WO2025137863A1 - Dna polymerase, and preparation method therefor and use thereof - Google Patents

Abstract

Provided are a DNA polymerase, and a preparation method therefor and the use thereof. The DNA polymerase has less than 42% sequence similarity to Taq DNA polymerase, and has polymerase activity, higher thermal stability, 5'-3' exonuclease activity and inhibition resistance.

Description

DNA polymerase and its preparation method and application Technical Field

The present invention belongs to the field of biotechnology and relates to DNA polymerase and its preparation method and application.

Background Art

In biological research, medical testing, forensic identification, agriculture and other related fields, nucleic acid amplification is a very important technology. At present, the most widely used nucleic acid amplification technology is the polymerase chain reaction (PCR) that relies on DNA polymerase. DNA polymerase has multiple functions and is an extremely important tool enzyme in molecular biology. For example, Taq DNA polymerase, which has high industrial value, is widely used in the field of molecular diagnosis. Taq DNA polymerase belongs to the A family DNA polymerase. In addition, members of this family also include Tth DNA polymerase with reverse transcriptase activity, Bst DNA polymerase with strand displacement activity, and bacteriophage T7 DNA polymerase that can bind to 2',3'-dideoxynucleotides.

Appropriate DNA polymerase is a necessary condition for the success of PCR reaction. Different DNA polymerases can be selected according to the requirements of different experiments for reaction conditions such as sensitivity, fidelity, fragment length, etc. In addition, A family DNA polymerases mainly have 5'-3' polymerization activity and 5'-3' exonuclease activity, which makes A family DNA polymerases, especially Taq DNA polymerase, widely used in molecular diagnosis.

At present, the widely used DNA polymerase is mainly Taq DNA polymerase of the A family DNA polymerase. The discovery and development of Taq DNA polymerase has a history of nearly 50 years. In recent years, due to its important role in the field of molecular diagnosis, the enzyme has been studied for many modifications, and many commercial enzymes are also developed based on this enzyme. However, with the continuous diversification of application scenarios, new requirements are constantly being put forward for the performance of the enzyme. Relying solely on traditional directed evolution methods for modification has limited space and is difficult to meet the growing demand for use.

In summary, in order to develop new molecular tools with a wider range of applications, it is still necessary to explore new A-family DNA polymerase skeletons.

Summary of the invention

The present application provides DNA polymerase and its preparation method and application, and explores new DNA polymerase.

In a first aspect, the present application provides a DNA polymerase, wherein the amino acid sequence of the DNA polymerase comprises:

(1) the sequence shown in SEQ ID NO.1 or SEQ ID NO.2; or,

(2) an amino acid sequence obtained by substituting, deleting or adding one or at least two amino acid residues of the sequence described in (1), and having the same or similar function as the sequence described in (1); or

(3) An amino acid sequence that has at least 90% sequence homology with the sequence described in (1) or (2) and has the same or similar function as the sequence described in (1).

In this application, based on the analysis of metagenomic sequencing data of deep-sea hydrothermal sediment samples, two protein sequences with the function of A family DNA polymerases were discovered (named 34°S-1 and 34°S-2). After sequence alignment, the sequence similarities with Taq DNA polymerase were only 40.92% and 41.94%, respectively, which are completely new sequence skeletons, indicating that these two proteins can be used as new types of DNA polymerases.

It can be understood that the present application found that the two proteins with amino acid sequences as shown in SEQ ID NO.1 or SEQ ID NO.2 have the function of A family DNA polymerase. Therefore, the enzyme obtained by replacing, deleting or adding one or at least two amino acid residues to the sequence shown in SEQ ID NO.1 or SEQ ID NO.2 using the general technical means in the art, and the function is the same or similar to the original protein, can also be expected to have the function of A family DNA polymerase.

In a second aspect, the present application provides a nucleic acid molecule encoding the DNA polymerase described in the first aspect.

Preferably, the nucleic acid sequence of the nucleic acid molecule includes the sequence shown in SEQ ID NO.3 or SEQ ID NO.4.

In a third aspect, the present application provides a recombinant vector, wherein the recombinant vector contains the nucleic acid molecule described in the second aspect.

In a fourth aspect, the present application provides a recombinant cell, wherein the recombinant cell contains the recombinant vector described in the third aspect.

