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DNA aptamer of small cell lung cancer marker gastrin releasing peptide precursor polypeptide fragment

A gastrin-releasing peptide and nucleic acid aptamer technology, applied in the field of chemical biology, can solve problems such as unseen screening

Inactive Publication Date: 2015-08-19
ZHENGZHOU UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Currently no screening of Pro-GRP 31-98 Related reports on DNA aptamer sequences

Method used

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  • DNA aptamer of small cell lung cancer marker gastrin releasing peptide precursor polypeptide fragment
  • DNA aptamer of small cell lung cancer marker gastrin releasing peptide precursor polypeptide fragment
  • DNA aptamer of small cell lung cancer marker gastrin releasing peptide precursor polypeptide fragment

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Embodiment

[0030] (1) In order to couple proteins on carboxyl-modified micron magnetic beads, first use 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxysulfosuccinimide (Sulfo-NHS) was activated; the activated magnetic beads were treated with 0.01M MES buffer (2.13g / L morpholine-ethanesulfonic acid·H 2 (0, 0.31g / L boric acid, pH=9.0) after washing, react overnight with the protein or polypeptide to be coupled in MES (0.01M, pH=9.0) solution on a molecular hybridization instrument at 30°C with shaking. After the reaction, the magnetic beads were magnetically recovered, and then resuspended in 0.05M phosphate buffer saline (PBS) (pH=7.4) to form a 1 mg / mL magnetic bead solution.

[0031] (2) Operation steps of DNA incubation and PCR amplification during SELEX screening:

[0032] First take 30 μL of Pro-GRP with a concentration of 1 mg / mL 31-98Coat the magnetic beads, wash thoroughly with Binding Buffer, and then resuspend them in 300 μL Binding Buffer for later use. Th...

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Abstract

The invention discloses a DNA aptamer sequence of a small cell lung cancer marker gastrin releasing peptide precursor polypeptide fragment Pro-GRP31-98, and relates to the technical field of chemicobiology. The DNA aptamer sequence is obtained by screening from a single-chain DNA library through an SELEX (Systematic Evolution of Ligands by Exponential Enrichment) screening method based on an affinity magnetic bead separation method, then carrying out PCR amplification on the screened DNA aptamer library, then separating a DNA aptamer contained in the DNA aptamer library through a gene clone technology by taking escherichia coli as a recipient cell and finally sequencing various DNA aptamers through a sequencing technology. An experimental result indicates that the DNA aptamer sequence disclosed by the invention is combined with a gastrin releasing peptide precursor fragment at high affinity and high specificity.

Description

technical field [0001] The invention relates to the technical field of chemical biology, more specifically to the sequence screening of the DNA nucleic acid aptamer of the small cell lung cancer marker gastrin releasing peptide precursor polypeptide fragment. Background technique [0002] Nucleic acid aptamers (Aptamers) are single-stranded DNA or RNA molecules selected from a large-capacity random oligonucleotide sequence library for non-nucleic acid target molecules that can bind to the target molecule with high affinity and specificity. Repeated adsorption, recovery, and re-amplification processes to achieve exponential enrichment of oligonucleotide sequences that bind to target molecules. This method of selecting nucleic acid aptamers is called SELEX (Systematic Evolution of Ligands by Exponential enrichment, systematic evolution of ligands through exponential enrichment), which was developed by Tuerk and Gold in 1990 (documents: C.Tuerk, L.Gold, Systematic evolution of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115
Inventor 崔惠芳李亚军王佳栗晓佳
Owner ZHENGZHOU UNIV
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