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A group of nattokinase nucleic acid aptamers and screening method thereof

A technology of soybean kinase nucleic acid and aptamer, which is applied in the field of genetic engineering, can solve the problems of difficult determination of nattokinase activity, good specificity, and high sensitivity, and achieve the effects of convenient chemical modification, high affinity, and strong affinity

Active Publication Date: 2022-04-08
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The screening method is the capillary electrophoresis method of ligand system evolution without exponential enrichment. The molecular recognition function of aptamer is used to accurately detect nattokinase. Difficulties in Determination of Soybean Kinase Activity

Method used

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  • A group of nattokinase nucleic acid aptamers and screening method thereof
  • A group of nattokinase nucleic acid aptamers and screening method thereof
  • A group of nattokinase nucleic acid aptamers and screening method thereof

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Experimental program
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Effect test

Embodiment

[0074] The screening method of a group of nattokinase nucleic acid aptamers provided in the embodiment of the present invention includes:

[0075] 1) Nucleic acid library and primer design

[0076] The nucleic acid primer design software Primer was used to design the sequence required for the experiment, and the secondary structure of the designed nucleic acid strand was evaluated by NUPACK software and The mfoldWeb Server.

[0077] 2) Separation of nattokinase, nucleic acids and complexes by capillary electrophoresis

[0078] The concentration of the immobilized fluorescently labeled oligonucleotide library is 0.5 μmol / L, and the concentration of nattokinase is 0.125 μmol / L. Mix the two, incubate at 30°C in the dark, take out every 10 min and shake gently. After 30min injection separation. The separation conditions are: capillary temperature 25°C, 0.5psi, 20s injection, separation voltage 20KV; before each injection for the first time, methanol, ultrapure water, 0.1mol / L hy...

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Abstract

The invention belongs to the technical field of genetic engineering, and discloses a group of nattokinase nucleic acid aptamers and a screening method thereof. The DNA sequences of the nattokinase nucleic acid aptamers are: SEQ ID NO: 1 to SEQ ID NO: 17; nattokinase Screening method of nucleic acid aptamer The purchased nattokinase is purified by chromatography and identified; the purified protein is used for screening of aptamers. The nucleic acid aptamer of the present invention has precise specificity, high affinity, and is convenient for chemical modification, and can be used as an effective molecular recognition tool for highly sensitive analysis of proteins. The recognition technology based on the nucleic acid aptamer is NK determination and Provide a basis for the development direction of high-efficiency separation and purification.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, in particular to a group of nattokinase nucleic acid aptamers and a screening method thereof. Background technique [0002] At present, the existing technologies commonly used in the industry are as follows: [0003] At present, the magnetic bead method is commonly used in the industry for screening, which requires the protein target to be immobilized on the magnetic beads, and the oligonucleotide molecules in the liquid phase diffuse freely. Corresponding target molecules that encounter the surface of the solid support can bind to it to remove unbound or bound weak oligonucleotide molecules. The oligonucleotide strand bound to the target molecule is eluted / isolated, followed by PCR amplification and the next cycle. This method requires multiple cycles, takes a long time, and is prone to nonspecific adsorption. [0004] Nattokinase (NK) as a thrombolytic agent has the characteristi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/115C40B50/06C12N15/10G01N33/68
CPCC12N15/115C40B50/06C12N15/1093C12N15/1089G01N33/68C12N2310/16C12Y304/21062C12N15/1048C12Q2531/107C12Q2541/101C12Q2565/125
Inventor 法芸赵海杰管明阳王琦刘会洲
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI