CN116286717A

CN116286717A - Hot start B-group DNA polymerase for fluorescence probe qPCR (quantitative polymerase chain reaction) and preparation method and application thereof

Info

Publication number: CN116286717A
Application number: CN202310241376.0A
Authority: CN
Inventors: 胡松青; 王艳茹; 刘光毅; 侯轶
Original assignee: South China University of Technology SCUT
Current assignee: South China University of Technology SCUT
Priority date: 2023-03-13
Filing date: 2023-03-13
Publication date: 2023-06-23

Abstract

The invention discloses a hot-start B-group DNA polymerase for fluorescence probe qPCR, and a preparation method and application thereof. The KOFU-pFEN1-mut DNA polymerase is modified by the way of substitution and directed evolution of a functional domain of wild KOD DNA polymerase and wild PfuDNA polymerase, fusion of flap endonuclease 1 at the C end and the like, and has higher thermal stability and amplification efficiency compared with the wild Taq DNA polymerase. After chemical modification and hot start, the thermal stability and sensitivity of the polymerase are further improved, and the polymerase is suitable for various conventional PCR, rapid PCR, long fragment PCR and hot start PCR. Meanwhile, the chimera has 5'-3' exonuclease activity and can be applied to a probe method qPCR. The chimeric has strong tolerance to PCR inhibitors, and is suitable for directly amplifying DNA from samples such as blood, soil and the like.

Description

Hot start B-group DNA polymerase for fluorescence probe qPCR (quantitative polymerase chain reaction) and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a hot-start B-group DNA polymerase for qPCR (quantitative polymerase chain reaction) by a fluorescent probe method, and a preparation method and application thereof.

Background

In 1983, the invention of the polymerase chain reaction (polymerase chain reaction, PCR) by Mullis et al uses a very small amount of DNA molecules as templates, so that a large amount of targets can be produced in an exponential amplification mode, and the method has the advantages of high specificity, high sensitivity, simplicity and convenience in operation, low cost and the like, and has been expanded to a plurality of fields of medical inspection, agricultural science, environmental science, food safety and the like. With the development of PCR technology, various new technologies based on PCR methods have been derived, such as: multiplex PCR, fluorescent quantitative PCR, digital PCR, and the like. The Real-time fluorescent quantitative PCR technology (Real-time quantitative PCR, qPCR) is based on the PCR technology, introduces fluorescent substances, converts amplified products into fluorescent signals, detects the amplified conditions in Real time, and overcomes the defects that the traditional PCR can only adopt an end-point method for observation and can not quantitatively analyze the products.

The proposal of the direct amplification qPCR technology enables rapid and accurate detection to be possible. Compared with the traditional qPCR technology, the technology reduces the nucleic acid extraction process of samples, effectively avoids the cross contamination of nucleic acid between samples and the loss of nucleic acid in the extraction process, shortens the detection time and improves the detection efficiency. However, samples typically contain large amounts of PCR inhibitors and nucleic acid molecules other than the target DNA, which can lead to decreased specificity and sensitivity of detection, and consequently, poor accuracy. The use of the hot start DNA polymerase can effectively optimize the amplified target product, inhibit the generation of nonspecific products, and improve the detection sensitivity, specificity and product yield. Wherein, the chemical modification preparation of the hot-start DNA polymerase can more completely block the activity of the DNA polymerase, has better specificity, but the activity of the polymerase can be greatly reduced by long-time heat shock. Therefore, the use of inhibitor-tolerant and thermostable DNA polymerases is critical to achieving direct amplification qPCR.

A variety of thermostable DNA polymerases have been developed in the PCR technology field, such as A-family Taq, tthDNA polymerase, and B-family Pfu, KODDNA polymerase, etc. Wherein the a-family DNA polymerase generally has three domains: a 5'-3' polymerase domain, a 5'-3' exonuclease domain and a 3'-5' exonuclease domain (generally less active or inactive); in contrast, family B has only two domains: a 5'-3' polymerase domain and a 3'-5' exonuclease domain. Studies have shown that the binding capacity of the A-family DNA polymerase to the template DNA is weaker than that of the B-family DNA polymerase, the fidelity of the amplified product is far lower than that of the B-family DNA polymerase, and the amplification length of the product of the B-family DNA polymerase is often higher than that of the A-family DNA polymerase. The B family DNA polymerase is also more tolerant to PCR inhibitors such as hemoglobin, humic acid, etc. present in blood, soil. Since the 90 s of the last century, researchers have randomly mutated and artificially evolved wild-type Taq DNA polymerase to explore the potential of application of Taq DNA polymerase, and although a variety of DNA polymerases with tolerance to inhibitors have been obtained, most of these DNA polymerases are unable to hydrolyze fluorescent probes due to lack of 5'-3' exonuclease activity and therefore cannot be applied in direct amplification fluorescent probe method qPCR. Studies have reported that amplification performance with KOD FX Neo DNA polymerase is significantly better than exonuclease-deficient Taq DNA polymerase in blood direct amplification. Group B DNA polymerases are naturally inhibitor tolerant and are a method of reversibly blocking DNA polymerase activity by chemical or antibody modification to achieve hot start.

In view of the foregoing, there is a need to develop a hot-start DNA polymerase with both inhibitor tolerance and high 5'-3' exonuclease activity for use in qPCR detection by the direct-amplification fluorescent probe method.

Disclosure of Invention

The primary object of the present invention is to overcome the disadvantages and shortcomings of the prior art and to provide a hot start group B DNA polymerase for fluorescent probe qPCR. The enzyme has high inhibitor tolerance and thermal stability, and can be used for a qPCR detection system and a multiplex PCR detection system by a hands-free fluorescence probe method. In some embodiments, the chimeric polymerases of the present invention are designed to have high amplification performance, inhibitor tolerance, thermostability, 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 invention combines the functional characteristics of different domains of different DNA polymerases, and utilizes the technology of substitution and directed evolution of the functional domains in the high-fidelity DNA polymerase to obtain the DNA polymerase with low fidelity, high amplification efficiency and strong inhibitor tolerance (named KOFU-mut DNA polymerase); the chimeric polymerase can be used for the qPCR technology of the fluorescent probe method by site-directed mutagenesis and fusion of the thermostable flap endonuclease 1 domain. The DNA polymerase of the present invention is a group B DNA polymerase having both thermostability, tolerance to PCR inhibitors and 5'-3' exonuclease activity.

Another object of the present invention is to provide a method for producing the above-mentioned hot-start group B DNA polymerase.

It is a further object of the present invention to provide the use of the above-described hot-start group B DNA polymerase.

The aim of the invention is realized by the following technical scheme:

a hot start B group DNA polymerase for fluorescent probe qPCR is named KOFU-pFEN1-mut, and the amino acid sequence of the hot start B group DNA polymerase is shown as SEQ ID NO. 1.

