T4 DNA ligase variants, their applications and kits

Abstract

The present invention provides a T4 DNA ligase variant, which is formed by deleting a part of the sequence of T4 DNA ligase. The deleted sequence is located within a Loop structure of wild-type T4 DNA ligase, and experimental results confirm that the thermal stability is effectively improved. On the basis of deleting some amino acid residues from wild-type T4 DNA ligase, the present invention further mutates to further improve the performance of T4 DNA ligase, including the improvement of one or at least two of the following characteristics: improved thermal stability, increased specific activity, reduced ligation ratio of adapter-adapter, increased DNA library construction yield, etc.

Description

Technical Field

[0001] The patent of this invention relates to a T4 DNA ligase variant, its application and kit, and belongs to the field of biotechnology. Background Art

[0002] DNA ligases, which seal phosphodiester bonds in broken nucleic acids, are essential enzymes in living organisms and are indispensable tools in life science research. T4 DNA ligase is the most commonly used DNA ligase. With the help of the ATP cofactor, T4 DNA ligase catalyzes the formation of diester bonds between 5'-phosphate and 3'-hydroxyl groups in duplex DNA or RNA, thereby repairing single-stranded gaps in duplex DNA, RNA, or DNA / RNA hybrids. It has a wide range of applications, including the ligation of DNA fragments in molecular cloning, automated circularization of linear DNA, and the ligation of DNA to adapter sequences in next-generation sequencing (NGS) sequencing.

[0003] Wild-type T4 DNA ligase, a gene-encoded product of the T4 bacteriophage, is a single-chain polypeptide with a molecular weight of approximately 55.23 kDa, composed of 487 amino acid residues, and a gene length of 1464 base pairs. Although T4 DNA ligase offers superior overall performance compared to other DNA ligases, existing T4 DNA ligases suffer from poor stability, with activity gradually decreasing over time. This leads to reduced ligation products during molecular cloning and reduced NGS library yield. Furthermore, during NGS sequencing, when T4 DNA ligase is used to connect DNA fragments to adapters, adapter-adapter self-ligation and DNA fragment-fragment self-ligation often occur. This results in fewer products of correctly ligated DNA fragments and adapters, ultimately reducing NGS library yield. Summary of the Invention

[0004] The object of the present invention is to provide a T4 DNA ligase variant with improved thermostability.

[0005] The technical solution adopted in the present invention is:

[0006] A T4 DNA ligase variant, which is a protein described in any one of the following a1-a3:

[0007] a1: based on the amino acid sequence of T4 DNA ligase as shown in SEQ ID No.1, at least one amino acid residue among amino acid residues at positions 220-254 is deleted; or based on the amino acid sequence of T4 DNA ligase as shown in SEQ ID No.1, 6-18 amino acid residues among amino acid residues at positions 220-254 are deleted; or based on the amino acid sequence of T4 DNA ligase as shown in SEQ ID No.1, at least one amino acid residue among amino acid residues at positions 224-247 is deleted; or based on the amino acid sequence of T4 DNA ligase as shown in SEQ ID No.1, 6-18 amino acid residues among amino acid residues at positions 224-247 are deleted;

[0008] a2: a protein having substantially the same enzymatic activity and performance obtained by replacing and / or deleting and / or adding one or more amino acid residues in the amino acid sequence shown in a1 except for the aforementioned mutation;

[0009] a3: A protein that has at least 90% sequence identity with the protein of a1 and has enzyme activity and performance that are substantially equivalent to those of the protein of a1.

[0010] In some embodiments of the present invention, the T4 DNA ligase variant provided by the present invention deletes 6AA, 12AA, 18AA or 24AA on the basis of the amino acid sequence of the T4 DNA ligase shown in SEQ ID No.1. In some embodiments of the present application, the T4 DNA ligase variant provided by the present application deletes amino acid residues 233-238, or deletes amino acid residues 230-241, or deletes amino acid residues 227-244, or deletes amino acid residues 224-247 on the basis of the amino acid sequence of the T4 DNA ligase shown in SEQ ID No.1. Deleting part of the amino acid residues in the present application refers to the deletion of the corresponding part of the amino acid residues relative to the wild-type sequence to form a truncated protein. Preferably, when the T4 DNA ligase variant provided by the present invention deletes amino acid residues 230-241 or deletes amino acid residues 233-238 on the basis of the amino acid sequence of the T4 DNA ligase shown in SEQ ID No.1, the thermal stability is significantly improved.

