阅读说明：本技术 一种kod dna聚合酶突变体及其制备方法与应用 (KOD DNA polymerase mutant and preparation method and application thereof ) 是由 胡松青 何贤蓉 刘光毅 侯轶 于 2021-08-18 设计创作，主要内容包括：本发明公开了一种KOD DNA聚合酶突变体及其制备方法与应用,属于生物技术领域。本发明的KOD DNA聚合酶突变体KOFU-S是将野生型KODDNA聚合酶进行缺失、定点突变、嵌合以及融合双链核酸结合蛋白Sso7d改造而成。本发明的KOD DNA聚合酶突变体与野生型KODDNA聚合酶相比,具有较高的保真度,同时扩增速率、延伸能力和耐抑制剂能力等PCR扩增性能提高,将拓宽该聚合酶在分子生物学领域的应用。(The invention discloses a KOD DNA polymerase mutant and a preparation method and application thereof, belonging to the technical field of biology. The KOD DNA polymerase mutant KOFU-S of the present invention is obtained by modifying a wild-type KODDNA polymerase to delete, site-directed mutate, chimera, or fuse double-stranded nucleic acid binding protein Sso7 d. Compared with the wild KODDNA polymerase, the KOD DNA polymerase mutant has higher fidelity, and simultaneously has improved PCR amplification performances such as amplification rate, extension capability, inhibitor resistance and the like, so that the application of the polymerase in the field of molecular biology is widened.)

1. A KOD DNA polymerase mutant characterized by: is named as KOFU-S, and the amino acid sequence of the amino acid sequence is shown as SEQ ID NO. 1.

2. A DNA molecule encoding the KOD DNA polymerase mutant according to claim 1.

3. The KOD DNA polymerase mutant DNA molecule according to claim 2, wherein: the nucleotide sequence of the DNA molecule is shown as SEQ ID NO. 2.

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

5. The method for producing a KOD DNA polymerase mutant according to claim 1, wherein the KOD DNA polymerase mutant comprises: the method specifically comprises the following steps:

1) inoculating the recombinant engineering cell strain of claim 4 in LB culture medium, culturing to obtain seed liquid;

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

3) adding IPTG (isopropyl thiogalactoside) into the obtained bacterial liquid until the final concentration is 0.1-0.5 mmol/L, inducing cells to express protein, and centrifugally collecting bacterial precipitates;

4) adding a lysis buffer solution into the obtained thallus precipitate to resuspend the thallus, carrying out ultrasonic crushing on the thallus, and centrifuging to obtain a supernatant;

5) incubating the obtained supernatant for 25-35 min at 70-80 ℃, centrifuging, filtering by a 0.22 mu m microporous filter membrane, and taking the supernatant;

6) and (3) carrying out nickel ion affinity chromatography, detecting each elution peak by protein denaturation electrophoresis, and collecting a sample containing the target protein to obtain the KOD DNA polymerase mutant.

6. The method for producing a KOD DNA polymerase mutant according to claim 5, wherein:

the LB culture medium in the step 1) and the step 2) contains 50 mu g/mL kanamycin;

the culture condition in the step 1) is shaking culture at 37 ℃ for overnight;

the culture conditions in step 2) were shaking culture at 37 ℃ to OD₆₀₀＝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 induction at 18 ℃ for 16-20 h;

the centrifugation condition in the steps 3) and 4) is that the centrifugation is carried out for 20-30 min at the rotating speed of 12000rpm at 4 ℃;

the dosage of the lysis buffer solution in the step 4) is calculated by adding 5mL in each gram of thallus precipitate;

the ultrasonic condition in the step 4) is that the power is 250W, the ultrasonic time is 5s, the interval is 5s, and the ultrasonic time lasts for 30 min;

the lysis buffer in step 4) comprises the following components: 50mmol/L Tris-HCl, 300mmol/L NaCl, 25mmol/L Imidazole, 5% Glycerol, pH 8.0;

the incubation condition in the step 5) is incubation for 30min at 75 ℃;

the components of the binding buffer used for the nickel ion affinity chromatography in the step 5) are as follows: 50mmol/L Tris-HCl, 300mmol/L NaCl, pH8.0; the elution buffer used had the following composition: 50mmol/L Tris-HCl, 300mmol/L NaCl, 500mmol/L Imidazole, pH 8.0.

7. A PCR reaction kit, which is characterized in that: comprising at least one of water for PCR, PCR reaction buffer and dNTPs, and the KOD DNA polymerase mutant according to claim 1.

8. The PCR reaction kit according to claim 7, wherein:

the PCR reaction buffer solution comprises the following components: 100 to 120mmol/L Tris-HCl, 2 to 3mmol/L MgCl₂、5～20mmol/L(NH₄)₂SO₄140-160 mmol/L KCl, 0.1-0.4 g/L BSA, 0.05-0.1% Triton X-100, and the pH value is 8-10;

the concentration of the KOD DNA polymerase mutant in a system is 0.01-0.05U/mu L;

the concentration of the dNTPs in the system is 50-300 mu mol/L.

9. Use of the PCR reaction kit according to claim 7 or 8 for amplifying a DNA sample, characterized in that:

the operation of the application is as follows: and carrying out PCR by using the PCR reaction kit and a sample containing the DNA sample to obtain a target gene product amplified by the PCR.

10. Use according to claim 9, characterized in that:

the sample further comprises one or more inhibitors; the inhibitor includes but is not limited to SDS, NaCl, ethanol, urea, humic acid, heparin and bile salt; further, the content of SDS is 0-0.01%, the content of NaCl is 0-100 mmol/L, the content of ethanol is 0-10%, the content of urea is 0-160 mmol/L, the content of humic acid is 0-80 ng/muL, the content of heparin is 0-0.03U/mL, and the content of bile salt is 0-0.8 mug/muL.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a KOD DNA polymerase mutant and a preparation method and application thereof.

Background

Polymerase Chain Reaction (PCR) is one of the most important basic research means in modern molecular biology, and has been recognized as a necessary technology for studying gene function. A key component in PCR technology is thermostable DNA polymerase.

Before the thermostable polymerase is used, Escherichia coli DNA polymerase which is not thermostable is used in the PCR procedure, and it is inactivated by denaturation temperature in the PCR reaction, so that it is necessary to add new polymerase at each cycle in the PCR process at that time, and the operation is complicated. The heat-resistant DNA polymerase still has polymerase activity at 95 ℃, so that the polymerase does not need to be added in each cycle of PCR, the operation of the PCR technology is greatly simplified, and the wide use of PCR is promoted. With the development of PCR technology, more and more DNA thermostable polymerases have been discovered and studied, and different polymerases have different reaction characteristics.

KODDNA polymerase is a thermostable DNA polymerase having high amplification ability, which is isolated from Thermococcus kodakaraensis KOD1, a hyperthermophilic archaea from the hot spring of the volcanic mouth of Kakkara prefecture (Kodakara), Japan. The amplification speed of the polymerase is 2 times of that of TaqDNA polymerase and 6 times of that of PfuDNA polymerase, and the amplification yield is also high; however, KOD DNA polymerase is not as fidelity as Pfu DNA polymerase, but only 1/3. Meanwhile, KOD DNA polymerase does not have the ability to amplify a long fragment, and when a DNA fragment of about 5kb is amplified, the yield begins to decrease. After the crystal structure of KODDNA polymerase was resolved in 2003, researchers began to molecularly modify the polymerase. In 2005, the Japanese scholaro Toshihiro et al found that 147 th amino acid located on the loop of KODDNA polymerase could regulate the exo-activity and polymerization activity, and they mutated this site to obtain a series of mutants with exo-activity of 9% -276% of wild type. The mutant H147K has 276% of exo-activity compared with the wild type, but has reduced extensibility and can not amplify a gene fragment of 3.6 kb. Therefore, further optimization and modification of KODDNA polymerase is necessary.

Disclosure of Invention

In order to improve the performance of the wild type KOD DNA polymerase, improve the DNA amplification capacity and inhibitor resistance capacity of a growing segment of the wild type KOD DNA polymerase and ensure the improvement of the fidelity of the wild type KOD DNA polymerase, the invention aims at providing a KOD DNA polymerase mutant. This is a mutant DNA polymerase with rapid extension ability, inhibitor resistance ability and high fidelity.

Another object of the present invention is to provide a method for producing the KOD DNA polymerase mutant.

Still another object of the present invention is to provide use of the KOD DNA polymerase mutant.

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

a KOD DNA polymerase mutant is named as KOFU-S, and the amino acid sequence of the mutant is shown in SEQ ID NO. 1.

Compared with the wild type KOD DNA polymerase with NCBI number of 1WNS _ A, the KOD DNA polymerase mutant has the following characteristics: 3 amino acids of alanine (Ala), serine (Ser) and alanine (Ala) are inserted into the 2 nd amino acid in sequence, the 332-;

on the basis of the insertion, the chimeric modification and the fusion modification, the KOD DNA polymerase mutant also has the following mutations of amino acid residues: the 150-bit histidine (His) was mutated to lysine (Lys).

The double-stranded binding protein fragment is preferably amino acid residues 2-64 of Sso7d, the NCBI number is P39476.2, and the amino acid sequence is shown as SEQ ID No. 3. Sso7d double-stranded binding protein from sulfolobusolfataricus thermophilus, 64 amino acids in total. Experimental research proves that the enzyme can be combined with Taq DNA polymerase and Pfu DNA polymerase to improve the extension capability and inhibitor resistance capability of the corresponding polymerase.

A DNA molecule encoding the KOD DNA polymerase mutant.

The nucleotide sequence of the DNA molecule is shown as SEQ ID NO. 2. The sequence is obtained by codon optimization according to the characteristics of an escherichia coli expression system, and the expression efficiency of a heterologous gene in host bacteria can be obviously improved.

An expression vector obtained by cloning a DNA molecule encoding the KOD DNA polymerase mutant into an expression vector.

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

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

The engineering cell is preferably an Escherichia coli cell; more preferably Escherichia coli BL21(DE3) cells. The recombinant engineering cell strain can express the expression vector quickly and solubly.

The preparation method of the KOD DNA polymerase mutant specifically comprises the following steps:

1) inoculating the recombinant engineering cell strain in an LB culture medium, and culturing to obtain a seed solution;

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

3) adding IPTG (isopropyl thiogalactoside) into the obtained bacterial liquid until the final concentration is 0.1-0.5 mmol/L, inducing cells to express protein, and centrifugally collecting bacterial precipitates;

4) adding a lysis buffer solution into the obtained thallus precipitate to resuspend the thallus, carrying out ultrasonic crushing on the thallus, and centrifuging to obtain a supernatant;

5) incubating the obtained supernatant for 25-35 min at 70-80 ℃, centrifuging, filtering by a 0.22 mu m microporous filter membrane, and taking the supernatant;

6) collecting the eluate containing the target protein by nickel ion affinity chromatography to obtain the KOD DNA polymerase mutant.

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

Preferably, the culturing conditions in step 1) are shaking culture at 37 ℃ overnight.

Preferably, the culturing conditions in step 2) are shaking culture at 37 ℃ to OD₆₀₀＝0.6～0.8。

Preferably, the seed liquid in the step 2) is inoculated in the LB culture medium according to the volume ratio of 1: 100.

Preferably, the induction condition in the step 3) is 18 ℃ for 16-20 h.

Preferably, the centrifugation conditions in the step 3) and the step 4) are centrifugation at 12000rpm for 20-30 min at 4 ℃.

Preferably, the amount of the lysis buffer in step 4) is 5 mL/g of the bacterial pellet.

Preferably, the ultrasonic condition in step 4) is that the power is 250W, the ultrasonic time is 5s, the interval is 5s, and the duration is 30 min.

Preferably, the lysis buffer described in step 4) has the following composition: 50mmol/L Tris-HCl, 300mmol/L NaCl, 25mmol/L Imidazole, 5% Glycerol, pH 8.0.

Preferably, the incubation in step 5) is performed at 75 ℃ for 30 min.

