Bst dna polymerase fused with single-strand dna binding protein and application thereof

Abstract

The application discloses a kind of BST DNA polymerase of fusion single-stranded DNA binding protein and application thereof, it is related to molecular biology and nucleic acid amplification technical field.Fusion protein successively includes artificial design of Escherichia coli SSB protein fragment with single-stranded DNA binding activity, flexible connecting peptide and artificial design of BST DNA polymerase fragment with strand displacement activity from N-terminal to C-terminal, by optimizing the length and amino acid composition of connecting peptide, BST polymerase and SSB protein realize functional synergy, significantly improve the amplification efficiency, high GC template amplification capacity and anti-inhibitor capacity of enzyme.Compared with the use of BST enzyme alone or the physical mixture of BST enzyme and SSB, the activity of the fusion enzyme SSB-BST of the application is 5 times and 700 times higher than that of NEB's SSB+BST and self-made BST according to the method of GB / T36755-2018 "BST DNA polymerase".

Description

Technical Field

[0001] This invention relates to the fields of molecular biology and nucleic acid amplification technology, and more specifically to a BST DNA polymerase fused with a single-stranded DNA-binding protein and its applications. Background Technology

[0002] Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid isothermal amplification technique developed in 2000 by Japanese scholars Notomi et al. It boasts advantages such as speed, sensitivity, high specificity, and the elimination of the need for expensive equipment, and has been widely applied in pathogen detection, clinical diagnosis, food safety, and environmental monitoring. The core enzyme in LAMP technology is BST DNA polymerase, derived from *Bacillus stearothermophilus*, which exhibits strong strand displacement activity and 5'→3' polymerase activity. Although BST polymerase demonstrates excellent performance in isothermal amplification, it is prone to false negatives for low-copy-number target nucleic acids. To address this issue, researchers have attempted to combine BST polymerase with single-stranded DNA binding protein (SSB). SSB protein specifically binds to single-stranded DNA, preventing its re-annealing and secondary structure formation, thereby improving the elongation efficiency of the DNA polymerase. However, existing technologies typically employ a physical mixing method between BST enzyme and SSB protein, which has the following drawbacks: the two proteins cannot achieve effective spatial synergy in the reaction system, resulting in limited effects; the concentration of SSB protein is difficult to optimize, and excessively high concentrations can inhibit the activity of BST polymerase; and the two proteins have different stabilizations, leading to a shortened shelf life of the kit.

[0003] Although there have been a few reports on SSB-BST fusion enzymes, these fusion enzymes generally suffer from problems such as severe loss of enzyme activity.

[0004] Therefore, there is an urgent need to develop a high-performance SSB-BST fusion enzyme to meet the growing demand for rapid nucleic acid detection. Summary of the Invention

[0005] In view of this, the present invention provides a BST DNA polymerase fused with a single-stranded DNA-binding protein and its applications. This fusion enzyme exhibits high enzyme activity and amplification efficiency.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A BST DNA polymerase fused with a single-stranded DNA-binding protein, wherein the fusion protein comprises, from N-terminus to C-terminus, an Escherichia coli SSB protein fragment with single-stranded DNA-binding activity, a flexible linker peptide, and an artificially designed BST DNA polymerase fragment with strand displacement activity.

[0008] The amino acid sequence of the Escherichia coli SSB protein fragment is shown in SEQ ID NO.1;

[0009] The amino acid sequence of the flexible linker peptide is shown in SEQ ID NO.2;

[0010] The amino acid sequence of the BST DNA polymerase fragment is shown in SEQ ID NO.3.

[0011] Furthermore, the amino acid sequence of the fusion protein is shown in SEQ ID NO.4.

[0012] Furthermore, the fusion protein also contains a purification tag located at the N-terminus or C-terminus; the purification tag is either His-tag or His-SUMO-tag.

[0013] The second objective of this invention is to provide a nucleic acid molecule encoding a BST DNA polymerase that encodes any of the above-mentioned fused single-stranded DNA-binding proteins.

[0014] Furthermore, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.5.

[0015] The third objective of this invention is to provide a recombinant expression vector containing the aforementioned nucleic acid molecule, wherein the nucleic acid molecule is operatively linked to a promoter; the promoter is a T7 promoter; and the expression vector is a pET series vector.

