ID NO: 11.

[0011] In some embodiments, the homologous sequence in A-family DNA polymerases is derived from the thumb domain.

[0012] In some embodiments, the homologous sequence in the A family DNA polymerase is selected from at least one of the following:

[0013] The amino acid sequence from position 546 to position 554 of the Bst DNA polymerase as shown in SEQ ID NO: 2;

[0014] The amino acid sequence from position 199 to position 209 of the Taq DNA polymerase as shown in SEQ ID NO: 12;

[0015] The amino acid sequence from position 276 to position 285 of the Escherichia coli DNA polymerase as shown in SEQ ID NO: 13; or

[0016] The amino acid sequence from position 249 to position 257 in the Escherichia coli Bsu polymerase is shown in SEQ ID NO: 14.

[0017] In some embodiments, the homologous sequence in the A family DNA polymerase is replaced with a sequence comprising, consisting essentially of, or consisting of the amino acid sequence shown in SEQ ID NO:3.

[0018] In some embodiments, the amino acid sequence of the recombinant protein is selected from at least one of SEQ ID NO:4, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17.

[0019] In some embodiments, the recombinant protein further comprises any of additional conservative mutations, additions, and deletions.

[0020] In some embodiments, the recombinant protein as a chimeric DNA polymerase has a higher processivity than the wild-type A-family DNA polymerase.

[0021] In a second aspect, an embodiment of the present invention provides a nucleic acid encoding a recombinant protein as described in any embodiment of the first aspect.

[0022] In some embodiments, the nucleotide sequence of the nucleic acid molecule is selected from at least one of SEQ ID NO:5, SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20.

[0023] In a third aspect, an embodiment of the present invention provides a vector comprising the nucleic acid as described in any embodiment of the second aspect.

[0024] In a fourth aspect, an embodiment of the present invention provides a kit comprising the recombinant protein as described in any embodiment of the first aspect, the nucleic acid as described in any embodiment of the second aspect, or the vector as described in any embodiment of the third aspect.

[0025] In a fifth aspect, an embodiment of the present invention provides a method for preparing the recombinant protein described in any embodiment of the first aspect, the method comprising: replacing the amino acid sequence obtained by the homologous sequence in the wild-type A family DNA polymerase with an amino acid sequence that can enhance the interaction between the DNA polymerase and the target DNA in the amplification reaction to obtain the recombinant protein.

[0026] In some embodiments, the method comprises: aligning multiple amino acid sequences from the wild-type A family DNA polymerase to determine a chimeric sequence; and replacing a homologous sequence in the wild-type A family DNA polymerase sequence with the chimeric sequence to obtain a candidate recombinant protein sequence.

[0027] In some embodiments, the method further comprises: performing computer simulation analysis on the candidate recombinant protein sequence to determine a recombinant protein sequence, wherein the recombinant protein sequence exhibits a reduced spatial configuration between the thumb domain and the finger domain.

[0028] In some embodiments, the candidate recombinant protein sequence is subjected to computer simulation analysis to obtain the recombinant protein sequence, including: performing structural prediction on the candidate recombinant protein sequence to obtain the tertiary structure of the recombinant protein; simulating the tertiary structure of the recombinant protein to obtain the molecular docking structure of the recombinant protein; performing ligand-based structural optimization on the molecular docking structure to obtain an optimized recombinant protein structure; and performing kinetic simulation on the optimized recombinant protein structure to determine the recombinant protein sequence.

[0029] In some embodiments, the method further comprises: expressing and purifying the recombinant protein sequence.

[0030] In some embodiments, the A-family DNA polymerase is selected from the group consisting of E. coli DNA polymerase I, T3 DNA polymerase, T5 DNA polymerase, T7 DNA polymerase, Taq DNA polymerase, Bsu DNA polymerase, and Bst DNA polymerase.

[0031] In some embodiments, the A family DNA polymerase is selected from at least one of a Bst DNA polymerase having the amino acid sequence shown in SEQ ID NO: 1, a Taq DNA polymerase having the amino acid sequence shown in SEQ ID NO: 9, an Escherichia coli DNA polymerase having the amino acid sequence shown in SEQ ID NO: 10, and a Bsu DNA polymerase having the amino acid sequence shown in SEQ ID NO: 11.

[0032] In some embodiments, the homologous sequence in the A family DNA polymerase is derived from the thumb domain sequence.

[0033] In some embodiments, the homologous sequence in the A family DNA polymerase is selected from at least one of the following:

[0034] The amino acid sequence from position 546 to position 554 of the Bst DNA polymerase as shown in SEQ ID NO: 2;

[0035] The amino acid sequence from position 199 to position 209 of the Taq DNA polymerase as shown in SEQ ID NO: 12;

[0036] The amino acid sequence from position 276 to position 285 of the Escherichia coli DNA polymerase as shown in SEQ ID NO: 13; or

[0037] The amino acid sequence from position 249 to position 257 in the Escherichia coli Bsu polymerase is shown in SEQ ID NO: 14.

[0038] In some embodiments, the homologous sequence in the A family DNA polymerase is replaced with a sequence comprising, consisting essentially of, or consisting of the amino acid sequence shown in SEQ ID NO:3.

[0039] In some embodiments, the amino acid sequence of the recombinant protein is selected from at least one of SEQ ID NO:4, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17.

[0040] In a sixth aspect, an embodiment of the present invention provides a method for amplifying a target DNA, wherein the target DNA is amplified using the recombinant protein described in any embodiment of the first aspect.

[0041] In some embodiments, the amplification is isothermal amplification, and the isothermal amplification is selected from rolling circle amplification RCA, multiple displacement amplification MDA, recombinase polymerase amplification reaction RPA, strand displacement amplification SDA, and loop-mediated isothermal amplification LAMP.

[0042] In some embodiments, the amplification is used to construct a DNA library or for sequencing.