[0011] Preferably, one or more of the following mutations are made based on the above protein: 16, 19, 40, 51, 117, 118, 148, 160, 163, 207, 251, 255, 296, 332, 333, 334, 338, 343, 371, 383, 391, 402, 403, 404, 405, 448, 449, 458, 470, 472, 486, 487. Among them, 117, 118, 160, 163, 255, 383, 391, 402, 403, 449, 457, 458. These sites are DNA substrates in the structure of T4 DNA ligase. Experimental results show that mutations at these sites can significantly improve the performance of T4 DNA ligase variants.

[0012] Preferably, one or more of the following mutations are made based on the above protein: K16E, Q19K or Q19E, S40D, P51S, A117S, S118T, K148E, A160Y, A163E, I207V, A251P, T255Y or T255W, F296S, K332E, V333K, I334F, Y338A, L343A or L343F Or L343H, D371C or D371W, R383S, G391C, K402V, V403G, N404K, A405V, D448G or D448T or D448R or D448Y or D448M or D448V or D448C or D448L, G449D, L458A or L458F, K470R, N472D, G486V, L487R.

[0013] In some embodiments of the present invention, one or more of the following mutations are made based on the aforementioned proteins: 40, 117, 332, 333, 334, 343, 371, 383, 391, 402, 403, 404, 405.

[0014] Preferably, one or more of the following mutations are made based on the aforementioned protein: S40D, A117S, K332E, V333K, I334F, L343A or L343F or L343H, D371C or D371W, R383S, G391C, K402V, V403G, N404K, A405V.

[0015] Preferably, it is a protein described in any one of the following b1-b3:

[0016] b1: A protein obtained by modifying the amino acid sequence of the T4 DNA ligase variant shown in SEQ ID No. 1 by any combination of the following changes:

[0017] 1)del233-238;

[0018] 2)del230-241;

[0019] 3)del227-244;

[0020] 4)del224-247;

[0021] 5)A117S / del230-241;

[0022] 6)A117S / del230-241 / D371C;

[0023] 7)A117S / del230-241 / R383S;

[0024] 8)A117S / del230-241 / S40D;

[0025] 9)del233-238 / R383S;

[0026] 10)del233-238 / L343H / K402V / V403G / N404K / A405V;

[0027] 11)del233-238 / R383S / S40D;

[0028] 12)del233-238 / R383S / A117S;

[0029] 13)del233-238 / R383S / D371C;

[0030] 14)del227-244 / K332E / V333K / I334F / D371C / G391C;

[0031] 15)del227-244 / L343H / R383S;

[0032] 16)A117S / del230-241 / R383S / D371C;

[0033] 17)A117S / del230-241 / S40D / D371C;

[0034] 18)A117S / del230-241 / S40D / R383S;

[0035] 19)del233-238 / R383S / D371C / A117S;

[0036] 20)del233-238 / R383S / S40D / A117S;

[0037] 21)del233-238 / R383S / D371C / S40D;

[0038] 22)A117S / del230-241 / S40D / R383S / D371C;

[0039] 23) del233-238 / R383S / D371C / A117S / S40D;

[0040] b2: a protein having substantially the same enzymatic activity and properties obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in b1 in addition to the aforementioned changes;

[0041] b3: A protein that has at least 90% sequence identity with the protein of b1 and has enzyme activity and performance that are substantially equivalent to those of the protein of b1.

[0042] The description of variants herein is a description recognized by those skilled in the art. For example, the sequence of one variant, A117S / del230-241, refers to a mutation of alanine (A) at position 117 to serine (S) in the amino acid sequence set forth in SEQ ID NO: 1, and deletion of amino acid residues 230 to 241, i.e., glutamic acid (E) at position 229 is linked to serine (S) at position 242. For ease of comparison with the wild-type, the amino acid code of the present invention is still based on the sequence of the wild-type T4 DNA ligase.

[0043] The T4 DNA ligase mutants provided herein also include amino acid sequences having at least 90% sequence identity to the deleted sequence or mutant sequence, or at least 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.8% sequence identity to the deleted sequence or mutant sequence.

[0044] As used herein, identity refers to amino acid sequence or nucleotide sequence identity. Percent sequence identity can be calculated by any method known in the art, for example using the BLOSUM62 matrix, as described by Henikoff et al. in PNAS, 89(22): 10915-10919 (1992).