[0016] The fourth objective of this invention is to provide a host cell comprising the above-mentioned recombinant expression vector; the host cell is Escherichia coli BL21(DE3), BL21(DE3)pLysS or Rosetta(DE3).

[0017] The fifth objective of this invention is to provide a method for preparing a BST DNA polymerase that fuses any of the above-mentioned single-stranded DNA-binding proteins, comprising the following steps:

[0018] (1) Construct a recombinant expression vector containing a nucleic acid molecule encoding a fusion protein;

[0019] (2) Transform the recombinant expression vector into host cells;

[0020] (3) Culture host cells to induce fusion protein expression;

[0021] (4) Collect and lyse host cells and purify to obtain fusion protein.

[0022] Furthermore, the conditions for inducing expression in step (3) are: when the bacterial culture OD 600When the concentration reaches 0.6-0.8, add IPTG to a final concentration of 0.1-1.0 mM and induce expression at 16-25℃ for 12-24 hours;

[0023] The purification method in step (4) is one-step purification by Ni-NTA affinity chromatography.

[0024] The sixth objective of this invention is to provide the application of any of the above-mentioned BST DNA polymerases fused with single-stranded DNA-binding proteins in nucleic acid amplification.

[0025] The seventh objective of this invention is to provide the application of any of the above-mentioned BST DNA polymerases fused with single-stranded DNA binding proteins in the preparation of detection kits.

[0026] Furthermore, the preferred expression vector is between pET-28a (+) vector NdeI and XhoI. The preferred host cell is Escherichia coli BL21 (DE3).

[0027] As can be seen from the above technical solution, compared with the prior art, this invention discloses a BST DNA polymerase fused with a single-stranded DNA binding protein and its application, achieving the following technical effects: This invention exhibits excellent high GC template amplification performance; by optimizing the length and amino acid composition of the linker peptide, this invention achieves perfect functional synergy between the BST polymerase and the SSB protein. Detection sensitivity is improved compared to using BST enzyme alone or a physical mixture of BST enzyme and SSB. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0029] Figure 1 The attached figure is a comparison diagram of the amino acid sequence design of the Escherichia coli SSB protein provided by the present invention before and after.

[0030] Figure 2 The attached figure is a comparison diagram of the amino acid sequence of the BST DNA polymerase provided by the present invention before and after optimization.

[0031] Figure 3The attached figure shows the expression and purification of the SSB-BST fusion protein provided by this invention. The meanings of each lane are as follows: SSB-BST: Marker, SSB-BST before purification, SSB-BST purified by Ni column; BST: Marker, BST purified product, BST purified by Ni column.

[0032] SSB: Marker, Marker, SSB Ni column purified product.

[0033] Figure 4 The attached figure is an activity detection diagram of the SSB-BST fusion protein provided by the present invention. Detailed Implementation

[0034] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0035] This invention discloses a BST DNA polymerase fused with a single-stranded DNA-binding protein and its applications.

[0036] In the examples, reagents and instruments not mentioned are all conventional, as shown in Tables 1 and 2:

[0037] Table 1

[0038] Acrylamide Sinopharm Chemical Reagent Co., Ltd. N,N'-Methylenebisacrylamide Sinopharm Chemical Reagent Co., Ltd. Concentrated hydrochloric acid (HCl) Sinopharm Chemical Reagent Co., Ltd. glycine Boao Tuoda Bromophenol Blue (BPB) Sinopharm Chemical Reagent Co., Ltd. glycerin Sinopharm Chemical Reagent Co., Ltd. methanol Sinopharm Chemical Reagent Co., Ltd. T4 DNA ligase Prometheus Ncol restriction endonucleases Prometheus Xhol restriction endonucleases Prometheus Bovine serum albumin Biological Center provides PEG20000 Biological Center provides P-300 Sinopharm Chemical Reagent Co., Ltd. sucrose Sinopharm Chemical Reagent Co., Ltd. PVP-30 Sinopharm Chemical Reagent Co., Ltd. Tetramethylethylenediamine (TEMED) Biomol Diaminobenzidine (DAB) Solarborg 30% hydrogen peroxide Yantai Shuangshuang Chemical Co., Ltd. <![CDATA[K2CO3]]> Sinopharm Chemical Reagent Co., Ltd. BSA Subfamily PEG-2W Sinopharm Chemical Reagent Co., Ltd. Sodium citrate Sinopharm Chemical Reagent Co., Ltd. Casein Sigma-Aldrich <![CDATA[Na2HPO4×12H2O]]> Sinopharm Chemical Reagent Co., Ltd. <![CDATA[NaH2PO4×2H2O]]> Sinopharm Chemical Reagent Co., Ltd. Tween-20 Sinopharm Chemical Reagent Co., Ltd. Coomassie Brilliant Blue R-250 Solarborg Anhydrous ethanol Sinopharm Chemical Reagent Co., Ltd. Agar powder Dongge Bio Green Safe DNA Dye Dongge Bio 10×Loading Buffer Dongge Bio Tris(hydroxymethyl)aminomethane (Tris) Sinopharm Chemical Reagent Co., Ltd. <![CDATA[Na2EDTA-2H2O]]> Sinopharm Chemical Reagent Co., Ltd. <![CDATA[Acetic acid (CH3COOH)]]> Sinopharm Chemical Reagent Co., Ltd. Sodium chloride Sinopharm Chemical Reagent Co., Ltd. Yeast extract Solarborg trypsin Solarborg Agar powder Solarborg Kanapine sodium Solarborg IPTG Solarborg Sodium hydroxide (NaOH) Sinopharm Chemical Reagent Co., Ltd. dNTP Mix Bao Ri Medical Products ammonium persulfate Sinopharm Chemical Reagent Co., Ltd. Coomassie Brilliant Blue Solarborg ethanol Sinopharm Chemical Reagent Co., Ltd. glacial acetic acid Sinopharm Chemical Reagent Co., Ltd. Magnesium chloride Sinopharm Chemical Reagent Co., Ltd. DTT Solarborg Ampicillin sodium Solarborg Sodium dodecyl sulfonate (SDS) Sinopharm Chemical Reagent Co., Ltd. β-Mercaptoethanol Sinopharm Chemical Reagent Co., Ltd. Acrylamide Sinopharm Chemical Reagent Co., Ltd.

[0039] Table 2

[0040] High-speed benchtop refrigerated centrifuge Jiangdong Instruments Clean bench Jin Xiao Ultrasonic cell disruptor Xinzhi Bio High current electrophoresis apparatus BIO-RAD Digital display constant temperature water bath Shanghai Yuejin peristaltic pump Lange Medical centrifuge Xiangyi Real-time quantitative PCR instrument BIO-RAD Rapid Mixer New Kang Vacuum drying oven Zhengzhou Shengyuan Instrument Co., Ltd. Ultra-micro UV spectrophotometer Thermo Fisher Scientific, Inc. One ten-thousandth electronic balance Ohaus pH meter Mettler Toledo Ultrapure water system Merck Millibote Chemical Imaging Analyzer BIO-RAD Electric thermostatic incubator Shanghai Jinghong Thermostatic Shaker SHIPING Example 1

[0041] Construction of BST, SSB, and SSB-BST fusion proteins

[0042] Single-stranded DNA binding protein was selected from NCBI (GenBank: CAA0273388.1), and a large fragment of Bacillus stearothermophilus BST DNA polymerase (WP_044730601.1) was selected. The natural N-terminal 5'-3' exonuclease domain of 1-300 amino acids was removed, and only the polymerase domain and strand substitution domain were retained.

[0043] The manual optimization process using online software involved: ExPASy ProtParam was used for manual analysis to screen for optimized fragments with an instability coefficient <40; SWISS-MODEL was used for 3D modeling, and flexible loop regions were manually identified. Site-directed mutagenesis was performed to reduce spatial folding steric hindrance and improve the stability coefficient; online codon optimization tools were used to optimize the fusion gene for E. coli codon preference, adapting it to the E. coli BL21 expression strain. The designed sequences were then synthesized by Shanghai Sangon Biotech and inserted between Nde I and XhoI in the pET-28a (+) vector, resulting in three protein expression vectors: SSB, BST, and SSB-BST.

