Fusion protein of Bst DNA polymerase and ferritin and its mutant and application

Abstract

The present invention discloses a fusion protein of Bst DNA polymerase and ferritin, and a mutant and application thereof. The present invention first provides a Bst DNA polymerase fusion protein obtained by connecting the C-terminus of a monomeric ferritin subunit to the N-terminus of a wild-type Bst DNA polymerase through a connecting peptide, and repeatedly and orderly displays a large fragment of the wild-type Bst DNA polymerase on the surface of the ferritin of Pyrococcus pyrococcus with strong heat resistance, thereby improving the enzyme activity and thermal stability of the fusion protein; the present invention further performs single-site and double-site mutations on the fusion protein, and compared with the wild-type Bst DNA polymerase and the fusion protein, the enzyme activity, specific activity and thermal stability of the obtained mutant are significantly improved or improved, and the LAMP specific amplification reaction can still be performed at a maximum of 85°C, which improves the rigor of the reaction and reduces the cost of enzyme use, and can be applied to loop-mediated isothermal amplification technology in more complex scenarios.

Description

Technical Field

[0001] The present invention relates to a fusion protein of DNA polymerase, in particular to a fusion protein obtained by fusing DNA polymerase and a ferritin subunit, as well as a mutant obtained by the fusion protein and an application thereof, belonging to the field of fusion proteins of DNA polymerase and ferritin and applications thereof. Background Art

[0002] Loopmediated isothermal amplification (LAMP) is an isothermal nucleic acid amplification technology proposed by Japanese scholar Notomi et al. in 2000. This technology uses multiple pairs of specific primers and a DNA polymerase with chain displacement activity to continuously self-cycle DNA synthesis at a single temperature without the need for heating to melt the chain and cooling to anneal, thereby achieving large-scale amplification of the target fragment under constant temperature conditions. Due to its advantages such as rapidity, sensitivity, simple operation, and no need for special instruments and equipment, it has broad application prospects in clinical scenario detection. Bst DNA polymerase is derived from Geobacillus stearothermophilus ( Geobacillus stearothermophilus , Bst), which can be hydrolyzed into two fragments by Bacillus subtilisin, of which the large fragment (Bst large fragment, Bst-LF) is also called Klenow fragment. It lacks the 5'-3' DNA exonuclease activity of the wild-type enzyme, but has 5'-3' DNA polymerase activity and strong strand displacement activity. It is widely used in constant temperature amplification experiments and has become the most mainstream enzyme in LAMP technology. Its performance is further optimized from the perspectives of its thermal stability and enzyme activity, and its amplification speed and applicability to high-temperature LAMP reaction types are improved, so that it can adapt to more complex application scenarios.

[0003] Ferritin nanoparticles are spherical hollow cage structures self-assembled from 24 ferritin subunits. Every 3 subunits form a trimer subunit, and 8 trimers are assembled into a spherical hollow nanocage structure with an outer diameter of 12nm and an inner diameter of 8nm. Based on the unique structure and excellent safety of ferritin, the target can be loaded inside ferritin or displayed on the outer surface through in vitro fusion expression and other methods. It is an ideal nanoplatform and has been widely used in drug delivery, antigen presentation, bioimaging, activity and high temperature resistance improvement and other fields. In addition, ferritin can maintain its natural structure unchanged under conditions of 80℃-100℃, pH3-10, etc. This high temperature resistance and strong acid and alkali resistance also lay the foundation for the stability modification of its delivery of active substances. Summary of the invention

[0004] One of the purposes of the present invention is to provide a fusion protein of Bst DNA polymerase and ferritin subunit, wherein the enzymatic activity and thermal stability of the fusion protein are significantly increased or improved compared with the wild-type Bst DNA polymerase;

[0005] The second object of the present invention is to provide a mutant of the fusion protein of the Bst DNA polymerase and the ferritin subunit;

[0006] The third object of the present invention is to provide a method for preparing a fusion protein or a mutant thereof of Bst DNA polymerase and ferritin subunit;

[0007] The fourth object of the present invention is to use the fusion protein of the Bst DNA polymerase and the ferritin subunit or its mutant as a DNA polymerase in a DNA polymerization reaction.

