Terminal deoxynucleic acid transferase mutant and application thereof
Patent Information
- Application Number
- CN202311863971.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-12-29
- Publication Date
- 2025-07-01
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biocatalysis. More specifically, the present invention relates to a terminal deoxynucleotidyl transferase mutant and its application. Background Art
[0002] DNA polymerases are important guardians of the genome during the orderly genetic and evolutionary processes of living organisms. Most DNA polymerases have high accuracy when performing genome replication and repair tasks. However, in order to ensure the orderly genetic and evolution of living organisms, low-conservation DNA polymerases have emerged. These low-conservation DNA polymerases greatly improve the flexibility of the templates they can utilize in some aspects, and they can replicate severely damaged or even completely informationless DNA fragments. Terminal deoxynucleotidyl transferase (TdT) is such a unique DNA polymerase. TdT is a template-independent DNA polymerase that can randomly add multiple deoxynucleoside triphosphates (dNTPs) to the protruding or blunt 3'-OH ends of DNA. This property makes TdT a general tool for detecting target DNA, RNA, metal ions, DNA-modifying enzymes, and apoptotic cells, and TdT can also be used for rapid amplification of cDNA ends.
[0003] Currently, based on the property that terminal deoxynucleotidyl transferase can extend the ends of template nucleic acid strands, a series of biosensing technologies have been constructed by integrating different signal output and amplification methods, such as electrochemical biosensors, fluorescence biosensors, surface plasmon resonance biosensors, etc. A series of biosensors designed according to the properties of TdT enzyme have the advantages of simplicity, rapidity, low cost, high sensitivity, good specificity, etc., and have realized the detection of metal ions, pathogens, proteins, etc.
[0004] However, the activity of wild-type terminal deoxynucleotidyl transferase (TdT) is low, and it cannot rapidly and efficiently extend the 3'-OH ends of single-stranded DNA, which is restricted when TdT extends the nucleic acid DNA end sequence. Summary of the Invention
[0005] The object of the present invention is to provide a terminal deoxynucleotidyl transferase mutant. Compared with the wild-type terminal deoxynucleotidyl transferase, the amino acids interacting with the substrate in its spatial structure are mutated, and its catalytic activity is changed. Therefore, it can efficiently catalyze the extension of the 3'-OH ends of single-stranded DNA with a high reaction yield.
[0006] In the first aspect of the present invention, a terminal deoxynucleotidyl transferase is provided. This terminal deoxynucleotidyl transferase:
[0007] (a) Compared with the amino acid sequence shown in SEQ ID NO: 1, its amino acid sequence has substitution mutations at at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or all 10 positions among the 16th, 187th, 390th, 421st, 422nd, 425th, 450th, 456th, 477th and 486th positions;
[0008] (b) is a protein derived from (a), which is formed by deletion, insertion and / or substitution of one or more amino acid residues in the amino acid sequence of (a), retains at least one substitution mutation described in (a), and has terminal deoxynucleotidyl transferase function;
[0009] (c) is a protein derived from (a), which has more than 80% identity with the amino acid sequence of (a) and has terminal deoxynucleotidyl transferase function, and this protein retains at least one substitution mutation described in (a).
[0010] In the second aspect of the present invention, there is provided a polynucleotide molecule, which has:
[0011] (1) a polynucleotide sequence encoding the terminal deoxynucleotidyl transferase according to any embodiment of the present invention; and
[0012] (2) the complementary sequence of the polynucleotide sequence in (1).
[0013] In the third aspect of the present invention, there is provided a nucleic acid construct, which contains the polynucleotide molecule according to any embodiment of the present invention; preferably, the nucleic acid construct is an expression cassette or a vector, such as a cloning vector or an expression vector.
[0014] In the fourth aspect of the present invention, there is provided a host cell, which: (1) expresses the terminal deoxynucleotidyl transferase according to any embodiment of the present invention, and / or (2) contains the polynucleotide molecule according to any embodiment of the present invention or the nucleic acid construct according to any embodiment of the present invention; preferably, the host cell is Escherichia coli.
[0015] In the fifth aspect of the present invention, there is provided an enzyme preparation, which contains the terminal deoxynucleotidyl transferase according to any embodiment of the present invention.
[0016] In the seventh aspect of the present invention, there is provided a method for preparing an extended oligonucleotide chain using oligonucleotide and dNTP as substrates in the absence of a template strand, the method comprising: using the terminal deoxynucleotidyl transferase according to any embodiment of the present invention to catalyze the reaction of oligonucleotide and dNTP, thereby adding dNTP to the 3'OH end of the oligonucleotide; wherein, the oligonucleotide is a single-stranded DNA with a length of 15 - 20bp.
[0017] In the eighth aspect of the present invention, a method for screening terminal deoxynucleotidyl transferase with improved function is provided. The method includes: preparing a terminal deoxynucleotidyl transferase mutant having substitution mutations at at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or all 10 positions among the 16th, 187th, 390th, 421st, 422nd, 425th, 450th, 456th, 477th, and 486th positions of its amino acid sequence relative to SEQ ID NO: 1, testing the terminal deoxynucleotidyl transferase function of the prepared mutant, and selecting a mutant whose terminal deoxynucleotidyl transferase function is higher than that of the terminal deoxynucleotidyl transferase shown in SEQ ID NO: 1.
[0018] Other aspects of the present invention will be apparent to those skilled in the art from the disclosure herein. Brief Description of the Drawings
[0019] Figure 1 It is a schematic diagram of the reaction of terminal deoxynucleotidyl transferase with a 15bp oligonucleotide probe substrate (SEQ ID NO: 2).
[0020] Figure 2 It is a schematic diagram of the reaction of terminal deoxynucleotidyl transferase with a 20bp oligonucleotide probe substrate (SEQ ID NO: 3).
[0021] Figure 3 It is a high-performance liquid chromatography diagram of the reaction solution after the reaction of terminal deoxynucleotidyl transferase mutant 89 with a 15bp oligonucleotide probe substrate (SEQ ID NO: 2). n represents the 15bp oligonucleotide probe substrate, and A represents the extended 1-nucleotide fragment.
[0022] Figure 4 It is a high-performance liquid chromatography diagram of the reaction solution after the reaction of terminal deoxynucleotidyl transferase mutant 89 with a 20bp oligonucleotide probe substrate (SEQ ID NO: 3). n represents the 20bp oligonucleotide probe substrate, and A represents the extended 1-nucleotide fragment.
[0023] Figure 5 It is a sequencing result diagram of mutant 89. Detailed Description of the Embodiments
[0024] In order to solve the technical problem that the wild-type terminal deoxynucleotidyl transferase has low activity and cannot rapidly and efficiently extend the 3'-OH end of single-stranded DNA, the present inventors have conducted in-depth research and provided a terminal deoxynucleotidyl transferase mutant. Compared with the wild-type terminal deoxynucleotidyl transferase, the amino acids interacting with the substrate in its spatial structure are mutated, and its catalytic activity is changed. Therefore, it can efficiently catalyze the extension of the sequence at the 3'-OH end of single-stranded DNA with a high reaction yield.
[0025] Terminal deoxynucleotidyl transferase mutant
[0026] Unless otherwise specified, as used herein, "terminal deoxynucleotidyl transferase mutant", "TdT mutant", "TdT enzyme mutant", "mutant TdT", "mutant TdT enzyme" can be used interchangeably and refer to a polypeptide corresponding to the wild-type terminal deoxynucleotidyl transferase (amino acid sequence shown in SEQ ID NO: 1, nucleotide sequence shown in SEQ ID NO: 5) with mutations. Specifically, the mutated sites can be selected from any one, any two, any three, any four, any five, any six or more, any seven, any eight, any nine or all ten positions of positions 16, 187, 390, 421, 422, 425, 450, 456, 477, 486 of SEQ ID NO: 1 with substitution mutations. In one or more embodiments, the terminal deoxynucleotidyl transferase mutant described herein also comes from the bird species Zonotrichia albicollis.
[0027] As used herein, "isolated terminal deoxynucleotidyl transferase mutant" means that the terminal deoxynucleotidyl transferase mutant is substantially free of other proteins, lipids, carbohydrates or other substances naturally associated with it. Those skilled in the art can purify the terminal deoxynucleotidyl transferase mutant using standard protein purification techniques. Substantially pure proteins can produce a single major band on non-reducing polyacrylamide gels.
[0028] As is well known in the art, substituting amino acids with similar or closely related physical and chemical properties usually results in a sequence that still retains its original biological function. In this article, the amino acids with similar or closely related physical and chemical properties can be classified according to Biochemistry (Second Edition) (edited by Zhilong Xiu), specifically including: (1) non-polar aliphatic amino acids, including glycine G, alanine A, valine V, leucine L, isoleucine I, proline P, and methionine M; (2) non-polar aromatic amino acids, including phenylalanine F, tyrosine Y, and tryptophan W; (3) polar uncharged amino acids, including serine S, threonine T, asparagine N, glutamine Q, and cysteine C; (4) polar positively charged (basic) amino acids, including lysine K, arginine R, and histidine H; (5) polar negatively charged (acidic) amino acids, including aspartic acid D and glutamic acid E.
[0029] In this article, the term "identity" refers to the degree of identity between two amino acid sequences when aligned for maximum consistency. The optimal alignment and percentage identity between two amino acid sequences can be determined using the BLASTP algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87: 2264-2268, 1993). On the NCBI website, these algorithms are incorporated into the BLAST program (http: / / www.ncbi.nlm.nih.gov / BLAST / ). To obtain a gapped alignment for comparison purposes, gapped BLAST can be used as described by Altschul et al. (Nucleic Acids Res. 25: 3389-3402, 1997). When using the BLAST and gapped BLAST programs, the default parameters of the respective programs are used. The percentage of sequence identity is determined by comparing two optimally aligned sequences within a comparison window, where the sequence portion in the comparison window may include additions or deletions (i.e., gaps) compared to the reference sequence used for the optimal alignment of the two sequences (which does not include additions or deletions). The percentage is calculated as follows: determine the number of positions where the same amino acids occur in the two sequences to obtain the number of matching positions, divide the number of matching positions by the total number of positions in the comparison window, and multiply the result by 100 to obtain the percentage of sequence identity.
[0030] In this article, the mutation at position 16 is preferably a mutation of Q to a polar uncharged amino acid, such as N, C, S, or T, or to a non-polar aromatic amino acid, such as F, Y, or W. Further preferably, the mutation at position 16 is a mutation of Q to N, C, S, T, or Y. More preferably, the mutation at position 16 is a mutation of Q to N or Y.
[0031] In this text, the mutation at the 187th position is preferably a mutation from C to a non-polar aliphatic amino acid such as G, A, V, L, I, P, or M, or to a polar uncharged amino acid such as S, T, N, Q, or C. Further preferably, the mutation at the 187th position is a mutation from C to S, T, or P. More preferably, the mutation at the 187th position is a mutation from C to T or P.
[0032] In this text, the mutation at the 390th position is preferably a mutation from R to a polar uncharged amino acid such as S, T, N, Q, or C. Preferably, the mutation at the 390th position is a mutation from R to N or Q. More preferably, the mutation at the 390th position is a mutation from R to N.
[0033] In this text, the mutation at the 421st position is preferably a mutation from E to a polar uncharged amino acid such as S, T, N, Q, or C. Preferably, the mutation at the 421st position is a mutation from E to S or T. More preferably, the mutation at the 421st position is a mutation from E to S.
[0034] In this text, the mutation at the 422nd position is preferably a mutation from Q to a non-polar aliphatic amino acid such as G, A, V, L, I, P, or M. Preferably, the mutation at the 422nd position is a mutation from Q to A, V, I, or L. More preferably, the mutation at the 422nd position is a mutation from Q to L.
[0035] In this text, the mutation at the 425th position is preferably a mutation from M to a polar uncharged amino acid such as S, T, N, Q, or C. Preferably, the mutation at the 425th position is a mutation from M to N or Q. More preferably, the mutation at the 425th position is a mutation from M to N.
[0036] In this text, the mutation at the 450th position is preferably a mutation from L to a polar uncharged amino acid such as S, T, N, Q, or C. Preferably, the mutation at the 450th position is a mutation from L to N or Q. More preferably, the mutation at the 450th position is a mutation from L to N.
[0037] In this text, the mutation at the 456th position is preferably a mutation from S to a polar uncharged amino acid such as T, N, Q, or C; preferably, the mutation at the 456th position is a mutation from S to N or Q; more preferably, the mutation at the 456th position is a mutation from S to N.
[0038] In this text, the mutation at the 477th position is preferably a mutation from N to a non-polar aliphatic amino acid such as G, A, V, L, I, P, or M. Preferably, the mutation at the 477th position is a mutation from N to A, G, I, L, or V. More preferably, the mutation at the 477th position is a mutation from N to A.
[0039] In the present text, the mutation at the 486th position is preferably a mutation from R to a polar basic amino acid, such as H or K. Preferably, the mutation at the 486th position is a mutation from R to H.
[0040] Preferably, compared with SEQ ID NO: 1, the mutations of the terminal deoxynucleotidyl transferase described herein are mutations at one or more of the following sites: (1) Q16N or Q16Y; (2) C187T or C187P; (3) R390N; (4) E421S; (5) Q422L; (6) M425N; (7) L450N; (8) S456N; (9) N477A; (10) R486H.