[0043] The recombinant protein in the embodiments of the present invention, as a chimeric A-family DNA polymerase, exhibits enhanced processivity and significantly superior amplification performance compared to wild-type A-family DNA polymerases. Furthermore, compared to B-family DNA polymerases, the recombinant protein in the embodiments of the present invention offers the following advantages: high thermal stability, convenient elution, and ease of storage. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] In order to more clearly illustrate the technical solutions of the embodiments of the present disclosure, the following briefly introduces the drawings required for use in the embodiments or descriptions of the prior art. Obviously, the drawings described below are only some embodiments of the present disclosure, and a person skilled in the art can also obtain other drawings based on these drawings.

[0045] FIG1 is a schematic diagram of the structure of the large fragment of Bst DNA polymerase.

[0046] FIG2 is a schematic diagram showing a sequence alignment of a large fragment of wild-type Bst DNA polymerase and a chimeric DNA polymerase according to an embodiment of the present invention.

[0047] FIG3 is a schematic diagram of the structures of chimeric DNA polymerases Taq-HS-1(a), Ecoli-HS-1(b), Bsu-HS-1(c) and Bst-HS-1(d) after docking with DNA molecules according to an embodiment of the present invention.

[0048] FIG4 is a schematic structural diagram of a complex of a chimeric DNA polymerase and a DNA molecule after kinetic simulation according to an embodiment of the present invention. DETAILED DESCRIPTION

[0049] The embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to be used to explain the present invention, but should not be understood as limiting the present invention.

[0050] Family A DNA polymerases include Escherichia coli polymerase I, Bst DNA polymerase, T7 DNA polymerase, and Taq DNA polymerase, the latter of which has been widely used in PCR. Bst DNA polymerase, derived from Geobacillus stearothermophilus, is a multifunctional enzyme characterized by high temperature resistance, strong strand displacement activity, and the ability to perform loop-mediated isothermal amplification. However, its sustainable amplification capability still needs to be improved. Bst DNA polymerase belongs to the family A DNA polymerase and has a typical right-handed structure, consisting of a thumb domain, a palm domain, a finger domain, and a 5'-3' exonuclease domain. During the polymerization reaction, the target DNA and part of the newly synthesized DNA are sandwiched between the thumb domain and the finger domain. In biotechnology applications, the 5'-3' exonuclease domain of Bst DNA polymerase is usually knocked out, leaving only the Bst polymerase large fragment (Bst-LF) containing the thumb domain, palm domain, and finger domain. Its structure is shown in Figure 1.

[0051] Currently, the DNA polymerases used in sequencing are primarily B-family strand-displacement enzymes, primarily phi29 polymerase. Derived from the bacteriophage phi29 of Bacillus subtilis, phi29 polymerase exhibits high processivity and strand-displacement activity, making it commonly used in RCA-based DNA cloning and MDA-based single-cell whole-genome amplification. Phy29 polymerase is a mesophilic enzyme with an optimal reaction temperature around 30°C, but is completely inactivated by incubation at 65°C for 10 minutes. The strong strand-displacement and high processivity of phi29 polymerase are primarily due to its TPR2 (terminal protein region 2) domain. During strand synthesis, the TPR2 domain unwinds the double-stranded template, allowing only the template strand to enter and trapping the template between the Exo domain and the TPR2 domain, preventing separation between the enzyme and template. This enables phi29 polymerase to achieve processivity exceeding 70,000 nt. However, phi29 polymerase has defects such as strict storage conditions, short shelf life, and large fluorescence background during sequencing.

[0052] Compared to B-family DNA polymerases (such as phi29, Pfu, and Kod polymerases), the thumb domain of A-family polymerases has a smaller region that binds to the DNA template and interacts less with the nascent DNA chain, resulting in weaker sustainable amplification capabilities for A-family polymerases, including Bst polymerase. Furthermore, because the 5'-3' exodomain has a DNA-binding effect, it prevents the complete dissociation of the polymerase from the template. When the 5'-3' exodomain is deleted, the processivity of A-family polymerases for large fragment synthesis decreases, and they are generally unable to effectively synthesize DNA chains greater than 10kb in length.

[0053] Based on the above problems, the inventors explored the structure of A family polymerases using Bst polymerase, Taq DNA polymerase, Escherichia coli DNA polymerase and Bsu DNA polymerase as examples, in order to provide a polymerase variant with high affinity for double-stranded DNA and high processivity. The recombinant protein provided in the embodiment of the present application, i.e., the chimeric A family polymerase, has achieved a structural transformation in which the thumb domain extends toward the finger domain, reducing the space between the thumb domain and the finger domain, so that the template chain and part of the newly synthesized DNA chain remain between the thumb domain and the finger domain, thereby playing an effect similar to that of the TPR2 domain in phi29 polymerase, strengthening the interaction between the thumb domain and the DNA double strand and forming a ring-shaped sliding clamp around the DNA template chain, thereby improving the processivity of the A family DNA polymerase.

[0054] According to one aspect of the present invention, a recombinant protein is provided, the amino acid sequence of which is obtained by replacing the homologous sequence in the wild-type A family DNA polymerase with an amino acid sequence that can enhance the interaction between the DNA polymerase and the target DNA in the amplification reaction.

[0055] It should be noted that the thumb domain of the recombinant protein provided in the embodiments of the present invention is replaced with a chimeric protein sequence. This sequence strengthens the interaction between the thumb domain and the DNA duplex and forms a circular sliding clamp around the DNA template strand, increasing the polymerase's affinity for the DNA duplex, thereby enhancing processivity. This recombinant protein also exhibits enhanced processivity, enhanced thermal stability, polymerization activity, and DNA duplex affinity, and can be used in a variety of DNA isothermal amplification reactions for library construction and sequencing.

[0056] It should be noted that the processivity of a polymerase can be defined as the number of nucleotides processed in a single incorporation. The processivity of a DNA polymerase generally reflects the rate and speed of synthesis, as well as the enzyme's affinity for its substrate. DNA polymerases with high processivity are suitable for amplifying long templates, sequences with secondary structure and GC-rich sequences, as well as blood and plant tissue samples in the presence of PCR inhibitors such as heparin, xylan, and humic acid.