The hot start B-group DNA polymerase has the following characteristics: the amino acid sequence at the 1-20 positions is a histidine tag (His-tag); the amino acid sequence at positions 21-788 is a chimeric (KOFU-mut) of a wild-type KOD DNA polymerase (NCBI accession No. 1WNS_A) and a wild-type Pfu DNA polymerase (NCBI accession No. AHH 82558.1); the 789-803 amino acid sequence is a linker composed of 15 amino acids shown in SEQ ID NO. 6; the amino acid sequence at positions 804-1130 is 1-327 amino acids of the flap endonuclease 1 domain (NCBI accession number WP_ 011012561.1) pFEN1 shown in SEQ ID NO. 5. Wherein the amino acid sequences of the KOFU-pFEN1-mut DNA polymerase at the positions 21-354 and 588-788 are the amino acid sequences of the wild-type KOD DNA polymerase at the positions 1-331 and 564-764, respectively, and the amino acid sequence of the wild-type Pfu DNA polymerase at the positions 355-587 is the amino acid sequence of the wild-type Pfu DNA polymerase at the positions 332-564; comprises two mutation sites, aspartic acid (Asp) at position 164 is mutated into asparagine (Asn); glu 166 is mutated to Glu (Gln).

Compared with the amino acid sequence of wild KOD DNA polymerase, the chimeric KOFU-mut DNA polymerase has the following characteristics: the 22 nd amino acid is inserted with 3 amino acids of alanine (Ala), serine (Ser) and alanine (Ala) in sequence; the 765-774 amino acid deletion from wild-type KOD DNA polymerase; aspartic acid (Asp) at position 170 to histidine (His); phenylalanine (Phe) at position 175 to serine (Ser); mutation of methionine (Met) at position 182 to threonine (Thr); alanine (Ala) at position 240 to valine (Val); aspartic acid (Asp) at position 608 to lysine (Lys); valine (Val) at position 660 to alanine (Ala); valine (Val) at position 670 to aspartic acid (Asp); glycine (Gly) at position 788 was mutated to alanine (Ala).

Compared with the wild type flap endonuclease 1 shown in SEQ ID NO.5, the flap endonuclease 1 domain pFEN1 has the following characteristics: aspartic acid (Asp) at position 830 to asparagine (Asn); mutation of phenylalanine (Phe) at position 1113 to lysine (Lys); arginine (Arg) at position 1117 to alanine (Ala); amino acids 328 to 340 of the wild type flap endonuclease 1 are deleted. Compared with the 5'-3' exonuclease activity of wild Taq DNA polymerase, the flap endonuclease 1 has stronger 5'-3' exonuclease activity. The flap endonuclease 1 can enable the fused protein to obtain 5'-3' exonuclease activity and hydrolyze fluorescent probes.

The N-terminal of the flap endonuclease 1 is connected with the C-terminal of the modified KOFU DNA polymerase through a connector shown in SEQ ID NO. 6.

A DNA molecule encoding the KOFU-pFEN1-mut described above, wherein the nucleotide sequence of the DNA molecule is shown in SEQ ID NO. 7. The sequence is obtained by codon optimization according to the characteristics of an escherichia coli expression system, and can remarkably improve the expression efficiency of heterologous genes in host bacteria.

A recombinant expression vector is obtained by cloning a DNA molecule encoding the above KOFU-pFEN1-mut into an expression vector.

The expression vector is preferably a prokaryotic expression vector; more preferably pET series vectors; most preferred is pET-28a.

A recombinant engineering cell strain is obtained by transforming the recombinant expression vector into engineering cells.

The engineering cell is preferably an escherichia coli cell; more preferably E.coli BL21 (DE 3).

The KOFU-pFEN1-mut can be obtained by a chemical synthesis method or by recombinant engineering cell strain induced expression and purification. From the cost point of view, the recombinant engineering cell strain is preferably prepared by inducing expression and purifying; the method specifically comprises the following steps:

1) Inoculating the recombinant engineering cell strain into an LB culture medium, and culturing to obtain seed liquid;

2) Inoculating the seed liquid into an LB culture medium, and culturing to obtain a bacterial liquid;

3) Adding IPTG to the obtained bacterial liquid to a final concentration of 0.025-0.4 mmol/L, inducing cells to express protein, and centrifugally collecting bacterial precipitate;

4) Adding a lysis buffer solution into the obtained bacterial precipitate to resuspend the bacterial, ultrasonically crushing the bacterial, and centrifuging to obtain a supernatant;

5) Incubating the obtained supernatant at 75 ℃ for 20-30 min, ice-bathing for 10-20 min, centrifuging, filtering with a 0.22 mu m microporous filter membrane, and taking the supernatant;

6) Collecting the sample eluent containing the target protein through nickel ion affinity chromatography and strong anion exchange chromatography, and obtaining the target protein.

Preferably, the LB medium described in step 1) and in step 2) contains 50. Mu.g/mL kanamycin sulfate.

Preferably, the culture conditions in step 1) are shaking culture at 37℃and 150 to 200 r/min.

Preferably, the culture conditions in step 2) are those of 37℃and 150-200 r/min shaking culture to OD ₆₀₀ ＝0.6～0.8。

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

Preferably, the induction conditions described in step 3) are induction at 25℃for 10-12 h.

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

Preferably, the centrifugation conditions in step 3) are centrifugation at 20000 Xg for 20-30 min at 4 ℃.

Preferably, the lysis buffer described in step 4) is added in an amount of 5mL per gram of bacterial pellet.

Preferably, the condition of the ultrasound described in step 4) is a power of 250W, ultrasound of 5.5s, interval of 5.5s, for 30min.

Preferably, the centrifugation conditions described in step 4) and step 5) are centrifugation at 20000 Xg for 10-20 min at 4 ℃.

Preferably, the composition of the lysis buffer described in step 4) is: 50mmol/L Tris-HCl, 50mmol/L NaCl, 5% (v/v) glycerol, pH8.0.

Preferably, the composition of the binding Buffer (Buffer a) used for the nickel ion affinity chromatography and the strong anion exchange chromatography described in step 6) is: 50mmol/L Tris-HCl, 50mmol/L NaCl, 5% (v/v) glycerol, pH 8.0; the elution Buffer (Buffer B) used for the nickel ion affinity chromatography comprises the following components: 50mmol/L Tris-HCl, 50mmol/L NaCl, 500mmol/L imidazole, 5% (v/v) glycerol, pH 8.0; the components of the elution Buffer (Buffer C) used for the strong anion exchange layer are: the components of the elution buffer used were: 50mmol/L Tris-HCl, 1mol/L NaCl, 5% (v/v) glycerol, pH8.0.

A hot-start DNA polymerase is obtained by specifically binding an acid anhydride compound to the amino group of the side chain of lysine at the active site of the polymerase of KOFU-pFEN1-mut. The principle is that the side chain amino on the amino acid residue is taken as a nucleophilic reagent to attack carbonyl carbon of an anhydride compound, nucleophilic attack reaction is generated, the reaction is reversible, and the enzyme activity of DNA polymerase is further blocked, so that the hot start modification of KOFU-pFEN1-mut is realized.