[0041] In some embodiments, compared with the amino acid sequence shown in SEQ ID NO: 1, the amino acid sequence of the terminal deoxynucleotidyl transferase described herein has the substitution mutations described herein at any one position, any two positions, any three positions, any four positions, any five positions, any six or more positions, any seven positions, any eight positions or all nine positions among the 16th, 187th, 390th, 421st, 422nd, 425th, 456th, 477th, 486th positions.
[0042] In some embodiments, the terminal deoxynucleotidyl transferase described herein is a single mutant, double mutant, triple mutant, quadruple mutant or quintuple mutant, that is, it has 1 substitution mutation, 2 substitution mutations, 3 substitution mutations, 4 substitution mutations or 5 substitution mutations compared with SEQ ID NO: 1.
[0043] In some embodiments, compared to SEQ ID NO: 1, the terminal deoxynucleotidyl transferase described herein includes substitution mutations at positions 16 and 187, and optionally one or more substitution mutations selected from positions 390, 421, 422, 425, 450, 456, 477, and 486. In some embodiments, compared to SEQ ID NO: 1, the terminal deoxynucleotidyl transferase described herein includes substitution mutations at positions 16, 187, and 390, and optionally one or more substitution mutations selected from positions 421, 422, 425, 450, 456, 477, and 486. In some embodiments, compared to SEQ ID NO: 1, the terminal deoxynucleotidyl transferase described herein includes substitution mutations at positions 16, 187, 390, and 421, and optionally one or more substitution mutations selected from positions 422, 425, 450, 456, 477, and 486; preferably, the terminal deoxynucleotidyl transferase further includes one or more substitution mutations selected from positions 422, 425, 450, and 456. In some embodiments, compared to SEQ ID NO: 1, the terminal deoxynucleotidyl transferase described herein includes substitution mutations at positions 16, 187, 390, and 422, and optionally one or more substitution mutations selected from positions 421, 425, 450, 456, 477, and 486; preferably, the terminal deoxynucleotidyl transferase further includes substitution mutations selected from positions 477 and 486. In some embodiments, compared to SEQ ID NO: 1, the terminal deoxynucleotidyl transferase described herein includes substitution mutations at positions 16, 187, 390, and 425, and optionally one or more substitution mutations selected from positions 421, 422, 450, 456, 477, and 486; preferably, the terminal deoxynucleotidyl transferase further includes one or more substitution mutations selected from positions 450, 456, 477, and 486. In some embodiments, compared to SEQ ID NO: 1, the terminal deoxynucleotidyl transferase described herein includes substitution mutations at positions 16, 187, 390, 425, and 456, and optionally substitution mutations at positions 477 and 486; preferably, the terminal deoxynucleotidyl transferase further includes the substitution mutation at position 477, or further includes the substitution mutations at positions 477 and 486.Preferably, in the above embodiments, the mutation at position 16 is Q16N or Q16Y, preferably Q16Y, the mutation at position 187 is C187T or C187P, preferably C187T, the mutation at position 390 is R390N, the mutation at position 422 is Q422L, the mutation at position 425 is M425N, the mutation at position 450 is L450N, the mutation at position 456 is S456N, the mutation at position 477 is N477A, and the mutation at position 486 is R486H.
[0044] In some embodiments, the terminal deoxynucleotidyl transferase described herein is a terminal deoxynucleotidyl transferase shown in Table 1. In some embodiments, the amino acid sequence of the terminal deoxynucleotidyl transferase is shown in SEQ ID NO:4.
[0045] In some embodiments, the terminal deoxynucleotidyl transferase described herein is formed by deleting, inserting and / or replacing the amino acid sequence of the above-mentioned terminal deoxynucleotidyl transferase through one or more amino acid residues, and has a protein having the function of the terminal deoxynucleotidyl transferase described herein, and the terminal deoxynucleotidyl transferase has a substitution mutation described in any embodiment of the present invention at the amino acid residues corresponding to positions 16, 187, 390, 421, 422, 425, 450, 456, 477 and / or 486 of SEQ ID NO: 1. In other words, the terminal deoxynucleotidyl transferase has at least one substitution mutation described herein. Preferably, the terminal deoxynucleotidyl transferase is also from the avian tribe (Zonotrichia albicollis). The deletion, insertion and / or substitution of the one or more amino acid residues is generally 1-20, preferably 1-10, more preferably 1-8, 1-5, 1-3, or 1-2 amino acids. The deletion and insertion may occur at the C-terminus and / or N-terminus. Preferably, the substitution is a conservative substitution using an amino acid having similar or similar physical and chemical properties as described herein, and such substitution does not change the function of the terminal deoxynucleotidyl transferase. The function of the terminal deoxynucleotidyl transferase described herein includes the function of catalyzing the reaction of an oligonucleotide and a dNTP in the absence of a template strand, thereby adding a dNTP to the 3'OH end of the oligonucleotide. It should be understood that the function of the terminal deoxynucleotidyl transferase provided herein is significantly stronger than that of the wild-type terminal deoxynucleotidyl transferase (SEQ ID NO: 1), for example, an increase of more than 10%, more than 20%, more than 30%, more than 50% or even higher.
[0046] In some embodiments, the terminal deoxynucleotidyl transferase described herein is a terminal deoxynucleotidyl transferase whose amino acid sequence has an identity of more than 80%, preferably more than 85%, more preferably more than 90%, more preferably more than 95%, more preferably more than 97%, more preferably more than 98%, more preferably more than 99% with the above-mentioned terminal deoxynucleotidyl transferase and has the function of the terminal deoxynucleotidyl transferase described herein. The terminal deoxynucleotidyl transferase has amino acid residues corresponding to positions 16, 187, 390, 421, 422, 425, 450, 456, 477, and / or 486 of SEQ ID NO:1 as in any embodiment herein. In other words, the terminal deoxynucleotidyl transferase has at least one substitution mutation described herein. Preferably, the terminal deoxynucleotidyl transferase also comes from the genus Zonotrichia (Zonotrichia albicollis).
[0047] The terminal deoxynucleotidyl transferase described herein can be a recombinant protein, a natural protein, or a synthetic protein, preferably a recombinant protein. The terminal deoxynucleotidyl transferase described herein can be a product of natural purification, a product of chemical synthesis, or produced using recombinant techniques from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, higher plants, insects, and mammalian cells).
[0048] Polynucleotide molecule
[0049] The present invention also provides polynucleotide molecules encoding the terminal deoxynucleotidyl transferase of the present invention and their complementary molecules.
[0050] The polynucleotide of the present invention can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand.
