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A nucleic acid aptamer of epithelial cell adhesion molecule epcam screened in human plasma and its preparation method and application

A technology for adhesion molecules and epithelial cells, applied in the fields of bioanalysis and clinical medicine, can solve problems such as poor affinity, and achieve low cost, improved capture efficiency, and high specificity

Active Publication Date: 2021-01-08
XIAMEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] The purpose of the present invention is to overcome the deficiencies of the prior art, providing a nucleic acid aptamer of epithelial cell adhesion molecule EpCAM screened in human plasma and its preparation method and application, aiming at the current traditional nucleic acid aptamer screening method The obtained nucleic acid aptamer has problems in capturing CTCs in a complex physiological environment. The present invention uses human plasma to simulate a complex physiological environment to construct an aptamer screening platform for the epithelial cell adhesion molecule EpCAM; in the process of nucleic acid aptamer screening and evolution , Eliminate sequences with poor affinity in complex physiological environments, enrich nucleic acid aptamer sequences with high specificity, high affinity and good stability in complex physiological environments, and obtain nucleic acid aptamers that can be directly applied to capture in peripheral blood sequence, so as to establish a new efficient nucleic acid aptamer screening method that can be used in complex systems

Method used

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  • A nucleic acid aptamer of epithelial cell adhesion molecule epcam screened in human plasma and its preparation method and application
  • A nucleic acid aptamer of epithelial cell adhesion molecule epcam screened in human plasma and its preparation method and application
  • A nucleic acid aptamer of epithelial cell adhesion molecule epcam screened in human plasma and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Example 1 Screening of nucleic acid aptamers of epithelial cell adhesion molecule EpCAM in human plasma

[0075] 1) Chemically synthesize an initial DNA library of 80 bases, as shown in SEQ ID No.8, the two ends of the initial DNA library are fixed sequences, including a random sequence of 40 bases in the middle, which is 5'- AGC GTC GAA TAC CAC TACAG-40nt-CTA ATG GAG CTC GTG GTC AG-3' with a library capacity of 10 15above. Take 5nmol DNA initial library dissolved in binding buffer (12mmol / L PBS, pH 7.4, 150mmol / L NaCl, 5mmol / L KCl, 0.55mmol / L MgCl 2 ), perform denaturation treatment: 95°C for 10 minutes, place on ice for 5 minutes, and then place at room temperature for 10 minutes;

[0076] 2) In the environment of human plasma, incubate the processed DNA initial library with Ni agarose microspheres at 37°C, carry out reverse screening, and collect the liquid that is not combined with Ni agarose microspheres; among them, the human plasma The preparation method is as...

Embodiment 2

[0081] Example 2 Investigating the Binding Ability of Single-Stranded DNA Enrichment Library and EpCAM Protein by Flow Cytometry

[0082] First, PCR amplify the 0, 4, 6, 8, 9, and 10 rounds of DNA enrichment libraries with fluorescent labels, using the forward primer (SEQ ID No.9): 5'-FAM-AGC GTC GAA TAC CAC TAC AG-3' and reverse primer (SEQ ID No.10): 5'-Biotin-CTG ACC ACG AGC TCC ATT AG-3', pre-denaturation at 94°C for 3 minutes, 94°C for 30s, 53°C for 30s, 72°C for 30s, Amplify for 10 cycles, and finally extend at 72°C for 5 min; the PCR product is double-stranded DNA with FAM at the 5' end and biotin at the 3' end, add streptavidin agarose microspheres, react at room temperature for 30 min, and then Use 0.1mol / L NaOH to carry out alkali denaturation and single-strandization, desalt and filter through NAP-5 column, and purify to obtain single-stranded DNA enrichment libraries for the 0th, 4th, 6th, 8th, 9th, and 10th rounds;

[0083] Use 100 μL of human plasma to prepare t...

Embodiment 3

[0085] Example 3 Determination of the dissociation constant between the eighth round of single-stranded DNA enrichment library and EpCAM protein obtained by flow cytometry

[0086] First, PCR amplifies the 8th round DNA enrichment library with fluorescent labeling, using the forward primer 5'-FAM-AGC GTCGAA TAC CAC TAC AG-3' (SEQ ID No.9): with the reverse primer (SEQ ID No. .10): 5'-Biotin-CTG ACCACG AGC TCC ATT AG-3', the PCR product is double-stranded DNA with FAM at the 5' end and biotin at the 3' end, add streptavidin agarose micro spheres, reacted at room temperature for 30 minutes, and then used 0.1mol / L NaOH to carry out alkali denaturation and single-strandization, desalted and filtered through NAP-5column, and purified to obtain the eighth round of single-stranded DNA enrichment library;

[0087] Use 0,5,10,50,75,100nmol / L concentration gradient of the eighth round of single-stranded DNA enrichment library and EpCAM protein agarose microspheres to determine the disso...

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Abstract

The invention discloses a nucleic acid aptamer of an epithelial cell adhesion molecule (EpCAM) screened in human plasma and a preparation method and application thereof. The nucleic acid aptamer capable of conducting highly specific recognition and high-affinity binding on the epithelial cell adhesion molecule (EpCAM) in the complex physiological environment of the human plasma is provided, a nucleic acid aptamer screening method making the human plasma simulate the complex physiological environment is developed, and the nucleic acid aptamer which is directly applied to capture of CTCs in peripheral blood can be obtained; the finally obtained aptamer has the advantages of being easy to obtain, low in cost, not prone to inactivation, free of immunogenicity or toxicity and the like, by meansof the sequences, specific recognition and capture of CTCs of tumor cell lines of EpCAM protein and expression EpCAM protein are achieved; in addition, the nucleic acid aptamer has high affinity in the human plasma, application of the nucleic acid in biological analysis and clinical medicine is ensured, and the aptamer is expected to serve as a molecule recognition tool of CTCs detection to be applied to liquid biopsies.

Description

technical field [0001] The invention belongs to the technical field of biological analysis and clinical medicine, and in particular relates to a nucleic acid aptamer of epithelial cell adhesion molecule EpCAM screened in human plasma, a preparation method and application thereof. The present invention specifically provides a nucleic acid aptamer capable of highly specific recognition and high affinity binding to the epithelial cell adhesion molecule EpCAM in the complex physiological environment of human plasma, its screening method and its application in biological analysis and clinical medicine, and is expected to be used as a circulating Molecular recognition tool for tumor cell detection applied to liquid biopsy. Background technique [0002] Cancer has seriously threatened human survival and health and has become the leading cause of death worldwide. Early diagnosis and prognosis assessment of cancer is an important guarantee to improve the survival rate of patients. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/115C12N15/10G01N33/569
CPCC12N15/10C12N15/115C12N2310/16G01N33/56966C12Q2531/113
Inventor 杨朝勇黄梦娇宋彦龄黄培烽陈小锋朱志
Owner XIAMEN UNIV
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