The anhydride compound is preferably maleic anhydride or citraconic anhydride; more preferably citraconic anhydride. The polyase modified by anhydride greatly reduces the possibility of non-specific product amplification and primer dimer formation in the PCR reaction process.

The preparation method of the hot start DNA polymerase comprises the following steps:

(1) Dialyzing the KOFU-pFEN1-mut into Tris-HCl buffer;

(2) Adding an anhydride compound, uniformly mixing and reacting;

(3) The reacted mixture was dialyzed into a storage buffer to obtain a stable hot-start DNA polymerase designated HS-KOFU-pFEN1-mut. The hot start DNA polymerase prepared by the method has excellent hot start performance, and no polymerase activity is released after incubation for 10min at 80 ℃; the activity can be completely released by heat shock at 95 ℃ for 14 min.

The Tris-HCl buffer solution is preferably Tris-HCl buffer solution with the concentration of 10-50 mmol/L, pH =8-9.

The preferable molar ratio of KOFU-pFEN1-mut to the anhydride compound is 1:2000-1:3000.

The reaction conditions are preferably 25-37 ℃ and 3-4 hours.

The formula of the storage buffer is preferably as follows: 50mmol/L Tris-HCl,0.1mmol/L EDTA,50% (v/v) glycerol, 2mmol/LDTT,0.002% (v/v) Tween-20,0.002% (v/v) IGEPAL CA 630, pH 9.0.

A PCR kit comprising at least one of water for PCR, a PCR reaction buffer, a primer, dNTPs, and KOFU-pFEN1-mut as described above.

Preferably, the composition of the PCR reaction buffer is as follows: 100 to 120mmol/L Tris-HCl, 10 to 30mmol/L KCl, 5 to 20mmol/L (NH) ₄ ) ₂ SO ₄ 、2～4mmol/L MgCl ₂ 0.025-0.10% (v/v) Triton X-100, pH 7.5-8.5.

Preferably, the concentration of the KOFU-pFEN1-mut enzyme activity in the system is 0.05-0.1U/. Mu.L.

Preferably, the concentration of dNTPs in the system is 100-300 mu mol/L.

Preferably, the concentration of the primer in the system is 0.2-0.4 mu mol/L.

The PCR kit is applied to DNA sample amplification.

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

Preferably, the qPCR reaction buffer has the following composition: 100 to 120mmol/L Tris-HCl, 10 to 30mmol/L KCl, 5 to 20mmol/L (NH) ₄ ) ₂ SO ₄ 、4～6mmol/L MgCl ₂ 0.025-0.10% (v/v) Triton X-100, pH 7.5-8.5.

Preferably, the enzyme activity concentration of the hot start DNA polymerase in the system is 0.01-0.05U/. Mu.L.

Preferably, the concentration of dNTPs in the system is 100-300 mu mol/L.

Preferably, the concentration of the primer in the system is 0.2-0.4 mu mol/L.

Preferably, the concentration of the probe in the system is 0.2-0.3 mu mol/L.

The application of the direct amplification qPCR kit in the direct amplification probe method qPCR.

The specific operation of the application is as follows: the direct amplification qPCR kit is adopted to directly amplify the biological sample (without nucleic acid extraction) so as to obtain the target gene product.

The KOFU-pFEN1-mut, the HS-KOFU-pFEN1-mut, the direct amplification PCR kit, or the direct amplification qPCR kit is applied to amplification and/or detection of biological samples.

The source of the DNA sample is not limited, and may contain at least one inhibitor selected from humic acid, hemoglobin, heparin sodium and the like. For example, a plant nucleic acid sample containing polysaccharide, a blood nucleic acid sample containing hemoglobin, a soil nucleic acid sample containing humic acid, and the like.

Compared with the prior art, the invention has the following advantages and beneficial effects:

1. the DNA polymerase (KOFU-pFEN 1-mut) is obtained by fusing the binding point mutation of the flap endonuclease 1 at the C end of the KOFU-mut on the basis of replacing and directionally evolving wild KOD DNA polymerase and wild PfuDNA polymerase domains and obtaining KOFU-mut with higher amplification efficiency and low fidelity, and has a platform-stage fluorescence value reaching the level of wild Taq DNA polymerase and higher inhibitor tolerance capability and thermal stability compared with the wild Taq DNA polymerase.

2. The hot-start DNA polymerase (HS-KOFU-pFEN 1-mut) has no activity release at 80 ℃ or below, reduces the possibility of non-specific product amplification in the qPCR reaction process, and improves the specificity and sensitivity of the qPCR reaction.

3. The hot start DNA polymerase of the invention has obviously improved resistance to PCR inhibitors existing in samples, and can tolerate various high-concentration inhibitors in qPCR detection by a fluorescent probe method, such as 8% ethanol, 0.01% SDS, 100mmol/L NaCl, 1% whole blood, 3 mug/uL bile salts or 600 ng/uL tannic acid. The complex genome DNA extraction and purification steps are not needed, so that the time cost and the material equipment cost are saved, and the cross contamination among samples during operation can be avoided, so that the detection is more convenient, quick and accurate.

Drawings

FIG. 1 is a graph showing the results of affinity chromatography purification of KOFU-pFEN 1-mut; wherein, lane M is protein Marker (10-180 kDa), lane 1 is cell disruption liquid of cells after induced expression, lane 2 is supernatant obtained by centrifugation after cell disruption, lane 3 is supernatant obtained by centrifugation of disrupted liquid supernatant at 75 ℃ for 30min, lane 4 is chromatographic effluent component, and lanes 5-14 are chromatographic elution component.

FIG. 2 is a graph showing the results of ion exchange purification of KOFU-pFEN 1-mut; wherein, lane M is protein Marker (10-180 kDa), lane 1 is affinity chromatography eluting sample, lane 2 is chromatography eluting component, and lanes 3-12 are chromatography eluting components.

FIG. 3 is a diagram showing the results of PCR electrophoresis of wild-type KOD DNA polymerase, wild-type Pfu DNA polymerase, KOFU-mut DNA polymerase and KOFU-pFEN1-mut DNA polymerase at different extension times, wherein lane M is a DNA Marker.

FIG. 4 is a diagram showing the results of electrophoresis 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 concentrations of NaCl, wherein lane M is a DNA Marker.

FIG. 5 is a graph showing the results of a test for 3'-5' exonuclease activity of KOFU-pFEN1-mut DNA polymerase.

FIG. 6 is a graph showing the results of amplification curves of KOFU-pFEN1-mut DNA polymerase in a probe method qPCR.