[0051] Polynucleotide molecules encoding the mature protein of the terminal deoxynucleotidyl transferase include: a coding sequence encoding only the mature protein; the coding sequence of the mature protein and various additional coding sequences; the coding sequence of the mature protein (and optional additional coding sequences) and non-coding sequences. In some embodiments, the polynucleotide molecule encoding the mature protein of the terminal deoxynucleotidyl transferase includes the nucleotide sequence shown in SEQ ID NO:6.
[0052] "Polynucleotide molecule encoding a protein" can be a polynucleotide including the polynucleotide encoding this protein, or a polynucleotide further including additional coding and / or non-coding sequences.
[0053] The polynucleotide molecules of the present invention can generally be obtained by PCR amplification, recombination, or artificial synthesis. For PCR amplification, primers can be designed based on the nucleotide sequences disclosed in the present invention, especially the open reading frame sequences, and a commercially available cDNA library or a cDNA library prepared by conventional methods known to those skilled in the art can be used as a template for amplification to obtain the relevant sequences. When the sequence is relatively long, it is often necessary to perform PCR amplification two or more times, and then splice the fragments amplified each time together in the correct order.
[0054] Nucleic acid construct
[0055] This article also relates to nucleic acid constructs comprising the polynucleotide molecules described herein. The nucleotide constructs generally contain one or more regulatory sequences operably linked to the polynucleotide molecules described herein.
[0056] As used herein, "operably linked" or similar descriptions refer to the arrangement of elements where the components are arranged in a certain configuration so as to perform their required functions. Thus, a given promoter operably linked to a coding sequence can enable the effective expression of the coding sequence in the presence of the correct transcription factors, etc. The promoter does not need to be adjacent to the coding sequence as long as it functions to direct the expression of the sequence. Thus, for example, sequences that are not involved in translation but are transcribed can be present between the promoter sequence and the coding sequence, as can transcribed introns; and the promoter sequence can still be considered "operably linked" to the coding sequence.
[0057] The polynucleotides encoding the present invention can be manipulated in various ways to ensure the expression of the TDT mutant or its conservative variant proteins. Manipulation of the polynucleotide molecule before its insertion into a vector may be desirable or necessary depending on the expression vector. Techniques for altering polynucleotide sequences using recombinant DNA methods are known in the art.
[0058] The regulatory sequence can be a suitable promoter sequence, a nucleotide sequence recognized by the host cell for expressing the polynucleotide molecules of the present invention. The promoter sequence contains transcriptional regulatory sequences for polypeptide expression. The promoter can be any nucleotide sequence that shows transcriptional activity in the selected host cell, including mutant, truncated, and hybrid promoters, and can be obtained from genes encoding extracellular or intracellular polypeptides homologous or heterologous to the host cell.
[0059] In Escherichia coli, suitable promoters include, but are not limited to, lac (lactose promoter), trp (tryptophan promoter), and tac (hybrid promoter of lactose and tryptophan).
[0060] The regulatory sequence can also be a suitable transcription terminator sequence, a sequence recognized by the host cell to terminate transcription. The terminator sequence is operably linked to the 3′ end of the nucleotide sequence encoding the polypeptide. Any terminator functional in the selected host cell can be used in the present invention. In some embodiments, the transcription terminator sequence is a terminator functional in Escherichia coli.
[0061] In some embodiments, the nucleic acid construct is an expression cassette that contains a promoter and a transcription terminator operably linked to the polynucleotide molecule described herein.
[0062] vector
[0063] The nucleic acid constructs described herein can also be vectors, including but not limited to expression vectors and cloning vectors.
[0064] In an expression vector, various nucleic acid molecules and regulatory sequences can be ligated together to produce a recombinant expression vector that includes one or more restriction sites that permit the insertion of the polynucleotide molecule or nucleic acid construct described herein at such sites. When constructing an expression vector, the polynucleotide molecule described herein is positioned in the vector such that it is operably linked to appropriate regulatory sequences.
[0065] The term "recombinant expression vector" refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses, or other vectors well known in the art. In general, any plasmid and vector can be used as long as it can replicate and be stable in the host. An important feature of an expression vector is that it usually contains an origin of replication, a promoter, a marker gene, and translation control elements.
[0066] A recombinant expression vector is any vector (such as a plasmid or virus) that can be conveniently subjected to recombinant DNA methods and can result in the expression of a nucleotide sequence of interest. The choice of vector generally depends on the compatibility of the vector with the host cell into which it is introduced. The vector can be linear or a closed circular plasmid.
[0067] The vector can be an autonomously replicating vector, i.e., an entity that exists as an extrachromosomal entity and whose replication is independent of chromosomal replication, such as a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector can contain any means for ensuring self-replication. Alternatively, the vector can be a vector that integrates into the genome when introduced into the host cell and replicates with the chromosome into which it has been integrated. In addition, a single vector or plasmid, or two or more vectors or plasmids, or a transposon that together contain the total DNA to be introduced into the genome of the host cell can be used.
[0068] The vector of the present invention may contain one or more selectable markers that allow for easy selection of transformed, transfected, transduced, etc. cells. Selectable markers are genes whose products provide resistance to antibiotics or viruses, resistance to heavy metals, prototrophy to auxotrophy, etc.
[0069] The vector of the present invention may contain elements that allow the vector to integrate into the genome of the host cell or the vector to replicate autonomously in the cell independently of the genome.
[0070] More than one copy of the polynucleotide of the present invention may be inserted into the host cell to increase the yield of the gene product. An increase in the copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the genome of the host cell or by including an amplifiable selectable marker gene and the polynucleotide, wherein cells containing the amplified copy of the selectable marker gene and thus containing additional copies of the polynucleotide can be screened by culturing the cells in the presence of an appropriate selection agent.
[0071] The expression vector of the present invention is more preferably a vector that can be used for expression in Escherichia coli.
[0072] The cloning vector containing the polynucleotide sequence of the present invention can be used to replicate a sufficient amount of the target plasmid. Therefore, the cloning vector of the present invention carries strong self-replicating elements such as an origin of replication, etc. Generally, the cloning vector of the present invention does not have expression elements.
[0073] Host cell
[0074] The present invention also encompasses host cells genetically engineered with the vector of the present invention or the terminal deoxynucleotidyl transferase coding sequence, as well as methods for producing the proteins described in the present invention by recombinant techniques. The host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a plant cell. Representative examples are: Escherichia coli, Bacillus subtilis, Streptomyces, Agrobacterium; eukaryotic cells such as yeast, plant cells, etc. In a specific embodiment of the present invention, Escherichia coli is used as the host cell.
