WO2025137876A1 - Isolated polypeptide, preparation method, and use - Google Patents

Abstract

Provided in the present application are an isolated polypeptide, a preparation method, and a use. The polypeptide has an amino acid sequence which is at least 91% homologous or 92% homologous or 93% homologous or 94% homologous or 95% homologous or 96% homologous or 97% homologous or 98% homologous or 99% homologous to SEQ ID NO: 1.

Description

Isolated polypeptide, preparation method and application Technical Field

The present application relates to the field of biotechnology, and in particular, to isolated polypeptides, preparation methods and applications thereof.

Background Art

In the field of molecular biology, PCR (polymerase chain reaction) technology is a key experimental method, commonly used for DNA amplification and cloning. The success of PCR is closely related to the DNA polymerase used, and DNA polymerases are divided into different families according to their polymerization and exolytic activities. Among them, family A and family B DNA polymerases have attracted much attention due to their special properties.

The DNA polymerases of the A family are derived from true bacteria, such as Taq (Thermus aquaticus), Tth (Thermus thermophilus), Tca (Thermus caldophilus), Tfl (Thermus flavus) from Thermus genus, and Bst (Bacillus stearothermophilus) from Bacillus genus. These polymerases mainly exhibit 5'-3' polymerization activity and 5'-3' exolytic activity.

The DNA polymerases of the B family originated from archaea, such as Tli (Thermococcus litoralis) of the genus Thermococcus, Pfu (Pyrococcus furiosus) and KOD (Thermococcus kodacaraensis) of the genus Pyrococcus, as well as Pwo (Pyrococcus woesei), Tgo (Thermococcus gorgonarius), Pab (Pyrococcus abyssi), etc. These polymerases not only have 5'-3' polymerization activity, but also exhibit unique 3'-5' nucleoside exonuclease activity, so they are called high-fidelity polymerases. This exonuclease activity gives the B family DNA polymerase a proofreading function, enabling it to ensure high fidelity of PCR amplification.

In PCR experiments, DNA fragments usually need to be tailed with an A tail at the 3' end for downstream applications (e.g., TA cloning). Currently, the selection of enzymes for DNA end addition is relatively limited.

Therefore, there is a need in the art to find an enzyme with stronger adaptability for adding A to DNA ends.

Summary of the invention

This application is completed by the inventor based on the discovery of the following problems and facts:

Currently, DNA end addition is mainly performed using Taq DNA polymerase or Klenow exo (3'-5'exo-). However, Taq DNA polymerase or Klenow exo (3'-5'exo-) has poor adaptability in different experimental scenarios and environments.

The present application aims to solve at least one of the technical problems existing in the prior art to a certain extent. To this end, the inventors aim to explore new enzymes for DNA end addition, so as to perform end modification of PCR products under different conditions, and provide more options for a wide range of application scenarios.

In view of this, in one aspect of the present application, the present application provides an isolated polypeptide. According to an embodiment of the present application, the polypeptide has an amino acid sequence that is at least 91% homologous, 92% homologous, 93% homologous, 94% homologous, 95% homologous, 96% homologous, 97% homologous, 98% homologous, or 99% homologous to SEQ ID NO: 1. According to an embodiment of the present application, the polypeptide has stable physicochemical properties. It can be applied to PCR amplification and PCR product modification (such as adding A at the end), and has better adaptability to different experimental environments.

In some examples of the present application, polypeptide sequences with higher homology are preferably selected.

According to an embodiment of the present application, the above-mentioned isolated polypeptide may also include at least one of the following technical features:

According to an embodiment of the present application, the polypeptide has the amino acid sequence shown in SEQ ID NO:1.

According to an embodiment of the present application, the polypeptide further has a purification tag sequence. The purification tag is used for affinity chromatography purification to obtain a high-purity polypeptide.

According to an embodiment of the present application, the polypeptide is a DNA polymerase, and the DNA polymerase has 5'-3' polymerization activity, 5'-3' exo-cleavage activity and terminal A-tailing activity.

In the second aspect of the present application, the present application proposes a nucleic acid molecule. According to an embodiment of the present application, the nucleic acid molecule encodes the isolated polypeptide described in the first aspect of the present application. According to an embodiment of the present application, the isolated polypeptide encoded by the nucleic acid molecule can be obtained by culturing in vivo or in vitro.