Fig. 7 shows the reaction of citraconic anhydride at various conditions: a graph of the relative enzyme activities of KOFU-pFEN1-mut polymerase before and after chemical modification at a KOFU-pFEN1-mut molar ratio;

FIG. 8 is a graph showing the results of KOFU-pFEN1-mut polymerase relative to enzyme activity before and after chemical modification at various temperatures;

FIG. 9 is a graph showing the results of relative enzyme activities after activation of KOFU-pFEN1-mut polymerase before and after chemical modification at different pH;

FIG. 10 is a graph showing the results of KOFU-pFEN1-mut polymerase relative to enzyme activity before and after chemical modification at various times;

FIG. 11 is a graph showing the threshold results of the fluorescence probe qPCR assay of the hot start KOFU-pFEN1-mut and Taq of the present invention with the addition of different concentrations of inhibitors; wherein A, B, C, D, E, F is a threshold result graph of PCR reaction when the inhibitor is ethanol, naCl, SDS, bile salt, whole blood or tannic acid;

FIG. 12 is the lowest 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 a hot start KOFU-pFEN1-mut DNA polymerase and B is a hot start Taq DNA polymerase.

Detailed Description

The invention is further illustrated below in conjunction with specific examples, which are cited merely to illustrate the invention and are not intended to limit the scope thereof. Other embodiments of the invention, which are based on the invention, will be apparent to those skilled in the art without undue burden, and are within the scope of the invention.

The various reagents and materials used in the present invention are commercially available or may be prepared by known methods unless otherwise specified.

Example 1: construction of recombinant vector containing nucleotide sequence encoding KOFU-pFEN1-mut

(1) According to the amino acid sequence (SEQ ID NO. 1) of the chimera, a synthetic splicing PCR method is adopted to artificially subclone and synthesize a DNA molecule for encoding the chimera, and the DNA molecule is specifically shown as SEQ ID NO. 7.

(2) The DNA molecule encoding KOFU-pFEN1-mut was subjected to homologous recombination with the expression vector pET-28 a. The chimeric amplification primer sequences were as follows:

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

KOFU-RP：5'-AAGCTTGTCGACTTATTTGATCGCCTTCTTCAGACGCT-3'(SEQ ID NO.9)。

the pET-28a linearized primer sequence is as follows:

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

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

the PCR was performed by adding 25. Mu.L of 2X Pfu Max HiFi PCR ProMix (product number P217A, ind. Biotechnology Co., guangzhou) and 1. Mu.L (10. Mu. Mol/L) of each of the upstream and downstream primers, respectively, using the artificially synthesized DNA molecule encoding KFU-pFEN 1-mut and pET-28a empty vector as templates, and adding water to 50. Mu.L. The amplification conditions of the chimeric DNA molecule were: 98 ℃ for 30s;98 ℃ for 10s, 58 ℃ for 30s and 68 ℃ for 2min, 25 cycles in total; and at 68℃for 5min. The plasmid linearization PCR amplification procedure was: 98 ℃ for 30s;98 ℃ for 10s, 58 ℃ for 30s and 68 ℃ for 2.5min, 25 cycles in total; and at 68℃for 5min. The full-length approximately 3.5kb chimeric DNA gene fragment was recovered by agarose gel electrophoresis, along with the approximately 5kb linearized pET-28a vector. The recovered products were subjected to homologous recombination in a system of 5. Mu.L 2X Hipro DNA Assembly Mix (K001A, biotech Co., ind., guangzhou) and 50ng of each recovered product was added, water was added to 10. Mu.L, and incubated at 50℃for 15min. After incubation, the recombinant product was transformed into DH 5. Alpha. Competent cells.

(3) And selecting single colony for colony PCR identification, sending positive monoclonal to sequencing company for sequencing verification, culturing and verifying correct competent cells, and extracting plasmids, wherein the obtained plasmids are recombinant vectors containing the KOFU-pFEN 1-mut.

Example 2: transformant preparation of KOFU-pFEN1-mut and identification of Small amount of expression

The recombinant vector obtained in example 1 was transformed into host cell E.coli BL21 (DE 3), single colonies were picked, inoculated into liquid LB (containing 50. Mu.g/mL kanamycin sulfate) medium and cultured to OD ₆₀₀ IPTG is added to a final concentration of 0.6-0.8, induction is carried out for 10h at 25 ℃, cells are collected by centrifugation at 20000 Xg at 4 ℃, a lysis buffer is added and ultrasonication and SDS-PAGE electrophoresis are carried out to detect the expression condition of the target protein, and as a result, the prepared transformant can be found to express the target protein efficiently.

Example 3: expression of KOFU-pFEN1-mut in recombinant E.coli

The positive transformant strain capable of expressing the chimera obtained in example 2 was inoculated into 60mL of LB medium containing 50. Mu.g/mL kanamycin sulfate, and placed in a shaking table at 37℃for shake culture overnight; inoculating overnight cultured seed solution at volume ratio of 1:100 into 1L LB medium containing 50 μg/mL kanamycin sulfate, shake culturing at 37deg.C in shaking table to OD ₆₀₀ 0.6-0.8; adding IPTG to a final concentration of 0.1mmol/L, and continuing oscillating at 25 ℃ for induction for 10-12h; after induction, the cells were collected by centrifugation and weighed, the wet weight of the cells was recorded, and stored at-20 ℃.

Example 4: purification of KOFU-pFEN1-mut

1. Ultrasonic disruption of induced expression cells

Taking the thallus frozen at the temperature of minus 20 ℃ after induced expression, adding 5mL of lysis buffer (50 mmol/L Tris-HCl, 50mmol/L NaCl, 5% (v/v) glycerol, pH 8.0) into each gram of thallus according to the wet weight of the thallus recorded in the example 3, and re-suspending the thallus, and lysing the thallus by an ultrasonic cytoclasis instrument under the following ultrasonic conditions: the power is 250W, the ultrasonic wave is stopped for 5.5s and the ultrasonic wave is stopped for 5.5s, and the power lasts for 30min. The lysed cells were centrifuged at 20000 Xg for 30min at 4℃and the supernatant was placed in 250mL sterile glass bottles. The supernatant was incubated in a thermostatic water bath at 75deg.C for 30min, centrifuged at 20000 Xg for 30min at 4deg.C, filtered through a 0.22 μm microporous filter membrane and the supernatant was taken to a 250mL sterile glass bottle. As a control, 20. Mu.L of each cell disruption solution before induction of expression was sampled after each treatment, and the results of protein expression were analyzed by SDS-PAGE, as shown in FIG. 1 (lanes 1-4).

2. Nickel ion affinity chromatography purification

The chromatographic column is HisTrap ^TM HP 5mL (from GE Healthcare), binding buffer was 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 8.0,0.22 μm membrane.

HisTrap is used ^TM After HP 5mL was connected to a peristaltic pump, the tube and column were rinsed with ultrapure water, the column was equilibrated with buffer A, and the supernatant from step 1 was loaded into the column. After loading, the column was washed with buffer A and then eluted with a gradient of 10% -100% with buffer B, and the eluted fractions were collected, 20. Mu.L of each fraction was sampled and tested by SDS-PAGE, and the results are shown in FIG. 1 (lanes 5-14).

