Nucleic acid aptamer of epithelial cell adhesion molecule and preparation method thereof

A technology for adhesion molecules and epithelial cells, applied in the field of nucleic acids, can solve the problems of unsatisfactory clinical test results, poor antibody specificity, and low binding force, and achieve the effect of simple and fast screening and detection, small molecular weight, and good permeability

Active Publication Date: 2012-12-19
苏州德运康瑞生物科技有限公司
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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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  • Nucleic acid aptamer of epithelial cell adhesion molecule and preparation method thereof
  • Nucleic acid aptamer of epithelial cell adhesion molecule and preparation method thereof
  • Nucleic acid aptamer of epithelial cell adhesion molecule and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 In vitro screening of nucleic acid aptamers that specifically bind to the 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 / 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;

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

[0049] 3) Incubate the liquid not bound to Ni microbeads with EpCAM Ni microbeads at 37°C for 40min;

[0050] 4) Wash the incubated EpCAM Ni microbeads with binding buffer, and then perform PCR reaction on the EpCAM Ni microbeads bound to oligonucleotides;

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

Embodiment 2

[0056] Example 2 Detection of the Binding Ability of the Obtained Single-Stranded DNA to 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 microbeads, react for 30min, then use 0.1mol / L NaOH for single-stranded DNA, and purify by desalting column That is, the FAM-labeled single-stranded DNA for flow analysis is obtained.

[0058] 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 resusp...

Embodiment 3

[0060] Example 3 Screening to obtain nucleic acid aptamers that specifically bind to various cell lines

[0061] 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;

[0062] 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.

[0063] 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 and6 ).

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Abstract

The invention relates to a nucleic acid, in particular to a nucleic acid aptamer of the epithelial cell adhesion molecule and a preparation method thereof. The invention provides a high-specificity and high-affinity nucleic acid aptamer of the epithelial cell adhesion molecule EpCAM and a preparation method and applications thereof. The nucleic acid aptamer of the epithelial cell adhesion molecule EpCAM is of the G-quadruplex structure or the stem-loop structure. The preparation method comprises the following steps: designing and synthetizing a single-stranded DNA (deoxyribonucleic acid) random oligonucleotide library, screening the target oligonucleotide sequence, and identifying the binding specificity and affinity of the sequence to the target protein through the flow cytometry. The screened nucleic acid aptamer is non-toxic, has small molecular weight and good permeability, is easy in synthesis and marking and can only perform specific identification to the EpCAM protein and perform specific identification and specific expression to the cell line of the EpCAM protein; and the nucleic acid aptamer can not identify the cell line which does not express the protein.

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 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, de Leij LM, et a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115C12N15/10C12Q1/68G01N33/68
Inventor 杨朝勇宋彦龄安源邹远张薇婷邬杰庄峙厦
Owner 苏州德运康瑞生物科技有限公司
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