According to an embodiment of the present application, the above-mentioned nucleic acid molecule may further include at least one of the following technical features:

According to an embodiment of the present application, the nucleic acid molecule has a nucleotide sequence that is at least 73% identical, or 74% identical, or 75% identical, or 76% identical, or 77% identical, or 78% identical, or 79% identical, or 80% identical, or 81% identical, or 82% identical, or 83% identical, or 84% identical, or 85% identical, or 86% identical, or 87% identical, or 88% identical, or 89% identical, or 90% identical, or 91% identical, or 92% identical, or 93% identical, or 94% identical, or 95% identical, or 96% identical, or 97% identical, or 98% identical, or 99% identical to SEQ ID NO: 2.

In some examples of the present application, nucleic acid sequences with higher identity are preferentially selected.

According to an embodiment of the present application, the nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO:2.

In the third aspect of the present application, the present application proposes an expression vector. According to an embodiment of the present application, the expression vector comprises the nucleic acid molecule described in the second aspect of the present application.

In some examples of the present application, the expression vector may further include a promoter, which is operably linked to the nucleic acid molecule.

According to an embodiment of the present application, the above expression vector may further include at least one of the following technical features:

According to an embodiment of the present application, the expression vector is a non-pathogenic viral vector.

According to an embodiment of the present application, the non-pathogenic viral vector includes an adenoviral vector or a retroviral vector.

In the fourth aspect of the present application, the present application proposes a recombinant cell. According to an embodiment of the present application, the recombinant cell expresses the isolated polypeptide described in the first aspect of the present application or carries the nucleic acid molecule described in the second aspect or the expression vector described in the third aspect. According to an embodiment of the present application, the recombinant cell is used to express or secrete the isolated polypeptide described in the first aspect of the present application.

According to an embodiment of the present application, the above-mentioned recombinant cell may further include at least one of the following technical features:

According to an embodiment of the present application, the recombinant cell is selected from Escherichia coli, yeast or mammalian cells.

In the fifth aspect of the present application, the present application proposes a recombinant strain. According to an embodiment of the present application, the recombinant strain expresses the isolated polypeptide described in the first aspect of the present application or the expression vector described in the third aspect. The polypeptide can be obtained quickly and in large quantities by culturing the recombinant strain.

In the sixth aspect of the present application, the present application proposes a method for obtaining an isolated polypeptide. According to an embodiment of the present application, the method comprises culturing the recombinant cell described in the fourth aspect of the present application or the recombinant strain described in the fifth aspect under conditions suitable for protein expression, so as to obtain the isolated polypeptide. This method can be used to rapidly culture and obtain a large amount of polypeptides in a short period of time.

In the seventh aspect of the present application, the present application proposes the polypeptide described in the first aspect, the polypeptide encoded by the nucleic acid molecule described in the second aspect, The use of the polypeptide expressed by the expression vector of the third aspect, the polypeptide expressed by the recombinant cell of the fourth aspect, the polypeptide expressed by the recombinant bacterium of the fifth aspect, or the polypeptide obtained according to the method of the sixth aspect as a DNA polymerase, wherein the DNA polymerase has 5'-3' polymerization activity, 5'-3' exo-cleavage activity, and terminal A-tailing activity. According to an embodiment of the present application, the polypeptide can be used as a DNA polymerase for PCR amplification, and deoxyadenylic acid (A) is added to the 3' end of the PCR amplification product.

In the present application, the 5'-3' polymerization activity refers to the activity of the DNA polymerase to add new nucleotide units one by one to the newly synthesized chain in the direction from the 5' end to the 3' end according to the nucleotide sequence on the template chain when synthesizing a new DNA chain.

In the present application, the 5'-3' exonuclease activity refers to the activity of the DNA polymerase to hydrolyze downstream nucleic acid fragments along the direction from the 5' end to the 3' end.

In the present application, the terminal A-tailing activity refers to the ability of DNA polymerase to add (attach) one or more deoxyadenylic acid (dATP) to the 3' end of a double-stranded DNA molecule. During the A-tailing process, DNA polymerase will add one or more deoxyadenylic acid to the 3' end of a double-stranded DNA fragment to form an A-tail. This A-tailed DNA fragment can be conveniently used for ligation to a vector with a T-tail to form a stable connection.

In the eighth aspect of the present application, the present application proposes a method for adding an A tail to the end of a double-stranded DNA. According to an embodiment of the present application, the method comprises: subjecting the double-stranded DNA to a terminal A tail addition reaction in the presence of the polypeptide described in the first aspect of the present application, to obtain a terminal A tail addition product.

