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

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

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 In vitro screening of aptamers that specifically bind to epithelial cell adhesion molecule EpCAM

[0047] 1) Dissolve the synthesized 5nmol single-stranded DNA nucleic acid library in binding buffer (12mmol / L PBS, 0.55mmol / L MgCl 2 ), heat treatment: heat at 95°C for 5 minutes, place on ice for 10 minutes, and then place at room temperature for 10 minutes;

[0048] 2) Incubate the processed single-stranded DNA nucleic acid library with Ni microbeads, and collect the liquid that is not bound to the Ni microbeads;

[0049] 3) Incubate the liquid that has not been combined with Ni microbeads together with EpCAM Ni microbeads at 37°C for 40 minutes;

[0050] 4) Wash the incubated EpCAM Ni beads with binding buffer, and then use the EpCAM Ni beads with oligonucleotides for PCR reaction;

[0051] The PCR reaction program is: 94°C pre-denaturation 3min, 94°C 30s, 53°C 30s, 68°C 30s, 10 cycles of amplification, and final extension at 68°C for 5 minutes,

[0052] Primer 1: 5'-FAM...

Embodiment 2

[0056] Example 2 Detect the binding ability of the obtained single-stranded DNA with the epithelial cell adhesion molecule EpCAM by flow cytometry

[0057] 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 a double-stranded DNA with FAM at the 5'end and biotin at the 3'end. Add streptavidin beads, react for 30 minutes, and then single-stranded with 0.1mol / L NaOH, and pass through a desalting column Purify and obtain FAM-labeled single-stranded DNA for flow analysis.

[0058] Use 0nmol / L, 5nmol / L, 10nmol / L, 20nmol / L, 50nmol / L, 100nmol / L, 200nmol / L concentration gradient of single-stranded DNA and target protein EpCAM Ni beads to determine the dissociation constant (Kd=22.8 ±6.0). Prepare the DNA solution of each concentration with 200 μl binding buffer, heat at 95°C for 5 min, place on ice for 10 min, and then at room temperature for 10 mi...

Embodiment 3

[0060] Example 3 Screening for specific binding of nucleic acid aptamers to various cell lines

[0061] 1) Dissolve the synthesized 5nmol single-stranded DNA nucleic acid in binding buffer (12mmol / L PBS, 0.55mmol / L MgCl 2 ), heat treatment: heat at 95°C for 5 minutes, place on ice for 10 minutes, and then place at room temperature for 10 minutes;

[0062] 2) Incubate the processed single-stranded DNA nucleic acid with 10,000 cells in a 24-well plate for 24 hours, and incubate at 37°C for 30 minutes or 4°C for 40 minutes.

[0063] 3) After incubation, remove the buffer solution and wash twice, then scrape the cells and dissolve in 200μL buffer solution, and use the BD company's FACSAria flow cytometer to measure the fluorescence of the beads (see Figure 5 with 6 ).

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Abstract

The invention provides an aptamer EpCAM (epithelial cell adhesion molecule) D 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 present invention relates to a nucleic acid, in particular to a preparation method of a nucleic acid aptamer of 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 and so on. EpCAM is a single transmembrane protein with a molecular weight of 30-40kDa encoded by tumor-associated calcium signal transducer1 (TACSTD1) gene. It is involved in regulating cell adhesion, mediating signal transduction, and 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 PM...

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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