3. Strong anion exchange chromatography purification

The chromatographic column is HiTrap ^TM Capto ^TM Q column 5mL (from GE Healthcare) binding buffer A:50mmol/L Tris-HCl, 50mmol/L NaCl, 5% (v/v) glycerol, pH 8.0; the elution buffer is buffer C:50mmol/L Tris-HCl, 1mol/L NaCl, 5% (v/v) glycerol, pH 8.0,0.22 μm membrane.

HiTrap is used for ^TM Capto ^TM After 5mL of the Q column is connected to a peristaltic pump, the pipeline and the chromatographic column are washed by ultrapure water, the column is balanced by buffer A, and then the supernatant obtained in the step 1 is loaded into the chromatographic column. After loading, the column was washed with buffer A, then eluted with a gradient of 10% -100% with buffer C, and the eluted fractions were collected, 20. Mu.L of each eluted fraction was sampled and detected by SDS-PAGE protein electrophoresis, and the results are shown in FIG. 2. The eluted fractions containing the target protein were mixed and gently mixed, and dialyzed overnight into storage buffer (50 mmol/L Tris-HCl,0.1mmol/L EDTA,50% (v/v) glycerol, 2mmol/L DTT,0.002% (v/v) Tween-20,0.002% (v/v) IGEPAL CA 630, pH 8.0).

Example 5: DNA polymerase Activity of KOFU-pFEN1-mut

KOFU-pFEN1-mut prepared in example 4 was taken and its polymerase activity was measured as follows. And (3) establishing a standard curve according to the amplification initial rates of different enzyme activities of the commercial DNA polymerase, and bringing the amplification initial rates of the DNA polymerase to be detected into the standard curve to determine the enzyme activities of the DNA polymerase to be detected. Primers (Test 1, test 2) complementary to the 3' ends are designed, annealing is performed to form primer extension substrates, dNTPs gradually permeate under the catalysis of DNA polymerase, and the substrates finally form double-stranded DNA (dsDNA) products. SYBR Green I is a highly sensitive fluorescent dye that binds double-stranded DNA and can bind double-stranded DNA to generate a Green fluorescent signal whose fluorescent signal intensity is positively correlated with the concentration of double-stranded DNA. Experiments have shown that during amplification, the initial slope of the amplification curve (curve of fluorescence intensity over 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 procedures were as follows:

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

Test 2：5'-ACACAGCGATGTGATGCTAATCTCAAAAAGCCAATCGTAGTCC-3'(SEQ ID NO.13)

(1) Preparing a reaction system: the primer annealed 9.6. Mu.L reaction system was 120mmol/L Tris-HCl, 20mmol/L KCl, 10mmol/L (NH) ₄ ) ₂ SO ₄ 、2mmol/L MgCl ₂ 0.025% TritonX-100, 60nmol/L Test 1, 60nmol/

L Test

2, 20. Mu. Mol/L dNTPs, 0.8X1YBR Green I, pH 7.8. The reaction procedure of primer annealing is 95 ℃ for 30s;65-45 ℃ (-5 ℃/cycle) for 30s. After the reaction is finished, the mixture is immediately placed on ice for cooling. Note that 3 replicates per condition were formulated to reduce errors in laboratory instrumentation and operation; 3 blank controls were prepared per reaction to subtract the real-time fluorescent background.

(2) Polymerase activity reaction: adding 0.4 mu L of polymerase into the system respectively, taking ultrapure water as a blank control, uniformly mixing and centrifuging, placing into a real-time fluorescence quantitative PCR instrument, and setting a reaction program: collecting SYBR fluorescent signals every 10s at 60 ℃ for 5 min; after the isothermal extension at 60℃was completed, the melting curve (60-95℃0.5℃5 s) was read.

(3) And (3) data processing: deriving amplification curveRaw fluorescence data of the line, subtracting the real-time fluorescence background, selecting representative 5 points with good linearity from the first 10 data points, and calculating the initial slope value (linear R ² >0.99)。

(4) Establishing a polymerase activity standard curve: the commercial enzyme KOD FX Neo (TOYOBO) with known enzyme activity concentration is selected as standard substance, diluted with storage buffer solution to enzyme activity concentration of 0.01U/. Mu.L, 0.015U/. Mu.L, 0.025U/. Mu.L, 0.03U/. Mu.L, 0.05U/. Mu.L, 0.06U/. Mu.L respectively, the initial slope of amplification curve of standard substance is determined, and then polymerase activity standard curve is drawn by software Origin, and regression equation y= 1852.2x (R) is fitted ² >0.99)。

(5) Determination of polymerase Activity: and (3) properly diluting the sample to be tested by using a storage buffer solution, preparing a system and performing polymerase activity reaction. Wherein the conditions of the reaction buffer of KOFU-pFEN1-mut are 120mmol/L Tris-HCl, 20mmol/L KCl, 10mmol/L (NH) ₄ ) ₂ SO ₄ 、2mmol/L MgCl ₂ 0.025% (v/v) TritonX-100, pH 7.8. And after the reaction is finished, calculating the initial slope of the amplification curve, substituting a regression equation of the polymerase activity standard curve, and calculating the enzyme activity concentration of the DNA polymerase to be detected. The results showed that the concentration of the polymerase activity of KOFU-pFEN1-mut was 125U/. Mu.L.

Example 6: amplification Properties of KOFU-pFEN1-mut DNA polymerase

The KOFU-pFEN1-mut DNA polymerase prepared in example 4 was used to determine the amplification performance as follows. In this example, 1 ng/. Mu.L of lambda.DNA was used as a template, and the 1kb fragment was amplified with wild-type KOD DNA polymerase, wild-type Pfu DNA polymerase, KOFU-mut DNA polymerase and KOFU-pFEN1-mut DNA polymerase. Wherein, 20 mu L of PCR reaction system is: 120mmol/L Tris-HCl, 20mmol/L KCl, 5mmol/L (NH) ₄ ) ₂ SO ₄ 、2mmol/LMgCl ₂ 0.025% (v/v) Triton X-100, 200. Mu. Mol/L dNTPs, 0.2. Mu. Mol/L upstream and downstream primers, 1U chimera, pH 7.8. The reaction procedure is: 94 ℃ for 3min;98℃for 10s,55℃for 30s,72℃for 1s/cycle, 10s/cycle, 30s/cycle, 60s/cycle for a total of 35 cycles; and at 72℃for 5min. Preparation of 1.5% agarose gel for PCR products And (5) detecting.

Primer information is as follows:

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

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

as shown in FIG. 3, KOFU-mut DNA polymerase was amplified under 1s/cycle extension conditions to give a large amount of 1kb fragments in the same reaction system. Indicating that substitution and truncation mutation generated by directed evolution improve the amplification performance of KOFU-mut DNA polymerase. In contrast, after the KOFU-mut DNA polymerase fused to the flap endonuclease 1 domain pFEN1 (KOFU-pFEN 1-mut DNA polymerase), the polymerase activity was inhibited, but it had a higher amplification performance depending on the past, a small amount of product was formed under the extension condition of 1s/cycle, similar to the wild-type KOD DNA polymerase, and significantly improved over the wild-type Pfu DNA polymerase.