[0075] By conventional recombinant DNA techniques, the polynucleotide sequence of the present invention can be used to express or produce recombinant terminal deoxynucleotidyl transferase mutants. Generally, the following steps are involved:
[0076] (1). Transform or transduce a suitable host cell with the polynucleotide (or variant) encoding the terminal deoxynucleotidyl transferase mutant of the present invention, or with a recombinant expression vector containing the polynucleotide;
[0077] (2). Culture the host cells in a suitable medium;
[0078] (3). Isolate and purify the protein from the medium or the cells.
[0079] Methods well known to those skilled in the art can be used to construct expression vectors containing the DNA sequence encoding terminal deoxynucleotidyl transferase and appropriate transcriptional / translational control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. The said DNA sequence can be effectively ligated to an appropriate promoter in the expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. The expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selecting transformed host cells.
[0080] Vectors containing the above-mentioned appropriate DNA sequence and appropriate promoter or control sequence can be used to transform appropriate host cells to enable them to express proteins.
[0081] Those of ordinary skill in the art are well aware of how to select appropriate vectors, promoters, enhancers, and host cells.
[0082] Enzyme preparation
[0083] In one or more embodiments, the present invention provides an enzyme preparation containing the terminal deoxynucleotidyl transferase described in any embodiment of the present invention. In one or more embodiments, the enzyme preparation further contains a buffer. Preferably, the buffer is selected from one or more of phosphate buffer (PBS), tris(hydroxymethyl)aminomethane buffer (TRIS), and triethanolamine buffer (TOEA), with a pH of 5.5 to 9, preferably 6 to 8, more preferably 6.5 to 7.5.
[0084] In one or more embodiments, the enzyme preparation is in powder form, such as lyophilized powder.
[0085] Application
[0086] The present invention also provides the application of the terminal deoxynucleotidyl transferase described in any embodiment herein in catalyzing the extension of an oligonucleotide chain without a template strand, or in preparing an extended oligonucleotide chain using an oligonucleotide and dNTP as substrates without a template strand.
[0087] In one or more embodiments, the oligonucleotide is a single-stranded DNA with a length of 15 to 20 bp. Exemplary sequences of oligonucleotide chains are shown as SEQ ID NO: 2 (5’-TAATACGACTCACTA-3’) or SEQ ID NO: 3 (5’-ATGGATCGCTGTACAGCGTA-3’).
[0088] It should be understood that the said single-stranded DNA can be functional DNA or non-functional DNA, as long as it can be catalyzed by terminal deoxynucleotidyl transferase, it belongs to the substrate of this enzyme.
[0089] After obtaining the information of the terminal deoxynucleotidyl transferase described herein, those skilled in the art will know how to use the terminal deoxynucleotidyl transferase to catalyze the extension of an oligonucleotide chain using dNTP and an oligonucleotide chain as substrates in the absence of a template strand. Various intracellular or extracellular catalytic synthesis methods are included in the present invention or can be applied to the present invention.
[0090] Therefore, the present invention also provides a method for preparing an extended oligonucleotide chain using an oligonucleotide and dNTP as substrates in the absence of a template strand, comprising: using the terminal deoxynucleotidyl transferase according to any embodiment of the present invention to catalyze the reaction of the oligonucleotide and dNTP, thereby adding dNTP to the 3'-OH terminus of the oligonucleotide. Generally, the oligonucleotide is a single-stranded DNA of 15-20 bp.
[0091] In the present invention, the dNTP is selected from one or more of dATP, dTTP, dGTP, and dCTP.
[0092] It should be understood that different products can be formed according to different dNTPs. For example, when the dNTP is dATP, the terminal deoxynucleotidyl transferase can catalyze the extension of the oligonucleotide chain and form a poly-A tail, i.e., (dA)n, also known as poly(A). Therefore, the products after the catalytic extension include poly(A), poly(T), poly(G), and poly(C).
[0093] In the present invention, the catalytic reaction can be carried out in a suitable buffer system. For example, one or more buffers selected from phosphate buffer (PBS), tris(hydroxymethyl)aminomethane buffer (TRIS), and triethanolamine buffer (TOEA) can be used to provide the buffer system. The pH of the reaction system can be 5.5-9.0, preferably 6-8, for example, 6.8. The reaction temperature can be 10-40°C, preferably 20-35°C, for example, 30°C.
[0094] In the method, the molar ratio of dNTP to the oligonucleotide chain in the reaction system can be (10-1):1, preferably (8-1):1; for example, 5:1. In the reaction system, the ratio of the terminal deoxynucleotidyl transferase to the oligonucleotide chain can be 1:100-1:500 mg / μM, such as 200-500 mg / μM or 300-500 mg / μM. In one embodiment, the ratio is 1:400 mg / μM.
[0095] Screening method
[0096] The present inventor has discovered sites in terminal deoxynucleotidyl transferase that are related to its catalytic enzyme activity. Therefore, in some embodiments, the present invention provides a method for screening terminal deoxynucleotidyl transferase with enhanced function, the method comprising: preparing a terminal deoxynucleotidyl transferase mutant having a substitution mutation at at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or all 10 positions among the 16th, 187th, 390th, 421st, 422nd, 425th, 450th, 456th, 477th, and 486th positions of its amino acid sequence relative to SEQ ID NO: 1, testing the terminal deoxynucleotidyl transferase function of the prepared mutant, and selecting a mutant whose terminal deoxynucleotidyl transferase function is higher than that of the terminal deoxynucleotidyl transferase shown in SEQ ID NO: 1. The mutant can be prepared by single-site saturation mutagenesis or multi-site saturation mutagenesis. Methods for preparing such mutants are well known in the art, including but not limited to chemical synthesis or repeated methods. In some embodiments, the method for preparing an extended oligonucleotide chain described herein can be used to test the terminal deoxynucleotidyl transferase function of the mutant.
[0097] Compared with the prior art, the progressive effect of the present invention is that the terminal deoxynucleotidyl transferase obtained in the present invention can efficiently catalyze the extension of oligonucleotide chains using oligonucleotides and dNTPs as substrates in the absence of a template strand. Compared with the wild type, after catalysis by the mutant, the number of extended fragments of the oligonucleotide chain increases and the yield increases.
[0098] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are generally carried out under conventional conditions or according to the conditions recommended by the manufacturer.
[0099] In the following examples, ultra-high performance liquid chromatography was used, and an ultra-high performance liquid chromatograph (Ultra Performance Liquid Chromatography, UPLC) from Agilent was used to detect and analyze the oligonucleotide extension sequence products.
[0100] Chromatographic column: ACQUITY UPLC OST C18, 2.1x50mm, 1.7μm.
[0101] The mobile phase is as follows:
[0102] (1) Aqueous phase: 1.3% n-hexylamine and 0.55% acetic acid dissolved in water
[0103] (2) Organic phase: a mixture of 50% aqueous phase and 50% acetonitrile.