[0008] In order to achieve the above object, the technical solution adopted by the present invention includes:

[0009] One aspect of the present invention is to provide a fusion protein of Bst DNA polymerase and ferritin subunit, wherein the fusion protein is obtained by connecting the C-terminus of a monomeric ferritin subunit to the N-terminus of a wild-type Bst DNA polymerase via a connecting peptide.

[0010] In a preferred embodiment of the present invention, the monomeric ferritin subunit comprises any one of a bacterial ferritin subunit, a plant ferritin subunit, an algae ferritin subunit, an insect ferritin subunit, a fungal ferritin subunit or a vertebrate ferritin subunit; preferably, the monomeric ferritin subunit of the present invention is a ferritin subunit of Pyrococcus furiosus ( Pyrococcus furiosus ferritin, The invention discloses a PfFn) monomer, wherein the arginine at position 64 in the amino acid sequence of the Pyrococcus furiosus ferritin monomer is mutated to lysine and the amino acids at positions 148 to 174 of the Pyrococcus furiosus ferritin monomer are deleted.

[0011] The wild-type Bst DNA polymerase large fragment described in the present invention is from Bacillus stearothermophilus ( Bacillus stearothermophilus ) DNA polymerase; the amino acid sequence number of the wild-type Bst DNA polymerase large fragment on NCBI is PDB: 1L3S_A.

[0012] In a most preferred embodiment of the present invention, the arginine at position 64 in the amino acid sequence of the ferritin monomer of Pyrococcus furiosus is mutated to lysine, and the amino acids at positions 148 to 174 of the ferritin monomer of Pyrococcus furiosus are deleted, and the C-terminus of the obtained amino acid sequence is connected to the N-terminus of the wild-type Bst DNA polymerase through a connecting peptide, so that the Bst DNA polymerase can be displayed on the surface of the ferritin nanoparticles, and a fusion protein with an amino acid sequence shown in SEQ ID No. 1 is obtained.

[0013] The amino acid sequence of the connecting peptide described in the present invention is preferably GGGSGGGGSGGGS (SEQ ID No. 2).

[0014] Another aspect of the present invention is to provide a gene encoding a fusion protein of the Bst DNA polymerase and the ferritin subunit; the present invention performs codon optimization on the encoding gene according to the codon preference of Escherichia coli to obtain a codon-optimized gene having a nucleotide sequence as shown in SEQ ID No.3:

[0015] The present invention finds that the expression amount of the fusion protein in the Escherichia coli host is not ideal when the fusion protein is expressed by fusion. In order to improve the expression amount of the fusion protein, the present invention optimizes and transforms the nucleotide sequence of the coding gene of the fusion protein according to the codon preference of Escherichia coli, optimizes and designs various related parameters affecting gene transcription efficiency, translation efficiency and protein folding, such as GC content, CpG dinucleotide content, codon preference, secondary structure of mRNA, mRNA free energy stability, RNA instability gene sequence, and repeat sequence, and keeps the final translated protein sequence unchanged. The nucleotide sequence obtained after optimizing the coding gene of the fusion protein shown in SEQ ID No.1 by the above-mentioned optimization means is the codon-optimized coding gene shown in SEQ ID No.3.

[0016] Another aspect of the present invention is to provide an expression cassette, an expression vector or a recombinant host cell containing the gene encoding the Bst DNA polymerase fusion protein; wherein the expression vector can be a prokaryotic expression vector or a eukaryotic expression vector, preferably a prokaryotic expression vector; and the recombinant host cell is preferably a recombinant Escherichia coli cell.

[0017] The present invention performs codon optimization on the coding gene of the fusion protein shown in SEQ ID No. 1 and then performs amino acid single-site mutation and double-site mutation on the fusion protein to further improve its enzyme activity, specific activity or thermal stability.