In a fifth aspect, the present application provides a method for preparing the DNA polymerase described in the first aspect, the preparation method comprising:

The coding gene of the DNA polymerase described in the first aspect is inserted into an expression vector to obtain a recombinant vector, the recombinant vector is introduced into a host cell to obtain a recombinant cell, the recombinant cell is cultured, and the cultured cells are collected for product purification to obtain the DNA polymerase.

Preferably, the expression vector comprises a pET28a vector.

Preferably, the host cell comprises Escherichia coli (E. coli) BL21.

Preferably, the method for purifying the product comprises purifying using a nickel column-ion column.

In a sixth aspect, the present application provides the use of the DNA polymerase described in the first aspect in the preparation of a nucleic acid amplification product.

In a seventh aspect, the present application provides use of the nucleic acid molecule described in the second aspect, the recombinant vector described in the third aspect, or the recombinant cell described in the fourth aspect in the preparation of DNA polymerase.

In an eighth aspect, the present application provides a nucleic acid amplification kit, the nucleic acid amplification kit comprising On the one hand, the DNA polymerase described.

In a ninth aspect, the present application provides the use of the DNA polymerase described in the first aspect in nucleic acid amplification.

In this application, the new DNA polymerase discovered has the function of A family DNA polymerase and can be widely used in nucleic acid amplification, such as PCR amplification sequencing, library construction, qPCR, TA cloning and other applications.

In a tenth aspect, the present application provides a method for nucleic acid amplification, comprising: mixing a nucleic acid template with a reaction system containing the DNA polymerase described in the first aspect, and performing an amplification reaction.

Compared with the prior art, this application has the following beneficial effects:

This application discovered a new type of DNA polymerase with 5’-3’ polymerization activity and 5’-3’ exolytic activity, which is consistent with the functional activity characteristics of A family DNA polymerases. The sequence similarity with Taq DNA polymerase is only 40.92% and 41.94%, which is a brand-new sequence skeleton with greater room for product transformation and provides a new polymerase skeleton for the development of new molecular tools with a wider range of applications.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the purification results of 34°S-1 DNA polymerase.

Figure 2 shows the purification results of 34°S-2 DNA polymerase.

Figure 3 shows the Tm value results of 34°S-1, 34°S-2 DNA polymerase and Taq DNA polymerase.

FIG. 4 is a schematic diagram showing the principle of determining the polymerization activity of DNA polymerase.

FIG. 5 is a schematic diagram showing the principle of determining the 5′-3′ exonuclease activity of DNA polymerase.

Figure 6 shows the 5’-3’ exo-activity results of 34°S-1, 34°S-2 DNA polymerase and Taq DNA polymerase.

Figure 7 shows the results of 34°S-1 DNA polymerase PCR amplification.

Figure 8 shows the results of 34°S-2 DNA polymerase PCR amplification.

Figure 9 shows the results of 34°S-2 DNA polymerase inhibition resistance test.

DETAILED DESCRIPTION

To further illustrate the technical means and effects adopted by the present application, the present application is further described below in conjunction with the embodiments and drawings. It is understood that the specific implementation methods described herein are only used to explain the present application, rather than to limit the present application.

If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. Regular products purchased through regular channels.

Example 1

This example performs sequence alignment.

The Clustal Omega online sequence alignment website was used to compare the novel DNA polymerases 34°S-1 (amino acid sequence SEQ ID NO.1) and 34°S-2 (amino acid sequence SEQ ID NO.2) with the existing Taq DNA polymerase (GenBank: AYJ71526.1). The comparison results showed that the sequence identities of 34°S-1 polymerase and 34°S-2 polymerase with Taq DNA polymerase were 40.92% and 41.94%, respectively. The sequence similarity between the novel DNA polymerases 34°S-1 and 34°S-2 was 56.84%, indicating that a completely new sequence framework has been discovered in this application, which provides greater room for product transformation.

Example 2

This example constructs a recombinant plasmid and expresses and purifies it.

Changzhou Xinyisheng Biotechnology Co., Ltd. was commissioned to synthesize the gene sequences of 34°S-1 polymerase and 34°S-2 polymerase (SEQ ID NO.3 and SEQ ID NO.4), and the genes were cloned into the pET28a expression vector with Nde I and Xho I cloning sites. The recombinant plasmids were transformed into Escherichia coli BL21 (DE3) competent cells and the plates were incubated at 37°C overnight for subsequent expression purification.