According to an embodiment of the present application, the 3' end of the A-tailed product contains a protruding deoxyadenylic acid. Adding A-tails to the DNA makes the subsequent ligation reaction easier to carry out.

In the ninth aspect of the present application, the present application proposes a polymerase chain reaction method. According to an embodiment of the present application, the method comprises: subjecting the DNA to be amplified to polymerase chain reaction (PCR) in the presence of the polypeptide described in the first aspect of the present application. According to an embodiment of the present application, the polypeptide can be used for PCR amplification and sequencing library construction.

In the tenth aspect of the present application, the present application proposes a kit. According to an embodiment of the present application, the kit includes: the polypeptide described in the first aspect of the present application, the polypeptide encoded by the nucleic acid molecule described in the second aspect, the polypeptide expressed by the expression vector described in the third aspect, the polypeptide expressed by the recombinant cell described in the fourth aspect, the polypeptide expressed by the recombinant bacteria described in the fifth aspect, or the polypeptide obtained according to the method described in the sixth aspect. According to an embodiment of the present application, the kit can be used for A-tailing reaction, PCR amplification, TA cloning, and sequencing library construction.

According to an embodiment of the present application, the above-mentioned kit may further include at least one of the following technical features:

According to an embodiment of the present application, the kit further comprises a DNA polymerase buffer. The kit may also comprise an instruction manual, double distilled water, etc.

According to an embodiment of the present application, the DNA polymerase buffer includes 8-15mM Tris-HCl, 80-120mM K ⁺ , 0.5-1.5mM DTT, 0.05-0.15mM EDTA, and 40-60% glycerol. In some examples of the present application, the Tris-HCl concentration is optionally 8mM, 9mM, 10mM, 11mM, 12mM, 13mM, 14mM, or 15mM; the K ⁺ concentration is optionally 90mM, 100mM, 110mM, 12mM, 13mM, 14mM, or 15mM. 110mM or 120mM; the DTT concentration is optionally 0.5mM, 0.6mM, 0.7mM, 0.8mM, 0.9mM, 1.0mM, 1.1mM, 1.2mM, 1.3mM, 1.4mM or 1.5mM; the EDTA concentration is optionally 0.05mM, 0.1mM or 0.15mM; the glycerol is optionally 40%, 50% or 60%.

In the eleventh aspect of the present application, the present application proposes the use of the polypeptide described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, the recombinant cell described in the fourth aspect or the recombinant strain described in the fifth aspect in the preparation of products related to terminal A-tailing, T-A cloning, polymerase chain reaction or sequencing library construction.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as implementation schemes) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described one by one here.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and/or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

FIG1 is a schematic diagram of the SDS-PAGE electrophoresis results of the DNA polymerase fusion protein according to an embodiment of the present application;

FIG2 is a schematic diagram of the polymerization activity detection principle according to an embodiment of the present application;

FIG3 is a schematic diagram of the exo-activity detection principle according to an embodiment of the present application;

FIG4 is a schematic diagram of the results of detecting the fluorescent signal of the 5′-3′ exo-active DNA polymerase according to an embodiment of the present application;

FIG5 is a schematic diagram of the principle of 3'-5' exo-activity detection according to one embodiment of the present application;

FIG6 is a schematic diagram of the results of detecting the fluorescent signal of the 3'-5' exo-activation of the DNA polymerase according to one embodiment of the present application;

Figure 7 is a schematic diagram of the test results of adding A tail to the DNA blunt end as described in an embodiment of the present application; wherein, NC is a blank control group using water instead of A pol_30 polymerase reaction, and Klenow (exo-) is a positive control group using commercialized Klenow fragment (exo-) instead of A pol_30 polymerase reaction.

DETAILED DESCRIPTION

Embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to be used to explain the present invention, but should not be understood as limiting the present invention.

In this application, unless otherwise specified, the terms "first" and "second" are used for descriptive purposes only and are not to be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of the features. In the description of the present invention, "plurality" means at least two, such as two, three, etc., unless otherwise clearly and specifically defined.

In this application, unless otherwise specified, the term "nucleotide" includes both nucleotides and nucleosides. Nucleosides, like nucleotides, contain a purine or pyrimidine base linked to a ribose or deoxyribose glycoside, but lack a phosphate residue. Synthetic and/or naturally occurring nucleotides have a 3' sugar hydroxyl group. Before modification, they are included in the definition.