Preferably, the components of the binding Buffer (Buffer a) used for the nickel ion affinity chromatography in step 5) are: 50mmol/L Tris-HCl, 300mmol/L NaCl, pH8.0; the elution Buffer (Buffer B) used had the following composition: 50mmol/L Tris-HCl, 300mmol/L NaCl, 500mmol/L Imidazole, pH 8.0.

A PCR reaction kit comprising at least one of water for PCR, PCR reaction buffer and dNTPs, and the KOD DNA polymerase mutant.

Preferably, the PCR reaction buffer has the following composition: 100 to 120mmol/L Tris-HCl, 2 to 3mmol/L MgCl₂、5～20mmol/L(NH₄)₂SO₄140-160 mmol/L KCl, 0.1-0.4 g/L BSA, 0.05% -0.1% Triton X-100, and the pH value is 8-10.

Preferably, the concentration of the KOD DNA polymerase mutant in the system is 0.01-0.05U/. mu.L.

Preferably, the concentration of the dNTPs in the system is 50-300 mu mol/L.

The PCR reaction kit is applied to the amplification of DNA samples.

The specific operation of the application is as follows: and carrying out PCR by using the PCR reaction kit and a sample containing the DNA sample to obtain a target gene product amplified by the PCR.

The sample may contain one or more inhibitors, including but not limited to SDS (sodium dodecyl sulfate), NaCl, ethanol, urea, humic acid, heparin, and bile salts; further, the content of SDS is 0-0.01%, the content of NaCl is 0-100 mmol/L, the content of ethanol is 0-10%, the content of urea is 0-160 mmol/L, the content of humic acid is 0-80 ng/muL, the content of heparin is 0-0.03U/mL, and the content of bile salt is 0-0.8 mug/muL.

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

1. the fidelity of the mutant KOFU-SDNA polymerase is twice that of the wild KOD DNA polymerase, and the improvement of the fidelity can reduce the base mutation probability in the PCR reaction process. Therefore, when the DNA product obtained by PCR amplification with the mutant KOFU-SDNA polymerase is further analyzed and identified, the obtained result is more real and reliable.

2. The PCR reaction performance of the mutant KOFU-SDNA polymerase is improved compared with that of a wild KOD DNA polymerase, the extension rate of the mutant KOFU-SDNA polymerase is up to 5s/kb at most, a DNA template with the length of 10kb can be amplified, and the PCR reaction with the amplified template nucleic acid concentration of 1 pg/mu L can be carried out. This makes the PCR reaction with the mutant KOFU-SDNA polymerase more efficient and time-saving.

3. The inhibitor tolerance of the mutant KOFU-S DNA polymerase is improved compared with that of the wild KOD DNA polymerase, and the mutant KOFU-S DNA polymerase can tolerate at least 0.01 percent of SDS, 100mmol/L of NaCl, 10 percent of ethanol, 160mmol/L of urea, 80 ng/mu L of humic acid, 0.03U/mL of heparin and 0.8 mu g/mu L of bile salt. The inhibitor resistance is remarkably improved, and the application range of the polymerase is expanded, so that the mutant KOFU-S polymerase is possible to be applied to direct amplification PCR.

4. The PCR reaction buffer solution can provide an optimal reaction environment for the mutant KOFU-SDNA polymerase, so that the polymerase can better exert good catalytic activity. Alternative polymerase with higher speed, high fidelity and sensitivity and PCR reaction buffer thereof are provided for more PCR reactions.

Drawings

FIG. 1 shows the purification results of mutant KOFU-SDNA polymerase; wherein lane M is (10-116kDa) prestained protein Marker; lane 1 is cell disruption; lane 2 is the supernatant after centrifugation of the cell disruption solution; lane 3 is the supernatant from centrifugation of the cell disruption solution, which was centrifuged after heating at 75 ℃ for 30 min; lanes 4-9 are mutant KOFU-SDNA polymerases from affinity chromatography after purification of imidazole eluents at different concentrations.

FIG. 2 is a graph showing the results of electrophoresis of the extension rate test of mutant KOFU-SDNA polymerase in example 7; wherein lane M is a DNA Marker; FIGS. (A), (B) and (C) are the results of electrophoresis with the extension times of the wild-type KODDNA polymerase and the KOFU-S polymerase set at 15S/kb, 30S/kb and 60S/kb, respectively, wherein the fragments of each set are 2kb, 4kb and 6kb in length, respectively; as a result of further examining the extension ability of KOFU-S polymerase, the fragments of 2kb, 4kb and 6kb were amplified with KOFU-S polymerase at rates of 5S/kb, 10S/kb, 15S/kb, 30S/kb and 60S/kb for each extension time.

FIG. 3 is a diagram showing the results of electrophoresis of PCR reactions for amplifying nucleic acid templates of different lengths using mutant KOFU-SDNA polymerase in example 8; wherein lane M is a DNA Marker.

FIG. 4 is a graph showing the results of electrophoresis of PCR reactions for amplifying nucleic acids of different concentrations by the mutant KOFU-SDNA polymerase in example 9; wherein lane M is a DNA Marker.

FIG. 5 is a graph showing a partial electrophoresis result of PCR reaction with the mutant KOFU-SDNA polymerase at different concentrations of PCR inhibitor tolerance in example 10; wherein lane M is a DNA Marker; FIGS. A, (B) and (C) are electrophoresis results of PCR reactions in which the inhibitors were SDS, NaCl and ethanol at different concentrations, respectively.

FIG. 6 is a graph showing a partial electrophoresis result of PCR reaction with the mutant KOFU-S DNA polymerase at different concentrations of PCR inhibitor tolerance in example 10; wherein lane M is a DNA Marker; FIGS. A, (B), (C) and (D) are electrophoresis results of PCR reactions in which the inhibitors were urea, humic acid, heparin and bile salt at different concentrations, respectively.

Detailed Description

The present invention will be further illustrated with reference to the following specific examples, but the present invention is only illustrated by the reference examples and is not limited to the scope of the present invention. Other embodiments of the invention based on the present invention, which can be made by a person skilled in the art without inventive step, belong to the scope of protection of the present invention.

