WO2024138762A1 - Recombinant protein and use thereof - Google Patents

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 by using the recombinant protein. As a chimeric family A DNA polymerase, the recombinant protein has improved processivity, the amplification performance of the recombinant protein is remarkably better than that of a wild-type family A DNA polymerase, and the thermal stability thereof is higher than that of a family B DNA polymerase.

Description

Recombinant proteins and their applications Technical Field

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 technique

In the DNB-SEQ sequencing method, phi29 DNA polymerase is usually used for rolling circle amplification (RCA) and multiple displacement amplification (MDA) reactions. Although phi29 DNA polymerase is a B family polymerase with extremely high chain displacement activity and sustainable synthesis ability, it has poor thermal stability and is easily inactivated. Therefore, it needs to be stored at low temperatures under harsh conditions and has a short shelf life. In addition, phi29 DNA polymerase has strong non-specific binding ability and is difficult to elute, resulting in a strong fluorescence background of phi29 DNA polymerase during sequencing.

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

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

Summary of the invention

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

To this end, the 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 in an amplification reaction, a nucleic acid encoding the recombinant protein, a vector comprising the nucleic acid, a kit comprising the above protein, nucleic acid or vector, a method for preparing the above recombinant protein, and a method for amplifying a target DNA using the recombinant protein. The recombinant protein provided by the embodiments of the present invention is a chimeric A family DNA polymerase with improved processivity and enhanced thermal stability, polymerization activity and DNA double-strand affinity, which can be used to amplify the target DNA.

In a first aspect, an embodiment of the present invention provides a recombinant protein whose amino acid sequence is obtained by replacing a homologous sequence in a 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.

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.

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.

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

In some embodiments, 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 in 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 257 in the Escherichia coli Bsu polymerase as shown in SEQ ID NO:14.

In some embodiments, 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.

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.

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

In some embodiments, the processivity of the recombinant protein as a chimeric DNA polymerase is higher than that of the wild-type A-family DNA polymerase.

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.

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.

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

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

In the 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.

In some embodiments, the method comprises: performing sequence alignment on 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.

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

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.

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

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.

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.

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

In some embodiments, 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 in 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 257 in the Escherichia coli Bsu polymerase as shown in SEQ ID NO:14.

In some embodiments, 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.

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.

In a sixth aspect, an embodiment of the present invention provides a method for amplifying a target DNA, using the recombinant protein described in any embodiment of the first aspect to amplify the target DNA.

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.

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

The recombinant protein in the embodiment of the present invention as a chimeric A family DNA polymerase has improved processivity, and its amplification performance is significantly better than that of the wild-type A family DNA polymerase. In addition, compared with the B family DNA polymerase, the recombinant protein in the embodiment of the present invention also has the following advantages: high thermal stability, convenient elution and easy storage.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the technical solutions of the embodiments of the present disclosure, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. 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.

Figure 1 is a schematic diagram of the structure of the large fragment of Bst DNA polymerase.

Figure 2 is a schematic diagram of the sequence comparison between the large fragment of wild-type Bst DNA polymerase and the chimeric DNA polymerase according to an embodiment of the present invention.

3 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.

FIG. 4 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 ways

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.

A family DNA polymerases include E. 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 from Geobacillus stearothermophilus is a multifunctional enzyme characterized by high temperature resistance, strong strand displacement activity and loop-mediated isothermal amplification, but its sustainable amplification ability still needs to be improved. Bst DNA polymerase belongs to the A family DNA polymerase and has a typical right-hand structure, which is divided into thumb domain, palm domain, finger domain and 5'-3' exonuclease domain, in which the target DNA and part of the newly synthesized DNA are sandwiched between the thumb domain and the finger domain in the polymerization reaction. 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, and its structure is shown in Figure 1.

The DNA polymerase currently used for sequencing is mainly the B family strand displacement enzyme, mainly phi29 polymerase. The phi29 polymerase is a polymerase from the bacteriophage phi29 of Bacillus subtilis. It has high continuous synthesis ability and strand displacement activity and is often used in RCA-based DNB preparation and MDA-based single-cell whole genome amplification. The phi29 polymerase is a mesothermal isothermal enzyme with an optimal reaction temperature of about 30°C, but it can be completely inactivated by incubation at 65°C for 10 minutes. The strong strand displacement ability and high continuous synthesis ability of the phi29 polymerase are mainly due to its TPR2 (terminal protein region 2) domain. The TPR2 domain can unwind the double-stranded chain of the template during the chain synthesis process, allowing only the template chain to enter and sandwiching the template between the Exo domain and the TPR2 domain, so that the enzyme and the template cannot be separated, thus enabling the phi29 polymerase to have a continuous amplification ability of >70,000nt. However, phi29 polymerase has defects such as strict storage conditions, short shelf life, and large fluorescence background during sequencing.