[0044] The amino acid sequence comparison of the E. coli SSB protein before and after design is shown in [link to design]. Figure 1 The optimized amino acid sequence is: MASRGVNKVILVGNLGDPKVRYMPNGGAVANITLATKESWRDKATGKMKQTETWHRVVLFGkLAEVAKKYLRKGRQVYIKGQLRTRKWTKQSGQDRYTTKVVVNVGGTMQMLGGRQGGGAPAGGNIKGGQPRGGWGQPRQPRGGNQFRGGAQSRPQQRAPAAPSKEPRMKFRKIPF, as shown in SEQ ID No. 1.

[0045] The optimized amino acid sequence comprehensively enhances the positive charge of the binding domain, reduces the negative charge, and increases the positive charge, such as Q→R and S→R next to G in the flexible region, which strengthens inter-chain anchoring. The terminal dfdddipf is continuously enriched with aspartic acid D, which strongly negatively repels ssDNA and is directly removed and replaced with kfrkipf. The acidic D part is replaced with basic K or R to reduce and eliminate electrostatic repulsion and anchor ssDNA.

[0046] The amino acid sequence of the flexible linker peptide is (GGS)n, where n=5~8, preferably n=6; amino acid sequence: GGSGGSGGSGGSGGSGGS, as shown in SEQ ID NO.2.

[0047] A comparison of the amino acid sequence of BST DNA polymerase before and after sequence optimization is shown in the figure. Figure 2 ;

[0048] Optimized amino acid sequence:

[0049] MAFTLADRVTEEMLADKAALVVEVVEENYHDAPIVPIAVVNEHGRFFLRPETALADPQFVAWLGDETKKKSMFDSKRAAVALKWKGIELCGVSFDLLLAAYLLDPAQGVDDVAAAAKMKQYEAVRPDEAVYGKGAKRAVPDEPVLAEHLVRKAAAIWELERPFLDKLRRNEQDRLLVELEQPLSSILAEMEFAGVKVDKKRLEQMGKELAEQLGTVEQRIYELAGQEFNINSPKQLGVILFEKLQLPVLKKTKTGYSTSADVLEKYAPYHEIVENILHYRQLGKLQSTYIEGLLKVVRPDTRKKVHTIFNQALTQTGRLSSTEPNLQNIPIRLEEGRKIRQAFVPSESDWLIFAADYSQIELRVLAHIAEDDNLMEAFRRDLDIHTKTAMDIFQVSAEDEVTPNMRRQAKAVNFGIVYGISDYGLAQDLNISRKEAAEFIERYFESFPGVKRYMENIVQEAAQKRGYVTTLLHRRRYLPDITSRRFNAVRSFAERMAMNTPIQGSAADIIKKAMIDLNARLKEERLQARLLLQVHDELILEAPKEEMERLCRLVPEVMEQAVTLRVPLKVDLHYHGPTWFDAK, as shown in SEQ ID NO.3.

[0050] In summary, the fusion protein comprises, from N-terminus to C-terminus, an artificially designed E. coli SSB protein fragment with single-stranded DNA binding activity, a flexible linker peptide, and an artificially designed BST DNA polymerase fragment with strand displacement activity, with the amino acid sequence as shown in SEQ ID NO.4.

[0051]

[0052] The fusion protein also contains a purification tag located at the N-terminus or C-terminus, which is selected from histidine tags (His-tag), His-SUMO tags, and preferably 6×His tags.

[0053] Example 2

[0054] Expression and purification of SSB-BST fusion protein (see...) Figure 3 )

[0055] Transformation and Induction of Expression: The recombinant expression vector pET-SSB-BST was transformed into *E. coli* BL21 (DE3) competent cells and plated on LB agar plates containing 50 μg / mL kanamycin. The plates were incubated overnight at 37°C. Single colonies were picked and inoculated into 5 mL of LB liquid medium containing kanamycin and cultured at 37°C with shaking until OD (out of control) was reached. 600 =0.6~0.8. Add IPTG to a final concentration of 0.5mM and induce expression at 16℃ for 16 hours.