In this application, unless otherwise specified, the term "reference genome" refers to the genome sequence of a species corresponding to a known sample, which can be a genome sequence obtained through public channels or obtained through sequencing assembly. It can be the entire sequence of the genome or a partial sequence of interest in the genome. For example, when analyzing human samples, multiple versions of human genome sequences provided by public databases, such as hg19, etc., can be used.

The inventors of the present application mined a peptide with a novel sequence from the metagenomic sequencing data of deep-sea hydrothermal sediment samples by sequence mining. After experimental testing, it was found that it has the conventional activity of DNA polymerases of the A family, such as 5'-3' polymerization activity, 5'-3' exo-cleavage activity and terminal A-tailing activity. Therefore, it is named A pol_30, and is sometimes referred to as A pol_30 DNA polymerase in this article.

In one aspect of the present application, the present application proposes an isolated polypeptide having an amino acid sequence that is at least 91% homologous, or 92% homologous, or 93% homologous, or 94% homologous, or 95% homologous, or 96% homologous, or 97% homologous, or 98% homologous, or 99% homologous to SEQ ID NO: 1. According to some preferred embodiments of the present application, the polypeptide has an amino acid sequence as shown in SEQ ID NO: 1.

According to the embodiments of the present application, the so-called "homology" refers to the similar or identical chemical properties of amino acids at corresponding positions in two or more protein sequences. In protein sequence comparison, homology means that the same or similar amino acid residues exist at specific positions of two proteins.

In some examples of the present application, the polypeptide also has a purification tag sequence. The purification tag sequence is a tag sequence used when the polypeptide (protein) is purified by affinity chromatography. It is usually divided into large tags and small tags. Large tags are usually tens of KD, such as GST, MBP, etc.; small tags are mostly composed of 6-10 amino acids, such as His (6-10), Strep (II), FLAG, etc. On the premise that it does not substantially affect the spatial structure of the protein, the purification tag sequence can be added to the N-terminus of the polypeptide or to the C-terminus of the polypeptide. Those skilled in the art can select a suitable purification tag sequence as needed to obtain a high-purity polypeptide.

In some examples of the present application, the polypeptide is a DNA polymerase, and the DNA polymerase has 5'-3' polymerization activity, 5'-3' exo-cutting activity and terminal A-tailing activity. The inventors of the present application have experimentally tested and found that the polypeptide has the conventional activity of DNA polymerases of the A family, such as 5'-3' polymerization activity, 5'-3' exo-cutting activity and terminal A-tailing activity. The polypeptide can be used as a DNA polymerase for A-tailing reaction, PCR amplification, TA cloning and sequencing library construction. In another aspect of the present application, the present application proposes a nucleic acid molecule encoding the above-mentioned isolated polypeptide.

It should be noted that, for the nucleic acid molecules mentioned in the specification and claims of this application, those skilled in the art should understand that they actually include any one or both of the complementary double strands. For convenience, in this specification and claims, although only one strand is given in most cases, the other strand complementary thereto is actually disclosed. In addition, the nucleic acid sequence in this application includes DNA form or RNA form, and disclosing one of them means that the other is also disclosed.

In some examples of the present application, the nucleic acid molecule has at least 73% identity, 74% identity, or 75% identity to SEQ ID NO: 2. In some preferred examples of the present application, the nucleic acid molecule has an amino acid sequence as shown in SEQ ID NO: 2.

In some examples of the present application, the nucleic acid sequence can be obtained by performing metagenomic sequencing data analysis on deep-sea hydrothermal sediment samples.

According to the embodiments of the present application, the so-called "identity" refers to the fact that the bases (adenine, guanine, cytosine or guanine) at corresponding positions in two or more nucleic acid sequences have the same base sequence. Nucleotide identity is usually expressed as a percentage, indicating the proportion of the number of identical nucleotides.

In another aspect of the present application, the present application proposes an expression vector comprising the aforementioned nucleic acid molecule. The type of expression vector here is not particularly limited, as long as it can replicate and express the corresponding mutant in the host cell. The expression vector may include an optional control sequence, which is operably connected to the nucleic acid molecule. Among them, the control sequence is one or more control sequences that can guide the expression of the nucleic acid molecule in the host. The expression vectors proposed in some specific embodiments of the present application can efficiently express proteins in suitable host cells, and the obtained proteins have the function of A family DNA polymerase.

