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A kind of double-stranded dna peptide ligase ddplasei and using method thereof

A technology of ligase and amino acid, applied in the field of ligase

Active Publication Date: 2022-07-12
黄种山
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are a variety of commercialized DNA ligases (such as T4 DNAlagase from Thermo Fisher) and modification enzymes (DNA methylase from NEB Company), which only connect and specifically modify nucleic acid fragments. Reports a ligase that can covalently link nucleic acid fragments and polypeptide fragments to each other

Method used

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  • A kind of double-stranded dna peptide ligase ddplasei and using method thereof
  • A kind of double-stranded dna peptide ligase ddplasei and using method thereof
  • A kind of double-stranded dna peptide ligase ddplasei and using method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1 Enzymes that can catalyze the linking of specific RNA single strands and specific polypeptides

[0021] The applicant used metagenomic sequencing technology to study the functional genome of the dry straw micro-ecosystem in a certain place in Fujian. Through the splicing analysis of the sequencing data, it was found that the upstream of more than a dozen different gene fragments contained similar special DNA structures. Enzyme (specifically refers to endoglucanase, EC.3.2.1.4, the main component of cellulase) gene as an example, the special DNA structure such as figure 1 shown. There are two ORFs (open reading frames) in the immediate front-end region of more than a dozen genes such as cellulase. Taking the cellulase gene as an example, the special DNA structure includes an X gene with a length of 1362 bp (the sequence of which is shown in SEQ ID NO. : 1) and a short peptide gene with a length of 24 bp (its sequence is shown in SEQ ID NO: 9). The polypeptide...

Embodiment 2

[0024] Example 2 Cloning and expression of X gene and exploration of X protein performance

[0025] Based on the X gene sequence (SEQ ID NO: 1), primers 5'-ATGCGGACGCGCCACAGC-3' and 5'-CTACATCTGACGTCGAAGG-3' were designed to amplify the X gene (refer to the conventional PCR system and conditions, the annealing temperature was 51 °C), using Japanese Takara The company's recombinant protein E. coli expression and purification system (pET Express & Purify Kits) clones and expresses the X gene according to the instructions, and uses the histidine tag attached to the fusion protein to separate and purify the X protein (the expression product encoded by the X gene). The final concentration of purified X protein measured by Qubit4.0 was 0.18 μg / ml, and its amino acid sequence is shown in SEQ ID NO: 2. It can be seen from Example 1 that X protein may be a ligase that can catalyze the connection of specific RNAs and specific polypeptides. Since X protein has been successfully expressed...

Embodiment 3

[0030] Example 3 Detection of reaction products of single-stranded RNA peptide ligase sRPlaseI and exploration of enzymatic properties

[0031]Examples 1 and 2 show that sRPlaseI is a ligase that can catalyze the ligation of a specific single-stranded RNA and a specific polypeptide. In order to further explore its enzymatic properties, a simple ligation reaction assay method should be established. Since the reaction product catalyzed by sRPlaseI ligase is a complex of covalently linked RNA single strand and polypeptide (O=P-C=O constructs a phosphorus-carbon bond), it is possible to try to measure its absorption spectrum characteristics in order to try to use a simple spectrophotometric method for the reaction Determination. Get each 3 μl of the reaction product in Table 1 of Example 2, and separate R9, P7 and blank reaction system, and use the Epoch microplate reader of Biotek Company in the United States to carry out full-wavelength scanning respectively in the range of 200n...

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Abstract

The present invention provides a novel double-stranded DNA peptide ligase dDPlaseI, which can be used to catalyze the covalent connection of a specific double-stranded DNA and a specific polypeptide, and its enzymatic properties and use methods. The amino acid sequence of the dDPlaseI enzyme is shown in SEQ ID NO: 6, and the corresponding gene coding sequence is shown in SEQ ID NO: 5; The 5'-terminal deoxyribonucleotide A is a specific DNA double-strand in the phosphorylated state and the C-terminal start is a specific polypeptide chain with 7 peptides of N'‑MANCEHL‑C', and catalyzes the covalent bond between the two Together; double-stranded DNA and polypeptide ligation products have characteristic absorption peaks at 360nm wavelength, which can be used for the determination of dDPlaseI ligase activity. dDPlaseI ligase buffer was composed of 450 mM Tris-HCl, 100 mM Mg, pH 7.8 2+ , 80mM NaCl, 20mM ATP and 8mM Triton X-100, the optimal reaction temperature range is 35℃-45℃, and the ligation reaction time is 3min-10min. The novel double-stranded DNA polypeptide ligase provided by the present invention can be used as a tool enzyme for genetic engineering and gene analysis.

Description

technical field [0001] The present invention relates to the field of molecular biology, in particular to a brand-new ligase that can connect a specific double-stranded DNA fragment and a specific polypeptide and a method for using the same. Background technique [0002] The field of genetic engineering is inseparable from various molecular biology tool enzymes. These tool enzymes can realize in vitro nucleic acid amplification, transcription or reverse transcription, digestion or excision, ligation and modification, and protein digestion or excision. They are widely used in Target gene amplification, nucleic acid sequence analysis, recombinant DNA preparation, vector construction, nucleic acid probe labeling and protein analysis, etc. For example, DNA polymerase is the core component of the PCR system, and the construction of cDNA library is inseparable from RNA polymerase . Commonly used tool enzymes in genetic engineering, mainly DNA polymerase, restriction endonuclease, ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/00C12P21/02
CPCC12N9/93C12P21/00C12P21/02C07K7/06
Inventor 黄志玲黄种山其他发明人请求不公开姓名
Owner 黄种山
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