[0056] Protein purification: Cells were collected by centrifugation at 8000 rpm for 10 minutes at 4°C and resuspended in lysis buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole). Cells were sonicated (300 W, 3 seconds on, 5-second intervals, 99 times in total), and the supernatant was collected by centrifugation at 12000 rpm for 30 minutes at 4°C. The supernatant was loaded onto a Ni-NTA affinity chromatography column pre-equilibrated with lysis buffer. Impurities were washed with wash buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 20 mM imidazole), and the target protein was eluted with elution buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 250 mM imidazole) to obtain high-purity SSB-BST fusion protein.

[0057] The eluted fraction containing the enzyme was collected and dialyzed at 4°C for 12 hours using dialysis buffer (containing 10 mM Tris-HCl, 50 mM potassium chloride, 0.1 mM EDTA, 50% glycerol, pH 8.0). Finally, the recombinant enzyme was dissolved 1:1 (v / v) in storage buffer (containing 10 mM Tris-HCl, 50 mM potassium chloride, 0.1 mM EDTA, 2 mM dithiothreitol (DTT), 0.15% Triton X-100, 50% glycerol, pH 8.0) and stored at -20°C. The concentrations of each chimeric protein were determined using a BCA protein assay kit (Beyotime).

[0058] Figure 3 The results showed that SSB-BST, BST, and SSB could all be purified by Ni-NTA affinity chromatography. Example 3

[0059] Activity assay of SSB-BST fusion protein

[0060] The activities of SSB-BST, SSB+BST, and BST were detected according to the method specified in GB / T36755-2018 "BST DNA Polymerase".

[0061] 1. Reagents:

[0062] The hairpin oligonucleotide sequence (T2) is as follows:

[0063] 5'-tagcgaaggatgtgaacctaatcccTGCTCCCGCGGCCGatctgcCGGCCGCGGGAGCA-3', dilute the hairpin oligonucleotide sequence (T2) powder to 100 μmol / L according to the instructions on the synthesis sheet, and store at -20°C. Before use, remove from -20°C and equilibrate to room temperature, mix on a vortex mixer for 10 s, and centrifuge on a microcentrifuge for a few seconds.

[0064] dNTPs, fluorescent nucleic acid dyes

[0065] 2. Preparation of reagent solutions

[0066] 1XTE solution: 10 mmol / L Tris-HCl, 1 mmol / L disodium ethylenediaminetetraacetate solution, pH 8.5 (25 °C).

[0067] PCR buffer: 250 mmol / L Tris-HCl, 50 mmol / L ammonium sulfate solution, 500 mmol / L potassium chloride solution, 1% (v / v) Triton X-100, pH 8.5 (25 °C).

[0068] Magnesium chloride solution: 25 mmol / L.

[0069] Enzyme dilution buffer: 10 mmol / L Tris-HCl, 0.1 mmol / L disodium ethylenediaminetetraacetate solution, 1 mmol / L DTT, 50% (v / v) glycerol, pH 8.0 (25°C).

[0070] 3. BST DNA polymerase gradient dilution solution: Dilute BST, SSB, and SSB-BST to 1 μg / mL using enzyme dilution buffer. Dilute the BST enzyme of NEB 10-fold. This step should be performed on ice.

[0071] Grouping:

[0072] 1 is 2.5 μl SSB-BST + 2.5 μl water;

[0073] 2 is NEB's BST 2.5μl + 2.5μl SSB;

[0074] 3 is a self-made BST 2.5μl + 2.5μl SSB;

[0075] 4 is NEB's BST 2.5μl + 2.5μl water;

[0076] 5 is a self-made BST mixture of 2.5 μl and 2.5 μl of water;

[0077] 6 is the blank control (enzyme dilution buffer).

[0078] The polymerization reaction solution (components shown in Table 3) was evenly distributed into microcentrifuge tubes. 5 μl of each enzyme dilution solution and a blank control (enzyme dilution buffer) were added to each tube. The tubes were vortexed for 10 seconds and then centrifuged for a few seconds. 25 μl of the mixture was dispensed into each of three 8-tube PCR aliquots, with three replicates per tube. The tubes were tightly capped and centrifuged for a few seconds before use.