Compared with the DNA polymerases of the B family (such as phi29, Pfu, and Kod polymerases), the thumb domain of the A family polymerases has a smaller area that binds to the DNA template and has a smaller interaction with the nascent DNA chain, resulting in weaker sustainable amplification capabilities of the A family polymerases, including Bst polymerase. In addition, since the 5'-3' exodomain has a DNA binding effect, it can prevent the complete dissociation of the polymerase from the template. When the 5'-3' exodomain is knocked out, the continuous synthesis ability of the large fragments of the A family polymerases will decrease, and they usually cannot synthesize DNA chains longer than 10kb very well.

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 implementation scheme of the present application, i.e., the chimeric A family polymerase, has achieved a structural transformation that allows the thumb domain to extend in the direction of the finger domain, reduces the space between the thumb domain and the finger domain, and keeps the template chain and part of the newly synthesized DNA chain between the thumb domain and the finger domain, thereby playing a role similar to 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 A family DNA polymerases.

According to one aspect of the present invention, 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.

It should be noted that the thumb domain of the recombinant protein provided in the embodiment of the present invention is replaced with a chimeric protein sequence. This sequence strengthens the interaction between the thumb domain and the DNA double strand, and forms a circular sliding clamp around the DNA template strand, so that the affinity of the polymerase for the DNA double strand is increased, thereby improving the effect of processivity. The recombinant protein also has improved processivity, enhanced thermal stability, polymerization activity, and DNA double strand affinity, and can be used for a variety of DNA isothermal amplification reactions in library construction and sequencing.

It should be noted that the processivity of a polymerase can be defined as the number of nucleotides processed in one incorporation. The processivity of a DNA polymerase generally reflects the rate and speed of synthesis, as well as the affinity of the enzyme for the substrate. DNA polymerases with high processivity are suitable for amplifying long templates, sequences with secondary structures 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.

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.

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.

The sequence of SEQ ID NO:1 is:

(加粗下划线为同源序列SEQ ID NO:2)

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

The sequence of SEQ ID NO:9 is:

(加粗下划线为同源序列SEQ ID NO:12)

(The bold underline is the homologous sequence SEQ ID NO: 12)

The sequence of SEQ ID NO: 10 is:

(加粗下划线为同源序列SEQ ID NO:13)

(The bold underline is the homologous sequence SEQ ID NO: 13)

The sequence of SEQ ID NO:11 is:

(加粗下划线为同源序列SEQ ID NO:14)

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

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

In some embodiments, 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 in the Bst DNA polymerase as shown in SEQ ID NO:2;

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

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

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

The sequence of SEQ ID NO:2 is: VLKKTKTGY.

The sequence of SEQ ID NO:12 is: AIGKTEKTGKR

The sequence of SEQ ID NO:13 is: PLKKTPGGAP

The sequence of SEQ ID NO:14 is: VVKKTKTGY

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. The sequence of SEQ ID NO: 3 is: PNREMKNQGSKKTLGSTRRGIDNGRKLRLGRQF.

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.

The sequence of SEQ ID NO:4 is:

(加粗下划线为嵌合序列SEQ ID NO:3)

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

The sequence of SEQ ID NO:15 is:

(加粗下划线为嵌合序列SEQ ID NO:3)

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

The sequence of SEQ ID NO:16 is:

(加粗下划线为嵌合序列SEQ ID NO:3)

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

The sequence of SEQ ID NO:17 is:

(加粗下划线为嵌合序列SEQ ID NO:3)

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

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

In some embodiments, the processivity of the recombinant protein as a chimeric DNA polymerase is higher than that of the wild-type A-family DNA polymerase.

According to yet 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.

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.

The sequence of SEQ ID NO:5 is:

The sequence of SEQ ID NO: 18 is:

The sequence of SEQ ID NO:19 is:

The sequence of SEQ ID NO:20 is:

According to still 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.

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.

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 an amplification reaction to obtain the recombinant protein.

In some embodiments, the method comprises: performing sequence alignment on 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.

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

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.

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

In some specific embodiments, the method specifically includes: searching through the sequence database of A family polymerase 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 DNA double strands and recombinant protein and (3) kinetic simulation analysis on the recombinant protein with a chimeric sequence according to an embodiment of the present invention; using a prokaryotic expression system to express and purify the recombinant protein determined by the simulation analysis, and determine its activity; and confirming the protein sequence by sequencing.

