WO2025137872A1 - Terminal deoxyribonucleoside transferase mutant, preparation method therefor and use thereof - Google Patents

Abstract

Disclosed are a terminal deoxyribonucleoside transferase mutant, a preparation method therefor and a use thereof. A wild-type terminal deoxyribonucleoside transferase is subjected to molecular modification, to improve modified nucleic acid monomer polymerization activity and modified nucleic acid monomer catalysis efficiency, and to realize efficient single-base addition efficiency. Multiple 3'-O-blocking modified dNTPs substrates can be added to a 3'-OH end of an oligonucleotide single chain without a template, so that a novel and effective tool enzyme can be provided for the enzymatic method from de novo synthesis of nucleic acid.

Description

Terminal deoxyribonucleoside transferase mutant and its preparation method and application Technical Field

The present application belongs to the field of genetic engineering technology and relates to a terminal deoxyribonucleoside transferase mutant and a preparation method and application thereof.

Background Art

Driven by high-throughput sequencing and gene editing technologies, synthetic biology has risen rapidly, and DNA synthesis has become an emerging industry. The demand for DNA synthesis is increasing in many fields of research and commercial activities. If DNA synthesis can be provided on a large scale and at low cost, it will promote the rapid development of technologies such as engineering biology, treatment, data storage and nanotechnology. After the discovery of the DNA structure, with the advancement of solid-phase synthesis technology, the de novo synthesis technology of DNA has achieved substantial milestones. At present, there are two main methods for synthesizing oligonucleotides from scratch: chemical synthesis (phosphoramidite synthesis) and biosynthesis (enzymatic synthesis). Phosphoramidite synthesis is the mainstream method for synthesizing short-chain DNA, which can reliably provide short DNA chains of <200 nucleotides. Phosphoramidite synthesis mainly completes a single base addition cycle through four steps of deprotection, coupling, capping and oxidation. The steps are cumbersome, and there are problems such as long single cycle time (6-8 minutes), high consumption of chemical reagents, and high cost. In addition, a large amount of toxic and flammable organic reagents are used in the reaction process, which causes great pollution. Currently, the synthesis of nucleotide sequences >200bp by phosphoramidite synthesis is still a heavy burden. For this reason, researchers have tried to develop alternative technologies such as Gibbs assembly to produce longer DNA chains.

Enzymatic de novo DNA synthesis is a promising technology that has attracted attention since the 1950s. Terminal deoxynucleotidyl transferase (TdT) is a DNA polymerase that is widely used and has obvious advantages in enzymatic de novo DNA synthesis technology. TdT can indiscriminately extend four natural nucleotides (A, T, C, G) to the 3' end of the starting chain without a template. Studies have shown that TdT can extend several nucleotides within 1 second and can extend the synthesized DNA to several thousand bases. The superior synthesis length and speed far exceed the range of commercially available phosphoramidite synthesis technology. However, TdT extends oligonucleotides in a promiscuous manner in the 5' to 3' direction of the starting chain. Since any of the four nucleotides will participate in each step of the reaction, it will eventually lead to the simultaneous formation of oligonucleotide sequences of different lengths and codes. To address this problem, an effective solution is to control the incorporation of nucleotides through a "reversible termination" mechanism: in the pentose sugar of the nucleotide, the incorporation of nucleotides is controlled by a "reversible termination" mechanism. A reversibly removable protecting group "Protecting Group, PG" is added to the 3rd position of the nucleotide to ensure that each reaction step is terminated after extending one nucleotide, and in the subsequent reaction, PG can be removed and restored to a hydroxyl group to extend the next required nucleotide (Figure 1). This DNA de novo synthesis strategy catalyzed by TdT only requires two steps of coupling and deprotection to complete a single base addition cycle. Compared with the phosphoramidite synthesis method, it greatly improves the possible limit of single-cycle efficiency, and the entire reaction is completed in the aqueous phase, with the characteristics of mild reaction conditions and greener. How to achieve efficient single-base addition efficiency is the key to the TdT-dominated enzymatic de novo DNA synthesis scheme. Since the scheme uses non-natural nucleotide monomers modified with PG, the activity of natural TdT on it is too low, resulting in low synthesis efficiency. Therefore, it is necessary to improve the polymerization activity of TdT on modified nucleotide monomers through molecular modification. For example, CN114921436A discloses a terminal deoxynucleotidyl transferase mutant with improved thermal stability, by mutating the sites of the amino acid sequence of the wild-type terminal deoxynucleotidyl transferase as shown in SEQ ID NO.1 to obtain a mutant with six mutations: N135P, S138H, Q229D, K232D, L234R, and V366M.