[0018] Thus, another aspect of the present invention is to provide a single-site mutant of a Bst DNA polymerase fusion protein, comprising: subjecting the fusion protein having an amino acid sequence as shown in SEQ ID No.1 to a single-site mutation; specifically, subjecting the amino acid shown in SEQ ID No.1 to a single-site mutation according to any one of F208L, M280L, F325S, E384K, G445D or P638S to obtain a plurality of single-site mutants; preferably, subjecting the amino acid shown in SEQ ID No.1 to a single-site mutation according to any one of F325S, E384K, G445D or P638S to obtain a single-site mutant, compared to the wild-type Bst DNA polymerase and the Bst DNA polymerase fusion protein, the enzyme activity and thermal stability of these mutants are significantly improved or improved.

[0019] The "F325S" single-site mutation in the present invention means that the phenylalanine at position 325 of the fusion protein shown in SEQ ID No. 1 is mutated to serine, and the meanings of the remaining single-site mutations are similar.

[0020] Another aspect of the present invention is to provide a double-site mutant of a Bst DNA polymerase fusion protein, comprising: a plurality of double-site mutants obtained by subjecting the fusion protein having an amino acid sequence as shown in SEQ ID No. 1 to any one of F325S-E384K, F325S-G445D, F325S-P638S, E384K-G445D, E384K-P638S or G445D-P638S double-site mutations, wherein the mutant obtained by F325S-E384K, E384K-P638S or G445D-P638S double-site mutation is preferred.

[0021] The "F325S-E384K" double-site mutation described in the present invention means that the phenylalanine at position 325 of the fusion protein shown in SEQ ID No. 1 is mutated to serine and the glutamic acid at position 384 is mutated to lysine at the same time, and the meanings of the remaining double-site mutations are similar.

[0022] Another aspect of the present invention is to provide a method for preparing the fusion protein or its mutant, comprising:

[0023] (1) constructing a prokaryotic expression vector or eukaryotic expression vector of the gene encoding the fusion protein or its mutant; (2) transforming the constructed prokaryotic expression vector or eukaryotic expression vector into a corresponding host cell, expressing the fusion protein in the host cell, and purifying and renaturing the fusion protein to obtain the fusion protein.

[0024] Preferably, the coding gene of the fusion protein or its mutant is cloned into a prokaryotic expression vector to construct a recombinant prokaryotic expression vector; the recombinant prokaryotic expression vector is used to infect Escherichia coli cells, and the infected Escherichia coli cells are cultured to express the corresponding fusion protein or its mutant, and then purified to obtain the fusion protein.

[0025] Another aspect of the present invention is to use the fusion protein or its mutant as a DNA polymerase in various DNA amplification reactions, especially as a DNA polymerase in loop-mediated isothermal amplification reactions.

[0026] The Bst DNA polymerase fusion protein (Ferritin-Bst-LF) provided by the present invention displays a large fragment (Bst-LF) of the wild-type Bst DNA polymerase in a repeated and orderly manner on the surface of the heat-resistant Ferococcus ferritin, thereby improving the enzymatic activity and thermal stability of the fusion protein; in addition, the present invention performs single-site and double-site mutations on the Bst DNA polymerase (Ferritin-Bst-LF) fusion protein, and compared with the wild-type Bst DNA polymerase and the Bst DNA polymerase fusion protein, the enzyme activity, specific activity and thermal stability of the obtained mutant are significantly increased or improved, and the LAMP-specific amplification reaction can still be performed at a maximum temperature of 85°C, and can be applied to loop-mediated isothermal amplification technology in more complex scenarios.

[0027] Definitions of terms used in this invention

[0028] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0029] The term "recombinant protein" refers to a protein produced by recombinant DNA technology, which can be used to clone and express genes to produce proteins in a variety of hosts including bacteria, mammalian cells, insect cells and plants.

[0030] The terms "mutation" and "mutant" have their ordinary meanings herein and refer to genetic, naturally occurring or introduced changes in nucleic acid or polypeptide sequences, and their meanings are the same as those generally known to those skilled in the art.

[0031] The term "host cell" or "recombinant host cell" means a cell comprising a polynucleotide of the present invention, regardless of the method used for insertion to produce a recombinant host cell, such as direct uptake, transduction, f-mating, or other methods known in the art. The exogenous polynucleotide may be maintained as a non-integrating vector such as a plasmid or may be integrated into the host genome.