Unless otherwise specified, the reagents and materials used in the present invention are commercially available products or products obtained by a known method.

EXAMPLE 1 construction of recombinant vector containing nucleotide sequence encoding KOFU-S DNA polymerase

(1) And (2) carrying out codon optimization of an escherichia coli expression system according to the amino acid sequence (SEQ ID NO.1) of the mutant KOFU-SDNA polymerase to obtain a DNA molecule capable of being efficiently expressed in escherichia coli, and artificially synthesizing the DNA molecule for coding the mutant KOFU-SDNA polymerase by using a splicing PCR method, wherein the DNA molecule is specifically shown as SEQ ID NO. 2.

(2) The DNA molecule encoding the mutant KOFU-SDNA polymerase was recombined with the expression vector pET-28 a. The primer sequences were synthesized as follows:

NdeI-FP：5'-GCGCCATATGATGTTAACCATC-3'(SEQ ID NO:4)；

XhoI-RP：5'-GCGCCTCGAGTTATTTTTTCTG-3'(SEQ ID NO:5)。

the mutant KOFU-SDNA polymerase DNA molecule was amplified by PCR using an artificially synthesized DNA molecule encoding the mutant KOFU-S polymerase as a template, to which 25. mu.L of 2 XPfu Max HiFi PCR ProMix (EnzyValley, Cat. P217, manufactured by Inzan Biotechnology Co., Ltd., Guangzhou), 1. mu.L (10. mu. mol/L) of each of the upstream and downstream primers Nde I-FP and Xho I-RP, and an appropriate amount of sterilized water were added to perform PCR amplification. The amplification conditions were: 30s at 98 ℃; 30 cycles of 98 ℃ for 10s, 60 ℃ for 30s and 72 ℃ for 30 s; 5min at 72 ℃. The target gene fragment of about 2.5kb in total length was recovered by agarose gel electrophoresis, and the target gene fragment and the vector pET-28a were digested with Nde I and Xho I, and ligated by T4DNA ligase, and the ligation product was transformed into DH 5. alpha. competent cells.

(3) Selecting a single colony for colony PCR identification, sending the positive single colony to a sequencing company for sequencing verification, verifying the correctness of the DNA molecule for coding the mutant KOFU-SDNA polymerase, culturing and verifying a correct competent cell, and extracting a plasmid, wherein the obtained plasmid is the recombinant vector containing the DNA molecule for coding the mutant KOFU-SDNA polymerase.

EXAMPLE 2 preparation of transformant expressing mutant KOFU-SDNA polymerase

The recombinant vector obtained in example 1 was transformed into a host cell e.coli BL21(DE3), and a single colony was picked up and inoculated into a liquid LB medium to OD₆₀₀And (3) adding IPTG (isopropyl-beta-thiogalactoside) to a final concentration of 0.1mmol/L, inducing at 18 ℃ for 16h, collecting thalli, carrying out ultrasonic disruption, and detecting the expression of a target protein through SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoresis.

EXAMPLE 3 expression and identification of mutant KOFU-SDNA polymerase

The positive transformant strain capable of expressing mutant KOFU-SDNA polymerase obtained in example 2 was inoculated into 60mL of LB medium containing 50. mu.g/mL kanamycin sulfate, and cultured overnight with shaking in a shaker at 37 ℃; the overnight-cultured seed solution was inoculated at 1:100 into 500mL of LB medium containing 50. mu.g/mL kanamycin sulfate, and shaking culture was continued in a shaker at 37 ℃ for 4 hours (OD₆₀₀0.6 to 0.8); adding IPTG into each bottle of bacterial liquid until the final concentration is 0.1mmol/L, and continuing to oscillate and induce for 18h at 18 ℃; centrifugally collecting the induced thalli, weighing, and recording the wet weight of the thalli; taking 1mL of bacterial liquid before and after induction respectively, centrifuging at 12000rpm for 1min, taking precipitate, adding 150 mu L of lysis buffer solution, oscillating uniformly, and lysing thallus by using an ultrasonic cell disruptor under the ultrasonic conditions: ultrasound for 4s, interval 4s, multiple cycles for 1 min. The supernatant was centrifuged and the pellet was added to 150. mu.L of lysis buffer and resuspended with shaking. Taking supernatant and sediment component suspension to carry out SDS-PAGE analysis, and observing protein expression conditions. The expression result was that the supernatant had a clear protein band at 98.3kDa, whereas the suspension of the precipitated fraction had no clear protein band at this point, confirming that the modification was soluble expression.

EXAMPLE 4 purification of mutant KOFU-SDNA polymerase

1. Ultrasonic crushing of recombinant thallus

Taking the induction expression thallus cryopreserved at the temperature of 20 ℃ below zero, adding 5mL of lysis buffer solution into each gram of thallus according to the wet weight of the thallus recorded in the example 3 for resuspending the thallus, and lysing the thallus by using an ultrasonic cell disruption instrument under the ultrasonic conditions: power 250W, ultrasound 5s, interval 5s, for 30 min. And (3) placing the lysed thalli into a high-speed refrigerated centrifuge, centrifuging at 12000r/min for 30min at the temperature of 4 ℃, and taking supernatant to a 250mL sterilized flask. Incubating the supernatant in a constant-temperature water bath at 75 ℃ for 30min, centrifuging at 12000r/min at 4 ℃ for 30min, filtering with a 0.22-micron microporous membrane, and taking the supernatant to a 250mL sterilized flask.

2. Nickel ion affinity chromatography purification

The chromatographic column is HisTrap^TMHP 5mL (from GE Healthcare), binding buffer a: 50mmol/L Tris-HCl, 300mmol/L NaCl, pH 8.0; the elution buffer was buffer B: 50mmol/L Tris-HCl, 300mmol/L NaCl, 500mmol/L Imidazole, pH 8.0; filtering with 0.22 μm filter membrane for use.