The affinity chromatography column used for protein purification is HisTrap FF 5mL. The specific protein expression and purification steps are as follows:

(1) Pick 5 single colonies with good growth on the plate, inoculate them into a 50/250mL LB liquid conical flask, and culture at 37°C for 6 hours until the _OD600 reaches 0.6-4.0; then inoculate the above bacterial liquid into 2L/5L LB medium at a 1% inoculum amount, culture at 37°C for 3 hours, and wait until the _OD600 reaches 0.8-1.0; precool the original shaker to 16°C, add IPTG to the culture medium to make the final concentration 0.5mM; in a shaker at 16°C, 220rpm, induce expression for 14 hours;

(2) The cells were collected by centrifugation at 8000 g for 30 min, and then Ni column affinity A buffer was added at a ratio of 1:20 to resuspend the cells. The cells were then broken by ultrasound in an ice bath environment.

(3) The ultrasonicated solution was centrifuged at 12000 rpm and 4°C for 60 min, and the supernatant was filtered through a 0.22 μM filter membrane as the sample for loading the purification column;

(4) The sample was loaded into the pretreated chromatography column (HisTrap FF 5 mL) at a rate of 3 mL/min; after loading, the Ni column affinity A solution (Ni column-A Buffer) was used to wash for 20 CV; then, the Ni column affinity B solution (Ni column-B Buffer) was used to linearly elute for 10.5 CV at a ratio of 0-70%; the eluted protein was collected when the UV absorption peak reached 50 mAu, and the collection was stopped when the UV absorption peak dropped to 100 mAu;

(5) The collected eluate was diluted 9 times with diluent and then loaded onto the pretreated Q column (HiTrap Q HP 5 mL). After loading, it was rinsed with Q column A solution (Q column-A Buffer) for 10CV until the baseline was stable at a flow rate of 5 mL/min. The target protein was gradient eluted with Q column B solution (0-100% Q column B solution, 10CV) at a flow rate of 5 mL/min. The collected samples were subjected to SDS-PAGE analysis to determine the protein purity.

The purified protein samples were dialyzed and the concentration was determined, then stored in enzyme storage solution for subsequent functional activity analysis.

The specific components of the buffer used in the purification process are as follows:

Ni column-A Buffer: 20mM Tris-HCl, 300mM NaCl, 20mM Imidazole, 5% Glycerol, pH 7.8;

Ni column-B Buffer: 20mM Tris-HCl, 300mM NaCl, 500mM Imidazole, 5% Glycerol, pH 7.8;

Q column-A Buffer: 20mM Tris-HCl, 50mM NaCl, 5% Glycerol, pH 8.0;

Q column-B Buffer: 20mM Tris-HCl, 1M NaCl, 5% Glycerol, pH 8.0;

Diluent: 20 mM Tris-HCl, 5% Glycerol, pH 8.0;

2× dialysis solution: 40mM Tris-HCl, 200mM KCl, 2mM DTT, 0.2mM EDTA, 5% Glycerol, pH 8.0;

Enzyme storage solution: 10mM Tris-HCl, 100mM KCl, 1mM DTT, 0.1mM EDTA, 50% Glycerol, Tween-20 0.5%, NP-40 0.5%, pH 8.0.

The purification results are shown in Figures 1 and 2, and the target protein of the expected size was purified.

Example 3

In this example, the thermal stability of a novel DNA polymerase was determined.

The protein stability was determined using the Protein Thermal Shift ^TM dye kit (ThermoFisher) according to the kit instructions. Taq DNA polymerase was used as a control. The results are shown in Figure 3. It can be seen that the Tm values of 34°S-1 and 34°S-2 DNA polymerases and Taq DNA polymerase (Sangon Biotechnology, catalog number B600001) are 95.3°C, 96.8°C, and 93.2°C, respectively, indicating that the new polymerase discovered in this application has higher thermal stability.

Example 4

In this example, polymerization activity was measured.

