Nucleic acid aptamer capable of specifically recognizing vimentin and application thereof

A nucleic acid aptamer and specific technology, applied in the biological field, can solve the problems of high immunogenicity, high production cost, instability, etc., and achieve the effect of simple reaction conditions, short incubation time, and high stability

Active Publication Date: 2019-02-19
THE FIRST AFFILIATED HOSPITAL OF WENZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Although the Vimentin monoclonal antibody developed in recent years has high affinity and specificity, the antibody itself has some defects, such as high immunogenicity, instability, difficult chemical modification, batch-to-batch variability, production Limited methods and high production costs, etc., make it subject to certain limitations in basic research and clinical application

Method used

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  • Nucleic acid aptamer capable of specifically recognizing vimentin and application thereof
  • Nucleic acid aptamer capable of specifically recognizing vimentin and application thereof
  • Nucleic acid aptamer capable of specifically recognizing vimentin and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Embodiment 1, the screening of nucleic acid aptamer

[0061] 1. Random library design and synthesis

[0062] Design and synthesize a random library with 18 fixed nucleotides at both ends and 40 nucleotides in the middle: 5'- ATCCAGAGTGACGCAGCA -N(40)- TGGACACGGTGGCTTAGT -3'; N(40) represents a random nucleotide sequence of 40 A, T, C or G.

[0063] 2. SELEX screening

[0064] The SELEX screening process is divided into two parts: positive screening and negative screening. Specific steps are as follows:

[0065] 1. Positive screening

[0066] Incubate 10 μg of recombinant human Vimentin protein (6X histidine tag) with an appropriate amount of Ni-Sepharose Beads (Ni-Sepharose Beads) at room temperature for 30 minutes to couple to Sepharose Nickel Beads, Binding Buffer Ⅰ (1% BSA, 0.1% Tween -20, 0.2mg / mL tRNA, DPBS, pH7.4) resuspended for use, washed to remove unbound substances, and used as the target for forward screening. The 10OD (about 14nmol) random library ...

Embodiment 2

[0082] Example 2, the binding ability of nucleic acid aptamer AptVim and Vimentin protein

[0083] 1. Flow cytometry experiment and Dot Blot experiment to detect the binding of nucleic acid aptamer AptVim and Vimentin protein

[0084] The 5'-end Cy3-modified AptVim sequence and the 5'-end biotin-modified AptVim sequence were synthesized according to the method in Example 1, respectively, and their binding to Vimentin protein was detected by flow cytometry and Dot blot experiments. The specific detection method is the same as the method in Step 3 of Example 1.

[0085] The flow detection results and Dot Blot detection results are as follows: Figure 4 and Figure 5 shown. The results showed that the nucleic acid aptamer AptVim obviously combined with Vimentin protein, but not with 6X histidine polypeptide.

[0086] 2. Affinity determination of nucleic acid aptamer AptVim

[0087] In order to determine the affinity between the nucleic acid aptamer AptVim and Vimentin protei...

Embodiment 3

[0099] Example 3. Nucleic acid aptamer AptVim induces Vimentin positive cells to undergo apoptosis

[0100] Panc-1 cells and BxPC-3 cells were seeded in 6-well plates and cultured overnight, and were divided into the following groups according to different treatment reagents:

[0101] AptVim+Turbofect treatment group: after the nucleic acid aptamer AptVim (adjust the concentration of the nucleic acid aptamer AptVim to 1 μM with serum-free DMEM) was packaged by Turbofect, the cells were treated for 24 hours;

[0102] Turbofect treatment group: Turbofect treated cells for 24 hours;

[0103] Turbofect+random library treatment group: random library (serum-free DMEM adjusted random library concentration to 1 μM) was packaged by Turbofect, and the cells were treated for 24 hours;

[0104] AptVim treatment group: Without Turbofect package, the cells were directly treated with the nucleic acid aptamer AptVim for 24 hours, and the final concentration of the nucleic acid aptamer AptVim...

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Abstract

The invention discloses a nucleic acid aptamer capable of specifically recognizing vimentin and an application thereof, wherein the sequence of the nucleic acid aptamer is shown in SEQ ID No. 1. The nucleic acid aptamer in AptVim of the present invention specifically binds to vimentin and has a Kd value of 70.9+ / - 12.3nM. After the terminal of AptVim is connected with biotin or a fluorescent molecule, the traditional vimentin antibody can be replaced, and the detection of cell expression or tissue expression of the vimentin protein usually can be completed in one step, so that the reaction conditions are simpler, the stability is higher, and the cost is lower. In addition, the nucleic acid aptamer of the present invention can induce apoptosis of tumor cells after the nucleic acid aptamer of the invention is introduced into tumor cells through a proper transfection reagent. Not only can the nucleic acid aptamer be used as an EMT diagnostic reagent for basic experiments and clinical cancers, but also induce the apoptosis of tumor cells for tumor treatment.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a nucleic acid aptamer specifically recognizing Vimentin and its application. Background technique [0002] Vimentin (Vimentin, Vim) is an intermediate filament protein mainly expressed in mesenchymal cells and mesoderm-derived cells. Vimentin is highly conserved in evolution and participates in a variety of physiological processes: maintaining cytoskeleton morphology, promoting cell adhesion and migration, regulating cell apoptosis, signal transduction, gene regulation, etc. Vimentin is the main secretory protein in the process of epithelial-mesenchymal transition (EMT) in tumor cells, so it has become an important marker for identifying EMT in recent years. A large number of studies have shown that Vimentin is overexpressed in a variety of epithelial malignant tumors. By participating in EMT and a variety of complex credit pathways, it accelerates tumor growth, promote...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115G01N33/68G01N33/574A61K31/711A61P35/00
CPCA61P35/00G01N33/57484G01N33/68A61K31/711C12N15/115C12N2310/16
Inventor 周蒙滔孙红光吴施佳张杰
Owner THE FIRST AFFILIATED HOSPITAL OF WENZHOU MEDICAL UNIV
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