In another aspect of the present application, the present application proposes a recombinant cell carrying the aforementioned nucleic acid molecule, expression vector or expressing the aforementioned isolated polypeptide. The recombinant cell is obtained by transfecting or transforming the expression vector. According to some specific embodiments of the present application, the recombinant cell can efficiently express the above-mentioned isolated polypeptide under appropriate conditions, and the polypeptide can be directly applied to A-tailing reaction, PCR amplification, TA cloning, and sequencing library construction, etc.

In some examples of the present application, the type of recombinant cells is not particularly limited, and may be, for example, Escherichia coli cells.

It should be noted that the "suitable conditions" referred to in the specification of this application refer to conditions suitable for the expression of the isolated polypeptide described in this application. It is easy for those skilled in the art to understand that the conditions suitable for the expression of the isolated polypeptide include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy host cell states, suitable host cell density, suitable cell culture environment, and suitable cell culture time. "Suitable conditions" are not particularly limited, and those skilled in the art can optimize the most suitable conditions for the expression of the isolated polypeptide according to the specific environment of the laboratory.

In another aspect of the present application, the aforementioned polypeptide is used as a DNA polymerase to perform an A-tailing reaction at the end of a double-stranded DNA fragment. Usually, it is the 3' end. After the reaction is completed, the 3' end of the obtained terminal A-tailed product contains one or more protruding deoxyadenylic acids. This polypeptide has similar performance to wild-type DNA polymerase and has no obvious preference when processing bases at the ends of DNA fragments.

In another aspect of the present application, the aforementioned polypeptide is used as a DNA polymerase to perform a polymerase chain reaction. The reaction comprises: subjecting the DNA to be amplified to a polymerase chain reaction (PCR) in the presence of the polypeptide described in the first aspect of the present application. It will be appreciated by those skilled in the art that the PCR amplification reaction should be performed under conditions suitable for the DNA polymerase, such as a suitable metal ion concentration (such as Mg ²⁺ , K ^+, etc.), appropriate pH (pH6.5-8.5), appropriate temperature, additives for stabilizing enzyme activity (such as dithiothreitol, bovine serum albumin, etc.), etc. In addition, the PCR amplification reaction system further includes primers that bind to the template chain in the DNA replication process and deoxynucleoside triphosphate substrates (dNTP mix) added to the new chain.

In another aspect of the present application, the present application proposes a kit, the kit comprising: the aforementioned polypeptide of the present application, the polypeptide encoded by the aforementioned nucleic acid molecule, the polypeptide expressed by the aforementioned expression vector, the polypeptide expressed by the aforementioned recombinant cell, the polypeptide expressed by the aforementioned recombinant bacterium, or the polypeptide obtained according to the aforementioned method. In some examples of the present application, the kit can be used for A-tailing reaction, PCR amplification, TA cloning, and sequencing library construction, etc. Those skilled in the art will appreciate that in some cases, the aforementioned kit may also include a DNA polymerase buffer to provide a reaction environment suitable for the reaction. In other cases, the aforementioned kit may also include instructions, double distilled water, etc., to facilitate the operation of those skilled in the art.

The present invention is described below with reference to examples, it should be noted that these examples are merely illustrative and do not limit the present invention in any way. Where specific techniques or conditions are not indicated in the examples, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used without indicating the manufacturer are all conventional products that can be obtained commercially.

It should be noted that in the present application, the isolated polypeptide can be used as a DNA polymerase. For the convenience of comparison, DNA polymerase will be used instead in the following examples. The A pol_30 DNA polymerase appearing in the following examples has the amino acid sequence shown in SEQ ID NO:1.

Example 1: Sequence identity comparison

In this example, the DNA polymerase in this application (SEQ ID NO:1, A pol_30) is aligned with the commercial Taq polymerase (SEQ ID NO:3) and Klenow fragment DNA polymerase (SEQ ID NO:4, Klenow (exo-)) using the Clustal Omega online sequence alignment website. The results show that the sequence consistency of SEQ ID NO:1 is 9.897779, 9.2202 and 9.726173 respectively.