[0032] The term "transfection" refers to the process by which a host cell acquires new genetic markers as a result of the incorporation of foreign DNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 To purify the wild-type Bst-LF, Ferritin-Bst-LF and Ferritin-Bst-LF mut .

[0034] Figure 2 shows the transmission electron microscopy observation of Ferritin-Bst-LF.

[0035] Figure 3 shows the gel electrophoresis analysis of LAMP amplification products. DETAILED DESCRIPTION

[0036] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, it should be understood that the embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications or replacements all fall within the protection scope of the present invention.

[0037] Example 1 Construction, expression and purification of the expression vector of the fusion protein of Bst DNA polymerase and ferritin and its mutant

[0038] The wild-type DNA polymerase large fragment Bst-LF (PDB: 1L3S_A) and the hyperthermophilic bacterium Pyrococcus furiosus ( Pyrococcus furiosus) ferritin monomer (GenBank sequence number on NCBI: AAL80866.1) amino acid sequence as the basis, Bst-LF was fused to the 147th amino acid at the C-terminus of the ferritin monomer subunit of Pyrococcus furiosus using the connecting peptide GGGSGGGGSGGGS (SEQ ID No. 2) (i.e., amino acids 1-146 were deleted), and at the same time, the 64th arginine (R) in the amino acid sequence of ferritin of Pyrococcus furiosus was mutated to lysine (K) to eliminate the potential cleavage site. The amino acid sequence of the obtained fusion protein of Bst DNA polymerase and ferritin is shown in SEQ ID No. 1, and the fusion protein is named Ferritin-Bst. (SEQ ID No. 1).

[0039] The amino acid sequence shown in SEQ ID No.1 was input into the Codon Optimizer software, and the E. coli genome sequence information was input at the same time. After codon optimization according to the codon preference of E. coli, the nucleotide sequence corresponding to the BstDNA polymerase fusion protein (Ferritin-Bst) was obtained as SEQ ID No.3:

[0040]

[0041] Introduce the upstream and downstream of SEQ ID No.3 Bam H Ⅰ / Eco The pUC18-Ferritin-Bst-LF plasmid (pUC18 with SEQ ID No. 3 inserted) was obtained by full gene synthesis after the R Ⅰ restriction site.

[0042] Using pUC18-Ferritin-Bst-LF plasmid as template, a large number of single-site mutations were performed using fusion PCR. In this experiment, only the effective single-site mutations and the corresponding mutation primers are listed in Table 1.

[0043] Table 1 Primer sequences used for effective point mutations

[0044]

[0045] The effective mutations (F208L, M280L, F325S, E384K, G445D or P638S) in the amino acid sequence shown in SEQ ID No. 1 were gradually subjected to single-site mutation by fusion PCR, and the resulting mutant was named Ferritin-Bst-LF mut 1 (F208L, M280L, F325S, E384K, G445D, P638S); Based on the effective single-point mutations, various combinations of double-site mutations were further performed, and the more excellent double-site combination mutant was named Ferritin-Bst-LF mut 2 (F325S-E384K, F325S-G445D, F325S-P638S, E384K-G445D, E384K-P638S, G445D-P638S).

[0046] The PCR reaction system is shown in Table 2.

[0047] Table 2 PCR reaction system

[0048]

[0049] PCR parameter settings: 95°C, 30 s; 95°C, 15 s, 64°C, 15 s, 72°C, 60 s, for a total of 29 cycles; 72°C, 5 min.

[0050] PCR product recovery: perform agarose gel electrophoresis on the PCR product, cut out the target band under ultraviolet light and place it in an EP tube, add 3 volumes of 6 M sodium iodide, and melt it in a 55°C water bath; add 8 μL of glass milk, mix well, and place in an ice bath for 10 min, shake every three minutes, centrifuge at 12000 r / min for 10 s, and discard the supernatant; add 800 μL of New Wash and flick to wash, repeat 3 times; discard the supernatant, dry in a 37°C oven for 5 min; add 20 μL of 0.1×TE, mix well, centrifuge at 12000 r / min for 5 min, take the supernatant, and store at -20°C.

