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Nucleic acid aptamer specifically recognizing vimentin and its application

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

Active Publication Date: 2022-06-28
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 specifically recognizing vimentin and its application
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  • Nucleic acid aptamer specifically recognizing vimentin and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1. Screening of nucleic acid aptamers

[0061] 1. Design and synthesis of random library

[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] 10 μg of recombinant human Vimentin protein (6X histidine tag) was incubated with an appropriate amount of nickel agarose beads (Ni-SepharoseBeads) for 30 minutes at room temperature, and then coupled to nickel agarose beads, Binding BufferⅠ (1%BSA, 0.1%Tween) -20, 0.2 mg / mL tRNA, DPBS, pH 7.4) resuspended for use, washed to remove unbound substances, and used it as the target for forward screening. The 10 OD (about 14 nmol) r...

Embodiment 2

[0082] Example 2. Binding ability of nucleic acid aptamer AptVim to Vimentin protein

[0083] 1. Flow cytometry and Dot Blot experiments to detect the binding of nucleic acid aptamer AptVim to 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 assay and Dot blot assay. The specific detection method is the same as the method in Step 3 of Embodiment 1.

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

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

[0087] In order to determine the affinity of the nucleic acid aptamer AptVim and Vimentin protein, the final workin...

Embodiment 3

[0099] Example 3. Aptamer AptVim induces apoptosis of Vimentin-positive cells

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

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

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

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

[0104] AptVim treatment group: cells were directly treated with nucleic acid aptamer AptVim for 24 hours without Turbofect wrapping, and the final concentration of nucleic acid aptamer AptVim was 1 μM.

[0105] NC contr...

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Abstract

The invention discloses a nucleic acid aptamer capable of recognizing Vimentin and its application, the sequence of which is shown in SEQ ID No.1. The nucleic acid aptamer AptVim of the present invention can specifically bind Vimentin, with a Kd value of 70.9±12.3nM. After attaching biotin or fluorescent molecules to the end of AptVim, it can replace the traditional Vimentin antibody. Usually, it only takes one step to complete the detection of cell expression or tissue expression of Vimentin protein. Not only the required reaction conditions are simpler, but also the stability is higher and the cost is lower. . In addition, after the nucleic acid aptamer of the present invention is introduced into tumor cells by a suitable transfection reagent, it can induce tumor cell apoptosis. Not only can it be used as an EMT diagnostic reagent for basic experiments and clinical cancers, but it can also induce apoptosis of tumor cells for tumor treatment.

Description

technical field [0001] The invention belongs to the field of biotechnology, and particularly relates to a nucleic acid aptamer that specifically recognizes Vimentin and its application. Background technique [0002] Vimentin (Vim) is an intermediate filament protein mainly expressed in mesenchymal cells and cells derived from mesoderm. Vimentin is highly conserved in evolution and participates in various physiological processes: maintaining cytoskeleton morphology, promoting cell adhesion and migration, regulating apoptosis, signal transduction, gene regulation, etc. Vimentin is the main secreted protein in the process of epithelial-mesenchymal transition (EMT) of tumor cells, so it has become an important marker for the identification of 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, promotes t...

Claims

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Application Information

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Patent Type & Authority Patents(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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