Example 7: tolerance of KOFU-pFEN1-mut DNA polymerase to NaCl

KOFU-pFEN1-mut DNA polymerase prepared in example 4 was taken and the tolerance of the polymerase to NaCl was determined as follows. In this example, 1 ng/. Mu.L of pUC19 plasmid was used as a template, and 0.4kb pUC19 plasmid was amplified with wild-type KOD DNA polymerase, wild-type Pfu DNA polymerase, KOFU-mut DNA polymerase and KOFU-pFEN1-mut DNA polymerase under different concentrations of NaCl. Experiments were performed according to the reaction system described in example 6, the reaction procedure being: 94 ℃ for 2min;98 ℃ for 10s,55 ℃ for 30s and 72 ℃ for 30s, and 35 cycles are total; and at 72℃for 5min. PCR products were detected by preparing 1.5% agarose gel.

Primer information is as follows:

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

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

in this example, the tolerance of four DNA polymerases to NaCl at a concentration of 0-125mM was examined. As can be seen from FIG. 4, KOFU-mut DNA polymerase with higher amplification efficiency has higher tolerance to NaCl in a wider concentration range, and the product yield does not change significantly. The tolerance properties of the wild-type KOD DNA polymerase and KOFU-mut DNA polymerase to NaCl are better than those of the wild-type Pfu DNA polymerase, but the polymerase activity is inhibited with increasing NaCl concentration, and the product yield is gradually decreased with increasing NaCl concentration. Thus, directed evolution generated mutations that made KOFU-mut DNA polymerase more tolerant to NaCl. In high salt buffer systems, DNA polymerase has a higher affinity for the template and can maintain high amplification efficiency, i.e. high salt tolerance is often correlated with high amplification efficiency.

Example 8: 3'-5' exonuclease Activity Studies of KOFU-pFEN1-mut in qPCR reactions

The KOFU-pFEN1-mut DNA polymerase and KOFU-mut DNA polymerase of the present invention are used to react with single-stranded probes, respectively, using the single-stranded probes as substrates. Wherein the substrate sequence is as follows:

ASFV probe: 5'-FAM-TCGATAAATTTCCATCAAAGTTCTGCAGCTC-BHQ1-3'. (SEQ ID NO. 18)

Wherein, 20 mu L of the reaction system is: 120mmol/L Tris-HCl, 20mmol/L KCl, 5mmol/L (NH) ₄ ) ₂ SO ₄ 、2mmol/L MgCl ₂ 0.025% (v/v) Triton X-100, 0.2. Mu. Mol/L probe, 0.4U chimera, pH 7.8. The reaction procedure was as follows: 3min at 95 ℃;95 ℃ for 15s, 60 ℃ for 30s and 40 cycles. And obtaining an amplification curve of each sample after the reaction is finished, and deriving original fluorescence data.

The experimental results are shown in FIG. 5, which shows that the fluorescence value increases linearly with time when KOFU-mut DNA polymerase is added to the reaction system. In contrast, the fluorescence value was not significantly changed compared with the control group by adding KFU-pFEN 1-mut DNA polymerase (3 '-5' exonuclease activity was completely removed) to the reaction system. The 3'-5' exonuclease can take the probe as a substrate, carry out hydrolysis reaction and release fluorescent signals. The random mutation generated by directed evolution and the amino acid mutation at 164 and 166 lead KOFU-pFEN1-mut DNA polymerase to completely lose 3'-5' exonuclease activity, so that a single-stranded probe is not taken as a substrate for cleavage to release a fluorescent signal.

Example 9: 5'-3' exonuclease Activity of KOFU-pFEN1-mut DNA polymerase in qPCR reaction at copy number concentration of 1X 10 ⁶ The novel coronavirus nucleocapsid protein gene nCoV-N recombinant plasmid (pMD-18T-nCoV-N, the insertion position of nCoV-N is restriction enzyme cutting site EcoR V) of cobies/. Mu.L is used as a template, and KOFU-pFEN1-mut DNA polymerase and KOFU-mut DNA polymerase in the invention are respectively used for amplifying genes, and the amplification primer probe and specific operation are as follows:

N-FP：5’-GGGGAACTTCTCCTGCTAGAAT-3’(SEQ ID NO.19)；

N-RP：5’-CAGACATTTTGCTCTCAAGCTG-3’(SEQ ID NO.20)；

N-probe：5’-HEX-TTGCTGCTGCTTGACAGATT-BHQ1-3’(SEQ ID NO.21)。

Wherein the 20 mu L qPCR reaction system of the DNA polymerase is as follows: 110mmol/L Tris-HCl, 20mmol/L KCl, 5mmol/L (NH) ₄ ) ₂ SO ₄ ，6mmol/L MgCl ₂ Triton X-100, 200. Mu. Mol/L dNTPs, 0.2. Mu. Mol/L upstream and downstream primer, 0.2. Mu. Mol/L probe, 0.4U hot start DNA polymerase, pH 7.8.qPCR reaction procedure was as follows: 3min at 95 ℃;95℃for 10s, 60℃for 30s,45 cycles. The results show that KOFU-mut DNA polymerase not fused to the flap endonuclease 1 domain pFEN1 lacks 5'-3' exonuclease activity, and thus cannot hydrolyze fluorescent probes simultaneously during primer extension, failing to form an S-type amplification curve. KOFU-pFEN1-mut DNA polymerase fused with the flap endonuclease 1 domain pFEN1 and having 3'-5' exonuclease activity removed hydrolyzes only the fluorescent probe annealed to the template in qPCR reaction and generates fluorescent signal exponentially with amplification of the product and gives a standard S-type amplification curve (FIG. 6).

Example 10: preparation of hot-start DNA polymerase

High purity KOFU-pFEN1-mut was dialyzed into 40mmol/LTris-HCl (pH 9.5) buffer and diluted to 1. Mu. Mol/L. Citraconic anhydride (11.06 mol/L) was diluted to 0.25, 0.20, 0.15, 0.10, 0.05mol/L with DMF, and equal amounts of citraconic anhydride at different concentrations were mixed with DNA polymerase after dialysis at a volume ratio of 1:100, and reacted at 42℃for 4 hours to determine the molar ratio of the fully blocked DNA polymerase activity. Chemical modification conditions, including modification temperature (0, 10, 25, 37, 42 ℃), modification pH (8,8.5,9,9.5, 10) and modification time (1, 2,3,4 h), were varied to determine the optimal hot-start DNA polymerase preparation method, respectively. The resulting hot-start DNA polymerase was dialyzed into a storage buffer (50 mmol/LTris-HCl,0.1mmol/L EDTA,50% (v/v) glycerol, 2mmol/L DTT,0.002% (v/v) Tween-20,0.002% (v/v) IGEPAL CA 630, pH 9.0), and the DNA polymerase activity of the hot-start DNA polymerase was determined as in example 5 after heat shock for 14min at 95 ℃. The results show that the group B DNA polymerase can be subjected to hot start modification by a chemical modification method, and the optimal condition for preparing the hot start KOFU-pFEN1-mutDNA polymerase is that the reaction molar ratio of citraconic anhydride to KOFU-pFEN1-mut is 2000:1 (FIG. 7), the reaction buffer pH is 9.0 (FIG. 9) after 25 ℃ (FIG. 8) incubation for 3h (FIG. 10), and the activity concentration of the polymerase after chemical modification is 5.8U/. Mu.L.

