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Group of nucleic acid aptamers for isozymes of creatine kinases and application of nucleic acid aptamers

A nucleic acid aptamer, creatine kinase technology, applied in genetic engineering, recombinant DNA technology, biochemical equipment and methods, etc., can solve problems such as unreported, and achieve the effect of high affinity and high binding specificity

Active Publication Date: 2017-09-15
BEIJING INSTITUTE OF TECHNOLOGYGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] So far, nucleic acid aptamers with high affinity and specificity to creatine kinase isozymes and rapid detection methods based on nucleic acid aptamers for creatine kinase isozymes have not been reported

Method used

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  • Group of nucleic acid aptamers for isozymes of creatine kinases and application of nucleic acid aptamers
  • Group of nucleic acid aptamers for isozymes of creatine kinases and application of nucleic acid aptamers
  • Group of nucleic acid aptamers for isozymes of creatine kinases and application of nucleic acid aptamers

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] 1. Selection and synthesis of single-stranded DNA library

[0063] The following 5 kinds of DNA were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.: a DNA library containing 81 bases, its nucleic acid sequence is: ATCCAGAGTGACGCAGCA(N 45)TGGACACGGTGGCTTAGT; "P1 biotin upstream": ATCCAGAGTGACGCAGCA; "P2 "Biotin downstream": ACTAAGCCACCGTGTCCA; "P3 upstream": ATCCAGAGGTGACGCAGCA; "P4 downstream": ACTAAGCCACCGTGTCCA. 1 OD DNA per tube, when needed, centrifuge at 12,000 rpm for 0.5 min before opening the cap; follow the instructions on the tube wall to prepare a 100 μM storage solution.

[0064] 2. Tosyl magnetic beads bound to creatine kinase isoenzyme

[0065] The tosyl magnetic bead (Dynal, 2mL) reagent bottle needs to be vortexed before use. Take 16.5μL (2×10 7 1) magnetic beads, wash twice with Buffer B (0.1M Na-phosphate buffer pH=7.4), add 1mL each time, after adding, vortex, place the 1.5mL centrifuge tube on the magnetic collector, after 1min, When t...

Embodiment 2

[0117] Get the creatine kinase isoenzyme nucleic acid aptamer described in embodiment 1, carry out the mensuration of dissociation constant respectively, concrete assay method is as follows:

[0118] (1) In a 96-well plate, 100 μL per well of creatine kinase isoenzyme (PROSPEC, 1 mg / 126.5 μL) solution at a concentration of 2000 ng / mL, incubated at 4°C overnight or at 37°C for 2 hours; creatine kinase isoenzyme Coating buffer (1.59gNa 2 CO 3 , 2.93 g NaHCO 3 , dissolved in 1L ultrapure water) for dilution.

[0119] (2) The liquid in the 96-well plate after the treatment in step (1) was emptied and patted dry, and washed twice with 200 μL phosphate Tween buffer, 1 min each time.

[0120] (3) Add 300 μL of blocking solution to each well of the 96-well plate treated in step (2), and incubate at 4°C for 1 hour; Prepared in saline buffer;

[0121] (4) Wash the 96-well plate treated in (3) three times with 200 μL phosphate Tween buffer, 1 min each time;

[0122] (5) Use phospha...

Embodiment 3

[0132] A method for preparing a colloidal gold-labeled A nucleic acid aptamer, the steps are as follows:

[0133] (1) A nucleic acid aptamer with the nucleotide sequence described in SEQ ID No.2, which is modified by sulfhydryl groups and has 20 thymine sequences attached to one end, is added to TE buffer solution (Tris-EDTA buffer) to form solution 1 , adding tris(2-carboxyethyl)phosphine (TCEP) to solution 1, and reacting for 2 hours, the molar ratio of TCEP to the sulfhydryl-modified nucleic acid aptamer of the present invention is 5:1, to obtain solution 2;

[0134] (2) Take 1 mL of colloidal gold solution, centrifuge at 8000 r / min for 30 min, discard the supernatant, add 100 μL of ultrapure water, mix 12 μL of solution 2, shake for 1 min, and overnight to obtain solution 3;

[0135] (3) Add 40 mM NaCl aqueous solution equal to the volume of solution 3 to solution 3 to obtain solution 4 with a concentration of 20 mM, shake for 1 min, and react at room temperature for 8 h; ...

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Abstract

The invention relates to a group of nucleic acid aptamers for isozymes of creatine kinases and application of the nucleic acid aptamers, and belongs to the field of biotechnologies. Nucleotide sequences of the nucleic acid aptamers are selected from SEQ ID No.1-SEQ ID No.10 in a nucleotide sequence table. The nucleic acid aptamers and the application have the advantages that the nucleic acid aptamers are high in binding specificity and affinity for the isozymes of the creatine kinases; the nucleic acid aptamers can be used for detecting, separating, purifying and immobilizing the isozymes of the creatine kinases, can be applied to preparing biosensors and analyzing and detecting target substances and is particularly applicable to preparing detection test paper in reagent kits for quickly detecting the isozymes of the creatine kinases; the nucleic acid aptamers have certain merits such as in-vitro screening, synthesis, modification easiness and high stability which are deficient in antibodies when the nucleic acid aptamers are used for detecting the isozymes of the creatine kinases.

Description

technical field [0001] The present invention relates to a group of creatine kinase isoenzyme nucleic acid aptamers and applications thereof, in particular to a method for preparing a group of creatine kinase isoenzyme nucleic acid aptamers using SELEX technology in molecular biology technology, that is, phylogenetic evolution index enrichment technology Kinase isozymes have high specificity and high affinity nucleic acid aptamers, and apply them. Belongs to the field of biotechnology. Background technique [0002] Creatine Kinase isoenzyme (Creatine Kinase-MB) is a clinically used biomarker for the diagnosis of acute myocardial infarction. Creatine kinase isoenzyme enters the blood at the 4th to 6th hour of acute myocardial infarction, reaches its peak at the 24th hour, and returns to normal levels after 48 to 72 hours. Rapid diagnosis in the early stage of the disease will help formulate a better treatment plan and achieve a better treatment effect. [0003] At present, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115G01N33/573
CPCC12N15/115C12N2310/16G01N33/573
Inventor 吕雪飞冯薇邓玉林
Owner BEIJING INSTITUTE OF TECHNOLOGYGY
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