In summary, the discovery and design of terminal deoxynucleotidyl transferases with high polymerization activity is of great significance in the field of nucleic acid synthesis.

Summary of the invention

The present application provides a terminal deoxyribonucleoside transferase mutant and a preparation method and application thereof, in order to solve the problems of low efficiency, reaction energy consumption, high pollution, etc. in synthesizing DNA by the traditional phosphoramidite synthesis method, as well as the low catalytic efficiency of wild terminal deoxyribonucleoside transferase.

In a first aspect, the present application provides a terminal deoxyribonucleoside transferase mutant, wherein the amino acid sequence of the terminal deoxyribonucleoside transferase mutant comprises any one of the following sequences:

(1) the following mutations occur based on the sequence shown in SEQ ID NO:2: any one or at least two of the following sequences: E459R, E459K, E459Q, E459V, R460Q, R460K, R460E, R460H, R460N, R460D, R460S, R460L, R460M, R460W, R460F, R460Y, R460C or R460G; or,

(2) a 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) A sequence having at least 90% sequence homology with the sequence described in (1) or (2) and having the same or similar functions as the sequence described in (1).

In this application, the terminal deoxyribonucleoside transferase GeTdT from Gecko was used to Molecular modification is used to adjust the polarity and spatial size of the active pocket so that it can accommodate non-natural nucleotides well, thereby improving its polymerization activity for modified nucleotide monomers, greatly improving the catalytic efficiency for modified nucleic acid monomers, and achieving efficient single-base addition efficiency. It can add a variety of 3'-O-blocked modified dNTPs substrates to the 3'-OH end of the oligonucleotide single chain without a template, providing a new and effective tool enzyme for the enzymatic synthesis of nucleic acids from scratch.

In the present application, the introduction of specific mutations into the wild-type terminal deoxyribonucleoside transferase can improve its polymerization activity for modified nucleotide monomers. It can be understood that based on the terminal deoxyribonucleoside transferase mutant, those skilled in the art can use the general technical means in the art to replace, delete or add one or at least two amino acid residues to obtain other sequences with the same or similar functions.

In this application, the term "homology" can be evaluated by the naked eye or by computer software (such as the software program described in Current Protocols in Molecular Biology by Ausubel et al. eds. (2007). When a position in the compared sequences is occupied by the same base or amino acid, the molecules are identical at that position. The homology between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the homology between related sequences. A polynucleotide sequence or amino acid sequence has a certain percentage (for example, 90%, 95%, 98% or 99%) of "sequence homology" with another sequence means that when the sequences are aligned, the percentage of bases or amino acids in the two compared sequences are the same.

In the present application, single-site mutation of E459 and combined mutation of two sites of E459 and R460 are involved. Preferably, (1) the mutation may include combined mutation of E459R and R460Q (which can be written as E459R/R460Q), combined mutation of E459R and R460K, combined mutation of E459R and R460E, combined mutation of E459R and R460H, combined mutation of E459R and R460N, combined mutation of E459R and R460D, combined mutation of E459R and R460S. mutation, E459R and R460L combined mutation, E459R and R460M combined mutation, E459R and R460W combined mutation, E459R and R460F combined mutation, E459R and R460Y combined mutation, E459R and R460C combined mutation or E459R and R460G combined mutation.

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

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 terminal deoxyribonucleoside transferase mutant according to the first aspect, the preparation method comprising:

Inserting a nucleic acid molecule encoding the terminal deoxyribonucleoside transferase mutant described in the first aspect into an expression vector to obtain a recombinant vector, and introducing the recombinant vector into a host cell, or directly integrating the nucleic acid molecule into the genome of the host cell to obtain a genetically engineered bacterium, and culturing and separating and purifying the terminal deoxyribonucleoside transferase mutant.

In a sixth aspect, the present application provides the use of the terminal deoxyribonucleoside transferase mutant described in the first aspect in the preparation of a product for nucleic acid synthesis.

In a seventh aspect, the present application provides a product for nucleic acid synthesis, wherein the product comprises the terminal deoxyribonucleoside transferase mutant described in the first aspect.

In an eighth aspect, the present application provides the use of the terminal deoxyribonucleoside transferase mutant described in the first aspect in nucleic acid synthesis.

