Nattokinase nucleic acid aptamer and screening method thereof
A technology of soybean kinase nucleic acid and screening method, which is applied in the field of nattokinase nucleic acid aptamer and its screening, can solve the problems of high sensitivity, difficulty in measuring nattokinase activity, good specificity, etc., achieve high affinity and facilitate chemical Modified, precise and specific effects
Active Publication Date: 2019-10-11
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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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
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Embodiment
[0074] The screening method of a group of nattokinase nucleic acid aptamers that the embodiment of the present invention provides comprises:
[0075] 1) Nucleic acid library and primer design
[0076] Use the nucleic acid primer design software Primer to design the sequence required for the experiment, and use NUPACK software and The mfoldWeb Server to evaluate the secondary structure of the designed nucleic acid chain.
[0077] 2) Separation of nattokinase, nucleic acid and complex by capillary electrophoresis
[0078] The concentration of the immobilized fluorescently labeled oligonucleotide library was 0.5 μmol / L, and the concentration of nattokinase was 0.125 μmol / L. Mix the two, incubate at 30°C in the dark, take it out every 10 minutes and shake it gently. After 30 min, the sample was injected and separated. Separation conditions are: capillary temperature 25°C, 0.5psi, 20s sample injection, separation voltage 20KV; before each injection for the first time, use methano...
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The invention belongs to the technical field of genetic engineering, and discloses a nattokinase nucleic acid aptamer and a screening method thereof. The DNA sequence of the nattokinase nucleic acid aptamer is shown in SEQ ID NO:1 to SEQ ID NO:17. The screening method of the nattokinase nucleic acid aptamer comprises the steps that purchased nattokinase is subjected to chromatographic purificationand identification; a purified protein is employed to screen the aptamer. The nucleic acid aptamer is precise in specificity and high in affinity, facilitates chemical modification, and can be used as an effective molecular recognition tool to be used for high-sensitivity analysis of the protein, and the nucleic acid aptamer-based recognition technology provides a basis for the development direction of NK determination and efficient separation and purification.
Description
technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates 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. The protein target needs to be immobilized on the magnetic beads, and the oligonucleotide molecules in the liquid phase diffuse freely. Corresponding target molecules encountering the surface of the solid support can bind to it to remove unbound or bound weak oligonucleotide molecules. The oligonucleotide strands bound to the target molecule are eluted / separated, followed by PCR amplification and the next cycle. This method requires multiple cycles, is time-consuming, and is prone to non-specific adsorption. [0004] Nattokinase (Nattokinase, NK) as a thrombolytic agent has...
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Patent Timeline
Login to View More 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