[0045] As used herein, "substantially the same" or "substantially equivalent" means that under identical testing conditions, the enzymatic activity and performance values ​​of the T4 DNA ligase variants deviate by no more than 20%. In some embodiments of the present invention, performance testing includes testing for thermal stability, specific activity, and adapter-adapter self-ligation.

[0046] The gene encoding the aforementioned T4 DNA ligase variant.

[0047] Expression vectors for the aforementioned T4 DNA ligase variants.

[0048] Host bacteria for the aforementioned T4 DNA ligase variants.

[0049] Use of the aforementioned T4 DNA ligase variants in DNA-DNA, DNA-RNA, and RNA-RNA ligations.

[0050] The present invention also discloses a kit containing the aforementioned T4 DNA ligase variant.

[0051] The present invention also discloses a sequencing library construction kit containing the aforementioned T4 DNA ligase variant.

[0052] T4 DNA ligase is an ATP-dependent ligase. The crystal structure of the complex between the enzyme and DNA shows that it has a DNA binding domain (DBD), a nucleotidyl transferase (NTase) domain, and an OB fold domain. Figure 1 This schematic diagram of the spatial folding of T4 DNA ligase provides a basic approach for its modification. This study targets the stability of T4 DNA ligase while also evaluating its performance, such as linker / fragment self-ligation and specific activity. By combining multiple protein modification approaches, we have achieved superior mutants with significantly improved stability and other properties.

[0053] Specifically, the present invention deletes a portion of the T4 DNA ligase sequence, located within a loop structure of the wild-type T4 DNA ligase. Experimental results confirm that this deletion effectively improves thermal stability. Furthermore, by performing further mutations based on the deletion of some amino acid residues in the wild-type enzyme, the present invention further improves the performance of T4 DNA ligase, including improvements in one or more of the following properties: increased thermal stability, increased specific activity, reduced linker-linker self-ligation ratio, and increased DNA library yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 Schematic diagram of the simulated spatial folding of wild-type T4 DNA ligase.

[0055] Figure 2 The middle shows the stability data of mutants generated after truncating the key loop.

[0056] Figure 3 The stability data of mutants generated by further modification methods such as random mutagenesis, DNA shuffling and site-directed combination. DETAILED DESCRIPTION

[0057] The specific embodiments of the present invention will be further described below with reference to the accompanying drawings, but the description of the embodiments does not limit the scope of protection of the present invention in any way.

[0058] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention pertains. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0059] Unless otherwise specified, the materials and instruments used in the following examples can be obtained from conventional commercial channels.

[0060] Example 1: Transformation and acquisition of mutant T4 DNA ligase

[0061] Wild-type T4 DNA ligase has drawbacks such as mild reaction conditions, temperature sensitivity, easy inactivation, instability, poor storage stability, and low DNA yield in NGS library construction applications. We have discovered that deleting a portion of the T4 DNA ligase sequence significantly improves its thermal stability. The deleted sequence is located within a loop structure of the wild-type T4 DNA ligase, and experimental results confirm that this effectively improves thermal stability. The loop structure is a common secondary structure in T4 DNA ligase, and T4 DNA ligase contains multiple loop structures. Structural analysis of T4 DNA ligase revealed the presence of approximately 28 loop structures. After comprehensively considering the functions of the loop structures, particularly whether they affect catalytic activity and DNA binding ability, we found that loop 220-254 is a domain that is independent of the core structure and is not expected to directly affect its structure. Furthermore, based on protein structural analysis, flexible loop structures typically have a high B-factor. Deleting the loop structure is likely to reduce the B-factor of the local structure and improve protein stability.

[0062] We used the ZymeEditor platform to modify the wild-type T4 DNA ligase protein. On the basis of the wild-type T4 DNA ligase, we adopted a method combining rational design and directed evolution for modification. Based on the analysis of the T4 DNA ligase protein structure, we first performed truncation deletions of varying lengths on Loop220-254. Mutants with better stability were selected as the starting parent for random mutagenesis for directed stability screening. We constructed a library of more than 10,000 random mutations. After determining the mutants with further improved stability, DNA Shuffling screening was performed, and finally the potential advantageous mutants were subjected to combined mutations to comprehensively judge the various aspects of the mutant's performance, such as stability, specific activity, linker-linker self-ligation ratio, and DNA library construction yield. The amino acid mutation types and numbers of the advantageous mutants obtained are shown in Table 1 below:

[0063] Table 1 Variants and types of changes

[0064]

[0065]

[0066] Example 2: Thermal stability assay of T4 DNA ligase variants

[0067] Dilute the T4 DNA ligase variant to 50 U / μL according to the enzyme activity, incubate at 42°C or 45°C for 0 or 10 min, and then add it to the following activity test reaction system:

[0068] name Volume / μL FAM DNA (5 μM) 1 BHQ-1 DNA (5 μM) 1 10×ligase buffer 10 T4 DNAligase 2 <![CDATA[ddH2O]]> 86

[0069] The fluorescence values ​​at 0 min and 10 min of incubation were monitored, and the thermal stability of the T4 DNA ligase variants was characterized by calculating the ratio of the fluorescence decrease.

[0070] Figure 2 The stability data of variants generated by truncating the key loop through a rational design approach are provided in the literature, which compares the residual activity values ​​of the parent and variants measured after incubation at 42°C for 10 minutes.

[0071] Figure 3 The authors present stability data for variants generated through further engineering methods, including random mutagenesis, DNA shuffling, and site-directed combination. This data compares the residual activity values ​​of the parent and variants after incubation at 45°C for 10 minutes. A higher residual activity value indicates greater thermal stability. Deletion of a portion of the sequence from 220 to 254 AA significantly improved stability compared to the wild-type. Further mutagenesis further enhanced the thermal stability of the resulting mutant.

[0072] Thermal stability testing at 42°C or 45°C is a common method used by those skilled in the art to study the thermal stability of T4 DNA ligase and its variants. Although the thermal stability testing in this application was conducted at 42°C or 45°C, this does not mean that the maximum heat resistance temperature of the variants in this application is 42°C or 45°C. A higher residual activity value indicates higher thermal stability and the ability to tolerate higher reaction temperatures and / or longer reaction times.

[0073] Example 3: Determination of T4 DNA ligase variant enzyme activity

[0074] The principle of T4 DNA ligase activity detection is to use a DNA fragment with a fluorescent group (FAM-DNA) and a DNA fragment with a fluorescence quencher (BHQ-1DNA) as substrates. In the presence of DNA ligase, the two DNA fragments are connected, and the fluorescence intensity gradually decreases. In other words, there is a certain relationship between fluorescence intensity and DNA ligase. The enzyme activity of T4 DNA ligase is detected by measuring the fluorescence intensity. The specific implementation method is as follows:

[0075] (1) Substrate preparation

[0076] FAM-DNA was prepared by mixing equal volumes of primers A (sequence: 5'-TAG / i6FAMdT / ACACTGTCCTCATTG-3') and B (sequence: 5'-CAATGAGGACAGTGT-3') and diluting them with TE buffer to a final concentration of 5 μM. The mixture was incubated at 37°C for 5 minutes, then incubated at room temperature for 10 minutes and stored at -20°C until use. BHQ-1 DNA was prepared by mixing equal volumes of primers C (sequence: 5'-CTCCTCGTTCATCTAC-3') and D (sequence: 5'-ACTAG / iBHQ1dT / AGATGAACGAGGAG-3') and prepared according to the FAM-DNA preparation method to a final concentration of 5 μM.

[0077] (2) Preparation of reaction system

[0078] name Volume / μL FAM DNA (5 μM) 1 BHQ-1 DNA (5 μM) 1 10×ligase buffer 10 T4 DNAligase 2 <![CDATA[ddH2O]]> 86

[0079] Add various concentrations of T4 DNA ligase to the reaction system, mix thoroughly, incubate at 25°C for 30 minutes, and read the fluorescence at 485nm / 535nm. The activity of the T4 DNA ligase mutant can be calculated based on the relationship between the fluorescence value and the activity of the T4 DNA ligase with known activity.