HisTrap^TMAnd (2) putting the HP 5mL into a column position valve of a rapid protein purification instrument, washing a system and a column by ultrapure water, balancing the column by using a buffer A, then loading the supernatant obtained in the step 1 by using a sample pump, washing the column by using the buffer A, then performing gradient elution by using an elution buffer B, and collecting an elution peak. Each peak was sampled at 20. mu.L and examined by SDS-PAGE protein electrophoresis, and the results are shown in FIG. 1. The results showed that the KOD DNA polymerase modified form a single band on the SDS-PAGE electrophoresis, and the molecular weight was 98.3 kDa.

Example 5 mutant KOFU-SDNA polymerase Activity and thermal stability test

The enzyme activity was measured by the following method using the mutant KOFU-SDNA polymerase prepared in example 4. At 74 ℃, using activated salmon sperm DNA as a template and a primer, adding into Tris-HCl 120mmol/L and MgCl 1.5mmol/L₂、10mmol/L(NH₄)₂SO₄150mmol/LKCl, 0.01g/L BSA, 0.05% Triton X-100, pH 8.0-An enzyme-catalyzed reaction was carried out in the reaction system of 10.0. Within 30min, the amount of polymerase catalyzing the incorporation of 10nmol dNTPs into the DNA is defined as 1U. The results showed that the concentration of polymerase activity of the chimera was 16U/. mu.L.

The mutant KOFU-SDNA polymerase was diluted to 5U/. mu.L and tested for thermal stability as described above. After incubating the diluted polymerases at 95 ℃ for 0, 1, 2, 3, 4, 5 and 6 hours respectively, sampling respectively and determining the DNA polymerase activity, the result shows that the mutant KOFU-SDNA polymerase has extremely high thermal stability and the half-life period at 95 ℃ is 4 hours.

Example 6 mutant KOFU-SDNA polymerase fidelity test

The mutant KOFU-SDNA polymerase prepared in example 4 was used to determine the fidelity as follows. A primer was designed based on the nucleotide sequence of plasmid pUC19, using plasmid pUC19 as a template and pUCl 9F, pUCl 9R as a primer. The primer sequences are as follows:

pUC19 F：5'-CCAGGCTTTACACTTTATGC-3'(SEQ ID NO:6)；

pUC19 R：5'-TGGCTTAACTATGCGGCATC-3'(SEQ ID NO:7)。

performing PCR amplification with KOFU-SDNA polymerase, wild-type KODDNA polymerase, and KOD commercial enzyme (TOYOBO, KOD FXneo), respectively; after homologous recombination of the amplified product, 5. mu.L of the ligation product was transformed into 50. mu.L of E.coli competent DH 5. alpha.; after rejuvenation at 37 ℃ for 1h, 100. mu.L of the bacterial solution, 800. mu. g X-gal, and 20. mu.L of 100mmol/L IPTG were applied to LB plate medium containing ampicillin resistance, and cultured in a 37 ℃ incubator for 12 hours or more to form single colonies. The number of blue colonies and white colonies on the plate medium were recorded, wherein the blue colonies were normal and the white colonies were variant. The fidelity of the mutant KOFU-SDNA polymerase was calculated to be 7.1X 10^-6Compared with wild KODDNA polymerase, the improved KODDNA polymerase is improved by 2 times and is equivalent to the commercial enzyme KOD FXneo.

Example 7 extension Rate of mutant KOFU-SDNA polymerase

The elongation rate was measured using the mutant KOFU-SDNA polymerase prepared in example 4 by the following method. In this example, fragments of 2, 4 and 6kb were amplified with KOFU-S and wild-type KODDNA polymerase using 10 ng/. mu.L of lambda DNA as a template at respective extension times of 15S/kb, 30S/kb and 60S/kb, respectively. The reaction procedure is as follows: 94 ℃ for 2 min; at 98 ℃ for 10s, at 55 ℃ for 30s, at 68 ℃ for 15s/kb, 30s/kb, 60s/kb for 30 cycles; 5min at 68 ℃. 1% agarose gel is prepared to detect PCR products.

Under the same reaction system, KOFU-S polymerase is used to amplify 2kb, 4kb and 6kb fragments, and the corresponding rates of each extension time are 5S/kb, 10S/kb, 15S/kb, 30S/kb and 60S/kb respectively. The reaction procedure is as follows: 94 ℃ for 2 min; at 98 ℃ for 10s, at 55 ℃ for 30s, at 68 ℃ for 5s/kb, 10s/kb, 15s/kb, 30s/kb, 60s/kb for 30 cycles; 5min at 68 ℃. 1% agarose gel is prepared to detect PCR products. Figure 2 results show that: KOFU-S has an elongation rate property higher than that of wild-type KOD DNA polymerase; in the figure, KOFU-S has obvious bands for fragments with different lengths, while the wild-type KOD DNA polymerase cannot amplify a band with 6kb at different extension rates, which indicates that the extension capability of the long fragment is limited, while the wild-type KOD DNA polymerase cannot amplify a band with 4kb in the case of 15S/kb, namely, in FIG. 2 (A). FIG. 2(D) is a result of further examining the elongation ability of KOFU-S. The fragments of 2kb, 4kb and 6kb were amplified with KOFU-S polymerase at respective extension times of 5S/kb, 10S/kb, 15S/kb, 30S/kb and 60S/kb. KOFU-S still amplified a 6kb band at 10S/kb, while a band of less than 4kb was amplified at 5S/kb. When a long fragment is amplified, the extension rate of KOFU-SDNA polymerase can reach 10S/kb at the fastest speed, and when a fragment below 4kb is amplified, the extension rate of KOFU-S DNA polymerase can reach 5S/kb at the fastest speed. The extension rate of the KOFU-S DNA polymerase is obviously improved compared with that of the wild-type KOD DNA polymerase.

EXAMPLE 8 Long fragment extension Capacity of mutant KOFU-SDNA polymerase

The long-fragment extension ability of the mutant KOFU-SDNA polymerase prepared in example 4 was measured by the following method. The long fragment extension capability refers to the capability of polymerase to extend an extended fragment in a PCR reaction, and the wild type KOD DNA polymerase has insufficient activity in the late extension stage of the PCR reaction, so that the extension capability is reduced, and only a template of 4-5kb can be extended. In practical applications, this limits the use of PCR for long-fragment amplification.