Among them, the amino acid sequences of commercial Taq polymerase and Klenow fragment DNA polymerase are shown below 47.18%, 41.99%;

Example 2: Recombinant plasmid construction, expression and purification

First, a DNA polymerase with the amino acid sequence shown in SEQ ID NO: 1 was synthesized (Beijing Liuhe BGI Gene Technology Co., Ltd.), and then the gene was cloned into the pET28A expression vector with cloning sites of Nde I and Xho I. The obtained recombinant plasmid was transformed into Escherichia coli BL21 (DE3) competent cells and left to stand overnight at 37°C for expression and purification. The affinity chromatography column used for protein purification was HisTrap FF×5mL.

The specific purification steps are as follows:

1. Cultivation and induction

A single colony with good growth was selected from the plate and inoculated into a conical flask containing 50 mL of kanamycin-resistant LB liquid medium, and cultured at 37°C for 5-7 hours until OD ₆₀₀ reached 0.6-4.0. Next, the above bacterial solution was transferred to 2L of kanamycin-resistant LB medium with a 1% inoculum and cultured at 37°C for 2-4 hours until OD ₆₀₀ reached 0.8-1.0. During this period, the shaker was cooled to 16°C in advance. IPTG was added to the culture medium to a final concentration of 0.5 mM. Then, the expression was induced at 220 rpm in a shaker at 16°C for 12-16 hours.

2. Collection and crushing of bacteria

The cells were collected by centrifugation at 8000 g for 30 minutes, and then suspended in Ni column affinity solution A at a ratio of 1:20, and disrupted by ultrasonication in an ice bath.

3. Sample loading and elution

The ultrasonic disrupted liquid was centrifuged at 12000rpm and 4℃ for 60 minutes, and the supernatant was filtered with a 0.22μM filter membrane to obtain the sample on the purification column. The sample was loaded at a rate of 3mL/min, and then washed with Ni column affinity A solution for 20 column volumes. Then, Ni column affinity B solution was used for linear elution. During the elution process, the target protein was collected when the UV absorption peak reached 50mAu. When the UV absorption peak decreased, Stop collecting when the value reaches 100 mAu.

4. Dialysis and storage

The purified protein samples were dialyzed, the concentration was measured, and they were stored in stock solution for subsequent functional activity analysis.

In the above purification process, the buffer components used are as follows:

Ni column-A buffer (Ni column affinity A solution): 20 mM Tris-HCl, 300 mM NaCl, 20 mM imidazole, 5% glycerol, pH 8.5;

Ni column-B buffer (Ni column affinity B solution): 20 mM Tris-HCl, 300 mM NaCl, 500 mM imidazole, 5% glycerol, pH 8.5;

Storage solution: 10mM Tris-HCl, 100mM KCl, 1mM DTT, 0.1mM EDTA, 50% glycerol, pH 8.0@25℃.

5. Purification results

The protein sample (A pol_30 polymerase) obtained in step 4 was subjected to SDS-PAGE electrophoresis detection, and the results are shown in Figure 1.

Example 3: Polymerization activity assay

The polymerization activity of A pol_30 polymerase was determined using primed M13 ssDNA substrate. The specific principle is shown in Figure 2. In the presence of polymerization activity, the primer on the primed M13 ssDNA will extend along the ssDNA (single-stranded DNA) in the 5’-3’ direction to produce dsDNA (double-stranded DNA), which can be quantitatively detected using Qubit dsDNA HS Assay Kits (Manufacturer: Thermo Fisher; Product Number: Q32854).

The method for preparing primed M13 ssDNA substrate is as follows: the M13 used is M13mp18 Single-stranded DNA (brand: NEB, product number: N4040S), the corresponding M13 annealing primer sequence is shown in Table 1, and the single-stranded M13 and the 7 primers shown are annealed by gradient cooling to obtain the primed M13 ssDNA substrate.

Table 1

The reaction system and components are shown in Table 2.

Table 2

After the reaction solution was reacted at 37°C, 55°C, and 72°C for 5 min (Table 3), 1 μL 0.5 M EDTA was added to terminate the reaction, and the dsDNA concentration was determined using the Qubit kit.

Table 3

The results showed that A pol_30 DNA polymerase had obvious polymerization activity at both 37°C and 55°C, and both synthesized dsDNA.