[0057] In some embodiments, the A-family DNA polymerase is selected from the group consisting of E. coli DNA polymerase I, T3 DNA polymerase, T5 DNA polymerase, T7 DNA polymerase, Taq DNA polymerase, Bsu DNA polymerase, and Bst DNA polymerase.

[0058] In some embodiments, the A family DNA polymerase is selected from at least one of Bst DNA polymerase having the amino acid sequence shown in SEQ ID NO: 1, Taq DNA polymerase having the amino acid sequence shown in SEQ ID NO: 9, Escherichia coli DNA polymerase having the amino acid sequence shown in SEQ ID NO: 10, and Bsu DNA polymerase having the amino acid sequence shown in SEQ ID NO: 11.

[0059] The sequence of SEQ ID NO: 1 is:

[0060] (The bold underline is the homologous sequence SEQ ID NO: 2)

[0061] The sequence of SEQ ID NO:9 is:

[0062] (The bold underlined sequence is SEQ ID NO: 12)

[0063] The sequence of SEQ ID NO: 10 is:

[0064] (The bold underlined sequence is SEQ ID NO: 13)

[0065] The sequence of SEQ ID NO:11 is:

[0066] (The bold underlined sequence is SEQ ID NO: 14)

[0067] In some embodiments, the homologous sequence in the A family DNA polymerase is derived from the thumb domain sequence.

[0068] In some embodiments, the homologous sequence in the A family DNA polymerase is selected from at least one of the following:

[0069] The amino acid sequence from position 546 to position 554 of the Bst DNA polymerase as shown in SEQ ID NO: 2;

[0070] The amino acid sequence from position 199 to position 209 of the Taq DNA polymerase large fragment as shown in SEQ ID NO: 12;

[0071] The amino acid sequence from position 276 to position 285 of the large fragment of Escherichia coli DNA polymerase as shown in SEQ ID NO: 13; or

[0072] The amino acid sequence from position 249 to position 257 of the large fragment of Escherichia coli Bsu polymerase is shown in SEQ ID NO: 14.

[0073] The sequence of SEQ ID NO: 2 is: VLKKTKTGY.

[0074] The sequence of SEQ ID NO: 12 is: AIGKTEKTGKR

[0075] The sequence of SEQ ID NO: 13 is: PLKKTPGGAP

[0076] The sequence of SEQ ID NO: 14 is: VVKKTKTGY

[0077] In some embodiments, the homologous sequence in the A-family DNA polymerase is replaced with a sequence comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 3. The sequence of SEQ ID NO: 3 is: PNREMKNQGSKKTLGSTRRGIDNGRKLRLGRQF.

[0078] In some embodiments, the amino acid sequence of the recombinant protein is selected from at least one of SEQ ID NO:4, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17.

[0079] The sequence of SEQ ID NO:4 is:

[0080] (The bold underline is the chimeric sequence SEQ ID NO: 3)

[0081] The sequence of SEQ ID NO: 15 is:

[0082] (The bold underline is the chimeric sequence SEQ ID NO: 3)

[0083] The sequence of SEQ ID NO: 16 is:

[0084] (The bold underline is the chimeric sequence SEQ ID NO: 3)

[0085] The sequence of SEQ ID NO: 17 is:

[0086] (The bold underline is the chimeric sequence SEQ ID NO: 3)

[0087] In some embodiments, the recombinant protein further comprises any of additional conservative mutations, additions, and deletions.

[0088] In some embodiments, the recombinant protein as a chimeric DNA polymerase has a higher processivity than the wild-type A-family DNA polymerase.

[0089] According to another aspect of the present invention, the present invention provides a nucleic acid encoding the recombinant protein as described in any embodiment of the first aspect.

[0090] In some embodiments, the nucleotide sequence of the nucleic acid molecule is selected from at least one of SEQ ID NO:5, SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20.

[0091] The sequence of SEQ ID NO:5 is:

[0092]

[0093] The sequence of SEQ ID NO: 18 is:

[0094]

[0095] The sequence of SEQ ID NO: 19 is:

[0096]

[0097] The sequence of SEQ ID NO:20 is:

[0098]

[0099] According to yet another aspect of the present invention, an embodiment of the present invention provides a vector comprising the nucleic acid as described in any embodiment of the second aspect.

[0100] According to another aspect of the present invention, an embodiment of the present invention provides a kit comprising the recombinant protein as described in any embodiment of the first aspect, the nucleic acid as described in any embodiment of the second aspect, or the vector as described in any embodiment of the third aspect.

[0101] According to another aspect of the present invention, an embodiment of the present invention provides a method for preparing the recombinant protein described in any embodiment of the first aspect, the method comprising: replacing the amino acid sequence obtained by replacing the homologous sequence in the wild-type A family DNA polymerase with an amino acid sequence that can enhance the interaction between the DNA polymerase and the target DNA in the amplification reaction to obtain the recombinant protein.

[0102] In some embodiments, the method comprises: aligning multiple amino acid sequences from the wild-type A family DNA polymerase to determine a chimeric sequence; and replacing a homologous sequence in the wild-type A family DNA polymerase sequence with the chimeric sequence to obtain a candidate recombinant protein sequence.

[0103] In some embodiments, the method further comprises: performing computer simulation analysis on the candidate recombinant protein sequence to determine a recombinant protein sequence, wherein the recombinant protein sequence exhibits a reduced spatial configuration between the thumb domain and the finger domain.

[0104] In some embodiments, the candidate recombinant protein sequence is subjected to computer simulation analysis to obtain the recombinant protein sequence, including: performing structural prediction on the candidate recombinant protein sequence to obtain the tertiary structure of the recombinant protein; simulating the tertiary structure of the recombinant protein to obtain the molecular docking structure of the recombinant protein; performing ligand-based structural optimization on the molecular docking structure to obtain an optimized recombinant protein structure; and performing kinetic simulation on the optimized recombinant protein structure to determine the recombinant protein sequence.