In this example, 10 ng/. mu.L of lambda DNA was used as a template. Under the same reaction system, fragments of 2kb, 4kb, 6kb, 8kb, 10kb and 12kb were amplified with KOFU-S and wild-type KOD DNA polymerase, and the extension time was set at a rate of 1 min/kb. The reaction procedure is as follows: 94 ℃ for 2 min; 30 cycles of 98 ℃ for 10s, 55 ℃ for 30s and 68 ℃ for 1 min/kb; 5min at 68 ℃. 1% agarose gel is prepared to detect PCR products. As can be seen from FIG. 3, KOFU-S amplified a fragment of up to 10kb, which is about 2.5 times that of the wild-type KOD DNA polymerase. Compared with wild-type KOD DNA polymerase, the long fragment extension capability of the modified KOFU-S is obviously improved.

Example 9 sensitivity of mutant KOFU-SDNA polymerase to different template concentrations

The amplification sensitivity of the mutant KOFU-SDNA polymerase prepared in example 4 to nucleic acid templates of different concentrations was determined as follows. In this example, 1 ng/. mu.L of lambda DNA was used as a template, and the template was subjected to 10-fold gradient dilution involving a total of 5 gradients (1ng, 100pg, 10pg, 1pg, 0.1pg), and 1kb of lambda DNA was amplified using KOFU-S, a wild-type KOD DNA polymerase in the same reaction system. The reaction procedure is as follows: 94 ℃ for 2 min; 30 cycles of 98 ℃ for 10s, 55 ℃ for 30s and 68 ℃ for 30 s; 5min at 68 ℃. 1% agarose gel is prepared to detect PCR products.

In this example, the influence of the lambda DNA concentration in the range of 1ng to 0.1pg on the PCR reaction was examined, and as can be seen from FIG. 4, KOFU-S polymerase was able to detect lambda DNA at a minimum of 1pg, while the wild-type KOD DNA polymerase had a low ability to amplify DNA at a trace concentration, and the target product could not be amplified when the lambda DNA was 10 p. The template sensitivity of the KOFU-S is 100 times of that of wild-type KOD DNA polymerase, and the nucleic acid template sensitivity of the modified KOFU-S is obviously improved compared with that of the wild-type KOD DNA polymerase.

Example 10 tolerance of mutant KOFU-SDNA polymerase to various inhibitors

The mutant KOFU-SDNA polymerase prepared in example 4 was used to determine the resistance of the polymerase to various inhibitors as follows. In this example, 5 ng/. mu.L of pUC19 plasmid was used as a template, and different concentrations of SDS (sodium dodecyl sulfate), NaCl, ethanol, humic acid, bile salt, heparin and urea were used as PCR inhibitors, respectively. Under the same reaction system, 0.7kb pUC19 plasmid was amplified with KOFU-S, wild-type KOD, wild-type Pfu polymerase. The reaction procedure is as follows: 94 ℃ for 2 min; 30 cycles of 98 ℃ for 10s, 55 ℃ for 30s and 68 ℃ for 30 s; 5min at 68 ℃. 2% agarose gel is prepared to detect the PCR product.

In this example, the degree of tolerance of KOFU-S polymerase to SDS at a concentration of 0 to 0.08% (W/V), NaCl at a concentration of 0 to 125mM, ethanol at a concentration of 0 to 20% (V/V), urea at a concentration of 0 to 240mM, humic acid at a concentration of 0 to 120 ng/. mu.L, heparin at a concentration of 0 to 0.48U/mL, and bile salt at a concentration of 0 to 1.6. mu.g/. mu.L was examined. As can be seen from FIGS. 5 and 6, KOFU-SDNA polymerase is resistant to at least 0.01% SDS, 100mmol/L NaCl, 10% ethanol, 160mmol/L urea, 80 ng/. mu.L humic acid, 0.03U/mL heparin and 0.8. mu.g/. mu.L bile salt. Whereas KODDNA polymerase can tolerate only 0.01% SDS, 50mmol/L NaCl, 5% ethanol, 120mmol/L urea, 40 ng/. mu.L humic acid, 0.03U/mL heparin and 0.2. mu.g/. mu.L bile salts. Meanwhile, Pfu DNA polymerase can tolerate only 0.01% SDS, 0mM NaCl, 10% ethanol, 120mmol/L urea, 40 ng/. mu.L humic acid, 0.03U/mL heparin and 0.4. mu.g/. mu.L bile salts.

Therefore, the tolerance of the mutant KOFU-SDNA polymerase to the PCR inhibitor is improved compared with that of KOD DNA polymerase.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

<110> university of southern China's science

<120> KOD DNA polymerase mutant and preparation method and application thereof

<160> 7

<170> SIPOSequenceListing 1.0

<210> 1

<211> 847

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> amino acid sequence of mutant KOFU-S polymerase