[0080] Table 2 provides a table of activities of T4 DNA ligase variants of specific sequences disclosed herein. In Table 2 below, the sequence numbers refer to the series of sequences following Table 1, where V0 refers to the unmodified parent, i.e., the wild-type sequence shown in SEQ ID No. 1, and V1-V23 are mutants obtained by modification based on V0. The modification type column provides modification strategies for obtaining different mutants. In the specific activity column, a single plus sign "+" indicates that the specific activity of the variant protein is 120% to 150% of the specific activity of the protein composed of the amino acid sequence represented by SEQ ID No. 1 in the sequence listing, i.e., 120% ≤ variant specific activity / wild-type specific activity < 150%. Two plus signs "++" indicate that the specific activity of the variant protein is 150% to 200% of the specific activity of the protein composed of the amino acid sequence represented by SEQ ID No. 1 in the sequence listing, i.e., 150% ≤ variant specific activity / wild-type specific activity < 200%. Three plus signs "+++" indicate that the specific activity of the variant protein is 200% to 250% of the specific activity of the protein composed of the amino acid sequence represented by SEQ ID No. 1 in the sequence listing, i.e., 200% ≤ variant specific activity / wild-type specific activity < 250%. Four plus signs "++++" indicate that the specific activity of the variant protein is 250% to 400% of the specific activity of the protein composed of the amino acid sequence represented by SEQ ID No. 1 in the sequence listing, i.e., 250% ≤ variant specific activity / wild-type specific activity < 400%.

[0081] Table 2 Variant specific activity values

[0082]

[0083]

[0084] Example 4: T4 DNA ligase variant linker residue determination

[0085] In this example, ultrasonically sheared calf thymus DNA was used as a fragmentation template. The YEASEN DNA Library Construction Kit (Cat. No. 12201) was then used to construct the library. The yield and adapter residue were then monitored using Qubit and Qsep. The specific implementation is as follows:

[0086] (1) End repair / dA tail addition

[0087]

[0088] The amplification procedure is as follows:

[0089] Temperature (heat cover 105℃) time 30℃ 30min 72℃ 30min 4℃ Hold

[0090] (2) Add connector

[0091]

[0092] The amplification procedure is as follows:

[0093] Temperature (heat cover off) time 20℃ 15min 4℃ Hold

[0094] (3) Magnetic bead purification (1×)

[0095] 1) Preparation: Hieff Remove DNA Selection Beads (12601) magnetic beads from the refrigerator and equilibrate at room temperature for at least 30 minutes. Prepare 80% ethanol.

[0096] 2) Vortex or invert the beads thoroughly to ensure thorough mixing.

[0097] 3) Pipette 110 μL Hieff Add DNA Selection Beads to the Adapter Ligation product, vortex or pipette gently to mix thoroughly, and incubate at room temperature for 5 minutes.

[0098] 4) Briefly centrifuge the PCR tube and place it on a magnetic rack to separate the beads and liquid. After the solution has clarified (approximately 5 minutes), carefully remove the supernatant.

[0099] 5) Keep the PCR tube in the magnetic rack and add 200 μL of freshly prepared 80% ethanol to rinse the magnetic beads. Incubate at room temperature for 30 seconds and carefully remove the supernatant.

[0100] 6) Repeat step 5 for a total of two rinses. Finally, use a 10 μL pipette tip to remove any remaining liquid.

[0101] 7) Keep the PCR tube in the magnetic rack at all times, open the lid and air-dry the magnetic beads until they just crack (no more than 5 minutes).

[0102] 8) Remove the PCR tube from the magnetic rack and directly add 21 μL of ddH2O. Vortex or gently pipette to mix thoroughly. Let stand at room temperature for 5 minutes. Briefly centrifuge the PCR tube and place it on the magnetic rack. Once the solution has cleared (approximately 5 minutes), carefully transfer 20 μL of the supernatant to the PCR tube, being careful not to touch the magnetic beads.

[0103] (4) Library amplification system

[0104] name Input Adapter Ligated DNA 20 μL 12201-E 25 μL Primer (25 μM) (12201-F) 5μL

[0105] Library amplification procedure

[0106] temperature time Number of cycles 98℃ 1min 98℃ 10s 12 60℃ 30s 72℃ 30s 72℃ 5min 4℃ Hold

[0107] (5) Magnetic bead purification (0.5×)

[0108] The purification steps are the same as those in step (3). The amplified product of the library was purified using DNA Selection Beads (0.5×, Beads:DNA=0.5:1) and eluted with 30 μL ddH2O.

[0109] (6) Concentration test

[0110] Before use, bring all components of the 1× dsDNA HS Assay Kit (YEASEN Catalog No. 12642) to room temperature and mix thoroughly by inverting the tube. Perform the Qubit calibration according to the kit instructions.