[0105] In some embodiments, the method further comprises expressing and purifying the recombinant protein sequence.

[0106] In some specific embodiments, the method specifically includes: searching the sequence database of A family polymerases and comparing homologous sequences to determine a candidate chimeric sequence for replacing the homologous sequence in the wild-type polymerase; using tools such as Alphafold, Autodock, and Gromacs to perform (1) structural prediction, (2) molecular docking simulation of the DNA double strand and the recombinant protein, and (3) kinetic simulation analysis on the recombinant protein with a chimeric sequence according to an embodiment of the present invention; expressing and purifying the recombinant protein determined by the simulation analysis using a prokaryotic expression system, and determining its activity; and confirming the protein sequence by sequencing.

[0107] It is understood that the sequence alignment in this method includes a variety of alignment strategies such as global alignment, local alignment, double sequence alignment, and multiple sequence alignment.

[0108] It is understandable that the structures of A family DNA polymerases are relatively conservative and similar. The method for preparing recombinant proteins can be applied to replace homologous sequences of various A family DNA polymerases and prepare the recombinant proteins.

[0109] In the embodiments of the present invention, the term "molecular docking" refers to the placement of a small molecule (ligand) in the binding region of a macromolecular target (receptor) through computer simulation, which allows the binding force and binding mode of the ligand and receptor to be calculated by adjusting physicochemical parameters, thereby obtaining the lowest energy conformation when the two are combined in their active regions. In the present invention, the ligand and receptor for molecular docking are chimeric polymerase and DNA, respectively. Simulating the interaction between molecules and proteins at the atomic level helps to discover strategies for further modification of chimeric polymerases.

[0110] In the embodiments of the present invention, the term "dynamic simulation" refers to the use of Newtonian classical mechanics to calculate the trajectory of molecules in space, solve the Newtonian equations of molecules or atoms in the system under the interaction of the potential energy of the interaction between molecules or atoms and the constraints imposed by the system, and simulate the microscopic process of the system advancing over time. The flexibility of proteins and ligands causes them to seek the most consistent and lowest energy conformation when binding to each other. Usually, after the docking is completed, when the accuracy of the ligand-protein complex obtained by the scoring function is not very high, it is necessary to use dynamic simulation to calculate the binding energy and verify the effective binding of the protein to the screened small molecule ligand.

[0111] In some embodiments, the A-family DNA polymerase is selected from the group consisting of Escherichia coli DNA polymerase I, T3 DNA polymerase, T5 DNA polymerase, T7 DNA polymerase, Taq DNA polymerase, Bsu DNA polymerase, and Bst DNA polymerase.

[0112] In some embodiments, the A family DNA polymerase is selected from at least one of Bst DNA polymerase having the amino acid sequence shown in SEQ ID NO: 1, Taq DNA polymerase having the amino acid sequence shown in SEQ ID NO: 9, Escherichia coli DNA polymerase having the amino acid sequence shown in SEQ ID NO: 10, and Bsu DNA polymerase having the amino acid sequence shown in SEQ ID NO: 11.

[0113] In some embodiments, the homologous sequence in the A family DNA polymerase is derived from its thumb domain.

[0114] In some embodiments, the homologous sequence in the A family DNA polymerase is selected from at least one of the following:

[0115] The amino acid sequence from position 546 to position 554 of the Bst DNA polymerase as shown in SEQ ID NO: 2;

[0116] The amino acid sequence from position 199 to position 209 of the Taq DNA polymerase as shown in SEQ ID NO: 12;

[0117] The amino acid sequence from position 276 to position 285 of the Escherichia coli DNA polymerase as shown in SEQ ID NO: 13; or

[0118] The amino acid sequence from position 249 to position 257 in the Escherichia coli Bsu polymerase is shown in SEQ ID NO: 14.

[0119] In some embodiments, the homologous sequence in the A family DNA polymerase is replaced with a sequence comprising, essentially consisting of, or consisting of the amino acid sequence shown in SEQ ID NO: 3.

[0120] In some embodiments, the amino acid sequence of the recombinant protein is selected from at least one of SEQ ID NO:4, SEQ ID NO:15, SEQ ID NO:16 and SEQ ID NO:17.

[0121] According to another aspect of the present invention, an embodiment of the present invention provides a method for amplifying a target DNA, wherein the target DNA is amplified using the recombinant protein described in any embodiment of the first aspect.

[0122] In some embodiments, the amplification is isothermal amplification, and the isothermal amplification is selected from rolling circle amplification RCA, multiple displacement amplification MDA, recombinase polymerase amplification reaction RPA, strand displacement amplification SDA, and loop-mediated isothermal amplification LAMP.

[0123] In some embodiments, the amplification is used to construct a DNA library or for sequencing.

[0124] Example

[0125] Example 1

[0126] In this example, based on the sequences of Bst polymerase, Taq DNA polymerase, Escherichia coli DNA polymerase, and Bsu DNA polymerase in the A family of DNA polymerases, database searches and MSA homologous sequence comparisons were performed to identify candidate chimeric sequences for replacing the homologous sequences in the wild-type polymerases. Tools such as Alphafold, Autodock, and Gromacs were used to perform structural predictions on the recombinant proteins with chimeric sequences in the examples of the present invention, as well as computational simulations such as molecular docking and kinetic simulation analysis of the DNA double strands and the recombinant proteins.

[0127] 1.1 Determination of chimeric sequences

[0128] Using the Protein-blast tool, homologous sequences of A-family polymerases were aligned in the NCBI protein sequence library, and the chimeric sequence (SEQ ID NO: 3) was identified as being homologous to amino acids 546-554 of the thumb domain of Bst DNA polymerase (SEQ ID NO: 2), amino acids 199-209 of the thumb domain of Taq DNA polymerase (SEQ ID NO: 12), amino acids 276-285 of the thumb domain of Escherichia coli DNA polymerase (SEQ ID NO: 13), and amino acids 249-257 of the thumb domain of Bsu DNA polymerase (SEQ ID NO: 14). The alignment results are shown in Figure 2. The chimeric sequence is derived from human pol theta (POLθ), and SEQ ID NO: 3 is approximately 24 amino acids longer than SEQ ID NO: 2, approximately XX amino acids longer than SEQ ID NO: 12, approximately XX amino acids longer than SEQ ID NO: 13, and approximately XX amino acids longer than SEQ ID NO: 14.