Example 11: thermal stability Studies of HS-KOFU-pFEN1-mut and KOFU-pFEN1-mut

The chimeras before and after citraconic anhydride modification were diluted to a polymerase activity concentration of 50U/mL and tested for heat stability as follows. The diluted enzymes were incubated at 95℃and 98℃for 0, 1, 2, 3, 4, 5 and 6 hours, respectively, and immediately cooled on ice, and the samples were taken to measure the DNA polymerase activity. As shown in Table 1, the results show that the chimera still has higher heat stability, the half-life time of the chimera at 95 ℃ is 250min, the half-life time at 98 ℃ is 70min, the heat-activated modification converts positively charged amino groups on the surface of the recombinase into negatively charged carboxyl groups, and the number of surface charges of the protein is increased in a weak alkaline buffer system, so that the heat stability of the chimera is improved.

TABLE 1 results of thermal stability studies

Example 12: PH stability study of HS-KOFU-pFEN1-mut and KOFU-pFEN1-mut

The chimera before and after citraconic anhydride modification was diluted to a polymerase activity concentration of 50U/mL, and the pH stability was investigated as follows. The diluted enzyme is respectively placed in buffers with different 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), and two buffer systems of PBS (pH=5.0-7.5) and Tris-HCl (pH=7.5-10.0) are selected according to the buffer ranges of different buffer systems to perform experiments, and incubated for 12 hours at 4 ℃. Samples were taken separately for DNA polymerase activity determination. The results show that KOFU-pFEN1-mut has a higher pH stability in neutral buffer systems, whereas HS-KOFU-pFEN1-mut has a higher pH stability in weakly acidic buffer systems.

Example 13: optimum reaction temperature of HS-KOFU-pFEN1-mut and KOFU-pFEN1-mut

The KOFU-pFEN1-mut and HS-KOFU-pFEN1-mut in the invention are used for amplifying genes simultaneously by taking pET-28a-Taq recombinant vector as a template, and the amplification primers and specific operations are as follows:

Taq-FP:5’-AGCGGTGGCGGCGGTTCTGGCGGTGGTGGCAGCATGCTGCCGCTTTTCGAG-3’(SEQ ID NO.22)；

Taq-RP:5’-AAGCTTGTCGACTTAGCTTTCAAGCAAACCAAACTCATGC-3’(SEQ ID NO.23)。

wherein the 20 mu L PCR reaction system of the chimera is as follows: 120mmol/L Tris-HCl, 20mmol/L KCl, 5mmol/L (NH) ₄ ) ₂ SO ₄ 、2mmol/L MgCl ₂ 0.025% (v/v) Triton X-100, 200. Mu. Mol/L dNTPs, 0.2. Mu. Mol/L upstream and downstream primers, 1U chimera, pH 7.8. The PCR reaction procedure was as follows: 98 ℃ for 3min/10min;98℃for 15s, 60℃for 30s, 72℃for 1min,35 cycles; and at 72℃for 5min. The PCR products obtained from the chimeras and the hot start DNA polymerase were subjected to 1.5% agarose gel electrophoresis. The result shows that the optimal reaction temperature of the chimera is 72-75 ℃, and the optimal reaction temperature of the chimera after hot start modification is 72-75 ℃.

Example 14: optimal reaction pH of HS-KOFU-pFEN1-mut and KOFU-pFEN1-mut

The chimera before and after citraconic anhydride modification was diluted to a polymerase activity concentration of 50U/mL, and its optimal reaction pH was measured as follows. The diluted enzyme was subjected to DNA polymerase activity measurement, the reaction buffer composition and concentration were kept unchanged, and the pH values of the reaction (7.2, 7.3, 7.4, 7.5, 7.6, 7.8, 8.0, 8.1, 8.2, 8.4, 8.5) were changed. The results showed that the optimum reaction pH for HS-KOFU-pFEN1-mut was 7.5 and that for KOFU-pFEN1-mut was 8.0.

Example 15: inhibitor tolerance study of KOFU-pFEN1-mut in qPCR reaction

Ethanol (0-10% (v/v)), naCl (0-125 mmol/L), SDS (0-0.15%o (w/v)), bile salts (0-4. Mu.g/. Mu.L), whole blood (0-1.5% (v/v)) and tannic acid (0-750 ng/. Mu.L) were added to the qPCR reaction to examine the tolerance of KOFU-pFEN1-mut to common amplification inhibitors. And simultaneously, a control group of wild type hot start Taq DNA polymerase is adopted.

Experiments were performed according to the reaction system (10. Mu.L) and the reaction procedure described in example 9, wherein the pre-denaturation time at 95℃was 10min. As shown in FIG. 11, the results show that the hot start KOFU-pFEN1-mut DNA polymerase can tolerate 8% ethanol, 100mmol/L NaCl, 0.01% SDS, 3. Mu.g/. Mu.L bile salts, 1% whole blood or 600 ng/. Mu.L tannic acid better than Taq's tolerance to common inhibitors. The results show that the tolerance of polymerase to common PCR inhibitors in the direct-amplification probe method detection can be significantly improved by only modifying the B-group DNA polymerase with higher amplification efficiency and higher inhibitor tolerance. Nucleic acid binding proteins can also be fused on this basis to further increase inhibitor tolerance of the polymerase.

Example 16: minimum limit of detection study of KOFU-pFEN1-mut

ASFV genomic DNA extracted by diluting whole blood of 2.5% healthy pigs, positive sample addition amounts in the system are respectively 50pg, 10pg, 5pg, 1pg, 0.5pg and 0.1pg, the lowest detection limit of KOFU-pFEN1-mut DNA polymerase is evaluated by hot start under the addition amount of 0.5% whole blood, sterilized ultrapure water is used as a negative control, and each condition is repeated for 3 times. The KOFU-pFEN1-mut DNA polymerase and Taq DNA polymerase of the present invention are used for simultaneous reaction, and the amplification primers and specific procedures are as follows:

ASFV-FP：5’-TTCCGTAACTGCTCATGGTATCAATCT-3’(SEQ ID NO.24)；

ASFV-RP：5’-CCTCCGTAGTGGAAGGGTATGTAAG-3’(SEQ ID NO.25)；

ASFV-probe：5’-ROX-TCGATAAATTTCCATCAAAGTTCTGCAGCTC-BHQ1-3’(SEQ ID NO.26)。

experiments were performed according to the reaction system and the reaction procedure described in example 9, wherein the pre-denaturation time at 95℃was 10min. Experiments were repeated 3 times for each condition. As a result, as shown in FIG. 12, when ASFV genomic DNA was detected at a whole blood content of 0.5%, KOFU-pFEN1-mut DNA polymerase could detect 0.5pg, while Taq DNA polymerase was completely inhibited, i.e., DNA polymerase having high amplification performance and salt ion tolerance had higher affinity for nucleic acid template, and the detection ability of target molecules in crude samples could be improved.

