A hot-start B-family DNA polymerase for fluorescent probe qPCR and its preparation method and application

Abstract

The present invention discloses a hot start B-family DNA polymerase for fluorescent probe method qPCR, and its preparation method and application. The KOFU-pFEN1-mut DNA polymerase of the present invention is modified by replacing and directed evolution of the functional domains of wild-type KOD DNA polymerase and wild-type PfuDNA polymerase, and fusing flap-shaped nuclease endonuclease 1 at the C-terminus. Compared with wild-type Taq DNA polymerase, it has higher thermal stability and amplification efficiency. After chemical modification and hot start, the thermal stability and sensitivity of the polymerase are further improved, and it is suitable for various types of conventional PCR, rapid PCR, long fragment PCR and hot start PCR. At the same time, the chimera has 5'-3' nuclease activity and can be applied to probe method qPCR. The chimera has strong tolerance to PCR inhibitors and is suitable for directly amplifying DNA from samples such as blood and soil.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a hot-start B-family DNA polymerase for fluorescent probe qPCR, and a preparation method and application thereof. Background Art

[0002] Invented by Mullis et al. in 1983, the polymerase chain reaction (PCR) can exponentially amplify a large number of targets using a very small number of DNA molecules as templates. With its high specificity, high sensitivity, ease of use, and low cost, PCR has been expanded to numerous fields, including medical testing, agricultural science, environmental science, and food safety. With the development of PCR technology, a variety of new PCR-based technologies have emerged, such as multiplex PCR, quantitative PCR, and digital PCR. Real-time quantitative PCR (qPCR) builds on PCR technology by introducing fluorescent substances to convert amplified products into fluorescent signals, enabling real-time detection of amplification activity. This overcomes the limitations of traditional PCR, which can only observe the product using endpoint analysis and cannot quantitatively analyze the product.

[0003] The introduction of direct amplification qPCR technology has made rapid and accurate detection possible. Compared with traditional qPCR technology, this technology reduces the nucleic acid extraction process of the sample, effectively avoids cross-contamination of nucleic acids between samples and loss of nucleic acids during the extraction process, can shorten the detection time, and improve the efficiency of the detection. However, the sample generally contains a large amount of PCR inhibitors and nucleic acid molecules other than the target DNA, which will lead to a decrease in the specificity and sensitivity of the detection, and the accuracy of the results is not high. The use of hot-start DNA polymerase can effectively optimize the amplified target product, while inhibiting the generation of non-specific products, thereby improving the sensitivity, specificity and product yield of the detection. Among them, chemical modification to prepare hot-start DNA polymerase can more completely block the activity of DNA polymerase and have better specificity, but long-term heat shock will greatly reduce the activity of the polymerase. Therefore, the use of inhibitor-tolerant and heat-resistant DNA polymerases is the key to achieving direct amplification qPCR.

[0004] Currently, a variety of thermostable DNA polymerases have been developed in the field of PCR technology, including A-family Taq and Tth DNA polymerases, and B-family Pfu and KOD DNA polymerases. A-family DNA polymerases generally have three domains: a 5'-3' polymerase domain, a 5'-3' exonuclease domain, and a 3'-5' exonuclease domain (generally with low or no activity). In contrast, B-family DNA polymerases have only two domains: a 5'-3' polymerase domain and a 3'-5' exonuclease domain. Studies have shown that A-family DNA polymerases bind template DNA more weakly than B-family DNA polymerases, resulting in amplification products with much lower fidelity than those produced by B-family DNA polymerases. The length of amplified products produced by B-family DNA polymerases is also often longer than that of A-family DNA polymerases. B-family DNA polymerases also have a higher tolerance to PCR inhibitors found in blood and soil, such as hemoglobin and humic acid. Since the 1990s, researchers have explored the potential of wild-type Taq DNA polymerase through random mutagenesis and artificial evolution. While several inhibitor-tolerant DNA polymerases have been generated, most lack 5'-3' exonuclease activity, preventing them from hydrolyzing fluorescent probes and, therefore, from being used in direct amplification (qPCR) using fluorescent probes. Studies have reported that KOD FX Neo DNA polymerase significantly outperforms exonuclease-deficient Taq DNA polymerases in direct amplification of blood. B-family DNA polymerases are naturally inhibitor-tolerant, and chemical or antibody-modified DNA polymerases for hot start are a method for reversibly blocking DNA polymerase activity.

[0005] In summary, there is an urgent need to research and develop a hot-start DNA polymerase that has both inhibitor tolerance and high 5'-3' exonuclease activity for application in direct amplification fluorescent probe qPCR detection. Summary of the Invention

[0006] The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide a hot-start B-family DNA polymerase for fluorescent probe qPCR. The enzyme has high inhibitor tolerance and thermal stability and can be used in extraction-free fluorescent probe qPCR detection systems and multiplex PCR detection systems. In some embodiments, the chimeric polymerase designed by the present invention has high amplification performance, inhibitor tolerance, thermal stability and 5'-3' exonuclease activity that hydrolyzes fluorescent probes. The first DNA polymerase of the present invention is KOD polymerase SEQ ID NO.2, and the second DNA polymerase is Pfu polymerase SEQ ID NO.3. The present invention combines the functional properties of different domains of different DNA polymerases, and utilizes the replacement of functional domains in high-fidelity DNA polymerases and directed evolution technology to obtain a DNA polymerase with low fidelity, high amplification efficiency and strong inhibitor tolerance (named KOFU-mut DNA polymerase); through site-directed mutagenesis and fusion of a thermally stable flap-shaped endonuclease 1 domain, the chimeric polymerase can be used in fluorescent probe qPCR technology. The DNA polymerase of the present invention is a B-family DNA polymerase which has thermal stability, tolerance to PCR inhibitors and 5'-3' exonuclease activity.

[0007] Another object of the present invention is to provide a method for preparing the hot-start B-family DNA polymerase.

[0008] Another object of the present invention is to provide an application of the hot-start B-family DNA polymerase.

[0009] The purpose of the present invention is achieved through the following technical solutions:

[0010] A hot-start B-family DNA polymerase for fluorescent probe-based qPCR is named KOFU-pFEN1-mut, and its amino acid sequence is shown in SEQ ID NO.1.

[0011] The hot-start B-family DNA polymerase has the following characteristics: the amino acid sequence at positions 1-20 is a histidine tag (His-tag); the amino acid sequence at positions 21-788 is a chimera (KOFU-mut) of wild-type KOD DNA polymerase (NCBI accession number 1WNS_A) and wild-type Pfu DNA polymerase (NCBI accession number AHH82558.1); the amino acid sequence at positions 789-803 is a linker consisting of 15 amino acids as shown in SEQ ID NO.6; and the amino acid sequence at positions 804-1130 is amino acids 1-327 of pFEN1, a flap endonuclease 1 domain as shown in SEQ ID NO.5 (NCBI accession number WP_011012561.1). Among them, the amino acid sequences of positions 21-354 and 588-788 of KOFU-pFEN1-mut DNA polymerase are the amino acid sequences of positions 1-331 and 564-764 of the wild-type KOD DNA polymerase, respectively, and the amino acid sequence of positions 355-587 is the amino acid sequence of positions 332-564 of the wild-type Pfu DNA polymerase; it contains two mutation sites, aspartic acid (Asp) at position 164 mutated to asparagine (Asn); glutamic acid (Glu) at position 166 mutated to glutamine (Gln).