[0111] Transfer 199 μL of 1× dsDNA assay solution to a 0.5 mL thin-walled centrifuge tube. Add 1 μL of the sample to be tested. Gently vortex for 2-3 seconds, avoiding bubbles. Read using a Qubit.

[0112] (7) Joint residue test

[0113] Based on the concentration tested by Qubit, the library yields of different T4 DNA ligase variants were diluted to 2 ng / μL, and the percentage of residual linkers was obtained using a Qsep analyzer.

[0114] Table 3 provides the results of an analysis of the adapter-to-adapter self-ligation ratio during NGS library construction using T4 DNA ligase. The lower the adapter-to-adapter self-ligation ratio, the more favorable it is for NGS library construction and the higher the yield of NGS library construction. In Table 3 below, the sequence numbers refer to a series of sequences in Table 1; in the adapter-to-adapter self-ligation ratio column, a minus sign "-" indicates that the adapter-to-adapter self-ligation ratio of the variant protein is higher than the adapter-to-adapter self-ligation ratio of the protein composed of the amino acid sequence represented by SEQ ID No. 1 in the sequence listing. A plus sign "+" indicates that the adapter-to-adapter self-ligation ratio of the mutant protein is comparable to the adapter-to-adapter self-ligation ratio of the protein composed of the amino acid sequence represented by SEQ ID No. 1 in the sequence listing, and is within ±10% of the adapter-to-adapter self-ligation ratio of the protein composed of the amino acid sequence represented by SEQ ID No. 1 in the sequence listing. Two plus signs "++" indicate that the adapter-to-adapter self-ligation ratio of the mutant protein is reduced by 20%-50% compared to the adapter-to-adapter self-ligation ratio of the protein composed of the amino acid sequence represented by SEQ ID No. 1 in the sequence listing. Three plus signs "+++" indicate that the linker-linker self-ligation ratio of the mutant protein is reduced by 50%-100% compared with the linker-linker self-ligation ratio of the protein composed of the amino acid sequence represented by SEQ ID No. 1 in the sequence listing.

[0115] Table 3 Analysis results of the self-connection ratio of variant joints-joints

[0116]

[0117] The applicant states that the present invention is intended to illustrate the detailed methods of the present invention through the above-described embodiments, but the present invention is not limited to the above-described detailed methods, that is, it does not mean that the present invention must rely on the above-described detailed methods in order to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions for various raw materials in the products of the present invention, addition of auxiliary ingredients, and selection of specific methods, etc., are all within the scope of protection and disclosure of the present invention.

Claims

1. A T4 DNA ligase variant, characterized in that: The protein described in b1 below: b1: A protein obtained by making any of the following changes based on the amino acid sequence of T4 DNA ligase shown in SEQ ID No. 1: 1)del233-238; 2)del230-241; 3)del227-244; 4)del224-247; 5) A117S / del230-241; 6)A117S / del230-241 / D371C; 7)A117S / del230-241 / R383S; 8)A117S / del230-241 / S40D; 9)del233-238 / R383S; 10) del233-238 / L343H / K402V / V403G / N404K / A405V; 11)del233-238 / R383S / S40D; 12)del233-238 / R383S / A117S; 13)del233-238 / R383S / D371C; 14)del227-244 / K332E / V333K / I334F / D371C / G391C; 15)del227-244 / L343H / R383S; 16)A117S / del230-241 / R383S / D371C; 17)A117S / del230-241 / S40D / D371C; 18)A117S / del230-241 / S40D / R383S; 19)del233-238 / R383S / D371C / A117S; 20) del233-238 / R383S / S40D / A117S; 21)del233-238 / R383S / D371C / S40D; 22)A117S / del230-241 / S40D / R383S / D371C; 23)del233-238 / R383S / D371C / A117S / S40D.

2. The T4 DNA ligase variant according to claim 1, characterized in that The variant comprises one or at least two of the following characteristics: improved thermal stability, improved specific activity, reduced linker-linker self-ligation ratio, or improved DNA library construction yield.

3. The coding gene, expression vector or expression host bacteria of the T4 DNA ligase variant according to claim 1 or 2.

4. Use of the T4 DNA ligase variant according to claim 1 or 2 in ligating DNA-DNA, DNA-RNA or RNA-RNA.

5. A kit, characterized in that Contains the T4 DNA ligase variant according to claim 1 or 2.

6. A sequencing library construction kit, characterized in that Contains the T4 DNA ligase variant according to claim 1 or 2.

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