In a ninth aspect, the present application provides a method for synthesizing nucleic acid, the method comprising:

The terminal deoxyribonucleoside transferase mutant described in the first aspect is used to catalyze the connection of dNTPs to the 3'-OH end of the oligonucleotide single chain.

Preferably, the dNTPs include 3’-O-blocking modified dNTPs, and the blocking modification includes a protecting group such as “Protecting Group, PG”.

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

The present invention molecularly modifies the wild-type terminal deoxyribonucleoside transferase to improve its polymerization activity for modified nucleotide monomers, greatly improves the catalytic efficiency for modified nucleic acid monomers, achieves efficient single-base addition efficiency, and is able to add a variety of 3'-O-blocked modified dNTPs substrates to the nucleotides without a template. Adding to the 3'-OH end of a single-stranded oligonucleotide provides a new and effective tool enzyme for enzymatic de novo synthesis of nucleic acids.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a flow chart of the terminal deoxyribonucleoside transferase-dominated enzymatic synthesis scheme.

FIG. 2 is an SDS-PAGE image of the purified protein of the GeTdT mutant.

Figure 3 Urea-PAGE of terminal deoxyribonucleoside transferase products.

FIG4 is a capillary electrophoresis diagram of the transformation products of GeTdT wild type and its E459R and E459R/R460Q mutants.

FIG5 is a Urea-PAGE image of the terminal deoxyribonucleoside transferase product.

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 techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

The overall scheme adopted in the specific embodiments of the present application is: design primers for site-directed mutagenesis → obtain the crude protein solution of TdT mutant through Escherichia coli expression system → obtain purified TdT mutant protein by nickel ion affinity chromatography → test the activity of protein → analyze the product by urea polyacrylamide gel (Urea-PAGE) and capillary electrophoresis (CE).

Example 1

Construction of terminal deoxyribonucleoside transferase GeTdT mutant.

The object of transformation is the terminal deoxyribonucleoside transferase GeTdT from Gecko, whose nucleotide sequence is shown in SEQ ID NO.1 and amino acid sequence is shown in SEQ ID As shown in NO.2, primers containing E459R, E459K, E459Q, E459V, E459R/("/" indicates sum)R460Q, E459R/R460K, E459R/R460E, E459R/R460H, E459R/R460N, E459R/R460D, E459R/R460S, E459R/R460L, E459R/R460M, E459R/R460W, E459R/R460F, E459R/R460Y, E459R/R460C, and E459R/R460G mutations were designed and synthesized according to the sequence, and site-directed mutagenesis of GeTdT was performed. To carry the partial peptide chain encoding GeTdT (amino acids G144 to A512 in SEQ ID NO.2, That is, the recombinant expression plasmid pGS-21a/GeTdT _{(G144—A512)} of the wild-type GeTdT gene described later was used as a template, and the mutation was introduced into the characteristic site by rapid PCR technology using the corresponding mutation primer pair shown in Table 1, and the coding gene of the GeTdT mutant was confirmed to be correct by Sanger sequencing. The complete peptide chain sequence expressed by the recombinant expression plasmid pGS-21a/GeTdT _{(G144—A512)} is shown in SEQ ID NO.3.

Table 1 Corresponding primers for constructing GeTdT mutants

The PCR reaction system was: 10 μL of 5×PS buffer, 4 μL of dNTPs Mix (2.5 mmol/L), 1 μL of forward primer (10 μmol/L), 1 μL of reverse primer (10 μmol/L), 1 μL of template DNA, 0.5 μL of Primer Star HS (Takara, 5 U/μL), and distilled water was added to 50 μL.

The PCR amplification program was set as follows: first, pre-denaturation at 94°C for 5 min; then 21 cycles (denaturation at 98°C for 10 s, annealing at 55°C for 5 s, extension at 72°C for 7 min 50 s); finally, extension at 72°C for 7 min, and insulation at 4°C. PCR products were detected by 1% agarose gel electrophoresis.

Dpn I was added to the PCR product verified by gel electrophoresis, and the template was degraded by water bath at 37°C for 2 hours, and then transformed into Escherichia coli JM109 competent cells (purchased from Tiangen Biochemical Technology Co., Ltd.). The transformation product was spread on LB solid medium containing 100 mg/L ampicillin, cultured at 37°C for 11 hours, and the clones were picked and inoculated into LB liquid medium and cultured at 37°C for 9 hours. The bacterial solution was subjected to Sanger sequencing to verify whether the mutation site was correctly introduced. The clones with correct sequencing were subjected to plasmid extraction and transformed into the expression host Escherichia coli BL21 (DE3) competent cells (purchased from Tiangen Biochemical Technology Co., Ltd.), and 19 recombinant Escherichia coli strains capable of expressing 18 mutants and GeTdT wild-type proteins were obtained.