[0012] Compared with the amino acid sequence of the wild-type KOD DNA polymerase, the chimeric KOFU-mut DNA polymerase has the following characteristics: three amino acids, alanine (Ala), serine (Ser), and alanine (Ala), are sequentially inserted at amino acid position 22; amino acids 765-774 derived from the wild-type KOD DNA polymerase are deleted; aspartic acid (Asp) at position 170 is mutated to histidine (His); phenylalanine (Phe) at position 175 is mutated to serine (Ser); methionine (Met) at position 182 is mutated to threonine (Thr); alanine (Ala) at position 240 is mutated to valine (Val); aspartic acid (Asp) at position 608 is mutated to lysine (Lys); valine (Val) at position 660 is mutated to alanine (Ala); valine (Val) at position 670 is mutated to aspartic acid (Asp); and glycine (Gly) at position 788 is mutated to alanine (Ala).

[0013] Compared to the wild-type flap-shaped endonuclease 1 shown in SEQ ID NO. 5, the flap-shaped endonuclease 1 domain pFEN1 has the following characteristics: aspartic acid (Asp) at position 830 is mutated to asparagine (Asn); phenylalanine (Phe) at position 1113 is mutated to lysine (Lys); arginine (Arg) at position 1117 is mutated to alanine (Ala); and amino acids 328 to 340 of the wild-type flap-shaped endonuclease 1 are deleted. Compared to the 5'-3' exonuclease activity of wild-type Taq DNA polymerase, flap-shaped endonuclease 1 has stronger 5'-3' exonuclease activity. Flap-shaped endonuclease 1 can impart 5'-3' exonuclease activity to the fused protein, enabling hydrolysis of the fluorescent probe.

[0014] The N-terminus of the flap-shaped endonuclease 1 is connected to the C-terminus of the modified KOFU DNA polymerase via a linker shown in SEQ ID NO.6.

[0015] The nucleotide sequence of the DNA molecule encoding the KOFU-pFEN1-mut is shown in SEQ ID NO. 7. This sequence is obtained by codon optimization based on the characteristics of the E. coli expression system, and can significantly improve the expression efficiency of heterologous genes in the host bacteria.

[0016] A recombinant expression vector is obtained by cloning the DNA molecule encoding the KOFU-pFEN1-mut into an expression vector.

[0017] The expression vector is preferably a prokaryotic expression vector; more preferably a pET series vector; most preferably pET-28a.

[0018] A recombinant engineered cell line is obtained by transforming the above recombinant expression vector into engineered cells.

[0019] The engineered cells are preferably Escherichia coli cells; more preferably Escherichia coli BL21 (DE3).

[0020] The KOFU-pFEN1-mut can be obtained by chemical synthesis or by induced expression and purification in a recombinant cell line. For cost considerations, it is preferably prepared by induced expression and purification in a recombinant cell line; the steps include:

[0021] 1) Taking the above-mentioned recombinant engineered cell line, inoculating it into LB medium, and culturing it to obtain a seed solution;

[0022] 2) inoculating the above seed solution into LB medium and culturing to obtain bacterial solution;

[0023] 3) Add IPTG to the resulting bacterial solution to a final concentration of 0.025-0.4 mmol / L to induce cell protein expression, and collect the bacterial precipitate by centrifugation;

[0024] 4) Add lysis buffer to the resulting bacterial pellet to resuspend the cells, disrupt the cells by ultrasonication, and centrifuge to obtain the supernatant;

[0025] 5) Incubate the resulting supernatant at 75°C for 20-30 min, place on ice for 10-20 min, centrifuge, filter through a 0.22 μm microporous filter, and collect the supernatant;

[0026] 6) Collecting the sample eluate containing the target protein through nickel ion affinity chromatography and strong anion exchange chromatography to obtain the target protein.

[0027] Preferably, the LB culture medium in step 1) and step 2) both contain 50 μg / mL kanamycin sulfate.

[0028] Preferably, the culture conditions in step 1) are 37° C. and shaking culture at 150-200 r / min.

[0029] Preferably, the culture conditions in step 2) are 37°C, 150-200 r / min shaking culture until OD 600 =0.6~0.8.

[0030] Preferably, the seed solution in step 2) is inoculated into LB medium at a volume ratio of 1:100.

[0031] Preferably, the induction condition in step 3) is 25° C. for 10 to 12 hours.

[0032] Preferably, the amount of the inducer IPTG added in step 3) is 0.1 mmol / L.

[0033] Preferably, the centrifugation condition in step 3) is 4° C. and 20,000×g for 20 to 30 minutes.

[0034] Preferably, the amount of lysis buffer in step 4) is calculated as 5 mL per gram of bacterial precipitation.

[0035] Preferably, the ultrasonic conditions in step 4) are as follows: power 250 W, ultrasonication 5.5 s, interval 5.5 s, and duration 30 min.

[0036] Preferably, the centrifugation conditions in step 4) and step 5) are 4° C. and 20,000×g for 10 to 20 minutes.

[0037] Preferably, the components of the lysis buffer in step 4) are: 50 mmol / L Tris-HCl, 50 mmol / L NaCl, 5% (v / v) glycerol, pH 8.0.

[0038] Preferably, the binding buffer (Buffer A) used for the nickel ion affinity chromatography and strong anion exchange chromatography in step 6) comprises: 50 mmol / L Tris-HCl, 50 mmol / L NaCl, 5% (v / v) glycerol, pH 8.0; the elution buffer (Buffer B) used for the nickel ion affinity chromatography comprises: 50 mmol / L Tris-HCl, 50 mmol / L NaCl, 500 mmol / L imidazole, 5% (v / v) glycerol, pH 8.0; the elution buffer (Buffer C) used for the strong anion exchange layer comprises: 50 mmol / L Tris-HCl, 1 mol / L NaCl, 5% (v / v) glycerol, pH 8.0.

[0039] A hot-start DNA polymerase is obtained by specifically binding an anhydride compound to the lysine side chain amino group in the polymerase active site of the KOFU-pFEN1-mut described above. The mechanism is that the side chain amino group on the amino acid residue acts as a nucleophile to attack the carbonyl carbon of the anhydride compound, resulting in a reversible nucleophilic attack reaction that blocks the DNA polymerase activity, achieving the hot-start modification of the KOFU-pFEN1-mut described above.

[0040] The acid anhydride compound is preferably maleic anhydride or citraconic anhydride, and more preferably citraconic anhydride. The anhydride-modified polymerase greatly reduces the possibility of nonspecific product amplification and primer dimer formation during the PCR reaction.

[0041] The preparation method of the hot-start DNA polymerase comprises the following steps:

[0042] (1) dialyzing the KOFU-pFEN1-mut into Tris-HCl buffer;

[0043] (2) adding an acid anhydride compound, mixing evenly, and reacting;

[0044] (3) The reaction mixture was dialyzed into storage buffer to obtain a stable hot-start DNA polymerase, named HS-KOFU-pFEN1-mut. The hot-start DNA polymerase prepared by this method has excellent hot-start performance. No polymerase activity was released after incubation at 80°C for 10 minutes, and the activity was completely released after heat shock at 95°C for 14 minutes.