[0079] Table 3

[0080]

[0081] Run the program

[0082] Set the following temperature program on the fluorescence PCR instrument: 65℃ for 16 seconds;

[0083] Fluorescence acquisition channel: SYBR;

[0084] 120 cycles.

[0085] See results Figure 4 :

[0086] It can be seen that the negative control showed no fluorescence signal. The mean CT values ​​of amplification curves 1-5 were 16.7, 19.1, 21.7, 23.6, and 26.2, respectively, with variances (STDs) of 0.485, 0.409, 0.214, 0.549, and 0.054, respectively. The differences in CT values ​​compared to curve 1 were 2.4, 5, 6.9, and 9.5, respectively. Compared to SSB-BST, the sensitivity of NEB's BST+SSB, the self-made BST+SSB, NEB's BST, and the self-made BST were 5.3, 32, 119.2, and 724.1 times, respectively.

[0087] Conclusion: The SSB-BST has a sensitivity that is 100 times higher than that of the SSB+BST, and a sensitivity that is 700 times higher than that of the self-made BST.

[0088] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0089] Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A BST DNA polymerase fused with a single-stranded DNA-binding protein, characterized in that, The BST DNA polymerase fused with a single-stranded DNA binding protein comprises, from N-terminus to C-terminus: an E. coli SSB protein fragment with single-stranded DNA binding activity, a flexible linker peptide, and an artificially designed BST DNA polymerase fragment with strand displacement activity. The amino acid sequence of the Escherichia coli SSB protein fragment is shown in SEQ ID NO. 1; The amino acid sequence of the flexible linker peptide is shown in SEQ ID NO. 2; The amino acid sequence of the BST DNA polymerase fragment is shown in SEQ ID NO.

3.

2. The BST DNA polymerase fused with a single-stranded DNA-binding protein according to claim 1, characterized in that, The amino acid sequence of the BST DNA polymerase fused with the single-stranded DNA-binding protein is shown in SEQ ID NO.

4.

3. The BST DNA polymerase fused with a single-stranded DNA-binding protein according to claim 1 or 2, characterized in that, The BST DNA polymerase fused with a single-stranded DNA-binding protein further includes a purification tag located at the N-terminus or C-terminus; the purification tag is a His-tag or a His-SUMO-tag.

4. A nucleic acid molecule encoding a BST DNA polymerase that fuses a single-stranded DNA-binding protein as described in any one of claims 1 to 3.

5. A recombinant expression vector, characterized in that, The expression vector comprises the nucleic acid molecule of claim 4, and the nucleic acid molecule is operatively linked to a promoter; the promoter is a T7 promoter; and the expression vector is a pET series vector.

6. A host cell, characterized in that, The recombinant expression vector of claim 5 is included; the host cell is Escherichia coli BL21(DE3), BL21(DE3)pLysS or Rosetta(DE3).

7. A method for preparing a BST DNA polymerase fused with a single-stranded DNA-binding protein as described in any one of claims 1 to 3, characterized in that, Includes the following steps: (1) Construct a recombinant expression vector containing a nucleic acid molecule encoding a BST DNA polymerase that encodes the fusion single-stranded DNA-binding protein; (2) Transform the recombinant expression vector into host cells; (3) Culturing the host cells to induce the expression of the BST DNA polymerase containing the fused single-stranded DNA binding protein; (4) Collect and lyse the host cells to purify the BST DNA polymerase containing the fused single-stranded DNA binding protein.

8. The method according to claim 7, characterized in that, The conditions for inducing the expression of BST DNA polymerase, which is a fusion single-stranded DNA binding protein, in step (3) are: when the bacterial culture OD 600 When the concentration reaches 0.6~0.8, add IPTG to a final concentration of 0.1~1.0 mM and induce expression at 16~25℃ for 12~24 hours; the BST DNA polymerase fused with single-stranded DNA binding protein obtained in step (4) is purified by Ni-NTA affinity chromatography in one step.

9. The use of the BST DNA polymerase fused with a single-stranded DNA-binding protein as described in any one of claims 1 to 3 in nucleic acid amplification.

10. The use of the BST DNA polymerase fused with a single-stranded DNA-binding protein according to any one of claims 1 to 3 in the preparation of a detection kit.

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