The embodiments described above are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the embodiments described above, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principles of the present invention should be made in the equivalent manner, and are included in the scope of the present invention.

Claims

1. A hot start group B DNA polymerase for fluorescent probe qPCR, characterized in that: the amino acid sequence of KOFU-pFEN1-mut is shown in SEQ ID NO. 1.

2. A DNA molecule encoding the KOFU-pFEN1-mut as claimed in claim 1, characterized in that: the nucleotide sequence of the DNA molecule is shown as SEQ ID NO. 7.

3. A recombinant expression vector or recombinant engineered cell line comprising the DNA molecule of claim 2.

4. The method for producing KOFU-pFEN1-mut as claimed in claim 1, wherein: the method comprises the following steps:

1) Inoculating the recombinant engineering cell strain in claim 3 into LB culture medium, and culturing to obtain seed solution;

2) Inoculating the obtained seed liquid into an LB culture medium, and culturing to obtain a bacterial liquid;

5) Incubating the obtained supernatant at 75 ℃ for 20-30 min, ice-bathing for 10-20 min, centrifuging to obtain supernatant, and filtering with a 0.22 mu m microporous filter membrane;

6) Performing nickel ion affinity chromatography, detecting the eluted products under each gradient by protein denaturation electrophoresis, and collecting a sample containing target proteins;

7) Performing strong anion exchange purification, detecting the eluted products under each gradient by protein denaturation electrophoresis, and collecting a sample containing target proteins to obtain the KOFU-pFEN1-mut;

wherein, the LB culture medium in the step 1) and the step 2) contains 50 mug/mL kanamycin sulfate;

the culture condition in the step 1) is that the temperature is 37 ℃ and the shaking culture is 150-200 r/min;

the culture condition in the step 2) is that the culture is carried out at 37 ℃ under the shaking culture condition of 150-200 r/min until the OD ₆₀₀ ＝0.6～0.8；

Inoculating the seed liquid in the step 2) into an LB culture medium according to the volume ratio of 1:100;

the induction condition in the step 3) is that the induction is carried out for 10 to 12 hours at 25 ℃;

the centrifugation condition in the step 3) is that centrifugation is carried out for 20-30 min at the temperature of 4 ℃ and the rotating speed of 20000 Xg;

the amount of the lysis buffer in the step 4) is 5mL per gram of bacterial precipitate;

the condition of the ultrasonic wave in the step 4) is that the power is 250W, the ultrasonic wave is 5.5s, the interval is 5.5s, and the ultrasonic wave lasts for 30min;

the centrifugation conditions in the step 4) and the step 5) are centrifugation at 20000 Xg for 10-20 min at 4 ℃;

the components of the lysis buffer described in step 4) are: 50mmol/L Tris-HCl, 50mmol/L NaCl, 5% v/v glycerol, pH8.0;

the components of the binding buffer used for the nickel ion affinity chromatography and the strong anion exchange chromatography described in step 6) are: 50mmol/L Tris-HCl, 50mmol/L NaCl, 5% v/v glycerol, pH8.0; the components of the elution buffer used for the nickel ion affinity chromatography are: 50mmol/L Tris-HCl, 50mmol/L NaCl, 500mmol/L imidazole, 5% v/v glycerol, pH8.0; the components of the elution buffer used for the strong anion exchange chromatography are: the components of the elution buffer used were: 50mmol/L Tris-HCl, 1mol/L NaCl, 5% v/v glycerol, pH8.0.

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

6. The method for producing a hot-start DNA polymerase according to claim 5, wherein: the method comprises the following steps:

(1) Dialyzing the KOFU-pFEN1-mut of claim 1 into Tris-HCl buffer;

(2) Adding an anhydride compound, uniformly mixing and reacting;

(3) Dialyzing the reacted mixed solution into a storage buffer solution to obtain stable hot start DNA polymerase;

the Tris-HCl buffer solution is 10-50 mmol/L, pH =8-9;

the molar ratio of KOFU-pFEN1-mut to the anhydride compound is 1:2000-1:3000;

the reaction conditions are that the temperature is 25-37 ℃ and the time is 3-4 hours;

the formula of the storage buffer solution is as follows: 50mmol/L Tris-HCl,0.1mmol/L EDTA,50% v/v glycerol, 2mmol/L DTT,0.002% v/vTween-20,0.002% v/vIGEPAL CA 630, pH 9.0.

7. A direct-amplification PCR kit is characterized in that: comprising at least one of water for PCR, a PCR reaction buffer, a primer and dNTPs, and KOFU-pFEN1-mut as described in claim 1 or a hot-start DNA polymerase as described in claim 5;

Wherein, the composition of the PCR reaction buffer solution is as follows: 100-120 mmol/LTris-HCl、10～30mmol/L KCl、5～20mmol/L(NH ₄ ) ₂ SO ₄ 、2～4mmol/L MgCl ₂ 0.025-0.10% (v/v) Triton X-100, pH 7.5-8.5;

the concentration of the KOFU-pFEN1-mut or the hot start DNA polymerase in the system is 0.05-0.1U/mu L;

the concentration of dNTPs in the system is 100-300 mu mol/L;

the concentration of the primer in the system is 0.2-0.4 mu mol/L.

8. A direct amplification qPCR kit is characterized in that: comprising at least one of qPCR water, qPCR reaction buffer, primers, probes, and dNTPs, and the hot start DNA polymerase of claim 5;

wherein, the qPCR reaction buffer solution comprises the following components: 100 to 120mmol/L Tris-HCl, 10 to 30mmol/L KCl, 5 to 20mmol/L (NH) ₄ ) ₂ SO ₄ 、4～6mmol/L MgCl ₂ 0.025-0.10% (v/v) Triton X-100, pH 7.5-8.5;

the enzyme activity unit of the hot start DNA polymerase in the system is 0.01-0.05U/mu L;

the concentration of dNTPs in the system is 100-300 mu mol/L;

the concentration of the primer in the system is 0.2-0.4 mu mol/L;

the concentration of the probe in the system is 0.2-0.3 mu mol/L.

9. The use of the direct amplification qPCR kit as set forth in claim 8 in a direct amplification probe method qPCR, characterized in that:

The operation of the application is as follows: and directly amplifying the biological sample by adopting the direct amplification qPCR kit to obtain a target gene product.

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