It should be noted that due to the principle of codon degeneracy, the nucleotide sequence for translating an amino acid sequence is not a unique constant sequence, and any nucleotide sequence that can encode the same amino acid sequence is a nucleic acid sequence within the scope of this patent.

Example 2

Expression and purification of terminal deoxyribonucleoside transferase GeTdT and its mutants.

The 19 recombinant E. coli obtained in Example 1 were inoculated into LB medium respectively, and after culturing at 37°C for 8h, they were transferred to 300mL TB fermentation medium with an inoculation amount of 5% of the volume of the fermentation medium. First, they were placed at 37°C and 200rpm for constant temperature culture for 3h. When the bacterial OD ₆₀₀ was 0.5-0.7, IPTG was added to a final concentration of 1μM and the culture was induced at 25°C and 200rpm for 24h. After the fermentation was completed, the fermentation broth was crushed by high-pressure homogenization (crushing conditions were 4°C and 80MPa) and then centrifuged (12000rpm, 30min, 4°C), and the supernatant was the crude enzyme solution of the terminal deoxyribonucleoside transferase mutant produced by the recombinant bacteria.

The enzyme solution sample prepared in the previous step was subjected to protein purification, as follows: the terminal deoxyribonucleoside transferase protein was purified by nickel ion affinity chromatography. The crude enzyme solution of the terminal deoxyribonucleoside transferase mutant was loaded onto a nickel ion affinity chromatography column (nickel column) respectively. The recombinant protein was purified by gradient elution method: purification buffer A and purification buffer B were used to prepare eluent 1 (containing 0% buffer B), eluent 2 (containing 5% buffer B), eluent 3 (containing 10% buffer B), eluent 4 (containing 20% buffer B), eluent 5 (containing 50% buffer B) and eluent 6 (containing 100% buffer B), and then the protein bound to the nickel column was gradually eluted with the eluents in the order of imidazole concentration from low to high. During the purification process, all eluents were precooled to 4°C to ensure that the recombinant protein maintained biological activity. The eluent 4 containing the purified target protein was ultrafiltered and concentrated using an ultrafiltration tube that could retain 30 kDa protein, and the purified eluent was replaced with 2× enzyme storage buffer. Finally, an equal volume of glycerol was added to the concentrated protein, mixed and stored at -20°C. The purity of the purified recombinant protein was analyzed by SDS-PAGE. In Figure 2, M represents protein standard; lanes 1-19 represent: 1, WT (wild type); 2, E459R; 3, E459K; 4, E459Q; 5, E459V; 6, E459R/R460Q; 7, E459R/R460K; 8, E459R/R460E; 9, E459R/R460H; 10, E459R/R460N; 11, E459R/R460D; 12, E459R/R460S; 13, E459R/R460L; 14, E459R/R460M; 15, E459R/R460W; 16, E459R/R460F; 17, E459R/R460Y; 18, E459R/R460C; 19, E459R/R460G, indicating that a relatively pure protein was obtained.

Purification buffer A (pH 8.0): 20 mM Tris-HCl, 500 mM NaCl, 10 mM imidazole.

Purification buffer B (pH 8.0): 20 mM Tris-HCl, 500 mM NaCl, 500 mM imidazole.

2× enzyme storage buffer (pH 7.2): 40 mM Tris-HCl, 400 mM NaCl, 5% glycerol.

Example 3

Semi-quantitative analysis of the activity of terminal deoxyribonucleoside transferase GeTdT and its mutants.

Using single-stranded Oligo(dT) ₁₈ (SEQ ID NO.40) with a polymerization degree of 18 as an oligonucleotide substrate, and using four nucleotides with azidomethyl modification at the 3' end (3'-O-Azidomethyl-dATP, 3'-O-Azidomethyl-dTTP, 3'-O-Azidomethyl-dCTP, 3'-O-Azidomethyl-dGTP) as nucleic acid monomer substrates (structures shown in Figure 3), the terminal transfer activity of the purified wild-type GeTdT and its mutants was determined. Oligo(dT) _18, an oligonucleotide substrate, was extended by one base under the action of terminal deoxyribonucleoside transferase to obtain the product Oligo(dT) _18- dNTP-3'-O-Azidomethyl. The reaction system for the activity test of the wild-type GeTdT enzyme and its mutants is shown in Table 2. After the reaction system is mixed, it is reacted at 37°C for 10 minutes. After the reaction is completed, it is heated at 95°C for 10 minutes to terminate the reaction. Urea polyacrylamide gel electrophoresis (20% denaturing gel) was used to semi-quantitatively analyze the reaction products of GeTdT wild type and its mutants, and the results are shown in Figure 3. The formula of the 5× reaction buffer used was: 0.5M Na-Cacodylate, 5mM CoCl ₂ , pH 7.2.

