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Cardiac troponin I specific nucleic acid aptamer and application thereof

A technology for cardiac troponin and nucleic acid aptamers, which can be used in the biological and medical fields to solve the problems of increased cost, low stability, suboptimal affinity and stability, etc.

Active Publication Date: 2020-09-15
UNIV OF SCI & TECH OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, limited by the existing nucleic acid aptamer screening technology, the obtained nucleic acid aptamer is not the simplest sequence to bind to the target, and the affinity and stability are not optimal.
In practical application, it will lead to increased cost and low stability, so there is a demand for cTnI nucleic acid aptamers with shorter sequences, higher affinity and higher stability

Method used

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  • Cardiac troponin I specific nucleic acid aptamer and application thereof
  • Cardiac troponin I specific nucleic acid aptamer and application thereof
  • Cardiac troponin I specific nucleic acid aptamer and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1: Synthesis of ssDNA nucleic acid aptamers specifically binding to cTnI protein

[0049] Synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. SEQ ID No.1 (cTnI-14S-7), SEQ ID No.2 (cTnI-14S-3), SEQ ID No.3 (cTnI-14S-16) The indicated nucleic acid aptamers.

Embodiment 2

[0050] Embodiment 2: surface plasmon resonance (SPR) detects the affinity of cTnI nucleic acid aptamer and cTnI protein

[0051] 1. The nucleic acid aptamers cTnI-14-7 (SEQ ID No.1), cTnI-14-3 (SEQ ID No.2), cTnI-14-16 (SEQ ID No.3) synthesized by Shanghai Sangon, Diluted with DPBS to: 0, 1.25nM, 2.5nM, 5nM, 10nM, 20nM, 50nM, 100nM;

[0052] 2. Coupling cTnI protein to the surface of CM5 chip (manufacturer: GE Healthcare, model: Biacore Sensor Chipseries CM5): first wash the chip with 50mM NaOH, inject 20ul at a flow rate of 10ul / min, and then use an equal volume of EDC (1 -(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; 0.4M aqueous solution) and NHS (N-hydroxysuccinimide; 0.1M aqueous solution) are mixed and injected into 50ul Activate the chip with a flow rate of 5ul / min. The cTnI protein was diluted with 10 mM sodium acetate at pH 4.5 to a final concentration of 50 μg / mL and then injected. The injection volume was 15 μL, the flow rate was 5 uL / min, and the cTn...

Embodiment 3

[0055] Embodiment 3: surface plasmon resonance (SPR) detects the binding situation of cTnI nucleic acid aptamer and interfering protein

[0056] 1. The nucleic acid aptamers cTnI-14-7 (SEQ ID No.1), cTnI-14-3 (SEQ ID No.2), cTnI-14-16 (SEQ ID No.3) synthesized by Shanghai Sangon, Dilute to 100nM with DPBS;

[0057] 2. Coupling cTnI protein, TNFα protein, BSA protein, and SA protein to the 1st, 2nd, 3rd, and 4th channels on the surface of the CM5 chip: first wash the chip with 50mM NaOH, inject 20ul at a flow rate of 10ul / min, and then With equal volumes of EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; 0.4M aqueous solution) and NHS (N-hydroxysuccinimide; 0.1M aqueous solution) After mixing the two reagents, inject 50ul of the activated chip at a flow rate of 5ul / min. Dilute the cTnI protein with 10mM sodium acetate at pH 4.5 to a final concentration of 50μg / mL and inject the sample. The injection volume is 15μL, the flow rate is 5uL / min, the cTnI protein ...

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Abstract

The invention provides a single-stranded DNA aptamer specifically binding to cardiac troponin I (hereinafter referred to as cTnI protein). The single-stranded DNA aptamer comprises a nucleotide sequence as shown in any one of SEQ ID Nos.1-3. The nucleic acid aptamer provided by the invention can also be various similar sequences with high homology or derivatives obtained from the sequences provided by the invention. The nucleic acid aptamer provided by the invention has stronger binding capacity with cardiac troponin I and has a shorter sequence, and also has the advantages of small molecularweight, easiness in synthesis, short production time, lower synthesis cost and the like. The invention also provides an application of the single-stranded DNA aptamer. The single-stranded DNA aptamercan be used for cTnI protein purification or cTnI protein detection independently or in combination. The invention also provides a kit for detecting the cTnI protein, and the kit comprises the single-stranded DNA aptamer provided by the invention.

Description

technical field [0001] The invention relates to the fields of biology and medicine, in particular to a nucleic acid aptamer capable of binding cardiac troponin I (Cardiactroponin I, cTnI) and its application. Background technique [0002] Cardiovascular disease is one of the important diseases affecting human health in today's society, and acute myocardial infarction is an important cause of death in cardiovascular diseases. Abnormal myocardial injury markers are one of the main basis for the diagnosis of myocardial infarction, so there are many diagnostic reagents for early myocardial injury serum markers at this stage. Cardiac troponin I (cTnI) is a specific protein of the myocardium, which has special clinical value for the detection of small myocardial damage. Normally, the level of cTnI in the circulation is very low. When the cardiomyocytes are damaged, cTnI It quickly enters the blood before other biochemical indicators, and rises within 3-5 hours. With the aggravati...

Claims

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Application Information

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IPC IPC(8): C12N15/115G01N33/68
CPCC12N15/115G01N33/68C12N2310/16G01N2800/325
Inventor 罗昭锋方晓娜何军林王进军张立云
Owner UNIV OF SCI & TECH OF CHINA
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