[0129] 1.2 Replacing the homologous sequence in wild-type A-family DNA polymerase with a chimeric sequence

[0130] 1.2.1 Based on the above homologous sequence alignment, the homologous sequence SEQ ID NO: 2 in the wild-type Bst DNA polymerase large fragment (Bst-LF WT) was replaced with the chimeric sequence SEQ ID NO: 3, thereby obtaining a chimeric Bst DNA polymerase having the sequence described in SEQ ID NO: 4.

[0131] Sequence of wild-type Bst DNA polymerase large fragment (Bst-LF WT) SEQ ID NO: 1:

[0132] >Bst-LF WT [Geobacillus stearothermophilus] amino acid sequence (293-831)

[0133] (The bold underline is the homologous sequence SEQ ID NO: 2)

[0134] Amino acid sequence of chimeric Bst DNA polymerase (Bst-HS-1) (SEQ ID NO: 4):

[0135] (The bold underline is the chimeric sequence SEQ ID NO: 3)

[0136] 1.2.2 Based on the above homologous sequence alignment, the homologous sequence SEQ ID NO: 12 in the wild-type Taq DNA polymerase large fragment (Taq-LF WT) was replaced with the chimeric sequence SEQ ID NO: 3, thereby obtaining a chimeric Taq DNA polymerase having the sequence described in SEQ ID NO: 15.

[0137] The sequence of wild-type Taq DNA polymerase large fragment (Taq-LF WT) is SEQ ID NO: 9:

[0138] >Taq-LF WT [Thermus aquaticus] amino acid sequence (1-529)

[0139] (The bold underlined sequence is SEQ ID NO: 12)

[0140] Amino acid sequence of chimeric Taq DNA polymerase (Taq-HS-1) (SEQ ID NO: 15):

[0141] (The bold underline is the chimeric sequence SEQ ID NO: 3)

[0142] 1.2.3 Based on the above homologous sequence alignment, the homologous sequence SEQ ID NO: 13 in the wild-type Escherichia coli DNA polymerase large fragment (Ecoli-LF WT) was replaced with the chimeric sequence SEQ ID NO: 3, thereby obtaining a chimeric Escherichia coli DNA polymerase having the sequence described in SEQ ID NO: 16.

[0143] The sequence of wild-type Escherichia coli DNA polymerase large fragment (Ecoli-LF WT) is SEQ ID NO: 10:

[0144] >Ecoli-LF WT [Escherichia coli] amino acid sequence (1-606)

[0145] (The bold underlined sequence is SEQ ID NO: 13)

[0146] Amino acid sequence of chimeric Escherichia coli DNA polymerase (Ecoli-HS-1) (SEQ ID NO: 16):

[0147] (The bold underline is the chimeric sequence SEQ ID NO: 3)

[0148] 1.2.4 Based on the above homologous sequence alignment, the homologous sequence SEQ ID NO: 14 in the wild-type Bsu DNA polymerase large fragment (Bsu-LF WT) was replaced with the chimeric sequence SEQ ID NO: 3, thereby obtaining a chimeric Bsu DNA polymerase having the sequence described in SEQ ID NO: 17.

[0149] The sequence of wild-type Bsu DNA polymerase large fragment (Bsu-LF WT) is SEQ ID NO: 11:

[0150] >Bsu-LF WT [Bacillus subtilis] amino acid sequence (1-603)

[0151] (The bold underlined sequence is SEQ ID NO: 14)

[0152] Amino acid sequence of chimeric Bsu DNA polymerase (Bsu-HS-1) (SEQ ID NO: 17):

[0153] (The bold underline is the chimeric sequence SEQ ID NO: 3)

[0154] 1.3 Structure prediction

[0155] Alphafold2 was used to predict the tertiary structure of recombinant proteins based on the sequences of chimeric A family DNA polymerases Bst-HS-1, Taq-HS-1, Ecoli-HS-1 and Bsu-HS-1.

[0156] 1.4 Molecular docking simulation

[0157] Based on the predicted structure, molecular docking simulations were performed using the Autodock tool on the DNA duplex with chimeric A-family DNA polymerases Bst-HS-1, Taq-HS-1, Ecoli-HS-1, and Bsu-HS-1 in the presence of dNTPs to confirm the binding strength of the modified chimeric DNA polymerases with extended thumb domains to the target DNA. As shown in Figure 3, the molecular docking simulations revealed that the chimeric sequence not only narrowed the space between the thumb and finger domains, thereby retaining the template strand between the thumb and finger domains, but also maintained a tight binding to the newly synthesized double-stranded DNA, thereby stabilizing the polymerase-DNA structure.

[0158] 1.5 Ligand structure optimization

[0159] The complexes of chimeric A family DNA polymerases Bst-HS-1, Taq-HS-1, Ecoli-HS-1 and Bsu-HS-1 with DNA molecules were docked using the Gromacs tool for ligand structure optimization.

[0160] 1.6 Dynamic simulation

[0161] The optimized structure was subjected to a 25 ns kinetic simulation at 330 K. As shown in Figure 4, the amino acid sequence NFN at positions 515 to 517 in the finger domain of the chimeric Bst DNA polymerase and K268 in the thumb domain maintain a constant distance of 4-6 angstroms, allowing the DNA template strand to be sandwiched in the pocket structure formed by the two. At the same time, the extended region can also maintain a tight bond with the newly synthesized double-stranded DNA.

[0162] In addition, experimental results show that additional mutations can be made to several sites in the chimeric sequence to enhance the thermal stability and polymerization activity of the chimera, and further improve the affinity of the chimeric polymerase for double-stranded DNA and its processivity.