<400> 1

Met Ala Ser Ala Ile Leu Asp Thr Asp Tyr Ile Thr Glu Asp Gly Lys

1 5 10 15

Pro Val Ile Arg Ile Phe Lys Lys Glu Asn Gly Glu Phe Lys Ile Glu

20 25 30

Tyr Asp Arg Thr Phe Glu Pro Tyr Phe Tyr Ala Leu Leu Lys Asp Asp

35 40 45

Ser Ala Ile Glu Glu Val Lys Lys Ile Thr Ala Glu Arg His Gly Thr

50 55 60

Val Val Thr Val Lys Arg Val Glu Lys Val Gln Lys Lys Phe Leu Gly

65 70 75 80

Arg Pro Val Glu Val Trp Lys Leu Tyr Phe Thr His Pro Gln Asp Val

85 90 95

Pro Ala Ile Arg Asp Lys Ile Arg Glu His Pro Ala Val Ile Asp Ile

100 105 110

Tyr Glu Tyr Asp Ile Pro Phe Ala Lys Arg Tyr Leu Ile Asp Lys Gly

115 120 125

Leu Val Pro Met Glu Gly Asp Glu Glu Leu Lys Met Leu Ala Phe Asp

130 135 140

Ile Glu Thr Leu Tyr Lys Glu Gly Glu Glu Phe Ala Glu Gly Pro Ile

145 150 155 160

Leu Met Ile Ser Tyr Ala Asp Glu Glu Gly Ala Arg Val Ile Thr Trp

165 170 175

Lys Asn Val Asp Leu Pro Tyr Val Asp Val Val Ser Thr Glu Arg Glu

180 185 190

Met Ile Lys Arg Phe Leu Arg Val Val Lys Glu Lys Asp Pro Asp Val

195 200 205

Leu Ile Thr Tyr Asn Gly Asp Asn Phe Asp Phe Ala Tyr Leu Lys Lys

210 215 220

Arg Cys Glu Lys Leu Gly Ile Asn Phe Ala Leu Gly Arg Asp Gly Ser

225 230 235 240

Glu Pro Lys Ile Gln Arg Met Gly Asp Arg Phe Ala Val Glu Val Lys

245 250 255

Gly Arg Ile His Phe Asp Leu Tyr Pro Val Ile Arg Arg Thr Ile Asn

260 265 270

Leu Pro Thr Tyr Thr Leu Glu Ala Val Tyr Glu Ala Val Phe Gly Gln

275 280 285

Pro Lys Glu Lys Val Tyr Ala Glu Glu Ile Thr Thr Ala Trp Glu Thr

290 295 300

Gly Glu Asn Leu Glu Arg Val Ala Arg Tyr Ser Met Glu Asp Ala Lys

305 310 315 320

Val Thr Tyr Glu Leu Gly Lys Glu Phe Leu Pro Met Glu Ala Gln Leu

325 330 335

Ser Arg Leu Val Gly Gln Pro Leu Trp Asp Val Ser Arg Ser Ser Thr

340 345 350

Gly Asn Leu Val Glu Trp Phe Leu Leu Arg Lys Ala Tyr Glu Arg Asn

355 360 365

Glu Val Ala Pro Asn Lys Pro Ser Glu Glu Glu Tyr Gln Arg Arg Leu

370 375 380

Arg Glu Ser Tyr Thr Gly Gly Phe Val Lys Glu Pro Glu Lys Gly Leu

385 390 395 400

Trp Glu Asn Ile Val Tyr Leu Asp Phe Arg Ala Leu Tyr Pro Ser Ile

405 410 415

Ile Ile Thr His Asn Val Ser Pro Asp Thr Leu Asn Leu Glu Gly Cys

420 425 430

Lys Asn Tyr Asp Ile Ala Pro Gln Val Gly His Lys Phe Cys Lys Asp

435 440 445

Ile Pro Gly Phe Ile Pro Ser Leu Leu Gly His Leu Leu Glu Glu Arg

450 455 460

Gln Lys Ile Lys Thr Lys Met Lys Glu Thr Gln Asp Pro Ile Glu Lys

465 470 475 480

Ile Leu Leu Asp Tyr Arg Gln Lys Ala Ile Lys Leu Leu Ala Asn Ser

485 490 495

Phe Tyr Gly Tyr Tyr Gly Tyr Ala Lys Ala Arg Trp Tyr Cys Lys Glu

500 505 510

Cys Ala Glu Ser Val Thr Ala Trp Gly Arg Lys Tyr Ile Glu Leu Val

515 520 525

Trp Lys Glu Leu Glu Glu Lys Phe Gly Phe Lys Val Leu Tyr Ile Asp

530 535 540

Thr Asp Gly Leu Tyr Ala Thr Ile Pro Gly Gly Glu Ser Glu Glu Ile

545 550 555 560

Lys Lys Lys Ala Leu Glu Phe Leu Lys Tyr Ile Asn Ala Lys Leu Pro

565 570 575

Gly Ala Leu Glu Leu Glu Tyr Glu Gly Phe Tyr Lys Arg Gly Phe Phe

580 585 590

Val Thr Lys Lys Lys Tyr Ala Val Ile Asp Glu Glu Gly Lys Ile Thr

595 600 605

Thr Arg Gly Leu Glu Ile Val Arg Arg Asp Trp Ser Glu Ile Ala Lys

610 615 620

Glu Thr Gln Ala Arg Val Leu Glu Ala Leu Leu Lys Asp Gly Asp Val

625 630 635 640

Glu Lys Ala Val Arg Ile Val Lys Glu Val Thr Glu Lys Leu Ser Lys

645 650 655

Tyr Glu Val Pro Pro Glu Lys Leu Val Ile His Glu Gln Ile Thr Arg

660 665 670

Asp Leu Lys Asp Tyr Lys Ala Thr Gly Pro His Val Ala Val Ala Lys

675 680 685

Arg Leu Ala Ala Arg Gly Val Lys Ile Arg Pro Gly Thr Val Ile Ser

690 695 700

Tyr Ile Val Leu Lys Gly Ser Gly Arg Ile Gly Asp Arg Ala Ile Pro

705 710 715 720

Phe Asp Glu Phe Asp Pro Thr Lys His Lys Tyr Asp Ala Glu Tyr Tyr

725 730 735

Ile Glu Asn Gln Val Leu Pro Ala Val Glu Arg Ile Leu Arg Ala Phe

740 745 750

Gly Tyr Arg Lys Glu Asp Leu Arg Tyr Gln Lys Thr Arg Gln Val Gly

755 760 765

Leu Ser Ala Trp Leu Lys Pro Lys Gly Thr Gly Thr Gly Gly Gly Gly

770 775 780

Ala Thr Val Lys Phe Lys Tyr Lys Gly Glu Glu Lys Glu Val Asp Ile

785 790 795 800

Ser Lys Ile Lys Lys Val Trp Arg Val Gly Lys Met Ile Ser Phe Thr

805 810 815

Tyr Asp Glu Gly Gly Gly Lys Thr Gly Arg Gly Ala Val Ser Glu Lys

820 825 830

Asp Ala Pro Lys Glu Leu Leu Gln Met Leu Glu Lys Gln Lys Lys

835 840 845

<210> 2

<211> 2544

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> mutant KOFU-S polymerase-optimized nucleic acid sequence