[0104] The duration of this method is 3 min. The concentration of the organic phase is 60% from 0 to 0.5 min, linearly increases after 0.5 min, becomes 85% at 2 min, remains at this level until 2.2 min, and then linearly decreases to 60% at 3 min. The flow rate is 0.5 mL / min, and the temperature during the operation is 60 °C.
[0105] Example 1. Terminal deoxynucleotidyl transferase mutant
[0106] Based on the three-dimensional structure and functional analysis of the wild-type TdT derived from Zonotrichia albicollis with the amino acid sequence shown in SEQ ID NO: 1 (the corresponding nucleotide sequence is shown in SEQ ID NO: 5), through computer-aided design, single-site saturation mutagenesis, and multi-site saturation mutagenesis, a recombinant plasmid of terminal deoxynucleotidyl transferase was constructed. After being transferred into Escherichia coli, it was induced to express, and the designed mutant was isolated and purified for functional verification.
[0107] The method for expressing and purifying terminal deoxynucleotidyl transferase protein is as follows:
[0108] (1) The constructed recombinant plasmid of terminal deoxynucleotidyl transferase was mixed and transferred into E. coli BL21(DE3). Positive clone screening was carried out using a kanamycin-resistant plate (Kan+, 50 μg / mL), and cultured overnight at 37 °C. A single clone was picked into 170 μL of LB liquid medium (Kan+, 50 μg / mL), cultured at 37 °C and 350 rpm for about 3 hours, then 20 μL of the bacterial solution in LB medium was transferred to 380 mL of TB medium (Kan+, 50 μg / mL), cultured at 37 °C and 450 rpm for about 3 hours, cooled to 25 °C, and IPTG was added to a final concentration of 0.1 mM to induce expression for about 16 h.
[0109] (2) The cultured bacterial solution was centrifuged (centrifuged at 4500 rpm for 20 min), the supernatant was removed, and the bacterial cells were left. The bacterial cells can be used to directly obtain a solution containing terminal deoxynucleotidyl transferase, or can be temporarily stored at -80 °C. The induced bacterial cells were resuspended with 250 μL of an aqueous solution containing 0.25 mg / mL of lysozyme and shaken at 25 °C and 1000 rpm for 2 hours. This step is to lyse the bacterial cells so that the TdT protein in the bacterial cells is fully exposed in the aqueous solution, and the obtained aqueous solution of TdT enzyme can be directly used for functional verification.
[0110] The obtained mutants and the mutation sites verified by sequencing are shown in Table 1. The sequencing of each mutant was commissioned to Azenta Life Sciences, Figure 5 The sequencing results of mutant 89 are shown.
[0111] Table 1: Terminal deoxynucleotidyl transferase mutants
[0112]
[0113]
[0114]
[0115]
[0116] Example 2: Functional verification of the extension of adenosine deoxyribonucleotides on a 15bp oligonucleotide probe substrate
[0117] In this example, adenosine deoxyribonucleotides (dATPs) and the 15bp oligonucleotide probe 5’-TAATACGACTCACTA-3’OH (SEQ ID NO:2) were used as substrates, and an aqueous solution without TdT enzyme was used as a control group to test the catalytic activity of some of the TdT enzyme mutants in Table 1.
[0118] Reaction system: (1) 50 μM of adenosine deoxyribonucleotide substrate, (2) 10 μM of 15bp oligonucleotide probe, (3) the aqueous solution of the TdT enzyme mutant obtained in Example 1, with a concentration of 2.5 mg / mL, and (4) buffer, where the buffer contains 250 μM CoCl2, 100 mM NaCl, and the buffer components shown in Table 2.
[0119] Reaction process: The reaction system was placed on a shaker at 1000 rpm for 1 hour. After the reaction, the enzyme was inactivated by heating in an 80°C water bath to stop the reaction. The supernatant obtained by centrifugation was used to detect and analyze the oligonucleotide extension sequence products using ultra-performance liquid chromatography (UPLC) produced by Agilent.
[0120] Result statistics: According to the UPLC chromatogram, the number of absorption peaks of the extended n-nucleotide fragments was counted to obtain the maximum value of the extended fragments, which was used as the number of extended fragments. The cumulative absorption peak areas of the experimental group with the addition of the aqueous solution of TdT enzyme and the control group without the addition of the aqueous solution of TdT enzyme were separately counted, and the yield was calculated: Yield = Cumulative absorption peak area of the experimental group / Cumulative absorption peak area of the control group.
[0121] Table 2: Reaction conditions for test examples and comparative examples
[0122]
[0123]
[0124]
[0125]
[0126]
[0127] Result Analysis
[0128] Taking Test Example 1 as an example, the reaction schematic diagram of the TdT enzyme catalyzing the generation of oligonucleotide extension fragments from dATPs and 15bp oligonucleotide probes is as Figure 1 shown. Among them, dA represents the extension of a 1-nucleotide fragment, and [dA]n represents the extension of an n-nucleotide fragment. The UPLC chromatogram of Test Example 88 is as Figure 3 shown.
[0129] The yields and numbers of extended fragments of the test examples and comparative examples are shown in Table 3. It can be seen that the terminal deoxynucleotidyl transferase mutants of the present invention can all catalyze the extension of 15bp oligonucleotide chains, and adenine deoxyribonucleotides (dATP) are extended at the 3'-OH terminus of the 15bp oligonucleotide sequence.
[0130] Table 3: Yields and Numbers of Extended Fragments of Test Examples and Comparative Examples
[0131]
[0132]
[0133]
[0134]
[0135] Example 3. Functional Verification of Extending Adenine Deoxyribonucleotides on a 20bp Oligonucleotide Probe Substrate
[0136] In this example, adenine deoxyribonucleotides (dATPs) and the 20bp oligonucleotide probe 5’-ATGGATCGCTGTACAGCGTA-3’ (SEQ ID NO:3) were used as substrates, and an aqueous solution without TdT enzyme was used as a control group to test the catalytic activity of the screened TdT enzyme mutants.
[0137] Reaction system: (1) 50 μM of the substrate adenine deoxyribonucleotide, (2) 10 μM of the 20bp oligonucleotide probe, (3) the aqueous solution of the TdT enzyme mutant obtained in Example 1, with a concentration of 2.5 mg / mL, and (4) buffer, where the buffer contains 250 μM CoCl2, 100 mM NaCl, and 500 mM phosphate buffer. The pH of the reaction system is 6.8.
[0138] Reaction process: The reaction system was placed in a shaker at 30 °C and reacted at 1000 rpm for 1 hour. After the reaction, the enzyme was inactivated by heating in a water bath at 80 °C to stop the reaction. The supernatant obtained by centrifugation was used to detect and analyze the oligonucleotide extension sequence products using an ultra performance liquid chromatography (UPLC) produced by Agilent.
