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Nucleic acid aptamer specifically binding to TIMP1 protein and application of nucleic acid aptamer

A nucleic acid aptamer and species-specific technology, applied in biochemical equipment and methods, instruments, analytical materials, etc., to achieve the effect of no batch difference, easy storage, and small molecular weight

Pending Publication Date: 2021-07-16
HANGZHOU GUANGKEANDE BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The sequence information of nucleic acid aptamers against TIMP1 has not been reported so far, so there is a need in this field for nucleic acid aptamers with high affinity to TIMP1

Method used

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  • Nucleic acid aptamer specifically binding to TIMP1 protein and application of nucleic acid aptamer
  • Nucleic acid aptamer specifically binding to TIMP1 protein and application of nucleic acid aptamer
  • Nucleic acid aptamer specifically binding to TIMP1 protein and application of nucleic acid aptamer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0102] Screening of nucleic acid aptamers specifically binding to TIMP1 protein

[0103] 1. The ssDNA sequence that specifically binds to TIMP1 was screened from the ssDNA library by magnetic bead method to obtain an enriched library. The protein screening kit used was purchased from Anhui Aptamy Biotechnology Co., Ltd. (Product No.: SEP -201903);

[0104] a. Coupling the carboxyl magnetic beads with the positive sieving target and the reverse sieving target substance respectively after activation treatment, to prepare positive sieving magnetic beads and reverse sieving magnetic beads;

[0105] b. Incubate and separate the anti-screening magnetic beads and the random nucleic acid library to obtain the supernatant;

[0106] c. incubating the supernatant obtained in step b with positive sieve magnetic beads, and then washing and eluting to obtain an eluent;

[0107] d. Take the eluate for PCR amplification, and then prepare DNA single strands to obtain a primary library;

[0...

Embodiment 2

[0113] Surface plasmon resonance (SPR) detection of binding of TIMP1 nucleic acid aptamer to TIMP1 protein

[0114] 1. Dilute the nucleic acid aptamer SEQ ID No: 1-8 to 1 μM with DPBS buffer;

[0115]2. Coupling TIMP1 protein and 8His polypeptide to the first and second channels on the surface of the CM5 chip: first wash the chip with 50 mM NaOH, inject 30 μL at a flow rate of 30 μL / min, and then use an equal volume of EDC (1-(3 -Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; 0.4M aqueous solution) and NHS (N-hydroxysuccinimide; 0.1M aqueous solution) were mixed and injected into 50 μL activated chip, Flow rate 10 μL / min. The TIMP1 protein and 8His polypeptide were diluted with 10mM sodium acetate at pH 4.5 to a final concentration of 50μg / mL before injection, the injection volume was 50μL, the flow rate was 10μL / min, the coupling amount of TIMP1 protein was 5500Ru, and the coupling amount of 8His polypeptide was 600 Ru. After the sample injection is completed, inj...

Embodiment 3

[0118] Nitrocellulose filter binding assay to verify the specificity of the nucleotide sequence shown in SEQ ID No.1-8 binding to TIMP1 protein

[0119] 1. Use PBS solution to dilute TIMP-1 protein, AFP protein, CA199 protein, CEA protein, BSA protein, and 8His polypeptide to 25 μg / mL;

[0120] 2. Use PBS solution to dilute FAM fluorescently modified to nucleic acid aptamers TIMP1-04, TIMP1-11, TIMP1-18, TIMP1-20, TIMP1-21, TIMP1-49, TIMP1-58, TIMP1-61 to 1 μM, Denaturation treatment: Denaturation at 95°C for 10 minutes, then immediately ice-bathed for 5 minutes, and then equilibrated at room temperature for 10 minutes;

[0121] 3. Mix the refolded nucleic acid aptamer with TIMP-1 protein, AFP protein, CA199 protein, CEA protein, BSA protein, and 8His polypeptide in equal volumes, and incubate at room temperature for 1 hour;

[0122] 4. Filter the incubated mixed solution through a nitrocellulose membrane (using a Bio-Dot SF microfiltration device), use an imaging system to t...

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Abstract

The invention discloses a nucleic acid aptamer specifically binding to TIMP1 protein and application of the nucleic acid aptamer. The nucleic acid aptamer sequence comprises at least one of the following four nucleotide sequences: A, a DNA sequence as shown in SEQ ID No.1-8; B, a DNA sequence having more than 60% of homology with the DNA sequence as shown in SEQ ID No.1-8; C, a DNA sequence hybridized with the DNA sequence as shown in SEQ ID No.1-8; D, an RNA sequence transcribed by the DNA sequence as shown in SEQ ID No.1-8; and wherein, the above four nucleotide sequences can be specifically combined with TIMP1 protein. The invention also discloses a nucleic acid aptamer derivative; and the invention also discloses application of the nucleic acid aptamer or the nucleic acid aptamer derivative. The nucleic acid aptamer of the invention can specifically bind to the TIMP1 protein, and has the advantages of small molecular weight, stable chemical property, easiness in synthesis, short production time, low cost, easiness in storage and marking, no batch difference, etc.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a nucleic acid aptamer specifically binding to TIMP1 protein and its application. Background technique [0002] TIMP1 protein is a glycoprotein that can be expressed from various tissues of organisms. This protein is an inhibitory enzyme of matrix metalloproteinases. The main secreting cells of TIMP-1 are a variety of connective tissue cells, which are considered to be the main regulators of MMPs activity in tissues. It can form a 1:1 complex with MMPs family members in a covalent manner, thereby exerting its inhibitory effect. Both work together to maintain extracellular matrix homeostasis. At present, many studies have shown that TIMP1 protein is related to the occurrence and development of various cancers and tumors such as colorectal cancer, lung cancer, and pancreatic cancer. Therefore, TIMP1 protein can be used as a diagnostic marker for related tumors and cancers and a targe...

Claims

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Application Information

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IPC IPC(8): C12N15/115G01N33/574
CPCC12N15/115G01N33/57484C12N2310/16
Inventor 高俊顺高俊莉李学明
Owner HANGZHOU GUANGKEANDE BIOTECHNOLOGY CO LTD
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