[0163] For all A family polymerases, such as Bst, Taq, Ecoli, BSU, etc., the chimeric fragment can be inserted into the corresponding thumb region, which can play a similar role to the modified structure on Bst, enhancing the affinity with the DNA double strand and the ability to continuously synthesize.

[0164] The following data compares the binding energy of four A-family polymerases and their chimeric mutants to double-stranded DNA, demonstrating that all polymerases enhance their affinity for double-stranded DNA after the addition of chimeric fragments:

[0165] 1.Bst-LF WT: -164.2kJ / mol

[0166] Bst-HS-1: -272.0 kJ / mol

[0167] ΔΔG=-107.8kJ / mol

[0168] 2.Taq-LF WT: -150.2kJ / mol

[0169] Taq-HS-1: -277.6 kJ / mol

[0170] ΔΔG=-127.4kJ / mol

[0171] 3.Ecoli-LF WT: -186.3kJ / mol

[0172] Ecoli-HS-1: -327.9kJ / mol

[0173] ΔΔG=-141.6kJ / mol

[0174] 4.BSU-LF WT: -178.4kJ / mol

[0175] BSU-HS-1: -283.3 kJ / mol

[0176] ΔΔG=-104.9kJ / mol

[0177] Example 2

[0178] In this example, the chimeric polymerase Bst-HS-1 was rationally designed and expressed and purified using a prokaryotic expression system to obtain the Bst-HS chimeric DNA polymerase for practical applications.

[0179] 2.1 Preparation of chimeric DNA polymerase plasmid

[0180] For the chimeric DNA polymerase sequence determined in Example 1 (i.e., SEQ ID NO: 4), upstream and downstream primers for introducing the embedded sequence (SEQ ID NO: 3) were designed. A plasmid containing a nucleic acid sequence (SEQ ID NO: 6) encoding wild-type Bst DNA polymerase (SEQ ID NO: 1) was used as a template. Point mutation PCR was performed using the Novagen point mutagenesis kit (C112-01) to obtain a nucleic acid sequence (SEQ ID NO: 5) encoding the DNA polymerase sequence (i.e., SEQ ID NO: 4).

[0181] The sequence of the nucleic acid sequence encoding wild-type Bst DNA polymerase (SEQ ID NO: 6) is:

[0182]

[0183] 2.1.1 Perform the above-mentioned point mutation PCR according to the PCR reaction system shown in Table 1 and the PCR reaction conditions shown in Table 2 to obtain a reaction solution containing the amplified product.

[0184] Table 1 PCR reaction system

[0185]

[0186] Upstream primer (SEQ ID NO: 7): TACTTTCAGGGCGCCGAGGGTGAAAAACCTCTG

[0187] Downstream primer (SEQ ID NO: 8): GGGTTAACCTTATTTAGCATCATACCAGGTCGGG

[0188] Table 2 PCR reaction conditions

[0189]

[0190] 2.1.2 Add 0.4 μl of dpn1 enzyme to 20 μl of the reaction solution containing the amplified product, and digest the amplified product at 37° C. for 3 hours to obtain a solution containing the digested product.

[0191] 2.1.3 Take 5 μl of the solution containing the digestion product to transform 100 μl of competent cells, and then spread the transformed competent cells on solid LB medium and incubate at 4°C overnight.

[0192] 2.1.4 Pick a single clone, shake it at 37°C, and then send it for sequencing to obtain the sequencing results.

[0193] 2.1.5 Compare the sequencing results to determine the plasmid containing the correct mutation.

[0194] 2.2 Inducible expression of Bst-HS chimeric mutants

[0195] 2.2.1 Use the Bst-HS chimeric mutant plasmid to transform BL21 competent cells to obtain cells transformed with the Bst-HS chimeric mutant.

[0196] 2.2.2 Add cells transformed with the Bst-HS chimeric mutant and 20 mL of fresh culture medium to a 50 mL centrifuge tube, and culture overnight at 37°C with shaking at 220 rpm to obtain the first bacterial liquid.

[0197] 2.2.3 Transfer 20 ml of the first bacterial solution to a 5 L conical flask containing 1 L of culture medium and culture at 37°C with shaking at 220 rpm until the OD value reaches 0.8-1 to obtain the second bacterial solution.

[0198] 2.2.4 Add IPTG inducer to the second bacterial solution to reach a final IPTG concentration of 0.2 mM, and incubate at 16° C. for 16 hours to obtain a third bacterial solution containing bacteria expressing the Bst-HS chimeric mutant.

[0199] 2.2.5 Centrifuge the third bacterial solution at 12,000 rpm for 10 min and collect the bacterial cells as the precipitate.

[0200] 2.3 Purification of Bst-HS chimeric mutant protein

[0201] 2.3.1 The precipitate was suspended in 40 ml of solution A and the cells were crushed using a high-pressure crusher at a pressure of 700 bar for 5 minutes to obtain the fourth bacterial suspension.

[0202] Solution A (pH 7.4)

[0203]

[0204] 2.3.2 Heat-treat the fourth bacterial solution in a water bath at 60°C for 15 minutes to obtain a final solution. Centrifuge the final solution at 13,000 rpm for 30 minutes, then filter the supernatant using a 0.22 μm filter membrane and collect the filtrate.

[0205] 2.3.3 Load the filtrate onto a nickel column for purification, eluting with 50% B solution to obtain the target protein.

[0206] Solution B (pH 7.4)

[0207]

[0208] 2.3.4 Replace Solution B containing the target protein with dialysate by dialysis to obtain the purified target protein.

[0209] Dialysate (pH 7.6)

[0210]

[0211] 2.3.5 Aliquot and store the purified target protein for subsequent activity determination.