[0139] Result statistics: According to the UPLC chromatogram, the number of absorption peaks of the extended fragments with n nucleotides was counted to obtain the maximum value of the extended fragments, which was used as the number of extended fragments. The cumulative absorption peak areas of the experimental group with added terminal deoxynucleotidyl transferase (TdT) aqueous solution and the control group without added TdT aqueous solution were counted separately, and the yield was calculated: Yield = Cumulative absorption peak area of the experimental group / Cumulative absorption peak area of the control group.
[0140] Result analysis
[0141] Taking Test Example A as an example, the reaction schematic diagram of TdT catalyzing dATPs and a 20bp oligonucleotide probe to generate oligonucleotide extension fragments is as Figure 2 shown. Among them, dA represents an extended fragment with 1 nucleotide, and [dA]n represents an extended fragment with n nucleotides. The UPLC chromatogram of Test Example A is as Figure 4 shown.
[0142] Table 4: Yields and numbers of extended fragments of test examples and comparative examples
[0143] Test Example / Comparative Example TdT enzyme Yield Number of extended fragments Comparative Example A Wild type 59％ 6 Test Example A Mutant 89 78％ 11 Test Example B Mutant 88 75％ 10
[0144] The results are shown in Table 4. It can be seen that the screened terminal deoxynucleotidyl transferase mutants 89 and 88 can catalyze the extension of a 20bp oligonucleotide chain, and adenine deoxyribonucleotide (dATP) is extended at the 3'-OH end of the 20bp oligonucleotide sequence.
[0145] The amino acid sequence and coding sequence of mutant 89 are shown in SEQ ID NO: 4 and 6 respectively.
[0146] SEQ ID NO:4
[0147] MDRFKAPAVISQRKR YKGLHSPKLSCSYEIKFSNFVIFIMQRKMGLTRRMFLMELGRRKGFRVESELSDSVTHIVAENNSYLEVLDWLKGQAVGDSSRFELLDISWFTACMEAGRPVDSEVKYRLMEQSQSLPLNMPALEMPAFIATKVSQYSCQRKTTLNNYNKKFTDAFEVMAENYEFKENEIF T LEFLRAASLLKSLPFSVTRMKDIQGLPCVGDQVRDIIEEIIEEGESSRVNEVLNDERYKAFKQFTSVFGVGVKTSEKWYRMGLRTVEEVKADKTLKLSKMQKAGLLYYEDLVSCVSKAEADAVSLIVKNTVCTFLPDALVTITGGFRRGKNIGHDIDFLITNPGPREDDELLHKVIDLWKKQGLLLYCDIIESTFVKEQLPS N KVDAMDHFQKCFAILKLYQPRVDNSTCNTSEQLE N AEVKDWKAIRVDLVITPFEQYPYALLGWTG N RQFGRDLRRYAAHERKMILD A HGLYDRRK H IFLKAGSEEEIFAHLGLDYVEPWERNA
[0148] SEQ ID NO:6
[0149]
[0150] The embodiments described above merely represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the patent for the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several variations and improvements can still be made, and these all fall within the protection scope of the present invention. Therefore, the protection scope of the patent for the present invention shall be subject to the appended claims. At the same time, all the documents mentioned in the present invention are cited in this application as references, just as if each document was cited separately as a reference.
Claims
1. A terminal deoxynucleotidyl transferase, the terminal deoxynucleotidyl transferase: (a) Compared with the amino acid sequence shown in SEQ ID NO:1, its amino acid sequence has substitution mutations at at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or all 10 positions among the 16th, 187th, 390th, 421st, 422nd, 425th, 450th, 456th, 477th and 486th positions; (b) is a protein derived from (a) formed by deletion, insertion and / or substitution of one or more amino acid residues, retaining at least one of the substitution mutations described in (a), and having the function of terminal deoxynucleotidyl transferase; (c) is a protein derived from (a) having more than 80% identity with the amino acid sequence of (a) and having the function of terminal deoxynucleotidyl transferase, and this protein retains at least one of the substitution mutations described in (a).
2. The terminal deoxynucleotidyl transferase according to claim 1, wherein: The mutation at the 16th position is a mutation of Q to a polar uncharged amino acid, such as N, C, S or T, or to a non-polar aromatic amino acid, such as F, Y or W; preferably, the mutation at the 16th position is a mutation of Q to N, C, S, T or Y; more preferably, the mutation at the 16th position is a mutation of Q to N or Y; The mutation at the 187th position is a mutation of C to a non-polar aliphatic amino acid, such as G, A, V, L, I, P or M, or to a polar uncharged amino acid, such as S, T, N, Q or C; preferably, the mutation at the 187th position is a mutation of C to S, T or P; more preferably, the mutation at the 187th position is a mutation of C to T or P; The mutation at the 390th position is a mutation of R to a polar uncharged amino acid, such as S, T, N, Q or C; preferably, the mutation at the 390th position is a mutation of R to N or Q; more preferably, the mutation at the 390th position is a mutation of R to N; The mutation at the 421st position is a mutation of E to a polar uncharged amino acid, such as S, T, N, Q or C; preferably, the mutation at the 421st position is a mutation of E to S or T; more preferably, the mutation at the 421st position is a mutation of E to S; The mutation at the 422nd position is a mutation of Q to a non-polar aliphatic amino acid, such as G, A, V, L, I, P or M; preferably, the mutation at the 422nd position is a mutation of Q to A, V, I or L; more preferably, the mutation at the 422nd position is a mutation of Q to L; The mutation at the 425th position is a mutation of M to a polar uncharged amino acid, such as S, T, N, Q or C; preferably, the mutation at the 425th position is a mutation of M to N or Q; more preferably, the mutation at the 425th position is a mutation of M to N; The mutation at the 450th position is a mutation of L to a polar uncharged amino acid, such as S, T, N, Q or C; preferably, the mutation at the 450th position is a mutation of L to N or Q; more preferably, the mutation at the 450th position is a mutation of L to N; The mutation at position 456 is a mutation from S to a polar uncharged amino acid, such as T, N, Q, or C; preferably, the mutation at position 456 is a mutation from S to N or Q; more preferably, the mutation at position 456 is a mutation from S to N; The mutation at position 477 is a mutation from N to a nonpolar aliphatic amino acid, such as G, A, V, L, I, P, or M; preferably, the mutation at position 477 is a mutation from N to A, G, I, L, or V; more preferably, the mutation at position 477 is a mutation from N to A; The mutation at position 486 is a mutation from R to a polar basic amino acid, such as H or K; more preferably, the mutation at position 486 is a mutation from R to H.