[0045] The Tris-HCl buffer solution preferably has a concentration of 10 to 50 mmol / L and a pH of 8 to 9.

[0046] The molar ratio of KOFU-pFEN1-mut to the acid anhydride compound is preferably 1:2000 to 1:3000.

[0047] The reaction conditions are preferably a temperature of 25°C to 37°C and a time of 3 to 4 hours.

[0048] The storage buffer preferably has a formula of: 50 mmol / L Tris-HCl, 0.1 mmol / L EDTA, 50% (v / v) glycerol, 2 mmol / L DTT, 0.002% (v / v) Tween-20, 0.002% (v / v) IGEPAL CA 630, pH 9.0.

[0049] A PCR kit comprises PCR water, a PCR reaction buffer, primers, at least one of dNTPs, and the KOFU-pFEN1-mut.

[0050] Preferably, the composition of the PCR reaction buffer is as follows: 100-120 mmol / L Tris-HCl, 10-30 mmol / L KCl, 5-20 mmol / L (NH4)2SO4, 2-4 mmol / L MgCl2, 0.025-0.10% (v / v) Triton X-100, and the pH value is 7.5-8.5.

[0051] Preferably, the enzyme activity concentration of KOFU-pFEN1-mut in the system is 0.05-0.1 U / μL.

[0052] Preferably, the concentration of the dNTPs in the system is 100-300 μmol / L.

[0053] Preferably, the concentration of the primer in the system is 0.2-0.4 μmol / L.

[0054] The use of the above PCR kit in DNA sample amplification.

[0055] A direct amplification qPCR kit comprises qPCR water, a qPCR reaction buffer, at least one of primers, probes and dNTPs, and the above-mentioned hot-start DNA polymerase.

[0056] Preferably, the composition of the qPCR reaction buffer is as follows: 100-120 mmol / L Tris-HCl, 10-30 mmol / L KCl, 5-20 mmol / L (NH4)2SO4, 4-6 mmol / L MgCl2, 0.025-0.10% (v / v) Triton X-100, and pH 7.5-8.5.

[0057] Preferably, the enzyme activity concentration of the hot-start DNA polymerase in the system is 0.01-0.05 U / μL.

[0058] Preferably, the concentration of the dNTPs in the system is 100-300 μmol / L.

[0059] Preferably, the concentration of the primer in the system is 0.2-0.4 μmol / L.

[0060] Preferably, the concentration of the probe in the system is 0.2-0.3 μmol / L.

[0061] Application of the direct amplification qPCR kit in direct amplification probe method qPCR.

[0062] The specific operation of the application is: using the direct amplification qPCR kit to directly amplify the biological sample (without nucleic acid extraction) to obtain the target gene product.

[0063] Use of the KOFU-pFEN1-mut, the HS-KOFU-pFEN1-mut, the direct amplification PCR kit, or the direct amplification qPCR kit in amplification and / or detection of biological samples.

[0064] The source of the DNA sample is not limited and may contain at least one of inhibitors such as humic acid, hemoglobin, and heparin sodium. For example, plant nucleic acid samples containing polysaccharides, blood nucleic acid samples containing hemoglobin, and soil nucleic acid samples containing humic acid can be used.

[0065] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0066] 1. The DNA polymerase (KOFU-pFEN1-mut) of the present invention is derived from wild-type KOD DNA polymerase and wild-type Pfu DNA polymerase through domain replacement and directed evolution, resulting in KOFU-mut with higher amplification efficiency and low fidelity. A flap-shaped endonuclease 1 binding point mutation is fused to the C-terminus of the KOFU-mut. The plateau fluorescence value of the KOFU-mut reaches that of wild-type Taq DNA polymerase, and the KOFU-mut exhibits higher inhibitor tolerance and thermal stability than wild-type Taq DNA polymerase.

[0067] 2. The hot-start DNA polymerase (HS-KOFU-pFEN1-mut) described in the present invention has no activity release at temperatures of 80°C and below, which reduces the possibility of nonspecific product amplification during the qPCR reaction and improves the specificity and sensitivity of the qPCR reaction.

[0068] 3. The hot-start DNA polymerase described in this invention exhibits significantly improved resistance to PCR inhibitors present in samples. In fluorescent probe-based qPCR assays, it can tolerate various high concentrations of inhibitors, such as 8% ethanol, 0.01% SDS, 100 mmol / L NaCl, 1% whole blood, 3 μg / μL bile salts, or 600 ng / μL tannic acid. This eliminates the need for complex genomic DNA extraction and purification steps, saving time and material and equipment costs while also preventing cross-contamination between samples during operation, making testing more convenient, rapid, and accurate. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1 The affinity chromatography purification result of KOFU-pFEN1-mut is shown; among them, lane M is protein Marker (10-180kDa), lane 1 is bacterial cell disruption solution after induced expression, lane 2 is supernatant obtained by centrifugation after cell disruption, lane 3 is supernatant of disruption solution after heating at 75°C for 30 minutes and centrifugation, lane 4 is chromatography outflow component, and lanes 5-14 are chromatography elution components.

[0070] Figure 2 The figure shows the ion exchange purification results of KOFU-pFEN1-mut; wherein, lane M is the protein marker (10-180 kDa), lane 1 is the affinity chromatography elution sample, lane 2 is the chromatography flow-through fraction, and lanes 3-12 are the chromatography elution fractions.

[0071] Figure 3 The electrophoresis results of PCR reactions with wild-type KOD DNA polymerase, wild-type Pfu DNA polymerase, KOFU-mut DNA polymerase, and KOFU-pFEN1-mut DNA polymerase at different extension times are shown, where lane M is a DNA Marker.

[0072] Figure 4 The electrophoresis results of PCR reactions of wild-type KOD DNA polymerase, wild-type Pfu DNA polymerase, KOFU-mut DNA polymerase, and KOFU-pFEN1-mut DNA polymerase under different NaCl concentrations are shown, where lane M is a DNA marker.

[0073] Figure 5The figure shows the test results of the 3'-5' exonuclease activity of KOFU-pFEN1-mut DNA polymerase.

[0074] Figure 6 This is the result of the amplification curve of KOFU-pFEN1-mut DNA polymerase in the probe method qPCR.

[0075] Figure 7 The results of the relative enzyme activities of KOFU-pFEN1-mut polymerase before and after chemical modification at different molar ratios of citraconic anhydride:KOFU-pFEN1-mut are shown in FIG.

[0076] Figure 8 The results of the relative enzyme activities of KOFU-pFEN1-mut polymerase before and after chemical modification at different temperatures are shown in the figure;

[0077] Figure 9 The results of the relative enzyme activity of KOFU-pFEN1-mut polymerase after activation before and after chemical modification at different pH values are shown in the figure;

[0078] Figure 10 The results of the relative enzyme activity of KOFU-pFEN1-mut polymerase before and after chemical modification at different times are shown;

[0079] Figure 11 This is a graph showing the threshold results of the fluorescent probe qPCR detection of the hot-start KOFU-pFEN1-mut and Taq in the presence of different concentrations of inhibitors; wherein A, B, C, D, E, and F are graphs showing the threshold results of the PCR reaction when the inhibitors are ethanol, NaCl, SDS, bile salts, whole blood, and tannic acid, respectively;

[0080] Figure 12 The minimum detection limit of hot-start KOFU-pFEN1-mut DNA polymerase and hot-start Taq DNA polymerase at 0.5% whole blood content; wherein A is hot-start KOFU-pFEN1-mut DNA polymerase and B is hot-start Taq DNA polymerase. DETAILED DESCRIPTION

[0081] The present invention will be further described below in conjunction with specific embodiments, but the examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Other embodiments based on the present invention made by professionals in this field without creative work are all within the scope of protection of the present invention.