Table 2 Activity test reaction system of wild-type GeTdT enzyme and its mutants

In Figure 3, the products were obtained by transforming four nucleotide monomer substrates with azidomethyl modification at the 3' end (3'-O-Azidomethyl-dATP, 3'-O-Azidomethyl-dTTP, 3'-O-Azidomethyl-dCTP, 3'-O-Azidomethyl-dGTP) by GeTdT and its mutants; NC represents Oligo(dT) ₁₈ ; PC represents Oligo(dT) ₁₉ ; Lanes 1-19 represent: 1, WT (wild type); 2, E459R; 3, E459K; 4, E459Q; 5, E459V; 6, E459R/R460Q; 7, E459R/R460K; 8, E459R/R460E; 9, E459R/R460H; 10, E459R/R460N; 11, E459R/R460D; 12, E459R/R460S; 13, E459R/R460L; 14, E459R/ R460M; 15, E459R/R460W; 16, E459R/R460F; 17, E459R/R460Y; 18, E459R/R460C; 19, E459R/R460G. The activities of the GeTdT mutants shown in the figure for the four nucleotides with azidomethyl modification at the 3' end are better than those of the wild type, among which the conversion rates of the mutants E459R, E459R/R460Q, E459R/R460K, E459R/R460E, E459R/R460H, E459R/R460N and E459R/R460D are greatly improved compared with the wild type.

Example 4

Conversion rate determination of terminal deoxyribonucleoside transferase GeTdT and its mutants.

Using 5'-ROX-Oligo(dT) ₉₈ (SEQ ID NO.41) with a single-stranded 5' end modified with ROX fluorescent group with a polymerization degree of 98 as an oligonucleotide substrate, and 3'-O-Azidomethyl-dTTP with an azidomethyl modification at the 3' end as a nucleic acid monomer substrate, the terminal transfer activity of the purified GeTdT wild type and its mutants was determined, and the conversion rate of the reaction product was quantitatively determined by capillary electrophoresis. The oligonucleotide substrate 5'-ROX-Oligo(dT) ₉₈ was extended by one base under the action of terminal deoxyribonucleoside transferase to obtain the product 5'-ROX-Oligo(dT) ₉₉ -3'-O-Azidomethyl. The reaction system for determining the conversion rate of the GeTdT wild type and its mutants is shown in Table 3. After the reaction system is mixed, the reaction is reacted at 37°C for 10 minutes. After the reaction is completed, the reaction is terminated by heating at 95°C for 10 minutes.

Table 3. Reaction system for determining the conversion rate of wild-type GeTdT enzyme and its mutants

The conversion rate of the terminal deoxyribonucleoside transferase reaction product was determined by capillary electrophoresis: the reaction product was diluted 200 times with ddH ₂ O, 1 μL was taken as a sample, and mixed with 9 μL of HiDi formamide mixed with internal standard, and the fragment length was analyzed by Applied Biosystems 3730xl Genetic Analyzer. The conversion rate of the reaction product can characterize the reaction activity, and the calculation formula is: conversion rate = fragment 99 peak area/(fragment 98 peak area + fragment 99 peak area). The activity test results of GeTdT wild type and mutant are shown in Table 4 and Figure 4, 98 represents the peak of substrate 5'-ROX-Oligo(dT) ₉₈ , and 99 represents the peak of product 5'-ROX-Oligo(dT) ₉₉ -3'-O-Azidomethyl.

Table 4 Activity test results of wild-type GeTdT enzyme and its mutants

The results show that the activities of the GeTdT mutants listed in the table towards 3’-O-Azidomethyl-dTTP are better than those of the wild type, and the conversion rates are increased from 45% of the wild type to more than 50%. Among them, the mutants E459R, E459R/R460Q, E459R/R460K, E459R/R460E, E459R/R460H, E459R/R460N and E459R/R460D are better mutants, with conversion rates greater than 90%.

Example 5

Activity testing of terminal deoxyribonucleoside transferase GeTdT and its mutants towards different 3’-O-blocked modified dTTPs.