It can be understood that the sequence alignment in this method includes various alignment strategies such as global alignment, local alignment, double sequence alignment, and multiple sequence alignment.

It can be understood that the structures of A family DNA polymerases are relatively conservative and similar, and the method for preparing recombinant proteins can be applied to replace homologous sequences of various A family DNA polymerases and prepare the recombinant proteins.

In the embodiments of the present invention, the term "molecular docking" refers to placing a small molecule (ligand) in the binding area of a macromolecular target (receptor) by computer simulation, so that the binding force and binding mode of the ligand and the receptor can be calculated by adjusting the physicochemical parameters, and the lowest energy conformation can be obtained when the two are combined in their active areas. In the present invention, the ligand and receptor of 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.

In an embodiment of the present invention, the term "dynamic simulation" refers to the use of Newton's classical mechanics to calculate the trajectory of molecules in space, solve the Newtonian equations of molecules or atoms in the system under the combined action potential energy and system constraints, and simulate the microscopic process of the system advancing over time. The flexibility of proteins and ligands enables them to seek the most consistent and lowest energy conformation when binding to each other. Usually, after 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 with the screened small molecule ligand.

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.

In some embodiments, 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.

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

In some embodiments, 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 in 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 257 in the Escherichia coli Bsu polymerase as shown in SEQ ID NO:14.

In some embodiments, 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.

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.

According to yet 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.

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.

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

Example

Example 1

This embodiment is based on the sequences of Bst polymerase, Taq DNA polymerase, Escherichia coli DNA polymerase and Bsu DNA polymerase in the A family DNA polymerase, and performs database search and MSA homologous sequence comparison to determine candidate chimeric sequences for replacing homologous sequences in wild-type polymerases; uses tools such as Alphafold, Autodock and Gromacs to perform structural prediction on the recombinant protein with chimeric sequence in the embodiment of the present invention, and performs computational simulations such as molecular docking and kinetic simulation analysis of the DNA double chain and the recombinant protein.

1.1 Determination of chimeric sequences

Protein-blast tool was used to compare homologous sequences of A family polymerases in the NCBI protein sequence library, and it was determined that the chimeric sequence (SEQ ID NO: 3) was homologous to amino acids 546-554 in the thumb domain of Bst DNA polymerase (SEQ ID NO: 2), amino acids 199-209 in the thumb domain of Taq DNA polymerase large fragment (SEQ ID NO: 12), amino acids 276-285 in the thumb domain of Escherichia coli DNA polymerase large fragment (SEQ ID NO: 13), and amino acids 249-257 in the thumb domain of Bsu DNA polymerase large fragment (SEQ ID NO: 14). The comparison results are shown in Figure 2. The chimeric sequence is derived from human DNA polymerase θ (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.

1.2 Replacement of homologous sequences in wild-type A-family DNA polymerases with chimeric sequences

1.2.1 According to 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 to obtain a chimeric Bst DNA polymerase having the sequence described in SEQ ID NO:4.

The sequence of wild-type Bst DNA polymerase large fragment (Bst-LF WT) SEQ ID NO: 1:

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

(加粗下划线为同源序列SEQ ID NO:2)

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

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

(加粗下划线为嵌合序列SEQ ID NO:3)

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

1.2.2 According to 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.

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

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

(加粗下划线为同源序列SEQ ID NO:12)

(The bold underline is the homologous sequence SEQ ID NO: 12)

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

(加粗下划线为嵌合序列SEQ ID NO:3)

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

1.2.3 According to 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.

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

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

(加粗下划线为同源序列SEQ ID NO:13)

(The bold underline is the homologous sequence SEQ ID NO: 13)

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

(加粗下划线为嵌合序列SEQ ID NO:3)

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

1.2.4 According to 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 to obtain a chimeric Bsu DNA polymerase having the sequence described in SEQ ID NO:17.

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

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

(加粗下划线为同源序列SEQ ID NO:14)

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

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

(加粗下划线为嵌合序列SEQ ID NO:3)

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

1.3 Structure prediction

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

1.4 Molecular docking simulation

Based on the predicted structure, the molecular docking simulation of the DNA double-stranded with the chimeric A family DNA polymerase Bst-HS-1, Taq-HS-1, Ecoli-HS-1 and Bsu-HS-1 was performed using the Autodock tool in the presence of dNTP to confirm the binding strength of the chimeric DNA polymerase with an extended thumb domain after modification to the target DNA. As shown in Figure 3, the molecular docking simulation shows that the chimeric sequence not only reduces the space between the thumb domain and the finger domain, thereby keeping the template chain between the thumb domain and the finger domain, but also can maintain a tight binding with the newly synthesized double-stranded DNA, thereby stabilizing the polymerase-DNA structure.