[0212] Example 3

[0213] In this example, the polymerization activity of the purified chimeric DNA polymerase Bst-HS and the wild-type DNA polymerase large fragment Bst-LF-WT was tested to compare their processivity. The enzyme solutions tested were a His6-tagged wild-type Bst DNA polymerase solution and a His6-tagged chimeric DNA polymerase Bst DNA solution prepared in Example 2.

[0214] 3.1 Synthetic capacity of chimeric DNA polymerase in RCA amplification

[0215] 3.1.1 Library Preparation

[0216] 40 μl of initial single-stranded circular DNA (self-synthesized Ecoli V3 single-stranded circular library, 1.82 ng / μl) was mixed with 1.06 μl of library complementary primer (5 μM) at a molar ratio of 1:1 and annealed according to the conditions described in Table 2 to obtain a library + primer mixture (concentration of approximately 1.77 ng / μl).

[0217] 3.1.2 RCA reaction

[0218] The library + primer mixture was prepared in the RCA reaction system as shown in Table 3 below, and incubated at 65° C. for 1 hour. The amplification reaction was terminated with 0.1 μl of 0.5 M EDTA to obtain the RCA reaction product.

[0219] Table 3 RCA reaction system

[0220]

[0221] 3.1.3 RCA product concentration detection

[0222] The Qubit ssDNA Assay Kit was used according to the manufacturer's instructions, and the RCA reaction product concentration was measured using a Qubit fluorometor 3.0. The results are as follows:

[0223] Bst-LF-WT:33.6ng / uL

[0224] Bst-HS-1:39.9ng / uL

[0225] This shows that in RCA isothermal amplification, the synthesis ability of the recombinant protein (i.e., the chimeric A family DNA polymerase) is higher than that of the wild-type A family DNA polymerase.

[0226] 3.2 Synthetic capacity of chimeric DNA polymerase in MDA amplification

[0227] 3.2.1 Preparation of DNB

[0228] 3.2.1.1 Mix single-stranded circular DNA (from a synthesized Ecoli V3 single-stranded circular library, concentration c) with DNB preparation buffer and TE buffer in a PCR tube and place on ice for approximately 0.5 hours. Vortex the tube for 5 seconds to mix thoroughly, centrifuge briefly, and place on ice until ready to use. Place the tube in a thermal cycler and run the following cycle: 95°C for 1 minute, 65°C for 1 minute, 40°C for 1 minute, with a heated lid at 102°C and a 4°C hold.

[0229]

[0230] 3.2.1.2 After the reaction, transfer the PCR tube to ice and add the volumes of DNB Polymerase Mix I and DNB Polymerase Mix II shown in the table below. Place the PCR tube back in the thermal cycler and perform the following program: 30°C for 30 minutes, heated lid at 35°C, 4°C hold.

[0231]

[0232] 3.2.1.3 Add 1 μl of DNB stop buffer to the PCR tube and vortex to mix to obtain DNB.

[0233] 3.2.2 MDA reaction

[0234] 3.2.2.1 Take the DNB prepared in 3.2.1 and mix them in a PCR tube according to the following reaction system. Perform the following procedure: 95°C for 1 minute, 65°C for 1 minute, 40°C for 1 minute, and the heated lid temperature is 102°C, maintained at 4°C.

[0235]

[0236] 3.2.2.2 After the reaction, transfer the PCR tube to ice and add the ingredients listed in the table below. After mixing, place the PCR tube back in the thermal cycler and perform the following program: 65°C for 30 minutes; heated lid at 70°C, maintained at 4°C.

[0237]

[0238] 3.2.3 MDA product concentration detection

[0239] The Qubit dsDNA Assay Kit was used according to the manufacturer's instructions, and the MDA product concentration was detected using a Qubit fluorometor 3.0.

[0240] The test results are as follows:

[0241] Bst-LF-WT:29.6ng / uL

[0242] Bst-HS-1:34.8ng / uL

[0243] This shows that in MDA isothermal amplification, the synthesis ability of the recombinant protein (ie, the chimeric A family DNA polymerase) is higher than that of the wild-type A family DNA polymerase.

[0244] In the present invention, the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of technical features indicated. Therefore, a feature specified as "first" or "second" may explicitly or implicitly include at least one of such features. In the description of the present invention, "plurality" means at least two, for example, two, three, etc., unless otherwise specifically defined.

[0245] In the present invention, the terms "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and the features of different embodiments or examples without contradiction.

[0246] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

[0247]

[0248]

[0249]

[0250]

[0251]

[0252]

[0253]

[0254]

[0255]

Claims

1. A recombinant protein, It is characterized in that The amino acid sequence of the recombinant protein is obtained by replacing the homologous sequence in the wild-type A family DNA polymerase with an amino acid sequence that can enhance the interaction between the DNA polymerase and the target DNA in an amplification reaction.

2. The recombinant protein according to claim 1, It is characterized in that The A family DNA polymerase is selected from the group consisting of Escherichia coli DNA polymerase I, T3 DNA polymerase, T5 DNA polymerase, T7 DNA polymerase, Taq DNA polymerase, Bsu DNA polymerase and Bst DNA polymerase.

3. The recombinant protein according to claim 1 or 2, It is characterized in that The A family DNA polymerase is selected from at least one of a Bst DNA polymerase having an amino acid sequence as shown in SEQ ID NO: 1, a Taq DNA polymerase having an amino acid sequence as shown in SEQ ID NO: 9, an Escherichia coli DNA polymerase having an amino acid sequence as shown in SEQ ID NO: 10, and a Bsu DNA polymerase having an amino acid sequence as shown in SEQ ID NO:

11.

4. The recombinant protein according to any one of claims 1 to 3, It is characterized in that The homologous sequences in the A family DNA polymerases are derived from their thumb domains.

5. The recombinant protein according to any one of claims 1 to 4, It is characterized in that The homologous sequence in the A family DNA polymerase is selected from at least one of the following: The amino acid sequence from position 546 to position 554 of the Bst DNA polymerase as shown in SEQ ID NO:2; The amino acid sequence from position 199 to position 209 of the Taq DNA polymerase as shown in SEQ ID NO:12; The amino acid sequence from position 276 to position 285 of the Escherichia coli DNA polymerase as shown in SEQ ID NO: 13; or The amino acid sequence from position 249 to position 257 in the Escherichia coli Bsu polymerase as shown in SEQ ID NO:

14.