[0082] Unless otherwise specified, all reagents and raw materials used in the present invention are commercially available products or products that can be prepared by known methods.

[0083] Example 1: Construction of a recombinant vector containing a nucleotide sequence encoding KOFU-pFEN1-mut

[0084] (1) Based on the amino acid sequence of the chimera (SEQ ID NO.1), a synthetic splicing PCR method was used to artificially subclone and synthesize a DNA molecule encoding the chimera, as specifically shown in SEQ ID NO.7.

[0085] (2) The DNA molecule encoding KOFU-pFEN1-mut was homologously recombined with the expression vector pET-28a. The chimera amplification primer sequences are as follows:

[0086] KOFU-FP: 5'-CGCGGCAGCCATATGGCTAGTGCGATACTTGATACTGA-3' (SEQ ID NO. 8);

[0087] KOFU-RP: 5'-AAGCTTGTCGACTTATTTGATCGCCTTCTTCAGACGCT-3' (SEQ ID NO. 9).

[0088] The pET-28a linearization primer sequences are as follows:

[0089] pET-28a-FP: 5'-TAAGTCGACAAGCTTGCGGCC-3' (SEQ ID NO. 10);

[0090] pET-28a-RP: 5'-CATATGGCTGCCGCGCG-3' (SEQ ID NO. 11).

[0091] PCR amplification was performed using a synthetic DNA molecule encoding KOFU-pFEN1-mut and a pET-28a empty vector as templates, respectively. 25 μL of 2× Pfu Max HiFi PCR ProMix (Guangzhou Yingzan Biotechnology Co., Ltd., Cat. No. P217A) and 1 μL of each upstream and downstream primer (10 μmol / L) were added to 50 μL of water. The chimeric DNA was amplified using the following conditions: 98°C for 30 s, followed by 25 cycles of 98°C for 10 s, 58°C for 30 s, and 68°C for 2 min, followed by 68°C for 5 min. The plasmid linearization PCR amplification protocol was as follows: 98°C for 30 s, followed by 25 cycles of 98°C for 10 s, 58°C for 30 s, and 68°C for 2.5 min, followed by 68°C for 5 min. The approximately 3.5 kb full-length chimeric DNA fragment and the approximately 5 kb linearized pET-28a vector were recovered by agarose gel electrophoresis. The recovered products were subjected to homologous recombination using a system consisting of 5 μL of 2× Hipro DNA Assembly Mix (Guangzhou Yingzan Biotechnology Co., Ltd., K001A) and 50 ng of each recovered product, diluted to 10 μL, and incubated at 50°C for 15 min. After incubation, the recombinant products were transformed into DH5α competent cells.

[0092] (3) Pick a single colony for colony PCR identification, and send the positive single clone to a sequencing company for sequencing verification. Cultivate and verify the correct competent cells, and extract the plasmid. The obtained plasmid is the recombinant vector containing the encoding KOFU-pFEN1-mut.

[0093] Example 2: Preparation of KOFU-pFEN1-mut transformants and identification of small-scale expression

[0094] The recombinant vector obtained in Example 1 was transformed into host cells E. coli BL21 (DE3), and a single colony was picked and inoculated into liquid LB medium (containing 50 μg / mL kanamycin sulfate) and cultured until the OD 600 The p-terminal pH value was 0.6-0.8, IPTG was added to a final concentration of 0.1 mmol / L, induced at 25°C for 10 h, and the cells were collected by centrifugation at 4°C and 20,000×g. Lysis buffer was added and the cells were ultrasonically disrupted. The expression of the target protein was detected by SDS-PAGE electrophoresis. The results showed that the prepared transformant could efficiently express the target protein.

[0095] Example 3: Expression of KOFU-pFEN1-mut in recombinant Escherichia coli

[0096] The positive transformant strains expressing chimeras obtained in Example 2 were inoculated into 60 mL of LB medium containing 50 μg / mL kanamycin sulfate and cultured overnight in a shaker at 37°C. The overnight cultured seed solution was inoculated into 1 L of LB medium containing 50 μg / mL kanamycin sulfate at a volume ratio of 1:100 and cultured in a shaker at 37°C until the OD 600 The pH value was 0.6-0.8; IPTG was added to a final concentration of 0.1 mmol / L, and the induction was continued at 25°C for 10-12 hours; the induced bacteria were collected by centrifugation and weighed, the wet weight of the bacteria was recorded, and the bacteria were stored at -20°C.

[0097] Example 4: Purification of KOFU-pFEN1-mut

[0098] 1. Ultrasonic disruption of induced expression cells

[0099] Take the bacteria frozen at -20℃ after induction of expression, and according to the wet weight of the bacteria recorded in Example 3, add 5mL of lysis buffer (50mmol / L Tris-HCl, 50mmol / L NaCl, 5% (v / v) glycerol, pH 8.0) per gram of bacteria to resuspend the bacteria, and use an ultrasonic cell disruptor to lyse the bacteria. The ultrasonic conditions are: power 250W, ultrasonic 5.5s, stop 5.5s, and continue for 30min. The lysed bacteria are centrifuged at 20000×g for 30min at 4℃, and the supernatant is taken to a 250mL sterilized glass bottle. The supernatant is incubated in a constant temperature water bath at 75℃ for 30min, centrifuged at 20000×g for 30min at 4℃, filtered with a 0.22μm microporous filter membrane, and the supernatant is taken to a 250mL sterilized glass bottle. The cell lysis solution of the bacteria before induction of expression was used as a control. 20μL of samples were taken after each step of treatment, and the protein expression results were analyzed by SDS-PAGE electrophoresis. The results are as follows. Figure 1 (lanes 1-4) are shown.

[0100] 2. Nickel ion affinity chromatography purification

[0101] The chromatography column used is HisTrap TM HP 5mL (purchased from GE Healthcare), the binding buffer is buffer A: 50mmol / L Tris-HCl, 50mmol / L NaCl, 5% (v / v) glycerol, pH 8.0; the elution buffer is buffer B: 50mmol / LTris-HCl, 50mmol / L NaCl, 500mmol / L imidazole, 5% (v / v) glycerol, pH 8.0, filtered through a 0.22μm filter membrane before use.

[0102] HisTrap TMAfter the HP 5mL is connected to the peristaltic pump, the pipeline and the chromatography column are cleaned with ultrapure water, and the column is equilibrated with buffer A. Then, the supernatant described in step 1 is loaded onto the chromatography column. After loading, the column is first washed with buffer A, and then a gradient elution of 10%-100% is performed with buffer B. The eluted fractions are collected, and 20 μL of each elution fraction is sampled for SDS-PAGE protein electrophoresis detection. The results are as follows Figure 1 (lanes 5-14).