[0051] Enzyme digestion: using restriction endonucleases Bam H Ⅰ and Eco The above PCR products and pET-28a (+) vector were double-digested with R Ⅰ, inactivated at 85℃ for 10 min, and stored at -20℃ for later use.

[0052] Ligation: Use T4 DNA ligase at 25°C for 10 min.

[0053] Table 3 Connection system

[0054]

[0055] Transformation and screening: Take the competent cells stored at -80℃, melt them to a semi-melted state, add the above-mentioned ligation product, and place them on ice for half an hour; place them in a 42℃ constant temperature water bath for 90 s, and quickly place them on ice for 2 minutes; add an appropriate amount of LB culture medium to the tube, and culture it in a 37℃ constant temperature incubator for 60 min; centrifuge, discard the supernatant, keep 200 μL of the supernatant to resuspend the bacteria, spread it on an LB plate (50 μg / mL Kana), and culture it upright in a 37℃ constant temperature incubator for 30 min, then invert and culture it overnight, pick a single colony for colony PCR identification, and sequence the positive single clone to verify the mutation results, and obtain the recombinant expression vector plasmids pET-28a-Ferritin-Bst-L and pET-28a-Ferritin-Bst-LF. mut 1 , pET-28a-Ferritin-Bst-LF mut 2 .

[0056] Induce expression: insert the correct recombinant plasmid pET28a-Ferritin-Bst-LF mut Transformed into E. coli expression strains E. coli1 mL of LB liquid medium was added to BL21(DE3), cultured at 37℃ for 1 h, centrifuged at 8000 r / min for 1 min, 200 μL of supernatant was retained to resuspend the bacteria, and evenly spread on LB solid plates (50 μg / mL, Kana), cultured at 37℃ for 30 min, and then inverted overnight. Pick a single colony from the plate, add 4.5 mL of LB liquid medium (50 μL / mL, Kana), and culture at 37℃, 220 r / min for 12 h. After 12 hours, the bacteria were inoculated into 200 mL of culture medium (50 μg / mL, Kana) at a ratio of 1:100, cultured at 37℃, 220 r / min, and cultured until OD600 was between 0.6-0.8, and IPTG was added at a final concentration of 1.0 mmol / L, and cultured at 16℃ with shaking at 160 rpm for 16h-18h. After the culture was completed, the cells were centrifuged at 12000 r / min for 5 min, and the cells were collected and resuspended in PBS for washing, and the process was repeated twice. After washing, the cells were resuspended in 20 mL of loading buffer (20 mM sodium phosphate, 0.5 M NaCl, 20 mM imidazole, pH 7.4), placed in an ice bath for 30 min, and ultrasonically disrupted. After centrifugation, the supernatant was collected and filtered through a 0.45 μm filter membrane for later use.

[0057] Purification: HisTrap FF Crude column affinity chromatography (purchased from GE) was used to purify the histidine-tagged protein on a protein purifier (AKTA purifier, purchased from GE) according to the operating manual. The loading buffer was 20 mM sodium phosphate, 0.5 M NaCl, 25 mM imidazole, pH 7.4; the elution buffer was 20 mM sodium phosphate, 0.5 M NaCl, 500 mM imidazole, pH 7.4. The eluted sample was desalted using an Amicon® Ultra-15 centrifugal filter tube with a specification of 30 kDa, and the recombinant protein was replaced into a universal enzyme storage buffer solution (100 mM KCl, 10 mM Tris-HCl, 0.1 mM EDTA, 1 mM DTT, 0.1% Triton ® X-100, 50% glycerol, pH 7.4). The harvested protein solution was sterile filtered through a 0.22 μm filter, quantified using a protein quantification kit (BCA method) (purchased from SINOPCR), diluted to 1 mg / ml with enzyme storage buffer, aliquoted under sterile conditions, and stored frozen at -80°C.

[0058] The purification results were analyzed by SDS-PAGE electrophoresis. Figure 1 As shown, it is proved that Ferritin-Bst-LF and Ferritin-Bst-LF mut with the expected band size of about 88Kda were successfully obtained. Figure 1 The band near 67 kDa in the middle is the unfused wild-type Bst-LF as a control.