1.5 Ligand structure optimization

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.

1.6 Dynamic simulation

The optimized structure was subjected to 25 ns kinetic simulation at a simulation temperature of 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 can be continuously maintained at a distance of 4-6 angstroms, allowing the DNA template chain to be clamped in the pocket structure formed by the two, while the extended region can also maintain a tight bond with the newly synthesized double-stranded DNA.

In addition, experimental structures have shown that additional mutations can be made at several sites in the chimeric sequence to enhance the thermal stability and polymerization activity of the chimera, and to further improve the affinity of the chimeric polymerase for double-stranded DNA and its ability to continuously synthesize.

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 helix and the continuous synthesis ability.

The following data is a comparison of the binding energy simulation calculations of four A-family polymerases and their chimeric mutants to double-stranded DNA. It can be seen that all polymerases can enhance their affinity with double-stranded DNA after adding chimeric fragments:

1.Bst-LF WT: -164.2 kJ/mol

Bst-HS-1: -272.0 kJ/mol

ΔΔG＝-107.8kJ/mol

2.Taq-LF WT: -150.2 kJ/mol

Taq-HS-1: -277.6 kJ/mol

ΔΔG＝-127.4kJ/mol

3.Ecoli-LF WT: -186.3 kJ/mol

Ecoli-HS-1: -327.9 kJ/mol

ΔΔG＝-141.6 kJ/mol

4.BSU-LF WT: -178.4 kJ/mol

BSU-HS-1: -283.3 kJ/mol

ΔΔG＝-104.9 kJ/mol

Example 2

In this example, based on the rationally designed chimeric polymerase Bst-HS-1, a prokaryotic expression system was used to express and purify the protein to obtain the Bst-HS chimeric DNA polymerase for practical application.

2.1 Preparation of chimeric DNA polymerase plasmid

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

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

2.1.1 The above-mentioned point mutation PCR was performed 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 an amplified product.

Table 1 PCR reaction system

Upstream primer (SEQ ID NO:7): TACTTTCAGGGCGCCGAGGGTGAAAAACCTCTG

Downstream primer (SEQ ID NO:8): GGGTTAACCTTATTTAGCATCATACCAGGTCGGG

Table 2 PCR reaction conditions

2.1.2 Add 0.4 μl dpn1 enzyme to a 20 μl 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.

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

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

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

2.2 Inducible expression of Bst-HS chimeric mutants

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.

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.

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.

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.

2.2.5 Centrifuge the third bacterial solution at 12000 rpm for 10 min and collect the bacterial cells as the precipitate.

2.3 Purification of Bst-HS chimeric mutant protein

2.3.1 Suspend the precipitate in 40 ml of solution A and crush the bacteria with a high-pressure crusher at a pressure of 700 bar for 5 minutes to obtain the fourth bacterial solution.

Solution A (pH 7.4)

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, and then filter the supernatant with a 0.22 μm filter membrane and collect the filtrate.

2.3.3 The filtrate was loaded onto a nickel column for purification, wherein 50% B solution was used for elution to obtain the target protein.

Solution B (pH 7.4)

2.3.4 The solution B containing the target protein is replaced with dialysate by dialysis to obtain the purified target protein.

Dialysate (pH 7.6)

2.3.5 The purified target protein is packaged and stored for subsequent activity determination.

Example 3

In this example, the purified chimeric DNA polymerase Bst-HS and the wild-type DNA polymerase large fragment Bst-LF-WT were tested for polymerization activity to compare their processivity. The wild-type Bst DNA polymerase solution with a His6 tag and the chimeric DNA polymerase Bst DNA solution with a His6 tag prepared in Example 2 were the enzyme solutions to be tested.

3.1 Synthetic capacity of chimeric DNA polymerase in RCA amplification

3.1.1 Library preparation

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 mixture of library + primer (concentration of approximately 1.77 ng/μl).

3.1.2 RCA reaction

Prepare the library + primer mixture in the RCA reaction system as shown in Table 3 below and incubate at 65°C for 1 hour. Use 0.1 μl of 0.5 M EDTA to terminate the amplification reaction to obtain the RCA reaction product.