Example 4: Exo-activity assay (5'-3' exo-activity)

In order to detect the exo-activity of A pol_30 polymerase, the Taqman probe method was used in this example. When the 5'-3' exo-activity acts, the downstream probe chain will be degraded by transferring the activity through the incision (the principle is shown in Figure 3). An ELISA instrument is used to detect the generated fluorescent signal, and the 5'-3' exo-activity is measured according to the change of the signal. The fluorescent probe sequence is as follows:

5'-Dabcyl-GCATCTGCTCGAGTCACGCGCTATGGCGATGCTTGATAGTGATGCTGTGTACAGAAAG(SEQ ID NO:12);

CCATAGCGCGTGACTCGAGCAGATGC-FAM-3'(SEQ ID NO:13);

CTTTCTGTACACAGCATCACTATCAAGCATCG(SEQ ID NO:14);

The three single-stranded DNA fragments were mixed in equal proportions and reacted at 90°C for 2 minutes, and then the power was turned off and the temperature was naturally cooled. The reaction system is shown in Table 4.

Table 4

The detection and fluorescence signal collection were performed using an ELISA instrument, wherein the excitation light was 492 nm and the emission light was 520 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 FIG4 , indicating that A poly_30 polymerase has 5'-3' exo-cleavage activity.

Example 5: Exo-activity assay (3'-5' exo-activity)

In this example, the terminal mismatch substrate probe method was used to measure the exosome activity of A pol_30 polymerase (the principle is shown in Figure 5).

The fluorescent probe sequences used are:

Primer with a quenching group at the 3’ end: ATCAGCAGGCCACACGTTAAACTGT 3’-BHQ2 (SEQ ID NO: 15); and primer with a fluorescent group at the 5’ end: TAGTCGTCCGGTGTGCAATTTCTGT 5’-Rox (SEQ ID NO: 16);

After annealing, complementary pairing forms a double-stranded substrate, which contains 4 mismatches at the ends.

The reaction system for detecting 3'-5' exo-activity is as follows, wherein the blank control is a reaction system without enzyme, the reaction is carried out at 30°C for 1 hour, and the fluorescence signal (FAM fluorescence, excitation peak is 492nm, emission peak is 520nm) is collected every 30 seconds. The reaction system is shown in Table 5.

Table 5

The test results are shown in Figure 6. A pol_30 polymerase did not produce obvious fluorescent signals, proving that the enzyme does not have 3’-5’ exolytic activity.

Example 6: DNA blunt end plus A tail test

In this example, the functional activity of A pol_30 polymerase in adding A to the blunt end of DNA was evaluated by detecting the change in the position of the band before and after the addition of A to the DNA fragment by denaturing gel electrophoresis. Two fluorescent probes (sequences are shown in Table 6, and the 5' end is labeled with fluorescence) were designed. When the functional activity of adding A takes effect, the length of each probe increases by one nt (nucleotide), and the activity of adding A tail is determined by the size of the Urea-PAGE band.

Table 6

Annealing of fluorescent probe substrate: react at 90°C for 2 min, then turn off the power and cool naturally. The reaction system is shown in Table 7, and a blank control group (NC) without enzyme and a positive control group with Klenow (exo-) DNA polymerase (brand: NEB, catalog number: M0212V) added are set up at the same time.

Table 7

Incubate at 37°C for 30 min. Place on ice immediately and add 1 μL 0.5 M EDTA solution to terminate the reaction.

The preparation method of 20% denaturing gel is shown in Table 8.

Table 8

The configuration method of 2X Gel loading solution is shown in Table 9.

Table 9

Mix 4 μL of reaction solution with an equal volume of Gel loading, heat at 80°C for 5 min, and immediately place on ice for sample loading.

During electrophoresis, use 1X TBE Buffer as the buffer. The denaturing gel needs to be preheated at 180V, 100mA for 30 minutes. Before loading the sample, blow away the urea in the gel holes with a syringe. Perform electrophoresis at 180V, 100mA for about 2 hours. End the electrophoresis when the indicator bromphenol blue is about 2cm away from the bottom of the gel.

The results are shown in Figure 7. Both A pol_30 and Klenow (exo-) of A family DNA polymerase have A-tailing activity.

In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

Although the embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as Without limiting the present invention, a person skilled in the art may make changes, modifications, substitutions and variations to the above-described embodiments within the scope of the present invention.