6. The recombinant protein according to any one of claims 1 to 5, It is characterized in that The homologous sequence in the A family DNA polymerase is replaced by a sequence comprising the amino acid sequence shown in SEQ ID NO:3, a sequence essentially consisting of the amino acid sequence shown in SEQ ID NO:3, or a sequence consisting of the amino acid sequence shown in SEQ ID NO:

3.

7. The recombinant protein according to any one of claims 1 to 6, It is characterized in that The amino acid sequence of the recombinant protein is selected from at least one of SEQ ID NO:4, SEQ ID NO:15, SEQ ID NO:16 and SEQ ID NO:

17.

8. The recombinant protein according to any one of claims 1 to 7, It is characterized in that The recombinant protein may further comprise any of additional conservative mutations, additions and deletions.

9. The recombinant protein according to any one of claims 1 to 8, It is characterized in that The processivity of the recombinant protein as a chimeric DNA polymerase is higher than that of the wild-type A family DNA polymerase.

10. A nucleic acid, It is characterized in that The nucleic acid encodes the recombinant protein according to any one of claims 1 to 9.

11. The nucleic acid according to claim 10, It is characterized in that The nucleotide sequence of the nucleic acid molecule is selected from at least one of SEQ ID NO:5, SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:

20.

12. A carrier, It is characterized in that The vector comprises the nucleic acid of claim 11.

13. A kit comprising the recombinant protein according to any one of claims 1 to 9, the nucleic acid according to claim 10 or 11, or the vector according to claim 12.

14. A method for preparing the recombinant protein according to any one of claims 1 to 9, It is characterized in that The method comprises: replacing the amino acid sequence obtained by replacing the homologous sequence in the wild-type A family DNA polymerase with an amino acid sequence capable of strengthening the interaction between the DNA polymerase and the target DNA in the amplification reaction to obtain the recombinant protein.

15. The method for preparing a recombinant protein according to claim 14, It is characterized in that The method comprises: performing a sequence alignment on a plurality of amino acid sequences from the wild-type A-family DNA polymerase to identify chimeric sequences; and The chimeric sequence is used to replace the homologous sequence in the wild-type A family DNA polymerase sequence to obtain a candidate recombinant protein sequence.

16. The method according to claim 15, It is characterized in that The method further comprises: Performing computer simulation analysis on the candidate recombinant protein sequence to determine the recombinant protein sequence, The recombinant protein sequence shown therein exhibits a reduced spatial configuration between the thumb domain and the finger domains.

17. The method according to claim 16, It is characterized in that Performing computer simulation analysis on the candidate recombinant protein sequence to obtain the recombinant protein sequence comprises: Performing structural prediction on the candidate recombinant protein sequence to obtain the tertiary structure of the recombinant protein; Simulating the tertiary structure of the recombinant protein to obtain a molecular docking structure of the recombinant protein; performing ligand-based structural optimization on the molecular docking structure to obtain an optimized recombinant protein structure; and The optimized recombinant protein structure is subjected to dynamic simulation to determine the recombinant protein sequence.

18. The method according to claim 16 or 17, It is characterized in that Also includes: The recombinant protein sequence is expressed and purified.

19. The method according to any one of claims 14 to 18, It is characterized in that The A family DNA polymerase is selected from the group consisting of Escherichia coli DNA polymerase I, T3 DNA polymerase, T5 DNA polymerase, T7 DNA polymerase, Taq DNA polymerase, Bsu DNA polymerase and Bst DNA polymerase.

20. The method according to any one of claims 14 to 19, It is characterized in that The A family DNA polymerase is selected from at least one of a Bst DNA polymerase having an amino acid sequence as shown in SEQ ID NO: 1, a Taq DNA polymerase having an amino acid sequence as shown in SEQ ID NO: 9, an Escherichia coli DNA polymerase having an amino acid sequence as shown in SEQ ID NO: 10, and a Bsu DNA polymerase having an amino acid sequence as shown in SEQ ID NO:

11.

21. The method according to any one of claims 14 to 20, It is characterized in that The homologous sequences in the A family DNA polymerases are derived from their thumb domains.

22. The method according to any one of claims 14 to 21, It is characterized in that The homologous sequence in the A family DNA polymerase is selected from at least one of the following: The amino acid sequence from position 546 to position 554 of the Bst DNA polymerase as shown in SEQ ID NO:2; The amino acid sequence from position 199 to position 209 of the Taq DNA polymerase as shown in SEQ ID NO:12; The amino acid sequence from position 276 to position 285 of the Escherichia coli DNA polymerase as shown in SEQ ID NO: 13; or The amino acid sequence from position 249 to position 257 in the Escherichia coli Bsu polymerase as shown in SEQ ID NO:

14.

23. The method according to any one of claims 14 to 22, It is characterized in that The homologous sequence in the A family DNA polymerase is replaced by a sequence comprising, substantially consisting of, or consisting of the amino acid sequence shown in SEQ ID NO:

3.

24. The method according to any one of claims 14 to 23, It is characterized in that The amino acid sequence of the recombinant protein is selected from at least one of SEQ ID NO:4, SEQ ID NO:15, SEQ ID NO:16 and SEQ ID NO:

17.

25. A method for amplifying a target DNA, It is characterized in that The target DNA is amplified using the recombinant protein according to any one of claims 1 to 9.

26. The method according to claim 25, It is characterized in that The amplification is isothermal amplification, and the isothermal amplification is selected from rolling circle amplification RCA, multiple displacement amplification MDA, recombinase polymerase amplification reaction RPA, strand displacement amplification SDA or loop-mediated isothermal amplification LAMP.

27. The method according to claim 25 or 26, It is characterized in that The amplification is used for constructing a DNA library or sequencing.

Images