Table 3 RCA reaction system

3.1.3 RCA product concentration detection

Use the Qubit ssDNA detection kit according to the instructions and use Qubit fluorometor 3.0 to detect the concentration of RCA reaction products. The results are as follows:

Bst-LF-WT:33.6ng/uL

Bst-HS-1:39.9ng/uL

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

3.2 Synthetic capacity of chimeric DNA polymerase in MDA amplification

3.2.1 Preparation of DNB

3.2.1.1 Mix the single-stranded circular DNA (self-synthesized Ecoli V3 single-stranded circular library, concentration c) with DNB preparation buffer and TE buffer in a PCR tube, and place the PCR tube on an ice box for about 0.5 hours. Use a vortex oscillator to shake the PCR tube for 5 seconds to mix the mixture evenly, centrifuge briefly and place on ice for later use. Place the PCR tube in a PCR instrument and perform the following program: 95℃ for 1min, 65℃ for 1min, 40℃ for 1min, the hot cover temperature is 102℃, and 4℃ is maintained.

3.2.1.2 After the reaction, transfer the PCR tube to ice and add the volumes of DNB polymerase mixture I and DNB polymerase mixture II as shown in the table below. Place the PCR tube in the PCR instrument again and perform the following program: 30℃ for 30 minutes, hot cover temperature 35℃, 4℃ hold.

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

3.2.2 MDA reaction

3.2.2.1 Take the DNB prepared in 3.2.1, mix them in a PCR tube according to the following reaction system, and carry out the following procedure: 95℃ for 1 minute, 65℃ for 1 minute, 40℃ for 1 minute, the hot cover temperature is 102℃, and maintained at 4℃.

3.2.2.2 After the reaction, transfer the PCR tube to ice and add the ingredients shown in the table below to the PCR tube. After mixing, place the PCR tube in the PCR instrument again and perform the following program: 65℃ for 30 minutes; hot cover temperature 70℃, maintained at 4℃.

3.2.3 MDA product concentration detection

The Qubit dsDNA Assay Kit was used according to the instructions, and the MDA product concentration was detected using Qubit fluorometor 3.0.

The test results are as follows:

Bst-LF-WT:29.6ng/uL

Bst-HS-1:34.8ng/uL

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

In the present invention, the terms "first" and "second" are used for descriptive purposes only and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In the description of the present invention, the meaning of "plurality" is at least two, such as two, three, etc., unless otherwise clearly and specifically defined.

In the present invention, the terms "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" etc. 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 representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the described specific features, structures, materials or characteristics may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine different embodiments or examples and features of different embodiments or examples described in this specification without contradiction.

Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims (27)

A recombinant protein, 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. The recombinant protein according to claim 1 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. The recombinant protein according to claim 1 or 2 is characterized in that 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. The recombinant protein according to any one of claims 1 to 3, characterized in that the homologous sequence in the A family DNA polymerase is derived from its thumb domain. The recombinant protein according to any one of claims 1 to 4, 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 in 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 257 in the Escherichia coli Bsu polymerase as shown in SEQ ID NO:14. The recombinant protein according to any one of claims 1 to 5, 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. The recombinant protein according to any one of claims 1 to 6, 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. The recombinant protein according to any one of claims 1 to 7, characterized in that the recombinant protein further comprises any one of additional conservative mutations, additions and deletions. The recombinant protein according to any one of claims 1 to 8, 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. A nucleic acid, characterized in that the nucleic acid encodes the recombinant protein according to any one of claims 1 to 9. The nucleic acid according to claim 10 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. A vector, characterized in that the vector comprises the nucleic acid according to claim 11. 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. A method for preparing a recombinant protein according to any one of claims 1 to 9, characterized in that the method comprises: replacing an amino acid sequence obtained by replacing a homologous sequence in a 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, so as to obtain the recombinant protein. The method for preparing a recombinant protein according to claim 14, 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. The method according to claim 15, 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. The method according to claim 16, characterized in that the computer simulation analysis of 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. The method according to claim 16 or 17 is characterized in that it also includes: expressing and purifying the recombinant protein sequence. The method according to any one of claims 14 to 18 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. The method according to any one of claims 14 to 19 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 shown in SEQ ID NO: 1, a Taq DNA polymerase having an amino acid sequence shown in SEQ ID NO: 9, an Escherichia coli DNA polymerase having an amino acid sequence shown in SEQ ID NO: 10, and a Bsu DNA polymerase having an amino acid sequence shown in SEQ ID NO: 11. The method according to any one of claims 14 to 20, characterized in that the homologous sequence in the A family DNA polymerase is derived from its thumb domain. The method according to any one of claims 14 to 21, 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 in 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 257 in the Escherichia coli Bsu polymerase as shown in SEQ ID NO:14. The method according to any one of claims 14 to 22, 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. The method according to any one of claims 14 to 23, 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. A method for amplifying a target DNA, characterized in that the target DNA is amplified using the recombinant protein described in any one of claims 1 to 9. The method according to claim 25 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. The method according to claim 25 or 26 is characterized in that the amplification is used for constructing a DNA library or sequencing.

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