Claims (22)

A separated polypeptide, characterized in that the polypeptide has an amino acid sequence that is at least 91% homologous, or 92% homologous, or 93% homologous, or 94% homologous, or 95% homologous, or 96% homologous, or 97% homologous, or 98% homologous, or 99% homologous to SEQ ID NO:1. The isolated polypeptide according to claim 1 is characterized in that the polypeptide has an amino acid sequence shown in SEQ ID NO:1. The isolated polypeptide according to claim 2, characterized in that the polypeptide also has a purification tag sequence. The isolated polypeptide according to any one of claims 1 to 3 is characterized in that the polypeptide is a DNA polymerase, and the DNA polymerase has 5'-3' polymerization activity, 5'-3' exolytic activity and terminal A-tailing activity. A nucleic acid molecule, characterized in that the nucleic acid molecule encodes the isolated polypeptide according to any one of claims 1 to 4. The nucleic acid molecule according to claim 5 is characterized in that the nucleic acid molecule has a nucleotide sequence that is at least 73% identical or 74% identical or 75% identical or 76% identical or 77% identical or 78% identical or 79% identical or 80% identical or 81% identical or 82% identical or 83% identical or 84% identical or 85% identical or 86% identical or 87% identical or 88% identical or 89% identical or 90% identical or 91% identical or 92% identical or 93% identical or 94% identical or 95% identical or 96% identical or 97% identical or 98% identical or 99% identical to SEQ ID NO:2. The nucleic acid molecule according to claim 5 is characterized in that the nucleic acid molecule has a nucleotide sequence shown in SEQ ID NO:2. An expression vector, characterized in that it comprises the nucleic acid molecule according to any one of claims 5 to 7. The expression vector according to claim 8 is characterized in that the expression vector is a non-pathogenic viral vector. The expression vector according to claim 9, characterized in that the non-pathogenic viral vector comprises an adenoviral vector or a retroviral vector. A recombinant cell, characterized in that it expresses the isolated polypeptide according to any one of claims 1 to 4 or carries the nucleic acid molecule according to any one of claims 5 to 7 or the expression vector according to any one of claims 8 to 10. The recombinant cell according to claim 11, characterized in that the recombinant cell is selected from Escherichia coli, yeast or mammalian cells. A recombinant strain, characterized in that it expresses the isolated polypeptide according to any one of claims 1 to 4 or carries the nucleic acid molecule according to any one of claims 5 to 7 or the expression vector according to any one of claims 8 to 10. A method for obtaining an isolated polypeptide, comprising: culturing the recombinant cell according to claim 11 or 12 or the recombinant strain according to claim 13 under conditions suitable for protein expression to obtain the isolated polypeptide. The polypeptide according to any one of claims 1 to 4, the polypeptide encoded by the nucleic acid molecule according to any one of claims 5 to 7, the polypeptide expressed by the expression vector according to any one of claims 8 to 10, the polypeptide expressed by the recombinant cell according to claim 11 or 12, the polypeptide according to claim 13 The polypeptide expressed by the recombinant bacteria or the polypeptide obtained according to the method of claim 14 is used as a DNA polymerase, and the DNA polymerase has 5'-3' polymerization activity, 5'-3' exo-cleavage activity and terminal A-tailing activity. A method for adding an A tail to the end of a double-stranded DNA, characterized in that it comprises: subjecting the double-stranded DNA to a terminal A tail addition reaction in the presence of the polypeptide described in any one of claims 1 to 4, to obtain a terminal A tail addition product. The method according to claim 16 is characterized in that the 3' end of the terminal A-tailed product contains a protruding deoxyadenylic acid. A polymerase chain reaction method, characterized in that the DNA to be amplified is subjected to polymerase chain reaction in the presence of the polypeptide according to any one of claims 1 to 4. A kit, characterized in that it comprises: A polypeptide according to any one of claims 1 to 4, a polypeptide encoded by a nucleic acid molecule according to any one of claims 5 to 7, a polypeptide expressed by an expression vector according to any one of claims 8 to 10, a polypeptide expressed by a recombinant cell according to claim 11 or 12, a polypeptide expressed by a recombinant bacterium according to claim 13, or a polypeptide obtained according to the method of claim 14. The kit according to claim 19, further comprising a DNA polymerase buffer. The kit according to claim 20, characterized in that the DNA polymerase buffer comprises 8-15 mM Tris-HCl, 80-120 mM K ⁺ , 0.5-1.5 mM DTT, 0.05-0.15 mM EDTA, and 40-60% glycerol. Use of the polypeptide according to any one of claims 1 to 4, the nucleic acid molecule according to any one of claims 5 to 7, the expression vector according to any one of claims 8 to 10, the recombinant cell according to claim 11 or 12, or the recombinant strain according to claim 13 in the preparation of products related to terminal A-tailing, TA cloning, polymerase chain reaction or sequencing library construction.