[0103] 3. Strong anion exchange chromatography purification

[0104] The chromatography column selected is HiTrap TM Capto TM A 5 mL Q column (purchased from GE Healthcare) was used, and the binding buffer was buffer A: 50 mmol / L Tris-HCl, 50 mmol / L NaCl, 5% (v / v) glycerol, pH 8.0; the elution buffer was buffer C: 50 mmol / L Tris-HCl, 1 mol / L NaCl, 5% (v / v) glycerol, pH 8.0, and filtered through a 0.22 μm filter membrane.

[0105] HiTrap TM Capto TM After the Q column 5mL is connected to the peristaltic pump, the pipeline and the chromatography column are cleaned with ultrapure water, and the column is equilibrated with buffer A. Then, the supernatant described in step 1 is loaded onto the chromatography column. After the loading is completed, the column is first washed with buffer A, and then a gradient elution of 10%-100% is performed with buffer C. The elution fractions are collected, and 20 μL of each elution fraction is sampled for SDS-PAGE protein electrophoresis detection. The results are as follows Figure 2 The eluted fractions containing the target protein were mixed and gently shaken, and dialyzed overnight into storage buffer (50 mmol / L Tris-HCl, 0.1 mmol / L EDTA, 50% (v / v) glycerol, 2 mmol / L DTT, 0.002% (v / v) Tween-20, 0.002% (v / v) IGEPAL CA 630, pH 8.0).

[0106] Example 5: DNA polymerase activity of KOFU-pFEN1-mut

[0107] Take the KOFU-pFEN1-mut prepared in Example 4, and determine its polymerase activity according to the following method. According to the initial amplification rates of different enzyme activities of commercial DNA polymerases, a standard curve is established, and the initial amplification rate of the DNA polymerase to be tested is brought into the standard curve to determine the enzyme activity of the DNA polymerase to be tested. Design primers with complementary 3' ends (Test 1, Test 2), anneal to form primer extension substrates, and under the catalysis of DNA polymerase, dNTPs gradually penetrate, and the substrate will eventually form a double-stranded DNA (dsDNA) product. SYBR GreenⅠ is a highly sensitive fluorescent dye that binds to double-stranded DNA. It can bind to double-stranded DNA to produce a green fluorescent signal, and its fluorescent signal intensity is positively correlated with the concentration of double-stranded DNA. Experiments show that during the amplification process, the initial slope of the amplification curve (the curve of fluorescence intensity changing with time) is positively correlated with the activity of the polymerase. Therefore, the activity value of the polymerase can be calculated from the amplification curve. The primers and specific operations are as follows:

[0108] Test 1: 5'-GGACTACGATTGGCTTTTTG-3'(SEQ ID NO.12)

[0109] Test 2: 5'-ACACAGCGATGTGATGCTAATCTCAAAAAGCCAATCGTAGTCC-3'(SEQ IDNO.13)

[0110] (1) Preparation of reaction system: The 9.6 μL reaction system for primer annealing consists of 120 mmol / L Tris-HCl, 20 mmol / L KCl, 10 mmol / L (NH4)2SO4, 2 mmol / L MgCl2, 0.025% Triton X-100, 60 nmol / L Test 1, 60 nmol / L Test 2, 20 μmol / L dNTPs, 0.8× SYBR Green I, pH 7.8. The reaction procedure for primer annealing is 95°C for 30 s, followed by 65-45°C (-5°C / cycle) for 30 s. After the reaction is completed, the reaction is immediately placed on ice for cooling. Note that three replicates should be prepared for each condition to reduce instrument and operator errors. Three blank controls should be prepared for each reaction to subtract the real-time fluorescence background.

[0111] (2) Polymerase activity reaction: 0.4 μL polymerase was added to the above system respectively, and ultrapure water was used as a blank control. After mixing and centrifugation, the mixture was placed in a real-time fluorescence quantitative PCR instrument. The reaction program was set as follows: 60°C for 5 min, and the "SYBR" fluorescence signal was collected every 10 s; after the 60°C isothermal extension was completed, the melting curve was read (60-95°C, 0.5°C / 5 s).

[0112] (3) Data processing: The original fluorescence data of the amplification curve was exported, the real-time fluorescence background was deducted, and 5 representative points with good linearity were selected from the first 10 data points to calculate the initial slope value of the amplification curve (linear R 2 >0.99).

[0113] (4) Establishment of polymerase activity standard curve: The commercial enzyme KOD FX Neo (TOYOBO) with known enzyme activity concentration was selected as the standard. It was diluted with storage buffer to enzyme activity concentrations of 0.01 U / μL, 0.015 U / μL, 0.025 U / μL, 0.03 U / μL, 0.05 U / μL, and 0.06 U / μL, respectively. The initial slope of the amplification curve of the standard was measured, and then the polymerase activity standard curve was drawn using the Origin software. The regression equation y = 1852.2x (R 2 >0.99).

[0114] (5) Determination of polymerase activity: The sample to be tested was appropriately diluted with storage buffer, and the system was prepared and the polymerase activity reaction was performed. The reaction buffer conditions for KOFU-pFEN1-mut were 120mmol / L Tris-HCl, 20mmol / L KCl, 10mmol / L (NH4)2SO4, 2mmol / L MgCl2, 0.025% (v / v) TritonX-100, pH 7.8. After the reaction was completed, the initial slope of the amplification curve was calculated and substituted into the regression equation of the polymerase activity standard curve to calculate the enzyme activity concentration of the DNA polymerase to be tested. The results showed that the polymerase activity concentration of KOFU-pFEN1-mut was 125U / μL.

[0115] Example 6: Amplification Performance of KOFU-pFEN1-mut DNA Polymerase

[0116] The amplification performance of the KOFU-pFEN1-mut DNA polymerase prepared in Example 4 was measured using the following method. In this example, a 1 kb fragment was amplified using wild-type KOD DNA polymerase, wild-type Pfu DNA polymerase, KOFU-mut DNA polymerase, and KOFU-pFEN1-mut DNA polymerase, using 1 ng / μL λ DNA as a template. The 20 μL PCR reaction system consisted of: 120 mmol / L Tris-HCl, 20 mmol / L KCl, 5 mmol / L (NH₄)₂SO₄, 2 mmol / L MgCl₂, 0.025% (v / v) Triton X-100, 200 μmol / L dNTPs, 0.2 μmol / L upstream and downstream primers, and 1 U of chimera, pH 7.8. The reaction program was as follows: 94°C for 3 min; 98°C for 10 s, 55°C for 30 s, 72°C for 1 s / cycle, 10 s / cycle, 30 s / cycle, 60 s / cycle, for a total of 35 cycles; 72°C for 5 min. PCR products were detected on a 1.5% agarose gel.

[0117] Primer information is as follows:

[0118] λ-FP:5'-CGTTTCCGTTCTTCTTCGTC-3'(SEQ ID NO.14)

[0119] λ-RP-1kb:5'-GGCTCAACGTGGGTTTTCA-3'(SEQ ID NO.15)

[0120] The experimental results are as follows Figure 3 As shown, under the same reaction system, KOFU-mut DNA polymerase can amplify a large number of 1kb fragments under an extension rate of 1s / cycle. This indicates that the substitution and truncation mutations generated by directed evolution have improved the amplification performance of KOFU-mut DNA polymerase. While the polymerase activity of KOFU-mut DNA polymerase is inhibited after fusion with the flap-like endonuclease 1 domain pFEN1 (KOFU-pFEN1-mut DNA polymerase), it still maintains high amplification performance, with a small amount of product formed under an extension rate of 1s / cycle, similar to that of wild-type KOD DNA polymerase and significantly improved compared to wild-type Pfu DNA polymerase.