Primed M13 ssDNA substrate was used to measure the polymerization activity. The specific principle is shown in Figure 4. In the presence of synergistic activity, the primers on the primed M13 ssDNA will extend along the ssDNA in the 5'-3' direction to produce dsDNA, which can be quantitatively detected by Qubit dsDNA HS Assay Kits (ThermoFisher). The specific reaction system and components are shown in Table 1.

Table 1

No polymerase was added to the NC group, and water was used to make up. After the above reaction solution was reacted at 72°C for 5 minutes, 1 μL of 0.5M EDTA was added to terminate the reaction, and the dsDNA concentration was determined using the Qubit dsDNA HS Assay Kits according to the instructions. The results are shown in Table 2, indicating that 34°S-1 and 34°S-2 DNA polymerases have polymerization activity at 72°C.

Table 2

Example 5

In this example, exo-activity assay (5'-3' exo-activity) was performed.

For the detection of exosome activity, the Taqman probe method was used. When the 5'-3' exosome activity was activated, the downstream probe chain would be degraded by transferring the activity through the incision (as shown in Figure 5). The generated fluorescent signal was detected by an ELISA instrument, and the 5'-3' exosome determination was performed based on the change in the signal.

Annealing of fluorescent probe substrate: Place the configured system into a PCR instrument and react at 90°C for 2 minutes, then turn off the power and allow to cool naturally.

The reaction system is shown in Table 3 below.

Table 3

The detection and fluorescence signal collection were performed using an ELISA instrument, where the excitation light was 494 nm and the absorption light was 522 nm. Specifically, the reaction was carried out at 37°C for 1 hour, and the fluorescence signal was collected once per minute. The results are shown in Figure 6, and it can be seen that 34°S-1 polymerase and 34°S-2 polymerase have 5'-3' exo-cleavage activity.

Example 6

This example performs PCR system optimization.

Fourteen representative PCR reaction buffers were used to test the potential of 34°S-1 polymerase and 34°S-2 polymerase for PCR. The components of the 14 reaction buffers used are as follows:

Buffer 1: 10mM Tris-HCl (pH 8.4), 25mM KCl, 1.5mM MgCl ₂ ;

Buffer 2: 10mM Tris-HCl (pH 8.4), 25mM KCl, 3.5mM MgCl ₂ ;

Buffer 3: 10mM Tris-HCl (pH 8.4), 75mM KCl, 1.5mM MgCl ₂ ;

Buffer 4: 10mM Tris-HCl (pH 8.4), 75mM KCl, 3.5mM MgCl ₂ ;

Buffer 5: 10mM Tris-HCl (pH 8.8), 25mM KCl, 1.5mM MgCl ₂ ;

Buffer 6: 10mM Tris-HCl (pH 8.8), 25mM KCl, 3.5mM MgCl ₂ ;

Buffer 7: 10mM Tris-HCl (pH 8.8), 75mM KCl, 1.5mM MgCl ₂ ;

Buffer 8: 10mM Tris-HCl (pH 8.8), 75mM KCl, 3.5mM MgCl ₂ ;

Buffer 9: 10mM Tris-HCl (pH 9.2), 25mM KCl, 1.5mM MgCl ₂ ;

Buffer 10: 10mM Tris-HCl (pH 9.2), 25mM KCl, 3.5mM MgCl ₂ ;

Buffer 11: 10mM Tris-HCl (pH 9.2), 75mM KCl, 1.5mM MgCl ₂ ;

Buffer 12: 10mM Tris-HCl (pH 9.2), 75mM KCl, 3.5mM MgCl ₂ ;

Buffer 13: 20mM Tris-HCl (pH 8.8), 10mM KCl, 10mM KCl, 10mM (NH ₄ ) ₂ SO ₄ , 1mg/mL BSA, 0.1% Triton;

Buffer 14: 20mM Tris-HCl (pH 8.8), 10mM KCl, 10mM KCl, 10mM (NH ₄ ) ₂ SO ₄ , 0.1% Triton.

5× Additive: 200 mM TMAC, 2.5 M Betain, 12.5% Glycerol, 0.25 mg/mL BSA, 0.5% Triton.