Using single-stranded Oligo(dT) ₁₈ (SEQ ID NO.40) with a degree of polymerization of 18 as an oligonucleotide substrate, and using four nucleotides (3'-O-Amide-dTTP, 3'-O-Allyl-dTTP, 3'-O-Methy-dTTP, 3'-O-Oxime-dTTP) with different protective groups (PG) modified at the 3' end as nucleic acid monomer substrates (structures shown in Figure 5), the terminal transfer activity of the purified wild-type GeTdT and its mutants was determined. Oligo(dT) ₁₈ , an oligonucleotide substrate, was extended by one base under the action of terminal deoxyribonucleoside transferase to obtain the product Oligo(dT) ₁₉ -3'-O-PG. The reaction system for the terminal transfer activity test of the wild-type GeTdT enzyme and its mutants is shown in Table 5. After the reaction system is mixed, it is reacted at 37°C for 10 minutes. After the reaction is completed, it is heated at 95°C for 10 minutes to terminate the reaction. The reaction products of GeTdT wild type and its mutants were semi-quantitatively analyzed by urea polyacrylamide gel electrophoresis (20% denaturing gel). The results are shown in FIG5 . The products were obtained by GeTdT and its mutants transforming four nucleotide monomer substrates with different 3'-end protective groups (Amide, Allyl, Methy and Oxime). NC represents Oligo(dT) ₁₈ ; Lanes 1-3 represent: 1, WT; 2, E459R; 3, E459R/R460E. The 5× reaction buffer used in this application is formulated as: 0.5M Na-Cacodylate, 5mM CoCl ₂ , pH 7.2.

Table 5. Terminal transfer activity test reaction system of wild-type GeTdT enzyme and its mutants

The results show that the activity of the GeTdT mutant shown in Figure 5 towards the four nucleotides modified with different protecting groups at the 3' end is better than that of the wild type.

In summary, the present application obtains advantageous TdT mutants by molecularly modifying natural TdT, thereby greatly improving the catalytic efficiency of modified nucleic acid monomers and achieving efficient single-base addition efficiency.

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 terminal deoxyribonucleoside transferase mutant, the amino acid sequence of which includes any one of the following sequences: (1) the following mutations occur based on the sequence shown in SEQ ID NO:2: any one or at least two of the following sequences: E459R, E459K, E459Q, E459V, R460Q, R460K, R460E, R460H, R460N, R460D, R460S, R460L, R460M, R460W, R460F, R460Y, R460C or R460G; or, (2) a 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) A sequence having at least 90% sequence homology with the sequence described in (1) or (2) and having the same or similar functions as the sequence described in (1). The terminal deoxyribonucleoside transferase mutant according to claim 1, wherein (1) the mutation comprises any one of a combination of E459R and R460Q, a combination of E459R and R460K, a combination of E459R and R460E, a combination of E459R and R460H, a combination of E459R and R460N, a combination of E459R and R460D, a combination of E459R and R460S, a combination of E459R and R460L, a combination of E459R and R460M, a combination of E459R and R460W, a combination of E459R and R460F, a combination of E459R and R460Y, a combination of E459R and R460C or a combination of E459R and R460G. A nucleic acid molecule encoding the glycosyltransferase mutant according to claim 1. A recombinant vector comprising the nucleic acid molecule according to claim 3. A recombinant cell comprising the recombinant vector according to claim 3. The method for preparing the terminal deoxyribonucleoside transferase mutant according to claim 1 or 2, comprising: Inserting a nucleic acid molecule encoding the terminal deoxyribonucleoside transferase mutant according to claim 1 or 2 into an expression vector to obtain a recombinant vector, and introducing the recombinant vector into a host cell, or directly integrating the nucleic acid molecule into the genome of the host cell to obtain a genetically engineered bacterium, and culturing and separating and purifying the bacterium to obtain the terminal deoxyribonucleoside transferase mutant. Use of the terminal deoxyribonucleoside transferase mutant according to claim 1 or 2 in the preparation of a product for nucleic acid synthesis. A product for nucleic acid synthesis, comprising the terminal deoxyribonucleoside transferase mutant according to claim 1 or 2. Use of the terminal deoxyribonucleoside transferase mutant according to claim 1 or 2 in nucleic acid synthesis. A method for synthesizing nucleic acid, comprising: The terminal deoxyribonucleoside transferase mutant according to claim 1 or 2 is used to catalyze the connection of dNTPs to the 3'-OH end of a single-stranded oligonucleotide.