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Application of aptamer in recognition of L selectin and combination with L selectin

A nucleic acid aptamer and selectin technology, applied in the fields of biotechnology and clinical medicine, to achieve the effect of strong specificity and high affinity

Active Publication Date: 2015-11-11
INST OF CHEM CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are only a few successful examples so far, and the bottleneck problem lies in the purification / identification of nucleic acid aptamer target molecules located on the cell membrane

Method used

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  • Application of aptamer in recognition of L selectin and combination with L selectin
  • Application of aptamer in recognition of L selectin and combination with L selectin
  • Application of aptamer in recognition of L selectin and combination with L selectin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1, Identification of nucleic acid aptamer Sgc-3b specifically recognizing and binding to L-selectin

[0060] 1. Preparation of nucleic acid aptamer Sgc-3b and its derivatives

[0061] 1. Synthesis of nucleic acid aptamer Sgc-3b

[0062] The nucleic acid aptamer Sgc-3b is synthesized by a DNA synthesizer, and the nucleotide sequence of the nucleic acid aptamer Sgc-3b is as follows: 5'-CTTATTCAATTCCCGTGGGAAGGCTATAGAGGGGGCCAGTCTATGAATAAG-3' (sequence 1), according to the needs of the test can be in the nucleic acid aptamer Sgc- Different molecules are marked on 3b to obtain derivatives of the nucleic acid aptamer Sgc-3b. Among them, the biotin-labeled nucleic acid aptamer Sgc-3b was selected in the following example 1; the fluorescein (FAM)-labeled nucleic acid aptamer Sgc-3b was selected in other examples.

[0063] 2. DNA deprotection: after deprotection with cold ammonia, then dissolve the DNA in TEAA solution;

[0064] 3. DNA purification: Purify by PAGE or h...

Embodiment 2

[0097] Example 2, flow cytometric analysis method to detect the binding ability of nucleic acid aptamer Sgc-3b and L-selectin

[0098] 1. Preparation of nucleic acid aptamer solution and treatment of cell lines

[0099] 1. Preparation of fluorescein-labeled nucleic acid aptamer Sgc-3b solution (Sgc-3b-FAM) (100nM)

[0100] The fluorescein-labeled nucleic acid aptamer Sgc-3b was obtained by coupling the fluorescein group to the 5' end of the nucleic acid aptamer Sgc-3b. Sgc-3b-FAM was dissolved in binding buffer, and the concentration was calibrated according to the ultraviolet absorption ( 100nM), heated at 95°C for 5min, placed on ice for 5min, and placed at room temperature for 15min.

[0101] 2. Preparation of PE-labeled anti-L-selectin antibody solution (anti-CD62L-PE)

[0102] The PE-labeled anti-L-selectin antibody is a product of eBioscience, and 0.125 μg was added to each test, with a concentration of 1.25 μg / mL.

[0103] 3. Preparation of fluorescein-labeled contro...

Embodiment 3

[0152] Example 3, Application of the nucleic acid aptamer Sgc-3b in determining the expression of L-selectin in different types of cells

[0153] 1. Preparation of fluorescein-labeled aptamer Sgc-3b solution (Sgc-3b-FAM)

[0154] Dissolve Sgc-3b-FAM in binding buffer, calibrate the concentration (100 nM) according to UV absorption, heat at 95° C. for 5 min, place on ice for 5 min, and place at room temperature for 15 min.

[0155] 2. Pretreatment of cell lines

[0156] Take one plate of each of the following 16 logarithmic growth cell lines: growing rat alveolar epithelial cells (RAEC), human embryonic lung fibroblasts (MRC-5), human alveolar epithelial cells (A549), human cervical cancer cells ( Hela), human liver cancer cells (Huh-7), human bladder cancer cells (T24), human hepatocellular carcinoma cells (SK-Hep-1), human breast cancer cells (MCF-7), drug-resistant human breast cancer cells ( MCF-7R), human ovarian cancer cells (SKOV-3), human breast cancer cells (MDA-MB-2...

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Abstract

The invention discloses application of an aptamer in recognition of L selectin and combination with L selectin. The condition that the aptamer Sgc-3b can specifically recognize L selectin and can be specifically combined with L selectin is discovered for the first time, and an L selectin detecting method is established by utilizing the combination of specificities of the aptamer Sgc-3b and L selectin. Experiments prove that the aptamer Sgc-3b has the characteristics of being high in affinity, strong in specificity, free of immunogenicity and toxicity, and the like, and the L selectin detecting method established based on the aptamer Sgc-3b can be used for detection of L selectin expression and diagnosis of related diseases.

Description

technical field [0001] The invention belongs to the technical fields of biotechnology and clinical medicine, and in particular relates to the application of nucleic acid aptamers in recognizing and binding L-selectin. Background technique [0002] Nucleic acid aptamer (aptamer) is a kind of single-stranded DNA, RNA, peptide nucleic acid or chemically modified nucleic acid sequence that can specifically interact with target substances, usually consisting of 15-80 nucleotides. Nucleic acid aptamers can form specific three-dimensional structures and bind to target molecules with high affinity, such as hairpins, pseudoknots, G-quadruplexes and other structures. Highly specific binding is through van der Waals forces, hydrogen bonds, electrostatic interactions, and hydrophobic interactions. achieved by intermolecular interactions. Nucleic acid aptamers are called "chemical antibodies" because of their high affinity, good specificity, non-immunogenicity, easy synthesis, transform...

Claims

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Application Information

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IPC IPC(8): G01N33/53
CPCG01N33/53
Inventor 上官棣华邴涛汪寅生刘祥军
Owner INST OF CHEM CHINESE ACAD OF SCI
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