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Aptamer EpCAM (epithelial cell adhesion molecule) Ccut of EpCAM and preparation method thereof

An adhesion molecule and epithelial cell technology, applied in the field of nucleic acids, can solve problems such as low binding force, poor antibody specificity, and unsatisfactory clinical test results, and achieve the effects of small molecular weight, good permeability, and simple and rapid screening and detection.

Active Publication Date: 2013-11-13
XIAMEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, due to the poor specificity and low binding force of EpCAM immunotherapy, the results of clinical trials are not satisfactory.

Method used

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  • Aptamer EpCAM (epithelial cell adhesion molecule) Ccut of EpCAM and preparation method thereof
  • Aptamer EpCAM (epithelial cell adhesion molecule) Ccut of EpCAM and preparation method thereof
  • Aptamer EpCAM (epithelial cell adhesion molecule) Ccut of EpCAM and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 In vitro screening of nucleic acid aptamers that specifically bind to epithelial cell adhesion molecule EpCAM

[0034] 1) Dissolve the synthesized 5nmol single-stranded DNA nucleic acid library in binding buffer (12mmol / L PBS, 0.55mmol / LMgCl 2 ), conduct heat treatment: heat at 95°C for 5 minutes, place on ice for 10 minutes, and then place at room temperature for 10 minutes;

[0035] 2) Incubate the processed single-stranded DNA nucleic acid library with Ni microbeads, and collect the liquid that is not bound to Ni microbeads;

[0036] 3) Incubate the liquid not bound to Ni microbeads with EpCAM Ni microbeads at 37°C for 40min;

[0037] 4) Wash the incubated EpCAM Ni microbeads with binding buffer, and then perform PCR reaction on the EpCAM Ni microbeads bound to oligonucleotides;

[0038] The PCR reaction program was: 94°C pre-denaturation for 3 minutes, 94°C for 30s, 53°C for 30s, 68°C for 30s, amplification for 10 cycles, and finally 68°C for 5 minutes. ...

Embodiment 2

[0043] Example 2 Detection of the binding ability of the obtained single-stranded DNA to the epithelial cell adhesion molecule EpCAM by flow cytometry

[0044] First PCR amplifies the fluorescently labeled single-stranded DNA, using primer 2: 5'-Biotin-CTG ACC ACG AGC TCC ATT AG-3' and primer 3: 5'-FAM-AGC GTC GAA TAC CAC TAC AG-3' , the PCR product is double-stranded DNA with FAM at the 5' end and biotin at the 3' end, add streptavidin microbeads, react for 30min, and then use 0.1mol / L NaOH for single-stranded DNA, pass through a desalting column Purify to obtain FAM-labeled single-stranded DNA for flow analysis.

[0045] The dissociation constant (Kd=22.8 ±6.0). Use 200 μl of binding buffer to prepare DNA solutions of the above concentrations, heat at 95°C for 5 minutes, place on ice for 10 minutes, and then place at room temperature for 10 minutes. Add 155nmol / L EpCAM microbeads and incubate at 37°C for 40min. Wash the beads 3 times with binding buffer, then resuspend t...

Embodiment 3

[0047] Example 3 Screening to obtain nucleic acid aptamers that specifically bind to various cell lines

[0048] 1) Dissolve the synthesized 5nmol single-stranded DNA nucleic acid in the binding buffer (12mmol / L PBS, 0.55mmol / L MgCl 2 ), conduct heat treatment: heat at 95°C for 5 minutes, place on ice for 10 minutes, and then place at room temperature for 10 minutes;

[0049] 2) Incubate the processed single-stranded DNA nucleic acid with 10,000 cells in a 24-well plate for 24 hours, at 37°C for 30 minutes or at 4°C for 40 minutes.

[0050] 3) After incubation, remove the buffer solution and wash twice, then scrape off the cells and dissolve them in 200 μL buffer solution, and use BD’s FACSAria flow cytometer to measure the fluorescence of the microbeads (see Figure 5 with 6 ).

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Abstract

The invention provides an aptamer EpCAM (epithelial cell adhesion molecule) Ccut of an EpCAM as well as a preparation method and application thereof, relating to nucleic acids. The aptamer has high specificity and affinity. The aptamer has a G tetramer structure or a stem-loop structure. The preparation method comprises the steps of designing and synthesizing a single-stranded DNA (deoxyribonucleic acid) random oligonucleotide bank, screening a target oligonucleotide sequence, and identifying the target protein binding specificity and affinity of the target oligonucleotide sequence by a flow analysis method. The screened aptamer does not have toxicity, has small molecular weight and good permeability, is easy to synthesize and label, only specifically identifies EpCAM proteins and cell lines expressing the EpCAM proteins, and does not have a function of identifying the cell lines which do not express the proteins.

Description

technical field [0001] The invention relates to a nucleic acid, in particular to a method for preparing a nucleic acid aptamer of an EpCAM (Epithelial cell adhesion molecule) epithelial cell adhesion molecule and its application in early detection, treatment and prognosis of tumors. Background technique [0002] EpCAM (Epithelial cell adhesion molecule) epithelial cell adhesion molecule belongs to the adhesion molecule family, also known as 17-A, ESA, EGP40, Trop-1, KSA, CD326, TACSTD1, CO17-1A, GA733-2, etc. EpCAM is a single transmembrane protein with a molecular weight of 30-40kDa encoded by the tumor-associated calcium signal transducer 1 (TACSTD1) gene, which is involved in regulating cell adhesion, mediating signal transduction, cell Functions such as migration, proliferation and differentiation (1. Kurtz JE, Dufour P. Adecatumumab: an anti-EpCAM monoclonal antibody, from the bench to the bedside[J]. Expert Opin BiolTher, 2010, 10(6):951-958; 2. Trzpis M, McLaughlin P...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115C12N15/10C12Q1/68
Inventor 杨朝勇宋彦龄安源邹远张薇婷邬杰庄峙厦
Owner XIAMEN UNIV
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