[0059] Experimental Example 1 Electron Microscope Detection Experiment of DNA Polymerase Fusion Protein (Ferritin-Bst-LF) Nanoparticles

[0060] The DNA polymerase fusion protein (Ferritin-Bst) obtained after purification in Example 1 was observed using an electron microscope. The specific observation method was carried out with reference to the operation method disclosed in the Chinese patent document (CN 112076315 A): the concentration of the purified DNA polymerase fusion protein (Ferritin-Bs-LF) was diluted to 0.1 mg / ml and dropped on a plastic film. The copper mesh was clamped with tweezers, and the side with the carbon support film was placed on the sample droplet to allow the copper mesh to adsorb the protein sample. After 3-5 minutes, excess water on the edge of the copper mesh was absorbed with absorbent paper, and then rinsed with sterile water and dried with absorbent paper. The copper mesh adsorbed with the fusion protein was then placed on a uranyl acetate stain for staining for 5 minutes, and finally placed on a clean bench to dry, and then observed under a transmission electron microscope HITACHI TEM-H7500.

[0061] Electron microscopy results show that the product size is consistent with the expected nanoparticles, and the diameter of the cage structure assembled by Ferritin-Bst-LF is about 30-40nm ( Figure 2 ).

[0062] Experimental Example 2 DNA polymerase fusion protein and its mutant enzyme activity detection test

[0063] According to the principles and methods disclosed in patent CN 117946997 A, the DNA polymerase activity and strand displacement activity of the DNA polymerase fusion protein (Ferritin-Bst) and its mutants obtained in Example 1 were detected, and the wild-type Bst-LF was used as a control. In this experiment, the mutants with obvious enzyme activity improvement effects among the DNA polymerase fusion protein mutants are listed in Table 4.

[0064] Table 4 Enzyme activities of Bst DNA polymerase fusion protein and its mutants

[0065]

[0066]

[0067] Experimental Example 3 Thermal Stability Determination of DNA Polymerase Fusion Protein and Its Mutants

[0068] This test reflects the thermal stability of Bst DNA polymerase by measuring the melting temperature (Tm) of the Bst DNA polymerase fusion protein and its mutants prepared in Example 1.

[0069] Specific principle: protein peptide chains fold in water, and their hydrophilic groups wrap the hydrophobic groups. When the temperature rises, the protein denatures and unfolds, exposing its hydrophobic groups. The hydrophobic groups can specifically bind to the hydrophobic dyes in the system and emit fluorescence. The Tm of different proteins can be calculated by the change of the fluorescence curve. Tm represents the temperature when the folded protein is equal to the unfolded protein. It reflects the thermal stability of the BstDNA polymerase fusion protein or its mutant to a certain extent, that is, the higher the Tm value, the better the thermal stability. For specific methods and parameters, refer to the methods and parameters disclosed in the Chinese patent document CN 118185902 A (invention name: Bst DNA polymerase mutants with enhanced thermal stability and their preparation methods and applications). The parameters of the fluorescence quantitative PCR instrument are set as follows: the heating rate is set to: 1℃ / 10s, the temperature range is: 25℃ to 99℃, the fluorescence signal is collected every 1-3℃ during the heating process, and the Tm is calculated based on the collected signal analysis.

[0070] Table 5 Melting temperatures of Bst DNA polymerase fusion proteins and their mutants

[0071]

[0072]

[0073] The Tm value of the wild-type Bst-LF is 71.2, and the Tm value of the Bst DNA polymerase fusion protein (Ferritin-Bst-LF) is 8°C higher than that of the wild-type Bst-LF. On this basis, the Tm values ​​of some dominant mutants of Ferritin-Bst-LF are more than 2°C higher than Ferritin-Bst-LF, reaching a maximum of 85.1. This indicates that the nanoparticles of the Bst DNA polymerase fusion protein and its mutants with ferritin as the skeleton obtained by the present invention have better thermal stability than the wild-type Bst-LF.

[0074] Experimental Example 4 LAMP detection test using DNA polymerase fusion protein and its mutants under different temperature conditions

[0075] Selection of conserved genes of bovine infectious rhinotracheitis virus B (GenBank Accession No.DQ006857.1) was used as the target, and the primers used for synthesizing LAMP are shown in Table 6.