[0121] Example 7: Tolerance of KOFU-pFEN1-mut DNA polymerase to NaCl

[0122] The KOFU-pFEN1-mut DNA polymerase prepared in Example 4 was used to determine its NaCl tolerance according to the following method. In this example, wild-type KOD DNA polymerase, wild-type Pfu DNA polymerase, KOFU-mut DNA polymerase, and KOFU-pFEN1-mut DNA polymerase were used to amplify a 0.4 kb pUC19 plasmid at varying NaCl concentrations, using 1 ng / μL of pUC19 as a template. The experiment was conducted using the same reaction system as described in Example 6, with the following protocol: 94°C for 2 minutes; 98°C for 10 seconds, 55°C for 30 seconds, and 72°C for 30 seconds, for a total of 35 cycles; and 72°C for 5 minutes. PCR products were examined on a 1.5% agarose gel.

[0123] Primer information is as follows:

[0124] 19S-FP:5'-GATGCCGCATAGTTAAGCCA-3'(SEQ ID NO.16)

[0125] 19S-RP:5'-TGGCTTAACTATGCGGCATC-3'(SEQ ID NO.17)

[0126] In this example, the tolerance of four DNA polymerases to NaCl concentrations of 0-125 mM was tested. Figure 4 As can be seen, the KOFU-mut DNA polymerase, which exhibits higher amplification efficiency, exhibits a higher tolerance to NaCl over a wide concentration range, with no significant change in product yield. Wild-type KOD DNA polymerase and KOFU-mut DNA polymerase exhibit better NaCl tolerance than wild-type Pfu DNA polymerase, but polymerase activity is inhibited as NaCl concentration increases, as evidenced by a gradual decrease in product yield. Therefore, mutations generated by directed evolution have conferred increased NaCl tolerance on the KOFU-mut DNA polymerase. In high-salt buffer systems, DNA polymerases exhibit a higher affinity for the template, maintaining high amplification efficiency. This indicates that high salt tolerance is often correlated with high amplification efficiency.

[0127] Example 8: Study on 3'-5' exonuclease activity of KOFU-pFEN1-mut in qPCR reaction

[0128] The single-stranded probe was used as a substrate and the KOFU-pFEN1-mut DNA polymerase and KOFU-mut DNA polymerase of the present invention were used to react with the single-stranded probe, respectively. The substrate sequence is as follows:

[0129] ASFV probe: 5'-FAM-TCGATAAATTTCCATCAAAGTTCTGCAGCTC-BHQ1-3'. (SEQ IDNO.18)

[0130] The 20 μL reaction system consisted of 120 mmol / L Tris-HCl, 20 mmol / L KCl, 5 mmol / L (NH₄)₂SO₄, 2 mmol / L MgCl₂, 0.025% (v / v) Triton X-100, 0.2 μmol / L probe, and 0.4 U chimera, pH 7.8. The reaction procedure was as follows: 95°C for 3 min, followed by 40 cycles of 95°C for 15 s and 60°C for 30 s. Amplification curves were generated for each sample after completion of the reaction, and raw fluorescence data were exported.

[0131] The experimental results are as follows Figure 5 As shown, the fluorescence value increased linearly with time when KOFU-mut DNA polymerase was added to the reaction system. However, when KOFU-pFEN1-mut DNA polymerase (with its 3'-5' exonuclease activity completely eliminated) was added to the reaction system, the fluorescence value did not change significantly compared to the control group. The 3'-5' exonuclease can hydrolyze the probe as a substrate, releasing a fluorescent signal. Random mutations generated by directed evolution and amino acid mutations at positions 164 and 166 completely abolished the 3'-5' exonuclease activity of KOFU-pFEN1-mut DNA polymerase, preventing it from cleaving the single-stranded probe as a substrate and releasing a fluorescent signal.

[0132] Example 9: Study on the 5'-3' exonuclease activity of KOFU-pFEN1-mut DNA polymerase in qPCR reaction. 6 The novel coronavirus nucleocapsid protein gene nCoV-N recombinant plasmid (pMD-18T-nCoV-N, nCoV-N is inserted at the restriction enzyme site EcoRⅤ) was used as a template, and the genes were amplified using the KOFU-pFEN1-mut DNA polymerase and KOFU-mut DNA polymerase of the present invention, respectively. The amplification primers, probes, and specific operations are as follows:

[0133] N-FP: 5'-GGGGAACTTCTCCTGCTAGAAT-3' (SEQ ID NO. 19);

[0134] N-RP: 5'-CAGACATTTTGCTCTCAAGCTG-3' (SEQ ID NO. 20);

[0135] N-probe: 5'-HEX-TTGCTGCTGCTTGACAGATT-BHQ1-3' (SEQ ID NO. 21).

[0136] The 20 μL qPCR reaction system consisted of 110 mmol / L Tris-HCl, 20 mmol / L KCl, 5 mmol / L (NH₄)₂SO₄, 6 mmol / L MgCl₂, 0.025% (v / v) Triton X-100, 200 μmol / L dNTPs, 0.2 μmol / L upstream and downstream primers, 0.2 μmol / L probe, and 0.4 U hot-start DNA polymerase, pH 7.8. The qPCR reaction protocol was as follows: 95°C for 3 min, followed by 45 cycles of 95°C for 10 s and 60°C for 30 s. The results showed that the KOFU-mut DNA polymerase, lacking the flap-like endonuclease 1 domain pFEN1, lacked 5'-3' exonuclease activity and, therefore, was unable to simultaneously hydrolyze the fluorescent probe during primer extension, resulting in a sigmoidal amplification curve. KOFU-pFEN1-mut DNA polymerase, which is fused with the flap endonuclease 1 domain pFEN1 and lacks 3'-5' exonuclease activity, will only hydrolyze the fluorescent probe annealed to the template in the qPCR reaction, and generate fluorescent signals in an exponential form as the product is amplified, resulting in a standard S-shaped amplification curve ( Figure 6 ).

[0137] Example 10: Preparation of hot-start DNA polymerase

[0138] Highly purified KOFU-pFEN1-mut was dialyzed into 40 mmol / L Tris-HCl (pH 9.5) buffer and diluted to 1 μmol / L. Citraconic anhydride (11.06 mol / L) was diluted to 0.25, 0.20, 0.15, 0.10, and 0.05 mol / L using DMF. Equal amounts of citraconic anhydride at varying concentrations were mixed with the dialyzed DNA polymerase at a 1:100 volume ratio and incubated at 42°C for 4 hours to determine the molar ratio required to completely block DNA polymerase activity. Chemical modification conditions were varied, including modification temperature (0, 10, 25, 37, and 42°C), modification pH (8, 8.5, 9, 9.5, and 10), and modification time (1, 2, 3, and 4 hours) to determine the optimal method for preparing the hot-start DNA polymerase. The resulting hot-start DNA polymerase was dialyzed into storage buffer (50 mmol / L Tris-HCl, 0.1 mmol / L EDTA, 50% (v / v) glycerol, 2 mmol / L DTT, 0.002% (v / v) Tween-20, 0.002% (v / v) IGEPAL CA 630, pH 9.0). After heat shock at 95°C for 14 minutes, the DNA polymerase activity of the hot-start DNA polymerase was measured according to the method of Example 5. The results showed that Group B DNA polymerases can be hot-start modified by chemical modification, and the optimal conditions for preparing the hot-start KOFU-pFEN1-mut DNA polymerase are a reaction molar ratio of citraconic anhydride to KOFU-pFEN1-mut of 2000:1 ( Figure 7 ), 25℃( Figure 8 ) incubated for 3 h ( Figure 10 ), the reaction buffer pH was 9.0 ( Figure 9 ), wherein the polymerase activity concentration after chemical modification was 5.8U / μL.