3. The terminal deoxynucleotidyl transferase according to claim 1 or 2, characterized in that, Compared with SEQ ID NO: 1, the mutations of the terminal deoxynucleotidyl transferase are mutations at one or more of the following sites: (1) Q16N or Q16Y; (2) C187T or C187P; (3) R390N; (4) E421S; (5) Q422L; (6) M425N; (7) L450N; (8) S456N; (9) N477A; (10) R486H.
4. The terminal deoxynucleotidyl transferase according to any one of claims 1 to 3, characterized in that: Compared with SEQ ID NO: 1, the terminal deoxynucleotidyl transferase comprises substitution mutations at positions 16 and 187, and optionally substitution mutations at one or more positions selected from positions 390, 421, 422, 425, 450, 456, 477, and 486; or Compared with SEQ ID NO: 1, the terminal deoxynucleotidyl transferase comprises substitution mutations at positions 16, 187, and 390, and optionally substitution mutations at one or more positions selected from positions 421, 422, 425, 450, 456, 477, and 486; or Compared with SEQ ID NO: 1, the terminal deoxynucleotidyl transferase comprises substitution mutations at positions 16, 187, 390, and 421, and optionally substitution mutations at one or more positions selected from positions 422, 425, 450, 456, 477, and 486; preferably, the terminal deoxynucleotidyl transferase further comprises substitution mutations at one or more positions selected from positions 422, 425, 450, and 456; or Compared with SEQ ID NO: 1, the terminal deoxynucleotidyl transferase comprises substitution mutations at positions 16, 187, 390, and 422, and optionally substitution mutations at one or more positions selected from positions 421, 425, 450, 456, 477, and 486; preferably, the terminal deoxynucleotidyl transferase further comprises substitution mutations at positions 477 and 486; or Compared with SEQ ID NO: 1, the terminal deoxynucleotidyl transferase includes substitution mutations at positions 16, 187, 390, and 425, and optionally substitution mutations at one or more positions selected from positions 421, 422, 450, 456, 477, and 486; preferably, the terminal deoxynucleotidyl transferase further includes substitution mutations at one or more positions selected from positions 450, 456, 477, and 486; or Compared with SEQ ID NO: 1, the terminal deoxynucleotidyl transferase includes substitution mutations at positions 16, 187, 390, 425, and 456, and optionally substitution mutations at positions 477 and 486; preferably, the terminal deoxynucleotidyl transferase further includes a substitution mutation at position 477, or further includes substitution mutations at positions 477 and 486; Preferably, the mutation at position 16 is Q16N or Q16Y, preferably Q16Y, the mutation at position 187 is C187T or C187P, preferably C187T, the mutation at position 390 is R390N, the mutation at position 422 is Q422L, the mutation at position 425 is M425N, the mutation at position 450 is L450N, the mutation at position 456 is S456N, the mutation at position 477 is N477A, and the mutation at position 486 is R486H; Preferably, the amino acid sequence of the terminal deoxynucleotidyl transferase is as shown in SEQ ID NO:
4.
5. A polynucleotide molecule having: (1) A polynucleotide sequence encoding the terminal deoxynucleotidyl transferase according to any one of claims 1-4; and (2) The complementary sequence of the polynucleotide sequence in (1).
6. A nucleic acid construct containing the polynucleotide molecule according to claim 5; preferably, the nucleic acid construct is an expression cassette or a vector, such as a cloning vector or an expression vector.
7. A host cell, which: (1) Express the terminal deoxynucleotidyl transferase according to any one of claims 1-4, and / or (2) contain the polynucleotide molecule according to claim 5 or the nucleic acid construct according to claim 6; preferably, the host cell is Escherichia coli.
8. An enzyme preparation containing the terminal deoxynucleotidyl transferase according to any one of claims 1-4; Preferably, the enzyme preparation further contains a buffer; more preferably, the buffer is selected from one or more of phosphate buffer (PBS), tris(hydroxymethyl)aminomethane buffer (TRIS), and triethanolamine buffer (TOEA); preferably, the pH of the buffer is 5.5-9, preferably 6-8, more preferably 6.5-7.5; Preferably, the enzyme preparation is in powder form, more preferably lyophilized powder.
9. Use of the terminal deoxynucleotidyl transferase according to any one of claims 1 to 4 in catalyzing the extension of an oligonucleotide chain in the absence of a template strand, or in the preparation of an extended oligonucleotide chain using an oligonucleotide and dNTP as substrates in the absence of a template strand; wherein, The oligonucleotide is a single-stranded DNA with a length of 15-20 bp; Preferably, the extension is: adding dNTP to the 3'OH end of the oligonucleotide; Preferably, the dNTP is selected from one or more of dATP, dTTP, dGTP, and dCTP.
10. A method for preparing an extended oligonucleotide chain using an oligonucleotide and dNTP as substrates in the absence of a template strand, the method comprising: The step of using the terminal deoxynucleotidyl transferase according to any one of claims 1 to 4 to catalyze the reaction of an oligonucleotide and dNTP, thereby adding dNTP to the 3'-OH end of the oligonucleotide; wherein the oligonucleotide is a single-stranded DNA with a length of 15 to 20 bp; Preferably, the dNTP is selected from one or more of dATP, dTTP, dGTP, and dCTP.
11. The method according to claim 10, characterized in that, The method has one or more of the following characteristics: The buffer of the reaction system for the catalytic reaction is selected from one or more of phosphate buffer (PBS), tris(hydroxymethyl)aminomethane buffer (TRIS), and triethanolamine buffer (TOEA); The pH of the catalytic reaction system is 5.5 to 9.0, preferably 6 to 8, more preferably 6.8; The temperature of the catalytic reaction system is 10 to 40 °C, preferably 20 to 35 °C, more preferably 30 °C; The molar ratio of dNTP to the oligonucleotide chain in the reaction system is (10 to 1):1, preferably (8 to 1):1; more preferably 5:1; The ratio of terminal deoxynucleotidyl transferase to the oligonucleotide chain in the reaction system is 1:100 to 1:500 mg / μM, preferably 1:400 mg / μM.
12. A method for screening terminal deoxynucleotidyl transferase with enhanced function, the method comprising: Preparing a terminal deoxynucleotidyl transferase mutant having substitution mutations at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or all 10 positions among positions 16, 187, 390, 421, 422, 425, 450, 456, 477, and 486 of its amino acid sequence relative to SEQ ID NO: 1, testing the terminal deoxynucleotidyl transferase function of the prepared mutant, and selecting a mutant whose terminal deoxynucleotidyl transferase function is higher than that of the terminal deoxynucleotidyl transferase shown in SEQ ID NO:
1.
13. The method according to claim 12, wherein: The mutant is prepared by single-site saturation mutagenesis or multi-site saturation mutagenesis; and / or The method according to claim 11 or 12 is used to test the terminal deoxynucleotidyl transferase function of the mutant.