<400> 2

atggcgtcgg cgatcttaga caccgattac attaccgagg acggcaagcc ggttatccgc 60

atcttcaaaa aagagaacgg cgagtttaag attgaatacg atcggacctt tgagccgtat 120

ttctatgcgt tgcttaagga cgattctgcg atagaagagg tcaaaaagat cacagcagag 180

cgtcacggca ccgtggttac cgtgaagcgg gtagaaaaag tacaaaaaaa gtttcttggg 240

cgtcctgtcg aagtttggaa gctgtatttt acccatccgc aagatgtgcc ggccatacgg 300

gacaagattc gtgaacatcc ggcggttatt gatatatacg agtacgatat cccctttgcg 360

aaacgctatc tgatagataa gggcctggtg ccgatggaag gcgatgagga actgaaaatg 420

cttgcgtttg acatagagac actgtataaa gagggggaag agttcgcgga aggcccgatc 480

ctgatgatat cttatgctga tgaagagggc gctcgcgtca ttacatggaa gaacgtggac 540

cttccttatg tggatgtcgt gagcaccgag cgtgaaatga ttaagcggtt tctgcgtgtt 600

gtgaaagaaa aggaccctga tgtactgatt acctataatg gggataattt cgactttgct 660

tacttaaaga aacgttgcga gaaattgggc atcaactttg cactgggacg tgatgggagc 720

gaaccgaaga ttcaacgtat gggcgatcgt tttgcggtag aagttaaagg ccgtatacat 780

tttgacttgt atccggtgat tcgtcgcacc attaacctgc cgacatatac cctggaagca 840

gtctacgaag cggtgttcgg ccaacccaag gaaaaggtat acgccgaaga gattactacc 900

gcgtgggaga caggcgagaa cctggaacgt gtggctcgtt atagtatgga ggacgcgaag 960

gtaacgtacg agctgggcaa ggagttcctg cctatggaag cgcagctgtc acgcctggtc 1020

ggtcagccgt tgtgggatgt ctcacgtagc agcaccggca atctggtgga atggttcttg 1080

ttgcgtaagg cgtacgaacg taatgaagtg gcgcccaata agccctccga ggaagaatat 1140

cagcgtcgtc tgcgggaatc ttacaccggc ggatttgtca aagaaccgga gaaaggcctg 1200

tgggaaaaca ttgtctactt agactttcgt gcgctgtacc cgtcgatcat aataacccat 1260

aacgtgagcc cagacacgtt aaatctggaa gggtgcaaga attatgacat tgccccccaa 1320

gtgggtcaca agttctgtaa ggacataccg ggctttatcc cgagcctgct gggtcatctg 1380

ttggaggaac gtcagaagat aaagacaaaa atgaaggaaa ctcaagatcc aatagagaaa 1440

attctgctgg attatcgtca aaaggcaatt aaactgctgg ctaactcgtt ttatggctat 1500

tacgggtatg ctaaagctcg ctggtattgc aaggaatgtg cggaaagcgt gacggcatgg 1560

ggccgtaaat acattgagct ggtgtggaag gagctggagg agaagttcgg cttcaaagta 1620

ctgtacatag ataccgatgg cttatatgcg acaattccgg gtggcgaaag cgaggagatt 1680

aagaaaaaag cgttggaatt tctgaagtac ataaatgcca agctgcctgg cgctctggaa 1740

ctggaatatg aaggcttcta taaacgtggc tttttcgtga ctaagaagaa gtacgcagta 1800

attgacgagg agggaaagat taccactcgc ggcttggaga ttgtgcgtcg tgattggtcg 1860

gaaattgcca aggagacaca agcgcgtgtg ttagaggcgc tgctgaaaga cggcgatgtc 1920

gaaaaggcgg tccgtattgt caaggaagtt accgaaaaac tgagcaagta cgaggtgcct 1980

cctgaaaagc tggttattca tgagcaaatt acccgtgacc tgaaggatta taaggctact 2040

ggcccacatg tagcagtggc caaacgtctg gccgcacgtg gcgtgaagat acgtccgggc 2100

actgtaatta gctacattgt gctgaagggc tctggtcgca ttggcgaccg tgcgattccg 2160

tttgatgaat ttgaccctac caaacataag tatgatgctg aatattacat cgagaatcag 2220

gttttaccgg cagttgaacg tatcctgcgt gcttttggct atcgtaagga ggatttgcgt 2280

taccaaaaaa cccgtcaggt aggcctgagc gcatggctga aaccaaaggg tactggcacc 2340

gggggtggtg gagcgactgt taagttcaaa tacaaggggg aggaaaaaga ggtggatatt 2400

agcaaaatca agaaggtttg gcgtgtgggg aaaatgataa gctttacata cgacgagggt 2460

ggaggcaaaa caggtcgtgg cgcggtgtcc gagaaagatg ccccgaaaga gttgttacag 2520

atgttagaaa agcagaagaa ataa 2544

<210> 3

<211> 64

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> amino acid sequence of double-stranded binding protein Sso7d

<400> 3

Met Ala Thr Val Lys Phe Lys Tyr Lys Gly Glu Glu Lys Glu Val Asp

1 5 10 15

Ile Ser Lys Ile Lys Lys Val Trp Arg Val Gly Lys Met Ile Ser Phe

20 25 30

Thr Tyr Asp Glu Gly Gly Gly Lys Thr Gly Arg Gly Ala Val Ser Glu

35 40 45

Lys Asp Ala Pro Lys Glu Leu Leu Gln Met Leu Glu Lys Gln Lys Lys

50 55 60

<210> 4

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> NdeI-FP

<400> 4

gcgccatatg atgttaacca tc 22

<210> 5

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> XhoI-RP

<400> 5

gcgcctcgag ttattttttc tg 22

<210> 6

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> pUC19 F

<400> 6

ccaggcttta cactttatgc 20

<210> 7

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> pUC19 R

<400> 7

tggcttaact atgcggcatc 20