[0139] Example 11: Thermal Stability Study of HS-KOFU-pFEN1-mut and KOFU-pFEN1-mut

[0140] The chimeras before and after citraconic anhydride modification were diluted to a polymerase activity concentration of 50 U / mL, and the thermal stability of the chimeras was tested as follows. The diluted enzymes were incubated at 95°C / 98°C for 0, 1, 2, 3, 4, 5, and 6 hours, immediately cooled on ice, and samples were taken for DNA polymerase activity assays. As shown in Table 1, the results show that the chimeras still have high thermal stability, with half-lives of 250 minutes at 95°C and 70 minutes at 98°C. The hot-start modification converts positively charged amino groups on the recombinant enzyme surface into negatively charged carboxyl groups, increasing the protein surface charge in a weakly alkaline buffer system and improving the thermal stability of the chimeras.

[0141] Table 1. Thermal stability study results

[0142]

[0143] Example 12: pH Stability Study of HS-KOFU-pFEN1-mut and KOFU-pFEN1-mut

[0144] The chimeras before and after citraconic anhydride modification were diluted to a polymerase activity concentration of 50 U / mL, and their pH stability was investigated as follows. The diluted enzymes were placed in buffers with varying pH values (5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, and 10.0). Two buffer systems, PBS (pH 5.0-7.5) and Tris-HCl (pH 7.5-10.0), were selected for the experiments, depending on the buffering range of the different buffer systems. The enzymes were incubated at 4°C for 12 hours. Samples were collected from each buffer for DNA polymerase activity assays. The results showed that KOFU-pFEN1-mut exhibited higher pH stability in neutral buffer systems, while HS-KOFU-pFEN1-mut exhibited higher pH stability in weakly acidic buffer systems.

[0145] Example 13: Optimal Reaction Temperature of HS-KOFU-pFEN1-mut and KOFU-pFEN1-mut

[0146] Using the pET-28a-Taq recombinant vector as a template, the KOFU-pFEN1-mut and HS-KOFU-pFEN1-mut of the present invention were used to simultaneously amplify the gene. The amplification primers and specific operations were as follows:

[0147] Taq-FP: 5'-AGCGGTGGCGGCGGTTCTGGCGGTGGTGGCAGCATGCTGCCGCTTTTCGAG-3' (SEQ ID NO. 22);

[0148] Taq-RP: 5'-AAGCTTGTCGACTTAGCTTTCAAGAAACCAAACTCATGC-3' (SEQ ID NO. 23).

[0149] The chimera PCR reaction system for 20 μL of the chimera consisted of 120 mmol / L Tris-HCl, 20 mmol / L KCl, 5 mmol / L (NH₄)₂SO₄, 2 mmol / L MgCl₂, 0.025% (v / v) Triton X-100, 200 μmol / L dNTPs, 0.2 μmol / L upstream and downstream primers, and 1 U of the chimera, pH 7.8. The PCR reaction protocol was as follows: 98°C for 3 min / 10 min; 35 cycles of 98°C for 15 s, 60°C for 30 s, and 72°C for 1 min; and 72°C for 5 min. The PCR products obtained from the chimera and the hot-start DNA polymerase were subjected to 1.5% agarose gel electrophoresis. The results showed that the optimal reaction temperature for the chimera was 72-75°C, and that for the chimera after hot-start modification was also 72-75°C.

[0150] Example 14: Optimal reaction pH of HS-KOFU-pFEN1-mut and KOFU-pFEN1-mut

[0151] The chimeras before and after citraconic anhydride modification were diluted to a polymerase activity concentration of 50 U / mL, and their optimal reaction pH was determined as follows. The diluted enzymes were then subjected to a DNA polymerase activity assay, maintaining the reaction buffer composition and concentration constant while varying the reaction pH (7.2, 7.3, 7.4, 7.5, 7.6, 7.8, 8.0, 8.1, 8.2, 8.4, and 8.5). The results showed that the optimal reaction pH for HS-KOFU-pFEN1-mut was 7.5, and the optimal reaction pH for KOFU-pFEN1-mut was 8.0.

[0152] Example 15: Study on Inhibitor Tolerance of KOFU-pFEN1-mut in qPCR Reaction

[0153] The qPCR reaction was performed with varying concentrations of ethanol (0-10% (v / v)), NaCl (0-125 mmol / L), SDS (0-0.15‰ (w / v)), bile salts (0-4 μg / μL), whole blood (0-1.5% (v / v)), and tannic acid (0-750 ng / μL) to assess the tolerance of KOFU-pFEN1-mut to common amplification inhibitors. A wild-type hot-start Taq DNA polymerase control was also used.

[0154] The experiment was carried out according to the reaction system (10 μL) and reaction procedure described in Example 9, wherein the pre-denaturation time at 95°C was 10 min. Figure 11As shown, the results showed that the hot-start KOFU-pFEN1-mut DNA polymerase exhibited excellent tolerance to 8% ethanol, 100 mmol / L NaCl, 0.01% SDS, 3 μg / μL bile salts, 1% whole blood, or 600 ng / μL tannic acid, exceeding the tolerance of Taq to common inhibitors. These results suggest that simply by modifying B-family DNA polymerases, which exhibit higher amplification efficiency and greater inhibitor tolerance, the polymerase's tolerance to common PCR inhibitors in direct amplification probe assays can be significantly improved. Furthermore, the inhibitor tolerance of the polymerase can be further enhanced by fusing it with nucleic acid-binding proteins.

[0155] Example 16: Study on the minimum detection limit of KOFU-pFEN1-mut

[0156] ASFV genomic DNA extracted from healthy porcine whole blood was diluted with 2.5% whole blood. The positive sample concentrations in the system were 50 pg, 10 pg, 5 pg, 1 pg, 0.5 pg, and 0.1 pg, respectively. The minimum detection limit of KOFU-pFEN1-mut DNA polymerase was evaluated by hot start at 0.5% whole blood. Sterile ultrapure water was used as a negative control. The experiment was repeated three times for each condition. The reaction was carried out simultaneously using the KOFU-pFEN1-mut DNA polymerase of the present invention and Taq DNA polymerase. The amplification primers and specific procedures were as follows:

[0157] ASFV-FP: 5'-TTCCGTAACTGCTCATGGTATCAATCT-3' (SEQ ID NO. 24);

[0158] ASFV-RP: 5'-CCTCCGTAGTGGAAGGGTATGTAAG-3' (SEQ ID NO. 25);

[0159] ASFV-probe: 5'-ROX-TCGATAAATTTCCATCAAAGTTCTGCAGCTC-BHQ1-3' (SEQ ID NO. 26).