The Escherichia coli genome was used as a template to amplify the 16S gene. The upstream and downstream primers used were E16S-27F and E16S-1492R. The primer sequences were 5’-AGAGTTTGATCCTGGCTCAG-3’ (SEQ ID NO. 5) and 5’-GGTTACCTTGTTACGACTT-3’ (SEQ ID NO. 6), respectively. The PCR reaction system is shown in Table 4.

Table 4

The PCR cycle was 95°C pre-denaturation for 2 min, then 95°C, 20 s; 58°C, 30 s; 72°C, 4 min; 30 cycles, and finally 72°C for 5 min. The PCR products were subjected to agarose gel electrophoresis. Figure 7 shows the electrophoresis results of PCR amplification by 34°S-1 DNA polymerase in buffer 6. The results show that 34°S-1 DNA polymerase has the ability to amplify the target DNA in buffer 6. Figure 8 shows the electrophoresis results of PCR amplification by 34°S-2 DNA polymerase in different reaction buffers. The results show that 34°S-2 DNA polymerase has the ability to amplify the target DNA in a variety of reaction buffers, among which buffer 14 is the most suitable reaction buffer.

Example 7

In this example, the inhibition resistance was measured.

Lambda DNA was used as a template to amplify a 2kb fragment. The upstream and downstream primers used were Anchor-λ F and λ-2 R (2kb). The primer sequences were 5’-CCTGCTCTGCCGCTTCACGC-3’ (SEQ ID NO. 7) and 5’-CCATGATTCAGTGTGCCCGTCTGG-3’ (SEQ ID NO. 8), respectively. The PCR reaction system is shown in Table 5.

Table 5

The PCR cycle was 95°C pre-denaturation for 3 min, followed by 95°C, 30 s; 58°C, 30 s; 72°C, 2 min; 30 cycles, and finally a supplementary reaction at 72°C for 5 min. The PCR product was subjected to agarose gel electrophoresis, and the electrophoresis results are shown in Figure 9. The results show that 34°S-2 has better inhibition resistance than Taq; 34°S-2 can tolerate 20 μM heme, 4 μg/mL humic acid, and 150 μg/mL tannic acid.

In summary, this application discovered two new types of DNA polymerases, whose sequence similarities with Taq DNA polymerase are only 40.92% and 41.94%, respectively. They have a completely new sequence backbone and polymerase activity. In addition, they have higher thermal stability, 5’-3’ exoactivity and inhibition resistance, providing a new polymerase backbone for the development of new molecular tools with a wider range of applications.

The applicant declares that the present application uses the above-mentioned embodiments to illustrate the detailed methods of the present application, but the present application is not limited to the above-mentioned detailed methods, that is, it does not mean that the present application must rely on the above-mentioned detailed methods to be implemented. The technicians in the relevant technical field should understand that any improvement to the present application, the equivalent replacement of the raw materials of the product of the present application, the addition of auxiliary components, the selection of specific methods, etc., all fall within the scope of protection and disclosure of the present application.

Claims (10)

A DNA polymerase, the amino acid sequence of which comprises: (1) the sequence shown in SEQ ID NO.1 or SEQ ID NO.2; or, (2) an amino acid sequence obtained by substituting, deleting or adding one or at least two amino acid residues of the sequence described in (1), and having the same or similar function as the sequence described in (1); or (3) An amino acid sequence that has at least 90% sequence homology with the sequence described in (1) or (2) and has the same or similar function as the sequence described in (1). A nucleic acid molecule encoding the DNA polymerase according to claim 1. The nucleic acid molecule according to claim 2, wherein the nucleic acid sequence of the nucleic acid molecule includes the sequence shown in SEQ ID NO.3 or SEQ ID NO.4. A recombinant vector comprising the nucleic acid molecule according to claim 2 or 3. A recombinant cell comprising the recombinant vector according to claim 4. Use of the DNA polymerase according to claim 1 in preparing a nucleic acid amplification product. Use of the nucleic acid molecule according to claim 2 or 3, the recombinant vector according to claim 4 or the recombinant cell according to claim 5 in the preparation of DNA polymerase. A nucleic acid amplification kit comprising the DNA polymerase according to claim 1. Use of the DNA polymerase according to claim 1 in nucleic acid amplification. A nucleic acid amplification method comprises: mixing a nucleic acid template with a reaction system containing the DNA polymerase according to claim 1 to perform an amplification reaction.