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CN-122012451-A - Wenchang fish tail end deoxynucleotidyl transferase and application thereof

CN122012451ACN 122012451 ACN122012451 ACN 122012451ACN-122012451-A

Abstract

The invention relates to a tail end deoxynucleotidyl transferase of Wenchang fish and application thereof, belonging to the technical field of genetic engineering. The invention provides a tail end deoxynucleotidyl transferase (BbeTdT) of Wenchang, the amino acid sequence of which is shown as SEQ ID NO. 2 or SEQ ID NO. 4. We have found that the terminal deoxynucleotidyl transferase (TdT) from Wenchang has not only terminal transferase activity under in vitro conditions, but also a nucleotide preference different from that of vertebrate TdT. This allows BbeTdT to have better selectivity for a particular sequence or terminal structure and reduces non-specific additions during practical use, thus complementing mammalian TdT, suitable for industrial processes requiring different preferences. In addition, we constructed BbeTdT mutants with higher terminal transferase activity, further optimizing the engineering potential of BbeTdT.

Inventors

  • YUAN SHAOCHUN
  • HUANG ZIWEN
  • YAN PEIYANG
  • HU YIHENG
  • Cai Zhenxi
  • WANG YING
  • XU ANLONG

Assignees

  • 中山大学

Dates

Publication Date
20260512
Application Date
20260206

Claims (10)

  1. 1. The tail end deoxynucleotidyl transferase of the Wenchang is characterized in that the amino acid sequence of the tail end deoxynucleotidyl transferase of the Wenchang is shown as SEQ ID NO. 2 or SEQ ID NO. 4.
  2. 2. The amphioxus terminal deoxynucleotidyl transferase according to claim 1, wherein the nucleotide sequence of the amphioxus terminal deoxynucleotidyl transferase is shown as SEQ ID NO. 1 or SEQ ID NO. 3.
  3. 3. A recombinant vector, characterized in that, the recombinant vector contains the nucleotide sequence according to claim 2.
  4. 4. A recombinant cell comprising the recombinant vector of claim 3, wherein the cell expresses the amphioxus terminal deoxynucleotidyl transferase of claim 1 or 2.
  5. 5. Use of the recombinant vector of claim 3 in the synthesis of nucleic acid molecules, of the amphioxus terminal deoxynucleotidyl transferase of claim 1 or 2.
  6. 6. The use according to claim 5, wherein the synthesis is the addition of deoxynucleotides at the 3' -end of the starting nucleic acid strand.
  7. 7. The use according to claim 6, wherein the preference for adding deoxynucleotides is dTTP > dGTP > dCTP > dATP.
  8. 8. A method for synthesizing a nucleic acid molecule, comprising the step of contacting a starting nucleic acid strand with at least one deoxynucleotide in the presence of the deoxynucleotidyl transferase at the tail end of Wenchang according to claim 1 or 2.
  9. 9. A kit comprising the amphioxus terminal deoxynucleotidyl transferase of claim 1 or 2, one or more nucleotides, and at least one starting nucleic acid strand.
  10. 10. The kit of claim 9, wherein the starting nucleic acid strand is single-stranded DNA.

Description

Wenchang fish tail end deoxynucleotidyl transferase and application thereof Technical Field The invention relates to the technical field of genetic engineering, in particular to a tail end deoxynucleotidyl transferase of Wenchang fish and application thereof. Background The terminal deoxynucleotidyl transferase (terminal deoxyribonucleotidyl transferase, tdT for short, terminal transferase) is a polymerase belonging to the PolX polymerase protein family. TdT is distinguished in that it is capable of adding deoxynucleotides to the 3' -end of a DNA strand independent of the template. This unique biochemical property makes it an irreplaceable role in a variety of biotechnology. TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) assay, developed in 1992, which uses TdT to label the end of a DNA double strand break, is a classical method for detecting programmed cell death (apoptosis). In RACE (rapid amplification of cDNA ends) techniques, tdT is used to add sequences to the cDNA ends, providing primer binding sites for subsequent PCR amplification. In addition, tdT is also an important tool for DNA synthesis in vitro. In 2018, palluk et al developed a controllable DNA synthesis strategy based on TdT-dNTP conjugates. The method shows high-efficiency nucleotide extension capability and provides an important basis for synthesizing DNA with high fidelity and high efficiency. In addition, tdT plays an important role in DNA information storage and cell lineage tracing. The TdT proteins commonly used today are mainly derived from mammals such as cattle and mice. Of the many mammalian TdT, bovine-derived TdT has become the first choice in nucleic acid tool development and related detection methods due to its more efficient DNA end-to-end capability during evolution, and commercial kits based on bovine TdT have also been widely marketed. Commercial TdT proteins are generally obtained by expressing a terminal transferase gene in E.coli, thereby obtaining an active TdT protein. TdT of mammalian origin has a strong terminal transferase activity, but has a significant base preference in the catalytic process. In mammals, tdT is usually preferred over four deoxynucleotides in order of dGTP > dCTP > dTTP > dATP. This preference affects the catalytic efficiency of TdT in practical applications, changing the relative proportions of the different products, and thus limiting to some extent the range of application of TdT in mammals. Disclosure of Invention The invention aims to overcome the defects of the prior art and provide a tail end deoxynucleotidyl transferase of the Wenchang and application thereof. In order to achieve the above purpose, the technical scheme adopted by the invention is as follows: In a first aspect, the invention provides a tail end deoxynucleotidyl transferase of the amphioxus, and the amino acid sequence of the tail end deoxynucleotidyl transferase of the amphioxus is shown as SEQ ID NO. 2 or SEQ ID NO. 4. As a preferred embodiment of the first aspect, the nucleotide sequence of the amphioxus terminal deoxynucleotidyl transferase is shown as SEQ ID NO. 1 or SEQ ID NO. 3. To solve the problem that mammalian-derived TdT proteins significantly favor polymerization of dGTP and dCTP during catalysis, we systematically screened TdT-like sequences from a variety of invertebrates. Finally, we identified a class of active TdT-like proteins (hereinafter BbeTdT) in invertebrate Wenchang (Branchiostoma belcheri, bbe). The protein is successfully expressed and purified in an escherichia coli system. By in vitro enzyme activity detection, bbeTdT was confirmed to have significant terminal transferase activity, and further found that the base preference was significantly different from that of vertebrate TdT, and BbetdT had a higher polymerization preference for dTTP. This property demonstrates that BbeTdT has potential advantages in dealing with the application limitations imposed by the preference of mammalian TdT bases, providing new possibilities for developing more controllable and widely applicable terminal transferases. In a second aspect, the present invention provides a recombinant vector comprising a nucleotide sequence according to the first aspect. In a third aspect, the present invention provides a recombinant cell comprising a recombinant vector according to the second aspect, the cell expressing a amphioxus terminal deoxynucleotidyl transferase according to the first aspect. In a fourth aspect, the invention provides the use of the amphioxus terminal deoxynucleotidyl transferase of the first aspect, the recombinant vector as described in the second aspect, in the synthesis of a nucleic acid molecule. As a preferred embodiment of the fourth aspect, the synthesis is the addition of deoxynucleotides at the 3' end of the starting nucleic acid strand. As a preferred embodiment of the fourth aspect, the preference of adding deoxynucleotides is dTTP > dGTP > dCTP > dATP. In a fifth aspect, the