[0076] Table 6 LAMP primer sequences

[0077]

[0078] In combination with Experimental Examples 3 and 4, mutants of Bst DNA polymerase fusion protein Ferritin-Bst-LF (F325S-E384K) and Ferritin-Bst-LF (G445D-P638S) with excellent thermal stability and high enzyme activity were selected for gradient temperature LAMP reaction, and wild-type Bst-LF and commercial brand polymerase (Bst 3.0 DNA polymerase, catalog number: M0374S) were used as controls.

[0079] Table 7 LAMP reaction system

[0080]

[0081] LAMP amplification conditions: several reaction temperatures were set at 65°C, 75°C, 80°C and 85°C. The reaction system without enzyme addition was used as a negative control. The reaction time was 30 minutes. Three sets of repeated tests were performed for each temperature condition. After the reaction, the amplified products were detected by agarose gel electrophoresis using 2.5% agarose gel electrophoresis.

[0082] The electrophoresis results are as follows Figure 3 As shown, wild-type Bst-LF, Ferritin-Bst-LF and its mutants as well as the positive control can form ladder-like DNA bands at 65°C, and the amplification effects can meet the requirements of LAMP detection; at 75°C, the two control groups of commercial enzyme and wild-type Bst-LF can no longer be effectively amplified, while the other groups can still produce obvious ladder-like bands; at 80°C, Ferritin-Bst-LF fusion protein and mutants can still be effectively amplified. When the temperature is raised to 85°C, only the mutants of Ferritin-Bst-LF fusion protein Ferritin-Bst-LF (F325S-E384K) and Ferritin-Bst-LF (G445D-P638S) can be effectively amplified.

[0083] Consistent with the test results in Experimental Example 3, this experiment further proves that the enzyme activity, specific activity and thermal stability of the Bst DNA polymerase fusion protein and its mutants are significantly improved compared with the wild-type Bst DNA polymerase, and are capable of LAMP reactions at high temperatures, which can improve the rigor of the reaction and reduce the cost of enzyme use, and expand the application of LAMP detection methods in more complex scenarios.

Claims

1. A fusion protein, characterized in that The fusion protein is obtained by connecting the C-terminus of a monomeric ferritin subunit to the N-terminus of a wild-type Bst DNA polymerase via a connecting peptide; the amino acid sequence of the fusion protein is shown in SEQ ID No.

1.

2. A gene encoding the fusion protein according to claim 1.

3. The coding gene according to claim 2, characterized in that The nucleotide sequence of the coding gene is shown in SEQ ID No.

3.

4. An expression cassette or recombinant expression vector containing the coding gene according to claim 2.

5. The mutant of the fusion protein according to claim 1, characterized in that The mutant is a single-site mutant obtained by subjecting the amino acid shown in SEQ ID No.1 to any one of F208L, M280L, F325S, E384K, G445D or P638S single-site mutation; or a double-site mutant obtained by subjecting the fusion protein shown in SEQ ID No.1 to any one of F325S-E384K, F325S-G445D, F325S-P638S, E384K-G445D, E384K-P638S or G445D-P638S double-site mutation.

6. The method for preparing the fusion protein according to claim 1, characterized in that: include: (1) constructing a prokaryotic expression vector or eukaryotic expression vector of the gene encoding the fusion protein; (2) transforming the constructed prokaryotic expression vector or eukaryotic expression vector into a host cell, expressing the fusion protein in the host cell, and purifying and renaturing the fusion protein to obtain the fusion protein.

7. The method for preparing a mutant according to claim 5, characterized in that: include: (1) constructing a prokaryotic expression vector or eukaryotic expression vector of the gene encoding the mutant; (2) transforming the constructed prokaryotic expression vector or eukaryotic expression vector into a host cell, expressing the fusion protein in the host cell, and purifying and renaturing the fusion protein to obtain the mutant.

8. The fusion protein according to claim 1 or the mutant according to claim 5 is used as a DNA polymerase in a DNA amplification reaction.

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