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DNA (Desoxyribonucleic Acid) ligand for specific binding with EPO (erythropoietin), preparation method thereof and application thereof

An aptamer, specific technology, applied in the field of protein new recognition element-DNA aptamer

Active Publication Date: 2011-09-21
INST OF PHARMACOLOGY & TOXICOLOGY ACAD OF MILITARY MEDICAL SCI P L A
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, there is no research work using SELEX technology to screen specific oligonucleotide aptamers against rHuEPO-α at home and abroad.

Method used

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  • DNA (Desoxyribonucleic Acid) ligand for specific binding with EPO (erythropoietin), preparation method thereof and application thereof
  • DNA (Desoxyribonucleic Acid) ligand for specific binding with EPO (erythropoietin), preparation method thereof and application thereof
  • DNA (Desoxyribonucleic Acid) ligand for specific binding with EPO (erythropoietin), preparation method thereof and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Example 1. Construction of random single-stranded DNA (ssDNA) library and its primers

[0078] (a) Construct a random ssDNA library with a length of 80 bases:

[0079] 5'-CTTCTGCCCGCCTCCTTCC-N 39 -GGAGACGAGATAGGCGGACACT-3' wherein N represents any one of the bases A, G, C, T (see Berezovski M, MusheevM, Drabovich A, Krylov SN.Non-SELEX Selection of Aptamers.J.Am.Chem.Soc.2006 , 128:1410-1411), the capacity of the library is 10 13 -10 15 .

[0080] (b) Synthesize the forward primer labeled with fluorescein (FAM) at the 5' end:

[0081] 5'-FAM-CTTCTGCCCGCCTCCTTCC-3' (SEQ ID NO: 121),

[0082] (c) Synthesize the reverse primer of the 5' terminal labeling biotin (biotin):

[0083] 5'-Biotin-AGTGTCCGCCTATCTCGTCTCC-3' (SEQ ID NO: 122).

[0084] The random ssDNA library and primers were synthesized by Shanghai Sangon Biotechnology Co., Ltd.

Embodiment 2

[0085] Example 2. Screening of DNA aptamers that specifically recognize rHuEPO-α

[0086] In order to screen out DNA aptamers with strong binding specificity to rHuEPO-α, a total of 8 rounds of screening were carried out by the SELEX method (8 SELEX cycles, see figure 1 ), the following takes the first round of screening as an example to describe this process in detail.

[0087] (1) SELEX method to screen the ssDNA library that specifically binds to rHuEPO-α

[0088] (a) Reverse screening treatment of wheat germ agglutinin coupling resin and ssDNA library

[0089] Take 2000 pmol of the random ssDNA library and dissolve it in 300 μL 1×SELEX screening buffer (1×SELEX screening buffer: 20 mM Tris-HCl, 140 mM NaCl, 5 mM MgCl 2 , 5mM KCl, PH=7.4), and placed in a water bath at 95°C for 5 minutes, then immediately placed in an ice bath for 10 minutes.

[0090] Then 220 μL of wheat germ agglutinin (WGA)-coupled agarose resin (particle size 200-300 μm, purchased from Sigma-Aldr...

Embodiment 3

[0143] Example 3. Electrophoretic mobility shift assay (EMSA) of screened oligonucleotide libraries

[0144] (1) Radioisotope labeling of ssDNA library

[0145] Add 2 μL of labeling buffer (10×T4 polynucleotide kinase buffer, TaKaRa Company), 30 pmol of ssDNA to be labeled with radioisotope (original ssDNA library and oligonuclei obtained from the 4th, 6th, and 8th rounds of screening) to the EP tube. nucleotide library), 4 μL water, 20 U (activity unit) of T4 polynucleotide kinase, 20 μL Ci[γ- 32 P]ATP, make up the volume to 20 μL with water, mix well, and incubate at 37°C for 1 hour. Immediately act at 68°C for 10 minutes to inactivate the activity of the enzyme, and filter the solution with a 30kDa ultrafiltration tube to remove free [γ- 32 P]ATP, finally dissolved in water to obtain 60μL [γ- 32 P] ATP-labeled ssDNA solution.

[0146] (2) EMSA determination

[0147] Take 25nM of [γ- 32 P] The ATP-labeled ssDNA library was incubated with 0, 25, 50, 75, 100, 200, ...

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Abstract

The invention relates to a DNA (Desoxyribonucleic Acid) ligand for specific binding with EPO (erythropoietin), a preparation method thereof and application thereof, specifically provides a DNA ligand capable of specifically binding with the EPO and a method for screening the ligand by an improved SELEX (Systematic Evolution of Ligands by Exponential Enrichment) technology, and provides EPO detecting elements with better detecting stability, higher sensitivity, convenience and quickness.

Description

technical field [0001] The present invention relates to the fields of proteinology, molecular biology and combinatorial chemistry. Specifically, the present invention relates to a novel protein recognition element-DNA aptamer (Aptamer), its preparation method and its use. Background technique [0002] Erythropoietin (EPO) is a functional glycoprotein that promotes the production of human red blood cells. When the human body is in a low blood oxygen concentration, the erythropoietin secreted by the kidney activates the erythropoietin receptor on the membrane of the erythropoietin cell, making it proliferate and differentiate into mature red blood cells, thereby regulating the number of peripheral red blood cells, so EPO is A potent stimulator of erythropoietin (Jelkmann W. Erythropoietin: structure, control of production, and function. Physiol Rev, 1992, 72(2):449-489). [0003] In 1985, people used gene recombination technology to synthesize recombinant human erythropoieti...

Claims

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Application Information

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IPC IPC(8): C12N15/115C12Q1/68G01N33/68C40B30/04
Inventor 谢剑炜张朝阳郭磊唐吉军
Owner INST OF PHARMACOLOGY & TOXICOLOGY ACAD OF MILITARY MEDICAL SCI P L A
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