[0160] The experiment was carried out according to the reaction system and reaction procedure described in Example 9, wherein the pre-denaturation time at 95°C was 10 min. The experiment was repeated 3 times under each condition. The results are shown in Figure 9. Figure 12 As shown, when detecting ASFV genomic DNA at a 0.5% whole blood content, KOFU-pFEN1-mut DNA polymerase can detect 0.5pg, while Taq DNA polymerase is completely inhibited, that is, DNA polymerases with high amplification performance and salt ion tolerance have higher affinity for nucleic acid templates and can improve the detection ability of target molecules in crude samples.

[0161] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A hot-start B-family DNA polymerase for fluorescent probe-based qPCR, characterized by: It was named KOFU-pFEN1-mut, and its amino acid sequence is shown in SEQ ID NO.

1.

2. A DNA molecule encoding the KOFU-pFEN1-mut of claim 1, characterized in that: The nucleotide sequence of the DNA molecule is shown in SEQ ID NO.

7.

3. A recombinant expression vector or recombinant engineered cell line containing the DNA molecule according to claim 2.

4. The method for preparing KOFU-pFEN1-mut according to claim 1, characterized in that: The steps include: 1) taking the recombinant engineered cell line described in claim 3, inoculating it into LB medium, and culturing it to obtain a seed solution; 2) The obtained seed solution was inoculated into LB medium and cultured to obtain bacterial solution; 3) Add IPTG to the resulting bacterial solution to a final concentration of 0.025-0.4 mmol / L to induce cell protein expression, and collect the bacterial pellet by centrifugation; 4) Add lysis buffer to the resulting bacterial pellet to resuspend the cells, disrupt the cells by ultrasonication, and centrifuge to obtain the supernatant; 5) Incubate the resulting supernatant at 75°C for 20–30 min, place on ice for 10–20 min, centrifuge, and filter through a 0.22 μm microporous membrane. 6) Perform nickel ion affinity chromatography and protein denaturing electrophoresis to detect the elution products at each gradient and collect samples containing the target protein; 7) performing strong anion exchange purification, detecting the elution products at each gradient by protein denaturing electrophoresis, and collecting samples containing the target protein to obtain the KOFU-pFEN1-mut; Wherein, the LB culture medium described in step 1) and step 2) both contains 50 μg / mL kanamycin sulfate; The culture conditions in step 1) are 37° C. and 150-200 rpm shaking; The culture conditions described in step 2) are 37°C, 150-200 rpm shaking culture until OD 600 =0.6~0.8; The seed solution described in step 2) is inoculated into LB medium at a volume ratio of 1:100; The induction conditions described in step 3) are 25°C for 10-12 hours; The centrifugation condition in step 3) is 20,000×g at 4° C. for 20 to 30 minutes; The amount of lysis buffer in step 4) is calculated as 5 mL per gram of bacterial pellet; The ultrasonic conditions in step 4) are as follows: power 250 W, ultrasonication 5.5 s, interval 5.5 s, and duration 30 min; The centrifugation conditions in step 4) and step 5) are 4°C and 20,000×g for 10 to 20 minutes; The lysis buffer described in step 4) comprises: 50 mmol / L Tris-HCl, 50 mmol / L NaCl, 5% v / v glycerol, pH 8.0; The binding buffer used in the nickel ion affinity chromatography and strong anion exchange chromatography described in step 6) comprises: 50 mmol / L Tris-HCl, 50 mmol / L NaCl, 5% v / v glycerol, pH 8.0; the elution buffer used in the nickel ion affinity chromatography comprises: 50 mmol / L Tris-HCl, 50 mmol / L NaCl, 500 mmol / L imidazole, 5% v / v glycerol, pH 8.0; and the elution buffer used in the strong anion exchange chromatography comprises: 50 mmol / L Tris-HCl, 1 mol / L NaCl, 5% v / v glycerol, pH 8.

0.

5. A hot-start DNA polymerase, characterized in that: The invention is obtained by specifically binding an anhydride compound to the amino group of the lysine side chain in the active site of the polymerase KOFU-pFEN1-mut described in claim 1; the anhydride compound is maleic anhydride or citraconic anhydride.

6. The method for preparing the hot-start DNA polymerase according to claim 5, characterized in that: The steps include: (1) dialyzing the KOFU-pFEN1-mut described in claim 1 into Tris-HCl buffer; (2) Add anhydride compound, mix well, and react; (3) The reaction mixture is dialyzed into storage buffer to obtain a stable hot-start DNA polymerase; The Tris-HCl buffer is a Tris-HCl buffer with a concentration of 10 to 50 mmol / L and a pH of 8 to 9; The molar ratio of KOFU-pFEN1-mut to the acid anhydride compound is 1:2000 to 1:3000; The reaction conditions are temperature 25°C to 37°C and time 3 to 4 hours; The storage buffer has a formula of: 50 mmol / L Tris-HCl, 0.1 mmol / L EDTA, 50% v / v glycerol, 2 mmol / L DTT, 0.002% v / v Tween-20, 0.002% v / v IGEPAL CA 630, pH 9.

0.

7. A direct amplification PCR kit, characterized in that: comprising at least one of PCR water, PCR reaction buffer, primers and dNTPs, and the KOFU-pFEN1-mut described in claim 1 or the hot-start DNA polymerase described in claim 5; The PCR reaction buffer is composed of 100-120 mmol / L Tris-HCl, 10-30 mmol / L KCl, 5-20 mmol / L (NH4)2SO4, 2-4 mmol / L MgCl2, 0.025-0.10% (v / v) Triton X-100, and a pH of 7.5-8.

5. The enzyme activity concentration of the KOFU-pFEN1-mut or hot start DNA polymerase in the system is 0.05-0.1 U / μL; The concentration of the dNTPs in the system is 100-300 μmol / L; The concentration of the primer in the system is 0.2-0.4 μmol / L.

8. A direct amplification qPCR kit, characterized in that: comprising water for qPCR, a qPCR reaction buffer, at least one of primers, probes, and dNTPs, and the hot-start DNA polymerase according to claim 5; The qPCR reaction buffer is composed of 100-120 mmol / L Tris-HCl, 10-30 mmol / L KCl, 5-20 mmol / L (NH4)2SO4, 4-6 mmol / L MgCl2, 0.025-0.10% (v / v) Triton X-100, and a pH of 7.5-8.

5. The enzyme activity unit of the hot start DNA polymerase in the system is 0.01 to 0.05 U / μL; The concentration of the dNTPs in the system is 100-300 μmol / L; The concentration of the primer in the system is 0.2-0.4 μmol / L; The concentration of the probe in the system is 0.2-0.3 μmol / L.

9. Use of the direct amplification qPCR kit according to claim 8 in direct amplification probe-based qPCR, characterized in that: The application operation is: using the direct amplification qPCR kit to directly amplify the biological sample to obtain the target gene product.

10. Use of the KOFU-pFEN1-mut described in claim 1, or the hot-start DNA polymerase described in claim 5, or the direct amplification PCR kit described in claim 7, or the direct amplification qPCR kit described in claim 8 in amplification and / or detection of biological samples.

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